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The Bioinorganic Chemistry of Mammalian MetallothioneinsClick to copy article linkArticle link copied!
- Artur KrężelArtur KrężelDepartment of Chemical Biology, Faculty of Biotechnology, University of Wrocław, Wrocław 50-383, PolandMore by Artur Krężel
- Wolfgang Maret*Wolfgang Maret*[email protected]Departments of Biochemistry and Nutritional Sciences, School of Life Course Sciences, Faculty of Life Sciences and Medicine, King’s College London, London SE1 9NH, U.K.More by Wolfgang Maret
Chemical Reviews
Cite this: Chem. Rev. 2021, 121, 23, 14594–14648
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Abstract
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The functions, purposes, and roles of metallothioneins have been the subject of speculations since the discovery of the protein over 60 years ago. This article guides through the history of investigations and resolves multiple contentions by providing new interpretations of the structure-stability-function relationship. It challenges the dogma that the biologically relevant structure of the mammalian proteins is only the one determined by X-ray diffraction and NMR spectroscopy. The terms metallothionein and thionein are ambiguous and insufficient to understand biological function. The proteins need to be seen in their biological context, which limits and defines the chemistry possible. They exist in multiple forms with different degrees of metalation and types of metal ions. The homoleptic thiolate coordination of mammalian metallothioneins is important for their molecular mechanism. It endows the proteins with redox activity and a specific pH dependence of their metal affinities. The proteins, therefore, also exist in different redox states of the sulfur donor ligands. Their coordination dynamics allows a vast conformational landscape for interactions with other proteins and ligands. Many fundamental signal transduction pathways regulate the expression of the dozen of human metallothionein genes. Recent advances in understanding the control of cellular zinc and copper homeostasis are the foundation for suggesting that mammalian metallothioneins provide a highly dynamic, regulated, and uniquely biological metal buffer to control the availability, fluctuations, and signaling transients of the most competitive Zn(II) and Cu(I) ions in cellular space and time.
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License Summary*
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Copyright © 2021 The Authors. Published by American Chemical Society
1. Introduction
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Three human generations have witnessed the development of the field of metallothioneins. It is crucial to start the narration with the early history to understand the facts and why and how contentions developed. In a discussion of discoveries seriatim, the etymology of “metallothionein” and the epistemology of the field will foster an understanding of how our present ideas about metallothionein (MT) were shaped. Trials and tribulations emerged from some assumptions in lieu of facts and from not interpreting chemistry in the context of biology. Resolutions of conjectures had to await additional discoveries, especially insights into the molecular basis of how cellular metal metabolism is controlled. MT is emblematic of the difficulties faced when trying to define functions of proteins without known activities. Seen with this perspective, the search for a function of an entire family of proteins turns out to be a quite educational lesson in the history of science. The article will start with the discovery of metallothioneins and definitions of the term with an account of the first 40 years of MT research culminating in the determination of three-dimensional (3D) structures of mammalian MTs. It will then address two significant developments that challenged the general perception about their structure, chemical reactivity, and metal complexation, proceed to discuss how the chemical properties of MTs are limited by the biological environment and how the highly dynamic properties of MTs relate to our current knowledge in mammalian metal biochemistry, and finally attempt a synthesis of our knowledge with the goal of advancing the understanding of their functions. By necessity, a scientific literature with over 20 000 original articles, numerous reviews, books, and conference proceedings will have to be reduced to ∼2% with representative articles. We hope that we do due diligence in citing key articles from a wide community of scientists whose disciplines have been and will be impacted by the fundamental roles of MTs.
1.1. Search for a Role of Cadmium in Biology: Discovery of Metallothionein
In the middle of the last century, an ongoing objective was to find biomolecules associated with the metal ions present in biological tissues (“biometals”). On the basis of reports in the late 1940s that cadmium is present in various living species, Margoshes and Vallee began to address the question whether cadmium is naturally present for an essential function or as a result of environmental contamination. Incidentally, the same question was posed for zinc 30 years earlier. (1) Among different animals screened, they chose equine kidney cortex because of its relatively high cadmium content. In 1957, they reported the isolation of a Cd(II)-binding protein from horse kidney. (2) They noted that the protein also contains zinc and that it does not absorb light at 280 nm, signifying the absence of aromatic amino acids. Further purification and characterization of the protein showed the presence of 2.9% cadmium, 0.6% zinc, and 4.7% sulfur (per gram of dry weight), the latter in the form of cysteine bound to the metal ions. (3) Since the protein contained different metal ions and is rich in sulfur, Kägi and Vallee named the holoprotein metallothionein and the apoprotein thionein. After clarifying onomastics, there is an issue of semantics from the beginning, namely, focusing the field on metallothionein (MT) with different metal ions and not on the protein thionein (T). On the ribosome, T is made, and whether or not MT is formed depends on the tightly controlled availability of metal ions. Further purification of MT using anion-exchange chromatography yielded two fractions and refined the composition of the major fraction to 5.9% Cd, 2.2% Zn, 0.2% Fe, 0.1% Cu, and 9.5% sulfur. (4) The electronic absorption of the protein at 250 nm was assigned to Cd-mercaptide bonds; 95% of the sulfur was found in the form of cysteine thiols. A comparison between MT from the liver and the kidneys of horses showed that the former contains mainly zinc. (5) Likewise, MT from adult human liver contains mostly zinc and small amounts of copper and cadmium. (6) In human and bovine fetal liver, however, a major Cu(I)-binding protein was identified as MT. (7,8) Thus, the metal composition depends on the tissue from which MT is isolated, containing mainly Zn(II), Cu(I), or Cd(II) or a combination of these metal ions. It also depends on the specific physiological and pathological condition of the tissue and its developmental stage. Ensuing work confirmed the presence of MT in basically all tissues from different mammalian species, albeit at different amounts. The conspicuous properties of binding rather high amounts of different metal ions, the large number of cysteines, and the absence of aromatic amino acids are “unusual and unconventional” for most proteins and guided the search for a function for years to come.
1.2. MT1 and MT2
Following initial reports of isolated horse kidney MT being heterogeneous with two or even three components detected by electrophoretic and chromatographic methods, two forms of liver metallothionein, MT-I and MT-II, were prepared from rabbits that received cadmium injections, which Magnus Piscator demonstrated to induce the protein. (9,10) The forms had pI values of 3.9 and 4.5 and showed slightly different amino acid and metal (Zn vs Cd) composition. Two fractions were obtained from human liver after anion-exchange chromatography at pH 8.6 and called MT1 (eluting first) and MT2 (eluting second) with the former having 6.05 total metal and the latter 7.24 total metal per protein. (6) These isolations, however, did not reveal the molecular identity of the forms. The difference between the two charge-separable forms MT1 and MT2 was later found to be due to an aspartate at position 11 in MT2.
1.3. MT1 Proteins
An additional purification step, that is, reversed phase high-performance liquid chromatography (RP-HPLC) at pH 7.5, demonstrated MT2 to be homogeneous but resolved five different forms of MT1 from human liver. (11) This observation was preceded by a report that the human genome contains at least a dozen MT genes. (12) The multiplicity and molecular identity of MT1 proteins will be addressed later in this article.
Two additional mammalian MTs were discovered later in 1991 and 1994 in specific tissues, one via a biological assay and the other through molecular cloning. They define a different branch in the evolutionary tree, and their discovery brought the number of expressed human MTs to at least 11. We will briefly describe these additional forms and then resume the chronology of discoveries.
1.4. MT3 (GIF)
A growth inhibitory factor (GIF) that is deficient in Alzheimer’s disease brain turned out to be a new metallothionein. (13) It has 68 amino acids, that is, seven additional amino acids compared to the MT1/MT2 proteins, with an additional amino acid (Thr) at position 5 near the N-terminus and a six amino acid, Glu-rich insert near the C-terminus. Otherwise the cysteines are conserved. The GIF activity, which refers to inhibiting the survival and neurite outgrowth of cortical neurons in vitro, is not shared by other MTs and abolished by specific mutations, in particular, replacing the prolines in the unique CPCP sequence starting at position 6. (14) Though GIF is the first MT found on the basis of a biological activity, the molecular basis for the GIF activity remains unknown. Isolated GIF contains Cu(I) and Zn(II), but the zinc protein is active as well. As a new member of the MT family, the protein was subsequently named MT3 and found not to be induced by metal ions or other common inducers of MTs. (15) It is, however, inducible, for example, by hypoxia, and this aspect of regulation is discussed below in section 5.1.
1.5. MT4
MT4 was discovered by molecular cloning. (16) Like MT3, it has an insertion at position 5 but, in this case, a glutamate instead of a threonine; it contains Cu(I) in addition to Zn(II) when isolated, and the positions of the cysteines are conserved. Its expression was found to be rather restricted to stratified squamous epithelia. It is believed to be important for the differentiation of these epithelia.
2. The Structures of Mammalian Zinc and Copper Metallothioneins
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2.1. Primary Structures
The first protein sequence determined was that of horse kidney MT1b. (17) It showed a special clustering of the cysteines in CC (3), CXC (7), and CXXC (2) motifs, and an absence of histidine and aromatic amino acids as noted earlier (Figure 1). The protein has 61 amino acids; thus, it is a relatively small protein, in agreement with earlier determinations of its molecular mass of 6000 Da. Noteworthy are the presence of seven Ser-Cys pairs and the association of cysteines with basic amino acids in four Cys-Lys, three Lys-Cys, and one Cys-Arg pair (Figure 1).
Figure 1
Figure 1. Amino acid sequence of horse MT1b with the Cys-containing motifs indicated, including CC (blue), CXC (red), CXXXC (green), SC (gray), and CK, KC, CR (underlined).
All sequences are referred to as metallothionein, though they represent the protein thionein only. The sequence per se does not indicate the types and numbers of bound metal ions, an issue to keep in mind as critical for the discussion to follow. Until today, investigators variably refer to the holoprotein in two ways, for example, copper metallothionein (employing the given name for the protein and indicating the type of metal ion bound) or copper thionein (employing the name of the protein and indicating the metal bound). Although the first term is a pleonasm, it is used more widely, similar to, for example, a zinc protein being called a zinc metalloprotein.
When sequences of small sulfur- and metal-rich proteins from invertebrates and fungi became available, it became obvious that the sequence similarity among so-called MTs is limited. A division into three classes was proposed: (18) class I─polypeptides with cysteine motifs closely related to horse kidney MT, class II─polypeptides with cysteine motifs distantly related to horse kidney MT, and class III─nontranslationally synthesized metal–thiolate polypeptides, such as phytochelatins. This division exacerbated the problem of nomenclature: What constitutes a metallothionein? This article focuses on mammalian class I MTs as the quintessential MTs for which sufficient information is available to discuss the structure–function relationships. Mammalian MTs are the prototypes on which most of the work has been performed, and they define the field.
The issue lingers why gene duplications for MT1 proteins occurred in some but apparently not all mammalian species. (19) The term MT1 has historic reasons to distinguish the protein(s) from MT2 on the basis of a charge difference due to Asp(D) in MT2 (at position 13 in the alignment) (Figure 2). MT3 and MT4 do not have an Asp at this position. All 20 cysteines are conserved in the human MT family. The total number of strictly conserved amino acids is 29.
From the standpoint of evolution, MT1 proteins and MT2 are in one phylogenetic branch, while MT3 and MT4 are in a different branch (Figure 3). Gene duplication occurred before speciation occurred, indicating functional differentiations. (22)
If one extends the analysis from mammalian MTs to examples of vertebrate MTs, at least three of the original tenets that define a prototype MT already break down (Figure 4). Only 16 out of 20 cysteines are strictly conserved, a His is found at the C-terminus of chicken MT, and an aromatic amino acid (Tyr) is found in African chameleon MT. This variability is important to note, as some human MTs were thought not to form functional proteins simply on the basis that they contain “unusual” amino acids.
2.2. Metal Composition: Zn(II), Cd(II), Cu(I)
It is essential to understand how MT is prepared for a chemical characterization in the ensuing work to determine its structure that serves as a pivotal point of reference. Several methods are employed, and they include harsh treatments. Since the metal-containing protein is rather heat-stable, a common assay, the so-called hemoglobin-binding assay, includes a step of boiling the tissue homogenate at 100 °C for 1 min. (25) A standard procedure for the isolation of the protein includes the precipitation of a tissue homogenate with a solution of 96% ethanol/chloroform (1.05:0.08). (26) The purification of MT is followed by an assay of its metal content. Hence any apoprotein (thionein) that may exist in tissues will not be detected unless it copurifies. Another matter─and a limitation─is that MT is prepared from cytosolic fractions. Therefore, MT present in cellular organelles is also not investigated routinely. Because of the harsh treatment, one cannot exclude that the heterogeneity of the isolated MT in terms of different bound metal ions, at least in part, is due to metal binding or metal swap during isolation. To prepare homometallic forms after isolation, T is prepared by removing Zn(II) or Cd(II) by an acidification of MT to pH 2 in the presence of dithiothreitol (DTT) to reduce any oxidized sulfhydryls either initially present or formed during isolation. In order to remove Cu(I), a short exposure to pH 1 or a treatment with diethyldithiocarbamate at pH 5 is necessary. (27) The protein is rather resistant to acid treatment, except for an Asp-Pro bond following the N-terminal Met. T thus obtained is then subjected to gel filtration and reconstituted with seven molar equivalents of divalent metal ions. (28) In this way of isolation and preparation, information about the natural metal composition and redox state of MT is lost, and one chooses intentionally a particular stoichiometry with a single metal ion based on an average content of approximately seven metal ions (sum of Zn(II), Cd(II), Cu(I)) generally found in isolated MTs and based on spectroscopic data that demonstrate a titration breakpoint when seven divalent metal ions are added. (28) Working with this stoichiometry, one assumes that metal ions are available in vivo to saturate the protein and to reach such a stoichiometry. This assumption is certainly not correct for Cd(II). To increase the yield of isolated MT, it is often induced with Cd(II) in laboratory animals, thus biasing the metal content toward the inducing metal ion. Even when rather high doses of Cd(II) cadmium are injected into an animal, the maximum incorporation of Cd(II) yields Cd5Zn2MT. (29) Cd7MT can be made in vitro but does not form in vivo. (30) A stoichiometry of seven metal ions has been referred to as the “magic number”. (31) Its significance is at the center of our discussion of structure and function later in this article.
Figure 2
Figure 2. Sequences of human MT1–4 with conservation of Cys residues. There is one exception, though: human MT1b has an extra cysteine, yielding a total of 21 Cys. Only some of the Lys (K) residues are conserved, one CKC motif in the N-terminal part and three (KCA, CKG, and KCS) in the C-terminal part─KCS is not conserved in MT1m and MT1b, though. The KK motif in the middle of the protein is conserved with the exception of MT4, where it is RK. Blue codes on the right denote UniProt entries. Alignment was performed using MAFFT software (20) and visualized using Jalview. (21) Yellow and black bars stand for conservation and consensus, respectively.
Figure 3
Figure 3. Phylogram (left) and cladogram (right) of human metallothioneins (based on protein structures, translation of pseudogenes is not included). MT2 is part of the MT1 branch, while MT3 and MT4 have a separate root. The tree was generated in Clustal Omega using the neighbor-joining clustering method and visualized by iTOL. (23,24) The bar indicates the number of changes per residue. The number 0.01 corresponds in length to a 1% difference in sequences.
Figure 4
Figure 4. Alignment of MT sequences from representative species of vertebrates. In this selection, 23 amino acids are strictly conserved, but among them only 16 Cys are strictly conserved. Besides Coelacanth and African chameleon MTs, all other MTs contain 20 Cys residues. Yellow and black bars below the alignment show the patterns of conservation and consensus. Blue codes on the right denote UniProt entries. Alignment was performed using MAFFT software (20) and visualized using Jalview. (21) Yellow and black bars stand for conservation and consensus, respectively.
2.3. 3D Structures
2.3.1. Structures of the Metal Sites
With 20 cysteines and seven divalent metal ions resulting in a metal–sulfur stoichiometry close to 1:3 and every metal ion bound in a tetrahedral coordination environment with sulfur donor ligands, (33) there are not enough cysteines to bind the metal ions individually, as 28 would be needed to do so. Therefore, a solution to the riddle of the coordination structure was not a straightforward matter. It turned out to be an exceptional, homoleptic coordination in bioinorganic chemistry in the form of “clusters” with bridging and terminal sulfurs of cysteines. (34) The cluster organization was known before the 3D structure of the protein was solved. On the basis of 113Cd NMR and the homonuclear coupling of the cadmium nuclei, it was proposed that the metal ions are organized in two clusters in rabbit liver MT, a three-metal cluster with three sulfur bridges and a total of nine cysteine sulfurs bound and a four-metal cluster with five sulfur bridges and a total of 11 cysteine sulfurs bound. (35) These stoichiometries result in an overall net charge of −3 for each cluster. MT1 and MT2 are indistinguishable in terms of the metal environments. The clusters can be described as cyclohexane-like (chair) and adamantane-like (tricyclic structure not fully formed) (Figure 5). (36) A limited proteolysis of the apoprotein led to the preparation of a C-terminal peptide in which four metal ions reside and the suggestion of a structure of MT with domains. (37) The reconstitution of this peptide with Cd(II) and a demonstration by 113Cd NMR that it contains the four-metal cluster established that the protein has two domains, with each one harboring one of the clusters. (38)
Remarkably, seven Cd(II) resonances are resolved in the 113Cd NMR spectra, demonstrating that each metal ion resides in a slightly different environment despite each Cd(II) ion being surrounded by four sulfurs in a tetrahedral geometry. The group of Kurt Wüthrich then went on to determine which cysteines are bound to each Cd(II) ion from the heteronuclear 113Cd–1H connectivities (Figure 5). (40) It revealed a criss-cross pattern of binding, in which the ligands are not employed linearly in the sequence and are therefore not predictable from (a) signature(s). MxCysy clusters are not restricted to MTs. A Zn3Cys9 cluster─characteristic for the β-domain of mammalian MT─is present in domains of histone lysine methyltransferase (41) and the E3 ubiquitin-protein ligase of male-specific lethal 2 (MSL2). (42) Further examples of Zn(II)/thiolate clusters in proteins will be discussed in section 5.1 with reference to Figure 26. The primary sequences of these proteins harboring the clusters, however, differ significantly from those of MTs in the pattern of cysteine residues and the presence of histidine and hydrophobic/aromatic residues that promote the formation of more stable secondary structures. In the crystal structure of the corresponding domain of the human histone methyltransferase ASH1L, only two Zn(II) were found to be bound to seven out of the nine Cys residues, indicating differences in the Zn(II) occupancy with functional implications yet to be determined. (43)
2.3.2. Structures of the Proteins
The protein is virtually devoid of a secondary structure in the absence of metal ions. In their presence, several half-turns and two 310 helix segments are formed, especially in the α-domain. With disorder-producing amino acids (G,S,P), no aromatic amino acids, and few aliphatic amino acids (I,V,L), both of which support the formation of a secondary structure, T classifies as an intrinsically disordered protein. Solution structures of rabbit, rat, and human Cd7MT2 were determined by NMR spectroscopy before a revised crystal structure of rat MT became available. (44,45) The peptide chain wraps around the clusters in either a left-handed (α-domain) or right-handed (β-domain) manner (Figure 6). With NMR spectroscopy, it was possible to obtain structures of the individual domains, but how the domains are positioned relative to each other remained unknown and had to await X-ray diffraction investigations in crystals of MT.
Figure 5
Figure 5. NMR assignments of Cd(II)/thiolate coordination environments in the MT clusters. Cluster A (or α) is in the C-terminal α-domain, and cluster B (or β) is in the N-terminal β-domain. Black and red numbers indicate Cys residues in the rabbit MT2 sequence and the Cd(II) ions assigned from 113Cd NMR (inset). (35,39)
Crystallography was performed on Cd5Zn2MT2 from rat liver, and the structure was refined to 2.0 Å (Figure 7). (47) The two Zn(II) ions reside in the β-domain cluster (N-terminal domain). The domains are aligned linearly and are connected by H bonds between the side chain of K31 and C21 and the sulfur atom of C19. This interaction includes a phosphate ion that interacts with the carbonyl of C19 and the side chain of K31. Besides H bonds between the domains, there are H bonds in each domain, and those between sulfur atoms and peptide amides (N–H···S) deserve special mention due to a charge compensation. Likewise, Ser residues in both domains are responsible for a stabilization of the structure by enhancing the H-bonding network, which is discussed further in section 3.2 with reference to Figure 15. Lys residues are an additional factor responsible for the charge compensation. They are conserved in the α-domain but not in the β-domain. The protein forms a dimer in the crystals, and there are several intermolecular contacts. A sodium ion binds to the carbonyl of C29 from one subunit, the carbonyl of A42 and the side chain oxygen of S45 of the other subunit, and three water molecules. Thus, in addition to describing the tertiary structure of the protein, crystallography also demonstrated that MTs can have a quaternary structure. The quaternary structure may have biological implications for the polymerization of the protein and its intermolecular metal exchange as discussed in sections 2.3 and 3.1.
X-ray crystal and NMR solution structures of rat MT2 are identical. (48) The 3D structures with Cd(II) and Zn(II) differ in cluster size only. The 3D structure with Cu(I) is different, however, demonstrating that not only the metal itself but also the type of metal determines the conformation of the protein.
An NMR solution structure of the α-domain of MT3 shows that the metal cluster is identical with that in MT2, but the additional amino acid residues in the α-domain of MT3 have a significant impact on the structure of an extended loop as observed in the individual domains and in the entire protein (Figure 8). (49,50)
Figure 6
Figure 6. NMR structures of mammalian α-domains (PDB: 1MRB, 1MRT, 1MHU) and β-domains (PDB: 2MRB, 2MRT, 2MHU) of MT2. (A) Comparison of the structures of β (left) and α (right) domains of rabbit (blue ribbon), rat (beige ribbon), and human (pink ribbon) Cd7MT2. (B) Sequences of both domains with criss-cross Cd(II) (M) binding sites. (44−46)
The solution structures of the individual domain peptides of murine MT1 were also determined by NMR spectroscopy (Figure 9). Cu(I) titration into the 3-Zn(II) or 4-Zn(II) containing domains yielded defined structures when three Cu(I) ions reside in the α-domain and four in the β-domain. (51) Principally, with Cd(II) isotopes, a heteronuclear coupling allowed a determination of how the sulfur donor ligands connect with the metal ions and hence provided structures of the clusters. Since Zn(II) and Cu(I) do not have this NMR property, neither the cluster structures nor the number of Zn(II) ions remaining bound could be determined in this copper MT. Importantly, the protein structure is significantly different from those with divalent metal ions bound, emphasizing the role of the metal ions in organizing structure. When the two domain peptides were mixed they retained their Cu(I) stoichiometry and did not exchange Cu(I). When more than the seven Cu(I) ions were titrated into the two domains, no defined structures were observed. However, species with a higher number of Cu(I) ions have been observed by spectroscopic methods, for instance, Cu12MT with six Cu(I) ions in the α- and β-domain (51,52) or Cu15MT as confirmed by mass spectrometry. (52−54) It indicates the variability in Cu(I) coordination in forming diagonal and trigonal geometries as shown in the crystal structure of yeast copper MT (Cup1). (55) A trigonal geometry is expected to predominate at a low-copper load, while the chances for a diagonal geometry increase with more Cu(I) ions bound to MT.
Figure 7
Figure 7. Structure of the entire rat MT2 molecule solved by X-ray crystallography. (A) Structure of Cd5Zn2MT2 with the indication of Cd(II) (beige color) and Zn(II) (gray color) ions in both domains. (B) Connectivities of the metal ions with the Cys residues in the β-cluster. (47)
A third method for visualizing a 3D structure is scanning tunnelling microscopy (STM). It provides a direct image of the molecule in a buffered solution and shows the compact structure of MT with its elongated shape and the bound metal ions
Figure 8
Figure 8. NMR structure of the α-domain of human MT3 in the complex with Cd(II). (A) Comparison of the MT2 (blue) and MT3 (beige) structures of the individual domains (PDB: 1MHU vs 2FJ4). (B) Comparison of the MT3 structures of the individual domain (beige) and the one in the entire protein (pink) (PDB: 2F5H). (50)
Figure 9
Figure 9. NMR structures of Cu(I)-containing mouse MT1 domains. (A) 20-structure family of ZnyCu4βMT1; (B) 20 best structures of ZnxCu3αMT1; (C) superposition of the mean Cd3βMT1 structure (red) and the structure family of ZnyCu4βMT1 (blue); (D) superposition of the mean Cd4αMT1 structure (red) and the structure family of ZnxCu3αMT1 (blue). (A, B) Gray, blue, and yellow colors represent the polypeptide backbone, cysteinyl side chain, and sulfur atoms, respectively. Figures were adopted with permission from ref (51), copyright 2007 by John Wiley and Sons.
Figure 10
The clusters are not static. They are thermodynamically stable and kinetically labile. A fluxional behavior allows a facile exchange of Cd(II) or Zn(II) intramolecularly─within the clusters and between the clusters, with metal ions in solution, and intermolecularly between MT molecules. Cd(II) exchange within the three-metal cluster occurs with rate constants of 0.2–2.7 s–1 (35 °C), while an exchange within the four-metal cluster was too slow to be detectable. (58) On the basis of first principles, Zn(II) exchange rates should be an order of magnitude lower. Zn(II) exchange between rabbit liver MT1 and MT2 is biphasic with rate constants of 5000 and 200 min–1 M–1 (pH 8.6, 25 °C) and was suggested to reflect the intermolecular exchange between three-metal clusters and four-metal clusters, respectively. (59) However, it could also reflect the exchange of four tightly bound metal ions and three less-tightly bound metal ions in the protein in a nondomain-specific way (see below). While the kinetic lability has been discussed primarily in terms of the lability of the divalent metal ions, it also signifies lability in the coordination of the sulfur ligands. Metal exchange requires the thiolate ligands to dissociate, which enhances the reactivity of the thiolates.
2.4. From Structure to Reactivity and Regulation
Starting from approximately the early 1980s, molecular biology and genetics began to contribute to the field of MTs and widened its scope significantly. (60) New MT genes were cloned, defining mammalian MT gene clusters, and the mapping of genes identified their coding and noncoding regions and resulted in a much improved understanding of the regulation of gene expression and silencing of expression by DNA methylation. While the induction of MTs by many endogenous and exogenous substances and conditions of stress was known, (36) the DNA recognition elements of MTs, the transcription factors that bind to MTs, and signaling pathways that control induction became known. Incidentally, the concept of zinc fingers in transcription factors developed at approximately that time. It significantly changed the understanding and scope of the then-known Zn(II) coordination environments in proteins by revealing the prevalence of Zn(II) coordination to the thiolate sulfur of cysteine. Many of the transcription factors that control the MT expression turned out to be zinc proteins with a Zn(II)/thiolate coordination. The pathway of metal induction of MTs was established with the discovery of the metal response element (MRE)-binding transcription factor-1, MTF-1. (61) MTF-1 has six zinc fingers that are thought to be involved in sensing an excess of cellular Zn(II). Perhaps the most important outcome of all the new insights from molecular biology and genetics was that it showed that MTs are integrated into many pathways of the cellular signal transduction network, underscoring the fundmental importance of MTs in many aspects of cell biology: proliferation, differentiation, and programmed cell death (apoptosis). (62,63) How changes in MT and zinc availability are associated in the proliferation, differentiation, and apoptosis of cells is discussed at the end of section 3.1. Until now in this article, we discussed the structure without a reference to function─the holy grail of MT research. The initial characterization of MT as a cadmium protein in horse kidney cortex did not establish a biological function but led to speculation about its roles in catalysis, storage, immune phenomena, or detoxification. (3) The idea that T rather than MT might be the functionally important species was advanced. (5) Subsequent findings of MT1/2 being mainly zinc proteins in many tissues and containing copper under some conditions, MT3/4 in particular, then focused research on the metabolism of these essential elements. Because of the association with multiple metals and the dynamic changes under a variety of conditions, the protein was considered multifunctional. After ∼30 years of research, a consensus developed in numerous review articles that the protein primarily has a role in zinc metabolism overlapping with copper metabolism under specific conditions and with cadmium metabolism under conditions of exposure. However, the molecular mechanism eluded all attempts to define it. Bert L. Vallee compared MTs to sphinxes that guard their riddles (and the career paths of many!). There was some expectation that the emerging gene deletion technology of molecular biology would answer the question. (60) The hope was dashed however when mice with genetic knockouts of MT1 and MT2 were found to be viable under laboratory conditions; that is, the mice reproduced and grew. (64,65) The interpretation of these genetic experiments is discussed in section 6.2. Molecular biology and genetics added a lot of important information about the number of MT genes and their induction, but naturally such investigations did not focus on the protein and therefore did not further our understanding of its mechanism of action. On the basis of the exquisite regulation of gene expression, it was concluded that MTs have a pivotal role in cellular metabolism under normal conditions and conditions of stress. (66) After these initial 30 years, knowledge about the structural and biophysical properties of MT left the scientific community with the specious impression that each MT is essentially one form as determined by the structure of Zn7MT, the elucidation of which undoubtedly was a feat and a culmination in the work on MTs. The work indicated that MT sequesters metal ions and stores them with high affinity. We will now re-examine these postulates after describing subsequent discoveries that were game-changers. Contemporaneous advances in the field of zinc and copper biology make it clear, retrospectively, why the conundrum of function could not have been solved at this earlier point in time. It had to linger for the next 30 years despite a myriad of investigations in both biology, describing conditions of changed gene expression of MTs in health and disease, and chemistry, characterizing the binding of many different metal ions to MT. We believe that the time has finally come to interpret this vast literature in the context of what is now known about zinc and copper metabolism, thus leading to a full exploration of the functional potential and potential functions of MT.
At least three discoveries changed the view about a static, passive role of MT in storing metal ions to a dynamic, active one, in which the protein serves as a metal donor and acceptor: first, the reactivity of its sulfurs, in particular, their redox activity; second, the realization that not all the metal ions are bound with the same high affinity; and third, the complex and dynamic regulation of multiple MTs. In addition to the cysteine sulfurs as ligand donors to metal ions, a series of investigations emphasized their reactivity with other agents as part of the mechanism of action of MTs. The work challenged the dogma of MT as a protein with seven divalent metal ions bound with higher affinity than in most other metalloproteins. At that time, it demonstrated that the prevailing view about the structure of MT and its reactivity was too simplistic and suggested how MTs can have a much more dynamic role in metal metabolism as metamorphic or multimorphic proteins, the structures of which depend on the redox state (section 3.1) and the availability of metal ions (section 3.3), both of which change in biology. MTs turned out to be remarkably reactive, engendering many more structures than implied by their 3D structures. The significance of these dynamic aspects was amplified through discoveries not concerning MT itself but how Zn(II) and Cu(I) ions are controlled and traffic in the cell. The work transiting from structure to reactivity was a turning point and provided different and new perspectives on structures and functions.
3. Reactivities and Metal Affinities of Metallothioneins
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The two properties of the cysteine sulfhydryls, metal coordination and reactions with electrophiles, are linked, because metals will dissociate when the sulfhydryls are chemically modified. The linkage is important because the reactivity of the sulfhydryls depends on the metal load of MTs. Free thiols are generally more reactive, but Zn(II)/thiolates can react, too.
3.1. Redox Chemistry and Biology of MT
The observation that MT reacts with hydroxyl radicals with a 340-fold higher rate constant than glutathione shifted interest from the metals to the reactivity of the sulfurs in MT. (67) It resulted in a significant number of investigations of MT as a scavenger of free radicals. (68) With a possible biological significance for leukocyte biology, it was shown that the three principal neutrophil oxidants, namely, hydrochlorous acid, superoxide, and peroxide, mobilize Zn(II) from MT. (69) The dissociation of Zn(II) was thought to cause cellular injury, but a beneficial role of this process was not ruled out.
Reactions of the thiolates in MT with organic and inorganic electrophiles were known. (70) They include a thiol/disulfide interchange with Ellman’s reagent (5,5′-dithiobis(2-nitrobenzoic acid) (DTNB)), which occurs with the metal-bound thiolates in the clusters, albeit with a slower rate than with free thiols (71) as supported by quenching the reactivity when four Zn(II) ions are added to thionein. (72,73) This quenching supports the hypothesis that four Zn(II) ions are tigthly bound across the protein to make it more resistant to electrophilic attack. While the reaction with MT is biphasic, the reaction with the individual α-domain peptide containing Cd(II) is monophasic, suggesting that each cluster reacts separately. (74) However, the reaction of both individual domain peptides containing Zn(II) with an excess of DTNB is biphasic. (75) At sub-stoichiometric amounts, DTNB generates inter- and intramolecular disulfides in MT. Mixed disulfides form only at stoichiometric and higher amounts of DTNB. (76) A biological disulfide, glutathione disulfide (GSSG), triggers the dissociation of Zn(II) from MT. (76,77) While the first report concluded that GSSG is unlikely to react with MT in vivo, the second suggested that such a reaction would link the metal content of MT to the cellular glutathione redox state. Indeed, other biological disulfides such as coenzyme A disulfide and cystamine react more rapidly than GSSG, suggesting that (i) such chemistry is feasible with biological oxidants in the cell, (ii) MT is a Zn(II) donor when the cellular redox state changes to more oxidizing conditions, and (iii) conditions of oxidative stress perturb metal metabolism with important consequences for the progression of many diseases associated with oxidative stress such as neurodegeneration. (78) A comparative investigation of chemical and biological oxidants with different redox potentials was then employed to estimate the redox potential of the thiols in MT. (79) The redox potential of MT was found to be quite low, demonstrating that MT is a redox protein that reacts readily with a host of biological oxidants in the cellular milieu. This novel redox biology of MT suggested a way of how Zn(II) is made available when its availability is low and linked it to the known induction of T to sequester Zn(II) when its availability is high, (80) essentially establishing how the cellular redox state controls the cellular Zn(II) availability via the MT/T couple (Figure 11). (81)
Figure 11
Figure 11. Function of the MT/T couple as a homeostatic Zn(II) system. An increase in the amount of available Zn(II) induces the synthesis of T through the action of Zn(II) on Zn(II)-dependent transcription factors and leads to the formation of MT and a sequestration of Zn(II) (left). When available Zn(II) is low and needed for the synthesis of zinc proteins, Zn(II) dissociates from MT and T is formed (right). For clarity, the effects of oxidants and reductants are omitted. The redox effects are illustrated in Figure 13.
Such redox activity is remarkable because Zn(II) is redox-inert in biology, and zinc sites in zinc proteins therefore had not been considered to participate in redox metabolism. Thus, while transition-metal ions such as Fe(II)/Fe(III) and Cu(I)/Cu(II) are involved directly in redox reactions, in the case of Zn(II), another d-block metal but not strictly a transition metal (Zn(II) has a filled d-shell ion and therefore is not considered a transition-metal ion per IUPAC recommendation), (82) the sulfur donor ligand in the coordination environment and not the central atom confers redox activity on the complex. The redox chemistry and reactivity of the sulfur donor ligands in MT add an important principle to redox biology, which hitherto was based on either redox-active transition metals or sulfur chemistry in the absence of metals. (83) The new principle is that, in seemingly redox-inert zinc proteins, the thiolate ligand has a direct redox function and the dissociated metal ion an indirect function on redox metabolism. It is a typical example of a context-specific biological chemistry that turned out to be different from the focus of purely chemical in vitro investigations of MT until that point in time. Since the Zn(II)/thiolate coordination is quite frequent in cellular proteins, in particular, in the large number of zinc finger type of proteins, the redox biology of MT also inspired investigations into the redox functions of other zinc proteins with Zn(II)/thiolate coordination environments, in which catalytic or structural sites in proteins were shown to be redox-modulated. In this way, these proteins can serve as redox sensors and transducers when Zn(II) dissociation and disulfide formation change their conformation. Thus, unlike in MT, where Zn(II) dissociation serves as a trigger of biological events, the changed protein conformation can become the trigger. The rather extensive subject matter of the redox activity of Zn(II)/thiolate coordination in proteins other than MT has been reviewed and is beyond the scope of this article. (84−86)
The redox chemistry and redox biology of MT suggested a set of new functions of the protein. In particular, it initiated the search for the metal-free protein (TR, thionein) and the oxidized protein (TO, thionin). It was known that the MT fraction in homogenates of neoplastic cells was able to bind additional Zn(II), suggesting that T exists in cells together with MT. (87) Further evidence for the existence of forms of MT that are not fully saturated with metal came from the investigations on rat tissues, in which commensurate amounts of MT and T were found by employing a differential modification assay with ABD-F (7-fluorobenz-2-oxa-1,3-diazole-4-sulfonamide) (Figure 12A). (88,89) Because of various Zn(II)-to-thionein affinities (see below), we now understand that T (the species entirely devoid of metal ions) cannot coexist with MT (the species that is saturated with metal ions), and that instead Zn7–xMT forms are present in cells. (73)
Figure 12
Figure 12. Differential modification of MT with ABD-F uncovers the presence of significant amounts of metal-depleted protein. (A) Schematic reaction of ABD-F with the sulfhydryl group resulting in a fluorescent adduct. (B) Amounts of MT and T (TR+TO) found in various rat tissues. (88) (C) Changes of MT and T (TR+TO) content in HT-29 cells pretreated with ZnSO4. (88) (D) Percentage of MT, T, TR, and TO in rat liver stored on ice for up to 30 h as determined with a modified differential ABD-F assay. (89) Gray, red, blue, and green bars correspond to MT, T (TR+TO), TR, and TO species. MT, T, TR, and TO correspond to fully metal-loaded, fully depleted, reduced and depleted, and oxidized MT, respectively.
3.1.1. TR (Thionein)
ABD-F is a fluorescent sulfhydryl labeling reagent that reacts rapidly with free sulfhydryls but only very slowly with the Zn(II)/thiolates in MT. It was employed for a differential labeling assay in which tissue extracts were analyzed by a modification with the reagent in the presence or absence of the chelating agent ethylenediaminetetraacetic acid (EDTA) and a subsequent chromatographic separation. (88) The assay is based on the observation that Zn(II) quenches the reactivity of cysteine residues with thiol-modifying reagents, whereas the removal of Zn(II) from MT with chelating agents restores the reactivity of the sulfhydryls. The incubation of MT-containing samples with ABD-F in the presence of a strong metal chelator such as EDTA and the reducing agent tris(2-carboxyethyl)phosphine (TCEP) results in a total metallothionein modification (MT+T) with ABD-F as manifested by a strong fluorescence at 512 nm when excited at 384 nm. A lower fluorescence observed when ABD-F reacts with MT in the absence of EDTA showed that a certain amount of protein must not be saturated with metal ions. A high-performance liquid chromatography (HPLC)-based quantification with fluorescence detection allowed a calculation of the MT amount as the difference between two reactions in the presence and absence of EDTA; that is, MT = (MT+T) – T. (88) Importantly, the terms “T” and “MT” in the assay refer to metal-free and metal-saturated protein because the only existing model at that time was one with highly cooperative Zn(II) binding, which would allow T and MT to coexist without any intermediates. When we consider later findings that this model is inadequate, the assay in fact examines an average of multiple species, namely, a ratio of modified and unmodified Cys residues in the assembly of MT proteins that includes partially metalated species (see below). Employing the assay, significant amounts of T were detected in biological tissues. The amounts found in rat liver, kidney, brain, and testis were 27, 54, 53, and 9% of total metallothionein (MT+T), respectively (Figure 12B). Similar analyses performed on HT-29 colorectal cancer cells showed the presence of T over total MT+T as 30%, an amount that decreased to 25 and 21% when cells where incubated with 100 and 200 μM ZnSO4, respectively, for 6 h prior to cell homogenization (Figure 12C). (89) Concurrently “free” Zn(II) concentrations increased from 0.8 pM in nontreated cells to 5.2 nM in cells incubated with 200 μM zinc sulfate. It demonstrated that T/MT molar ratios correlate with freely available Zn(II) and indicated a fundamental role of the ratios in the buffering of cellular zinc. (89,90)
3.1.2. TO (Thionin)
A second differential modification assay was developed to interrogate the redox state of the cysteine thiols in MT and to distinguish TO from TR, that is, oxidized from reduced species not fully saturated with metal ions. It uses a derivatization of the thiols with the fluorescent probe 6-iodoacetamide-fluorescein and a colorimetric determination of the product. (91) In the presence and absence of the nonsulfhydryl-based reducing agent tris(2-carboxyethyl)phosphine (TCEP), the assay gives the amounts of TO and TR, respectively. Similar to the reaction with ABD-F, the assay in the presence and absence of EDTA then gives the total amount of the protein (MT + TR) and TR, respectively, and thus allows the determination of all three species MT, TO, and TR. In this work, chemical (DTNB) and enzymatic (sulfhydryl oxidase (SOX)) oxidations were used. Likewise, a modified ABD-F method employing quenching of the sulfhydryl groups with N-ethylmaleimide allowed the determination of the three species in homogenized rat tissues and demonstrated an increase of TO over time (Figure 12D). (90) Thus, oxidized forms of the protein indeed exist in vivo. Disulfides form in both domains when MT is overexpressed in the heart of mice and increase under conditions of oxidative stress. (92) Cysteines 44 and 48, which are ligands to metal number 5, are involved (Figure 5). Also, the human ovarian surface epithelial (HOSE) cell line was used together with the same cell line transformed with an H-rasv12 oncogene, which produces reactive oxygen species, to determine the oxidation state of MT. In the parent cell line, less than 10% of the thiols were found to be oxidized, an amount that corresponds to the formation of one disulfide bridge. But in the transformed cell line, a threefold increase in oxidation was observed. (90)
Chemical oxidation in vitro generates intra- and intermolecular disulfides in MT. (76,93) MT polymers form in the reaction with DTNB. (76) Multimers are found when the protein is oxidized in vitro and analyzed electrophoretically after fluorescence labeling with 5-iodoacetamide-eosine. (94) In this oxidative polymerization, MT polymers from dimers all the way to decamers were detected. Arrangements of MT molecules as dimers in the crystal structure and a possible trimer interface suggest how this polymerization proceeds structurally. (47,95)
With the finding of TR and TO forms, it became clear that MT undergoes a redox cycle (Figure 13) and that there are minimally two species in addition to MT, an oxidized form, thionin, TO, and a reduced form, thionein, TR. The redox cycle is extended to a dual cycle to account for the later finding of partially metalated species (see below).
Figure 13
Figure 13. MT and T redox cycles. In this expanded dual cycle, TR and TO represent the fully reduced (thionein) and oxidized (thionin) metal-free protein. Zn7MT, Zn7–xMTR, and Zn7–xMTO refer to fully loaded, partially Zn(II)-depleted reduced, and partially Zn(II)-depleted oxidized protein, respectively. It is presumed that thionein/thionin can serve its own role as a redox couple in vivo under conditions of restricted metal ion availability.
From prior investigations, it had been known that MT is a rather reactive protein, prone to oxidation during protein isolation, purification, and storage, but this information was not related to its biology. One may ask why additional forms, TR and TO, had not been discovered earlier when MT was isolated from biological tissue. There are several answers. Because the protein lacks aromatic amino acids and there are no assays based on a biological activity, MT was largely isolated by following its metal content, thus precluding the detection of other forms that do not contain metals. The discovery of an efficient induction by heavy metal ions changed significantly the procedure of MT preparation and purification. To increase the yield of the protein prepared from livers, rats or rabbits are injected with sublethal doses of Zn(II) or Zn(II) and Cd(II) salts for several days. (5,96) With this procedure, the protein is forced into the metal-bound form. Yet, isolated MT remained heterogeneous. The metal-to-protein ratios did not always indicate a full saturation of the protein with seven divalent ions, demonstrating that partially saturated MT may be present in some tissues even when the protein is metal-induced. Also, MT was mostly isolated from liver, the hepatocytes of which normally do not proliferate and are rich in metals, but rarely from other sources. Nowadays, overproduction of a specific MT in a bacterial expression system is the most straightforward method of MT preparation. It requires a cloning of the cDNA encoding MT into an appropriate vector, for example, the plasmids pETYB or pET, and the subsequent induction of thionein production with isopropylthio-β-galactoside (IPTG). (97) Although metals are not inducers in these constructs, they may increase the yield of protein during purification. Heterologous expression increases protein purity due to the production of only one MT isoform, but conditions are different in terms of redox state and metal ion availability from native tissues as metal ions are added to bacterial growth media. Also in this case, the isolated MT can be partially oxidized and not fully saturated by metal ions, and the expressed MT is not a chemically pure species regardless of the method of isolation or induction, though using Cd(II) instead of Zn(II) for induction increases the metalation of induced MT due to its higher affinity for the protein. (28) Thus, MT isolated from either natural sources or bacterial cells is heterogeneous. In order to obtain homogeneous MT a reconstitution procedure is required. It includes the reduction of any oxidized thiols in the protein, removing the bound metal ions and, subsequently, a controlled reconstitution of the fully reduced T with the desired metal ion. (98)
In the search for the biological oxidant(s), many proposals were made for the redox biology in vivo. Glutathione (GSH)/glutathione disulfide (GSSG) received particular attention as a biological redox pair interacting with MT and metal ions. Changing the GSH/GSSG ratio affects the Zn(II) dissociation from MT and Zn(II) transfer to the apoforms of zinc proteins. (99) In addition to GSSG releasing Zn(II) from MT, GSH modulates the transfer. GSH binds to invertebrate copper MT and rabbit liver Zn(II),Cd(II)-MT. (100) For the latter species, the binding constant is 14 ± 6 μM. Molecular modeling and energy minimization suggests a stabilization of MT and a prevention of the dissociation of Zn(II) when the GSH sulfur replaces the sulfur ligand of Cys26 in the N-terminal domain of MT. (101) The biological significance of the interaction is not entirely clear, but it could indicate that the metal affinities of MT are further modulated by a ternary complex formation. GSH inhibits the Zn(II) transfer, and a Cu(I)/GSH complex has been postulated to be in equilibrium with copper MT. (99,102) The role of Zn(II)/GSH complexes in cellular zinc trafficking is even less clear than in the case of Cu(I), though the binding equilibria have been well-characterized. (103,104) The putative role of GSH in copper metabolism is addressed in section 4.4.
Another type of reaction is the S-glutathionylation and S-homocysteinylation of MT. Glutathionylation was investigated with commercial rabbit liver MT in vitro with either S-nitrosoglutathione or GSH and hydrogen peroxide or diamide and detection of the glutathionylated protein with an anti-GSH antibody. (105) Glutathionylated, highly aggregated MT was also found in peripheral blood mononuclear cells (PBMCs) in vivo. Solvent-accessible Cys7, Cys13, and Cys59 were implicated in the reaction. (105) Cys7 and Cys13 have been shown to be involved in the nitrosylation of MT. (106) The homocysteinylation of MT was detected in human aortic endothelial cells (HAECs) when using 35S-homocysteine. (107) The intermolecular disulfide bond is stable in the presence of 10 mM GSH. This stability is based on the higher sulfhydryl pKa value of l-homocysteine (10.0) compared to l-cysteine (8.3). The proposed biological significance of this reaction is that it provides a pathway of how increased blood levels of homocysteine, which are a risk factor for cardiovascular disease, effect metal redistribution.
Numerous investigations showed that a subjection of cells to various types of oxidative stress increases the availability of Zn(II) ions in the cell. It includes disulfide stress. 2,2′-Dithiodipyridine, a cell-permeable disulfide, leads to Zn(II) dissociation. (108)
Enzymes have been shown to be involved in the redox chemistry of MT in vitro, but the significance of these reactions in vivo remains to be shown. One example is avian sulfhydryl oxidase, a flavoenzyme of the quiescin sulfhydryl oxidase family of proteins that introduce disulfide bonds into cellular and extracellular proteins. (109) A kinetic characterization of the reaction demonstrated that T, but not MT, is one of the best substrates of this enzyme with a kcat/Km value of 2.1 × 105 M–1 s–1. (91,94) Another protein involved in the disulfide formation in proteins is protein disulfide isomerase (DsbA). It reacts with MT under stoichiometric conditions. (80) Further kinetic characterization of the reaction also demonstrated a rather high (103 higher than glutathione) rate constant of 4.4 × 105 M–1 s–1. (110) Last but not least, MT thiols are a substrate for the selenium enzyme glutathione peroxidase. (111)
Biology uses selenium, the congener of sulfur in the periodic system of the elements, in redox biology. The redox chemistry of selenium compounds with the thiolates in MT has been investigated. (111) It is remarkable, because, in addition to reacting stoichiometrically in higher oxidation states than the selenol─the equivalent of a thiol─selenium compounds catalyze reactions of MT with reactive disulfides and the subsequent reduction of the oxidized proteins. (112,113) It suggests a relationship between the metals carried by MT and the biologically essential micronutrient selenium and its anti-inflammatory and anticarcinogenic actions.
The redox chemistry of MT and the presence of MT species that are not saturated with metal ions changed the field from a perception of MT as a protein storing metal ions to one being involved dynamically in metal and redox metabolism. A characterization of isolated MT and investigations of its state in cells and tissues demonstrate that MT is unlike any other zinc or copper protein with catalytic or structural sites that are generally fully occupied with the metal ion to maintain function. MT’s metal content is variable, and MT has a dynamic structure that depends on the metal availability, type of metal ions, and redox conditions in its biological environment at extracellular and intracellular locations. A correlation between the cellular redox state, the redox state of “MT”, and the concentrations of free Zn(II) has been observed in four defined states of the same human cell line (growth-arrested, proliferating, differentiated, and apoptotic). (89) Thus, observations in both isolated systems and cells suggest that the redox state of MT is one factor determining the availability of cellular Zn(II) ions.
A limited number of investigations have suggested alternative or additional functions of the redox chemistry of MT and T, namely, that Zn(II) controls their redox function rather than their redox function controlling Zn(II). For example, in the presence of EDTA, MT assists the protein disulfide isomerase-catalyzed formation of native pancreatic ribonuclease A in vitro. (114) In another interaction with enzymes, it was shown that TR/TO could be part of a redox chain between thioredoxin and methionine sulfoxide reductase, essentially linking nicotinamide adenine dinucleotide phosphate (NADPH) to the reduction of methionine sulfoxide. (115) In a search for a reducing factor for human methionine sulfoxide reductases (Msr) B2 and B3, a heat-stable factor from bovine liver was isolated, shown to serve as such as a reductant in the presence of EDTA, and identified as MT. It was then shown that TR can reduce human MrsB3 and TO can be reduced by the thioredoxin system. Selenocystamine or selenocystine increases the potency of thioredoxin or TR as reducing agents. (116)
In terms of redox chemistry, MT is often discussed as an antioxidant. Any such antioxidant function must consider the metal load of MT and the role of the dissociated Zn(II) ions, which are potent effectors of protein function and have their own indirect effects on redox biology. (117) Depending on concentrations of dissociated Zn(II) ions, they can have either pro-antioxidant or pro-oxidant functions in the cell.
For the reasons discussed here, we link both MT and T primarily to zinc homeostasis rather than redox homeostasis, namely, that global redox changes or changes of particular redox pairs determine the availability of cellular Zn(II) (and Cu(I)). This link has gained further support when experiments in cells examined how signaling with with reactive species targets MT and affects Zn(II) dissociation to generate “zinc signals” as potent effectors of biological processes. These observations were critically important for the emerging field of zinc signaling (see below), as they demonstrated that the Zn(II)/thiolate clusters in MTs are transducers of redox signals into zinc signals. The observation that MT reacts with nitric oxide (NO) followed by a Zn(II) dissociation continued the focus on the sulfhydryl reactivity of MT. (118) The possibility that MT is a target of redox signaling became apparent when it was shown that cellular NO formation triggered by hormonal stimulation of nitric oxide synthase reacts with MT and increases concentrations of cytosolic and nuclear Zn(II) ions. (119,120) To bring about specificity in terms of which sulfhydryls are targeted, transnitrosation is a mechanism. For example, MT3 is the only MT that has an acid–base sequence motif indicative for S-nitrosylation by transnitrosation at Cys22 and Cys42, and it reacts with S-nitroso-l-cysteine faster than other MTs. (121) On the basis of observations that extracellular S-nitroso-albumin and a cell surface protein disulfide isomerase are involved in transnitrosation of intracellular proteins, MT was identified as a protein of such transnitrosation in endothelical cells. (122) Thioredoxin and lipoic acid then could be involved in the denitrosation. (123) It was suggested that oxygen is needed for the S-nitrosothiol formation because an MT-SNOH radical intermediate forms under anaerobic conditions. (124) Reactions of the sulfhydryls of MT are not restricted to species with bound Zn(II), as redox signals can also lead to the dissociation of bound Cd(II) or Cu(I). (125,126) These reactions were mostly studied in vitro. Analyses in which Cys thiol(ates) are nitrosylated in vivo have yet to be performed. Uncertainties remain about these reactions under normal conditions versus conditions of redox stress.
Another type of modification of the sulfhydryls in MT is alkylation. Reactive carbonyls react with the sulfhydryls in MT with a concomitant Zn(II) dissociation. (127) Reactants can include endogenous aldehydes, aldehydes formed under carbonyl stress such as observed in lipid peroxidation, and xenobiotic aldehydes in the diet or the environment. For example, oxidized dopamine products react with MT. (128,129) Alkylation has also been observed with pharmaceuticals, in particular, with highly reactive compounds used in cancer chemotherapy. The important message is that all these reactions affect the metal redistribution or perturbation of the control of metal homeostasis with implications for many mechanisms of cytoprotection or cytotoxicity. (130)
A determination of the redox state of MTs, their polymerization and modifications, the presence of Cu(I)/Cu(II) and Zn(II) under some conditions, ligand binding, and the existence of multiple MTs makes speciation a hugely challenging analytical problem that has not been resolved to this date. What the species MT, TR, and TO are molecularly is a different matter, as all of them are not exactly one species but rather an assembly of species (see below), making the name “metallothionein” very unspecific in terms of what it actually refers to─a key consideration when associating functions with molecules that have a generic name only. And the species undergo dynamic changes, which introduce biological time and space as yet other variables. With the assays described above the metal load of MTs is measured, the MT/T ratio, which is the averaged sum of different forms. Finding a given percentage of T indicates that the protein is not fully saturated with metal ions. Metal binding depends on metal affinity and metal availability in addition to biological factors such as the rates of synthesis and degradation of the proteins. Assays of liver homogenates in the absence and presence of added Zn(II) ions demonstrate that the proteins indeed do not carry a full complement of seven Zn(II) ions because the metal load increases when metal ions are added. (89) The interpretation of highly cooperative binding needed to be revised when affinities of the seven Zn(II) ions for human MT2 were found to differ, (73) and this is the next development to be discussed, which further increases the number of species that the epithet “MT” refers to.
Much as we made the point before that redox biology added to the chemistry of MT in the biology milieu, the opposite is equally important, namely, how chemistry adds to biology as now discussed by chemical investigations on the affinity of MT for Zn(II) ions. To emphasize this aspect, a quote from Professor Colin Thorpe’s webpage (University of Delaware) is provided: “Biology cannot ignore chemistry, much as I wish it could” (John Maynard Smith).
3.2. General Notes on Metal Affinities and the Essence of Metal/Thiolate Coordination
Metal binding is at the center of the properties of MTs. The determination of the affinities of metals for proteins is challenging. (131) In the case of MT, the challenge is even greater due to the presence of multiple and alternative coordination sites. The affinity of metal ions for MTs depends on numerous factors. Definitely the most important one is the tendency of a particular metal ion to form a metal–thiolate complex. According to the hard and soft acids and bases (HSAB) concept, soft metal ions prefer binding to soft bases such as sulfur donors. (132) Therefore, the softest metal ions such as Hg(II) and Cu(I) have the highest affinities toward MTs, although their exact stability constants are still under scrutiny. Cd(II) has a much softer character than Zn(II) and therefore forms significantly more stable complexes. Zn(II) has a moderately soft character, resulting in binding to nitrogen and oxygen donors in addition to cysteinyl sulfur in proteins. Neither nitrogen nor oxygen donors are involved in metal ion coordination in mammalian MTs. Only sulfur donors are responsible for efficient but not necessarily equal binding in terms of affinity. The presence or absence of sulfur donor bridges in MT may or may not impact affinity depending on the particular metal ion. Metal ions such as Cu(I) and Cd(II) have a higher tendency for thiolate cluster formation, whereas the tendency of others such as Zn(II) or Co(II) is lower. (98,133,134) One question in the history of MT research always has been what the significance is for the formation of thiolate clusters.
The interaction of MTs with various metal ions has been investigated with multiple techniques, mostly different types of spectroscopies and mass spectrometric aproaches, among which only a few have provided limited information about metal-to-protein affinities. More sensitive methods have added to our knowledge and define the order of metal ion affinity in MT (Figure 14). This order is not absolute, as other chemical and biological factors may affect the range of affinities.
Figure 14
Figure 14. Affinity series of metal ions binding to metallothioneins. The order of affinities is based on experimental data and estimations from exchange experiments and model studies. (28,135−142) The series also defines the tendency of free metal ion concentrations buffered by MTs. Its implication for the metalation of proteins is discussed in section 4.1.
Affinity data have been collected with multiple methods at various conditions. Considering only the essential metal ions Zn(II) and Cu(I), the Kd values in Figure 14 correlate well with published cellular free metal ion concentrations, (89,143−145) suggesting that MTs function as a buffering system for these metal ions. Moreover, the range of affinities also should be commensurate with fluctuations of free metal ions, as Zn(II) ions are signaling ions, and more recently Cu(I) ions also have been suggested to have signaling functions. At this time, our knowledge of the stability of metal complexes is limited to a few MTs with a single metal ion, and investigations on affinity constants of mixed metal MTs are lacking. Given the different affinities and stereochemical preferences of Zn(II) and Cu(I), it is likely that a partial occupation with one metal ion will affect the affinity of the other one. Since mixed MT complexes (Zn(II)/Cu(I), Zn(II)/Cd(II), or Cd(II)/Cu(I)) form and are found in tissues, it is an important issue awaiting scientific inquisitiveness to be addressed. In addition to serving as a dual metal buffer for the two most competitive metal ions, MTs could have a role in keeping strict ratios between these metal ions or, perhaps more importantly, in changing these ratios depending on metabolic or energetic demands of cells. Therefore, much more emphasis needs to be placed on the properties of mixed MT complexes, especially with essential metal ions such as Zn(II) and Cu(I). It is insufficient to base our knowledge about MTs on the binding properties of only one metal ion because pools of many different metal ions exist in the biological environment. How the metal ions cooperate in the cell will determine how they interact with MTs. Since MTs bind various metal ions with affinities covering a wide range, it is important to understand how the formation of mixed-metal complexes affects buffering and hence the availability of metal ions. How this control occurs from a molecular and cellular point of view is a critical question to be addressed experimentally. Do the mixed metal complexes still serve as metal donors and acceptors in the same way or does one metal ion inhibit or enhance the availability of the other? How does the cellular redox status affect the buffering properties of such mixed-metal complexes? Do MTs retain one metal ion under certain conditions and tune the availability of another when needed? How would such subtle control work? Do MTs function as Zn(II)-modulated Cu(I) chaperones?
Because MTs do not form stable secondary or tertiary structures in the absence of metal ions, the most important factor that drives particular metal ion affinities is the enthalpy of the metal–sulfur bond formation. While the thiophilicity is an overriding factor in explaining why Hg(II) forms more stable complexes than Cd(II) and the Cd(II) complex is more stable than that of Zn(II), several other factors are critically important for the metal-to-protein affinity, for example, differences of metal affinities provided by the different primary structure of the domains and particular coordination environments of each site in the two domains.
In principle, the difference in stabilities between metal ions with filled d-shells, that is, Zn(II), Cd(II), Hg(II), and Cu(I), and those with partially filled d-orbitals derives from the ligand field stabilization energy (LFSE) in the latter. For instance, Zn(II) and Co(II) binding to Cys-rich sites is often accompanied by a transition from an octahedral geometry in water to a tetrahedral geometry in protein sites. With Co(II) (d7), a change of geometry leads to a thermodynamic penalty due to the LFSE. The penalty arises from different splittings of the d-orbital energy levels in an octahedral versus a tetrahedral ligand field. In the case of Zn(II), the d10 shell leads to the same average electronic energy in either ligand field. The different splitting of d-orbitals into eg and t2g levels in both geometries gives Co(II) a preference for an octahedral geometry compared to Zn(II), which, on the basis of model complexes, amounts to a difference of −4.5 kcal/mol in free energy. Indeed, experimental data, mostly for zinc finger proteins, show that formation constants of the Zn(II) complexes are 2–4 orders of magnitude higher than the ones for Co(II). (135,146,147) Another important factor that affects the coordination number of metal ions is the overall net charge of the complex formed. The formation of complexes with higher coordination numbers becomes increasingly more unfavorable energetically due to a Coulomb repulsion. The higher stability of Zn(II) over Co(II) complexes is also due to the greater charge/size ratio, as the Zn(II) cation is smaller than Co(II). Data on metal affinities of Co(II) or Ni(II) toward MTs are fragmentary, for example, using only the α-domain of MT2 by spectroscopically monitored titrations. (148) Therefore, the order Zn(II) > Co(II) > Ni(II) is based not only on these limited data sets but on LFSE rules and metal binding affinities of other Cys-rich sites (Figure 14).
For metal complexation in MT, several specific factors need to be considered. One effect that is mostly neglected in discussions of metal-to-protein affinity is proton dissociation from binding amino acid residues driven by metal ion coordination. From a biological perspective, it is a factor to contend with, as pH values vary in different cellular compartments. Metal binding to MT causes several energetically important events at the same time. Besides water molecules dissociating from the metal ion and the formation of the coordination bonds between the metal ion and the sulfur donors, the ionization of R-SH groups impacts the energetic cost of the whole process. The proton disscociation is endergonic, while the proton association to solution components is exergetic. The pKa values of thiols therefore have an impact on the process. Deprotonated thiol groups clearly do not contribute to the energetics of proton dissociation, and in this case the enthalpy of the metal–ligand bonds is the most important component of metal-binding thermodynamics. Overall, the more acidic the cysteine thiol the more likely is the formation of a more stable binding site. There is limited information about the acid–base properties of T. The average reported pKa value of the cysteine thiols in MT is 8.6–8.9, depending on the investigation. (141) However, the acid–base properties of individual thiol groups in T remain unknown. Various Cys motifs in the MT sequences and variable environments of particular cysteines are expected to fine-tune the acidity of the thiol groups. Thiols and thiolates can interact with amide protons. (149,150) Fluorescence resonance energy transfer (FRET) probes, molecular modeling, and mass spectrometric investigations have shown that T does not adopt an absolute random coil conformation and that electrostatic interactions including hydrogen bonding determine its conformation. (151−153) All of the above-mentioned energetic effects are important for the metal complexation in individual or clustered sites in MTs. In individual sites, four Cys residues are involved in complexation, while the formation of an M2Cys6 cluster requires the deprotonation of three Cys thiols per one metal ion. The coordination of a second metal ion to a preformed MCys4 core requires only two deprotonations. One cannot neglect entropy, which changes during metal complexation to the protein depending on the metalation mechanism. The significance of entropy is even more obvious, as the enthalpy of binding of each metal ion is very similar in the same coordination spheres. Thus, entropy is a determining effect in the thermodynamics of the binding process. (154) In T, there are no preorganized coordination environments, and protein folding, that is, the spatial organization of the protein’s main chain, is coupled to the metal binding with a potentially very large conformational space in the process. There will be significant differences between the binding of the first couple of metal ions, which serve a role in nucleation of protein folding, and the binding of the last metal ions, which bind to an already mostly folded protein. Protein folding therefore is a very important factor in the overall affinity of the metal ions. Since the only secondary structure elements in MT are two 310 helical segments and a few half-turns, the formation of a secondary structure does not seem to be critical. Numerous hydrogen bonds have been observed in both domains of the crystal structure of rat Cd5Zn2MT2 (Figure 15)
Figure 15
Another important issue concerns the conditions of the experimental procedures employed to determine the metal-to-protein affinities. Notably, MT is not a simple ML complex, in which a metal ion binds to a strictly defined binding site with a stoichiometry of 1:1. In a simple metal complexation such as in a zinc finger (ZF) domain, the binding is represented by one event, for example, at neutral pH, and depends on the relative ratios of the reactants, the M-ZF complex, and the apo-ZF present. The pZn (−log[Zn(II)]) dependence of complex formation is characterized by only one binding isotherm with an inflection point corresponding to the −log Kd value of the M-ZF complex. If the point is shifted to a slightly lower or higher pH, only one event will be observable in the pZn graph (Figure 16); however, conditional dissociation constants are shifted due to their dependence on pKa values of the ligand donors of amino acid residues. How significant the shift of affinity as a function of pH is will depend on the composition of the coordination sphere. For example, if the metal binding occurs in a mixed coordination environment of His (H) and Cys (C) ligands, the pH-dependent shift of affinity will be different from that of a site with only Cys ligands because His is mainly deprotonated close to neutral pH.
Figure 16
Figure 16. Illustration of how the metal-to-protein affinity shifts when the pH increases or decreases. The case corresponds to a model metal site composed of Cys residues with a −log Kd value = 11.5. Inflection points of the binding isotherms numerically correspond to −log Kd values under the conditions used.
In three different metal binding sites (CCCC, CCHH, and HHHH) one obtains three quite different relationships between the pH value and the stability of the complex formed. Assuming that the Zn(II) coordination system has the same Kd value at pH 7.4 in all cases, the site with His residues will only form the most stable complexes at lower pH and the weakest above pH 7.4 in comparion to Cys residues. Morever, the Kd value remains constant in the alkaline pH range, because one N–H in the imidazole ring is completely deprotonated at pH ≈ 7 or above, and metal binding above this pH does not cause a deprotonation of the second N–H. The scenario is different if only Cys ligands are involved in the Zn(II) binding site. In this case, a pH-dependent increase of the complex stability occurs in the alkaline pH range until a pH value above 10 is reached, where all thiol groups are ionized (Figure 17). A mixed coordination sphere of Cys and His ligands behaves in between the two discussed examples. (135,159−161) A determination of the stability at different pH values, therefore, undoubtedly will result in major differences of metal affinities. A shift of only one pH unit will result in ∼4 orders of magnitude difference in Kd values. Clearly, this pH dependence makes a comparison of Kd values determined at different pH values problematic, a fact to consider in experimental procedures and discussions of MT thermodynamics. In conclusion, these considerations illustrated in Figure 17 resolve a long-standing issue about the exclusive coordination to Cys ligands in mammalian MTs. There are at least three purposes for the use of sulfur. In addition to their redox activity, the sulfur donors impart a higher affinity and hence selectivity for the more thiophilic metal ions, in particular, the competitive essential metal ions Zn(II) and Cu(I) and a pH sensitivity of the affinity in the range of physiological pH values with yet to be fully explored biological consequences for the pH control of metal coordination.
Figure 17
Figure 17. Influence of the composition of the coordination environment in zinc sites on the metal affinity and the pH dependence of conditional dissociation constants. With HySS software, the relationships were simulated based on the assumed protonation and stability constants in such a way that all formed complexes reach −log Kd = 12 at pH 7.4. (162) The pKa values of Cys thiols and His imidazoles have been limited to the 8.6–8.9 and 6.1–6.4 ranges, respectively, based on previously determined data. (135,160,161)
With the clusters, MTs are a much more complicated coordination system than zinc fingers. (135) The next chapter will discuss the metal affinities and metal binding mechanism of MTs, which was the subject of debate for several decades. New evidence was provided that Zn(II) binding occurs differently from that of Cd(II) and Cu(I) and with much less cooperativity than assumed hitherto. Retrospectively, these findings are understandable because the clusters in MT are not as symmetrical as clusters in inorganic models, and metal binding occurs in a specific order determined by energy minima influenced by the folding of the peptide chain. The main reason why results suggested that Zn(II) binding to MTs occurs with an indistinguishable affinity is that investigators examined the binding process as an average of affinities. The observation of metal binding events in metal-controlled media is much more robust and avoids easy-to-miss differences in affinities or an overestimation of stability constants. To illustrate step-by-step the origin of errors in relying on data that are averaged, one needs to dissect the binding process molecularly and eschew the assumption that Zn(II) binding is highly cooperative. UV absorption is followed in a pH titration of MT to determine affinities because Zn(II)/thiolate and Cd(II)/thiolate coordination results in several ligand-to-metal charge transfer (LMCT) bands in the UV (followed at 218 nm for Zn(II) and at 254 nm for Cd(II)). In the case of the Zn7MT molecule, there are 28 Zn–S bonds; 12 are present in the β-domain, and the remaining 16 are in the α-domain. Clearly, it is impossible to distinguish the contribution of the terminal Zn–S bonds and the bridging Zn–S–Zn bonds to the UV absorption. All Zn–S bonds are expected to differ from each other, but it would require quantum-chemical calculations to resolve the contributions from the protein’s environment. Considering that the first four bound Zn(II) ions are distributed across the protein (in the α- and β-domains), they generate up to 16 Zn–S bonds. This maximal number assumes total independent events with the same high affinity, but a more likely scenario includes some bridges between metals as well. The binding therefore is observable as one event and causes a huge increase in absorption amounting to ∼60% of the total absorbance in the UV range (Figure 18A). The remaining three Zn(II) ions bind to the protein by docking to already coordinated metal ions, mostly by generating bridged connectivities that result in the formation of the maximally remaining 12 Zn–S bonds. Because those three Zn(II) ions bind to the protein with lower affinities, pH-dependent events in the pH titration are shifted to higher pH values. This shift is readily noticeable. The fifth and the sixth Zn(II) ions bind with only slightly lower (moderate) affinity compared to the first four, resulting in a small shift, and all the effects from 0 to 6 Zn(II) ions overlap each over. A careful analysis of titration curves shows that they are not symmetrical at both ends. The curve has a sharp turn at low pH when metal binding begins and a much broader turn above the inflection point. Such asymmetry is further increased when the weakest, seventh Zn(II) ion binds. The binding causes a deprotonation of two SH groups (Cys21 and possibly Cys5) and the formation of four Zn–S bonds, including two bridged ones. (15) It results in a significant pH shift of the titration curve with a relatively small effect on absorbance (4 bonds over 28 giving a maximal 14% change of total absorbance). As a result, the last Zn(II) binding is almost indistinguishable in the pH titration curve. To observe the binding of the last Zn(II) ion, one needs to titrate the protein to at least pH 7 and analyze the shape of the curve. It requires enough signal/noise to observe the increase in absorption. Figure 18A illustrates an experimental Zn7MT2 titration curve over a wide pH range with an indication of the curve’s asymmetry. As a confirmation of the discussed effects, Figure 18B shows simulations of the absorbance increase and includes the experimental binding model (solid line) with gradually lowered affinities of Zn(II) toward MT (73) and a theoretical binding model (dotted line) that assumes all Zn(II) ions to be bound with the same high affinity. The comparison indicates that the simulated curve (solid line) is similar to the experimental one (Figure 18A), despite the assumption in the simulation that the thiols have the same pKa values; that is, affinities are differentiated, but thiol pKa values are averaged. The situation is different when the pH-dependent formation of Cd7MT2 is considered. 113Cd-NMR spectroscopic investigations have shown that Cd(II) binding occurs in such a way that the α-cluster in the C-terminal domain is formed first at a lower pH and then the β-cluster in the N-terminal domain is formed with the remaining three metal ions at a higher pH. (141,157,163) This binding can also be observed when UV absorbance is followed in pH titrations. The experimental curve (Figure 18C) demonstrates two independent regions of metal binding events, the first with an inflection point at pH ≈ 3 and the second with one at ∼3.8. Doubtlessly, the two steps confirm the conclusions from the NMR experiments and clearly prove that the four Cd(II) ions in the C-terminal domain bind with significantly higher affinity (∼2 orders of magnitude) than the three Cd(II) ions in the N-terminal domain. The steepness of the observed curve at the first stage of metalation shows that the affinities of all metal ions in the α-cluster are identical or rather similar. Cd(II) ions in the β-cluster also bind with very similar affinities; however, the shape indicates some differences as concluded previously. (164)
Figure 18
Figure 18. Experimental and simulated UV-monitored pH titrations of Zn7MT2 and Cd7MT2. (A) Experimental pH titration of 1 μM Zn7MT2 from pH 3 to 7. (B) The simulations of pH-dependent saturation of the protein with Zn(II) (see main text for details). The dashed line corresponds to a case where all seven Zn(II) are bound with the same affinity (−log Kd = 11.7). The solid line indicates a case where four sites are bound with high affinity and the remaining three with gradually lower affinities. (73) (C) Experimental pH titration of 1 μM Cd7MT2 from pH 3 to 7.
To summarize, it is very important to understand the pH-dependent equilibria in MTs as a structural component of their functions, especially to realize the significance of homoleptic coordination with only sulfur donors endowing such a complex system with a strong pH dependence around physiological pH and multiple pH-dependent equilibria, which we are unable to describe ab initio with the ionization constants of each of the 20 thiol groups. These considerations illustrate why pH control is critical for the purification and investigation of MTs and a discussion of their affinities for metal ions. For instance, size-exclusion chromatography at neutral or slightly acidic conditions of Zn(II)-reconstituted proteins results in the preparation of proteins that are not fully saturated with metal ions and leads to drawing the wrong conclusions about reactivity or function. Only purification steps at higher pH, for example, pH 8.6, warrant the preparation of fully saturated proteins.
Another source of overestimation of metal-to-protein affinities in MTs is the assumption that all metal ions bind with the same affinity. Inasmuch as pH titration of T provides average pKa values of the thiols, a pH titration of MT provides average pKa′ values of the thiols that compete with metal ions. In the latter case, the inflection point in Figure 18A is shifted to lower pH values, and the magnitude of the shift is an indication of the affinity of the metal to the protein. The assumptions that all metal ions bind with the same affinity and that one metal ion causes a deprotonation of three Cys residues allows obtaining an average −log Kd value for all metal ions. In such a procedure, weaker metal binding sites are averaged and dominated by high-affinity sites, and, due to the assumption of indistinguishable −log Kd values and pKa values of thiol groups, the average −log Kd value is overestimated and does not provide information about variation of stabilities of individual sites. The only robust way to address the actual metal-to-protein affinities of such a complex system is an analysis of the metal binding process using changes of free metal concentrations instead of changes of pH. It allows a resolution of small and significant differences in stabilities due to the wide range of free metal concentrations used in such investigations. In other words, one needs to use a competitor or a set of competitors that have the sensitivity and affinity toward the same metal ion to allow a monitoring of what happens with the protein when the free metal ion concentrations change systematically. To the best of our knowledge, such a procedure has been used only twice for MTs, and the results are highly significant because they show that the metal ions bind to MTs with a wide range of affinities. When highly sensitive fluorescent FluoZin-3 or RhodZin-3 probes were used, the affinities of Zn(II) to MT2 were demonstrated to differ and range from nanomolar (∼10–8 M) to picomolar (∼10–12 M). (73) With isothermal titration calorimetry (ITC), it was shown that Zn(II) and Pb(II) bind to MT3 with a wide range of affinities, similar to MT2; however, the investigations were performed at a lower pH. (137)
In other approaches to determine metal affinities, colorimetric (nonfluorescent) competitors were employed to monitor titrations of T with a metal ion. Fractional saturations─depending on reactant concentrations─are directly converted to the average Kd of MT based on a stoichiometric model and known affinities of the metal-chelating probes. An inherent issue with such an approach is the model’s assumption in treating all metal ions as indistinguishable and the narrow range of metal sensing of commonly employed chromophoric agents. (165−170) Focusing on narrow ranges only and not monitoring free metal ion concentrations results in an overestimation of the Kd because the contributions of weaker sites are not resolved. If all sites in ZnMT2 were to have the same affinities, all fractional saturations would report the same average value. With nano- to picomolar affinities of Zn(II) to MT2 (see below), the resulting Kd value reports average affinities of various sites. For instance, if the probe is added at a concentration that would allow a removal of ∼30% of Zn(II) from MT2, the average constant calculated will correspond to the Kd value of moderate sites because 30% of total Zn(II) corresponds to 2.1 metal equivalents. If the transfer is ∼45%, then the resulting Kd reports affinities from moderate to high-affinity sites, since 3.1 equiv of Zn(II) are transferred to the probe during equilibrium. Any higher fractional saturation mainly reports high affinities. To achieve detailed information on a weaker site, one would need to focus on only a few percent Zn(II) transfer, since the affinity of weak and moderate sites will be averaged above 14% transfer. Such a low Zn(II) transfer is usually not examined due to the limited response of the chelating probe, and therefore there is a high chance that the obtained value is significantly biased. A detailed analysis in this case would require several probes with various Zn(II) affinities and a presentation of total Zn(II) transfer against free metal ion concentrations.
3.3. Partially Metalated Zinc MT Species
3.3.1. Partially Metalated MT Species as Components of a Cellular Zinc Buffer
Although a function of MT as a zinc buffer was postulated repeatedly, the actual buffer would be T, which would have to be present in different states of metalation for effective buffering. The lack of further insight into MT metalation and speciation and the range in which cellular Zn(II) must be buffered led to the very unsatisfactory situation that research on MT remained phenomenological and correlative for at least 50 years. The then prevailing dogma that MT binds all seven Zn(II) with a low picomolar affinity was at odds with the increasing number of observations that eukaryotic cells keep free Zn(II) concentrations in the high picomolar to nanomolar range, far above the affinity of MT for Zn(II). (89,171) The assumed high cooperativity of Zn(II) binding and the then widely accepted overall dissociation constant would allow MTs to buffer free Zn(II) only in a narrow range around Kd (eqs 1 & 2; Figure 19, left panel). This range is far from the reported fluctuations of cellular free Zn(II) and the T/(T+MT) ratios found in many tissues (orange dashed box).
(1)
(2)
Figure 19
Figure 19. Speciation of zinc metallothionein (Zn7MT, MT) according to three thermodynamic models in the literature. (left) Model with all seven Zn(II) ions bound with the same affinity (Kd1–7 = 10–11.8 M). (141) (middle) Stepwise model with Kd values ranging from 10–11.8 to 10–7.8 M for human MT2. (73) (right) Stepwise model with Kd values close to each other in the range from 10–12.5 to 10–11.4 M for a modified human MT1a. (172) The upper panels show the relationship between ratios of the apo-form (T) over total protein and free Zn(II) concentrations (−log[Zn(II)]). (lower) Speciation of the apo-form, partially and fully Zn(II)-saturated species. The heuristic value of the models is discussed in the main text.
The situation drastically changed when human Zn(II)-MT2 was analyzed with the highly sensitive fluorescent zinc probes FluoZin-3 and RhodZin-3, and new insights into the affinity of MT for Zn(II) were gained. (73) It was discovered that the seven Zn(II) ions bind with various affinities ranging from picomolar to nanomolar. Four Zn(II) ions indeed bind with a low, highly similar, picomolar affinity forming a Zn4MT species. Whether or not the formation of this species is cooperative requires a high-resolution mapping and further insights into the metal-coupled protein folding. The binding of the remaining three Zn(II) ions results in the stepwise formation of Zn5MT, Zn6MT, and Zn7MT species with lower metal-to-protein affinity. The investigation with fluorescent probes and investigations performed later with other chelators and mass spectrometry showed that this process is reversible, meaning that dissociation (Kd7 – Kd1–4) of Zn(II) from Zn7MT also occurs stepwise as indicated in eqs 3–6.
(3)
(4)
(5)
(6)
The existence of several partially saturated species allows T to buffer Zn(II) in a much wider range (Figure 19, middle panel)─almost 4 orders of magnitude─than in the presumed case of MT binding all seven Zn(II) with the same affinity, as indeed would be required for the myriad of cellular functions of Zn(II) and its fluctuations as a signaling ion. The relationship between free Zn(II) and the concentration of particular species (eq 7) shows that free Zn(II) concentrations depend on the concentrations of not only the metal-free species but all partially metalated forms.
(7)
Using the above equations and the dissociation constants (Kd1–4 = 6.3 × 10–47.2, Kd5 = 3.5 × 10–11, Kd6 = 1.1 × 10–10, and Kd7 = 2.0 × 10–8 M) one can calculate the concentrations of any particular species as a function of free Zn(II) concentration(s). (73)
Taking into account the fluctuations of free Zn(II) established in numerous investigations of eukaryotic cell lines (143,144,171,173−176) it became clear that partially metal-saturated species such as Zn5MT or Zn6MT could serve as both Zn(II) donors and acceptors (orange box in the middle panel of Figure 19). Depending on Zn(II) concentrations, cell types, and the physiological or pathological state with increased or decreased concentrations of free Zn(II), the Zn7MT and Zn4MT species are expected to be present as well and to participate in buffering.
The different models of Zn(II) binding to MT are discussed in more depth because of their implications and the controversies they engendered. The state of the protein could be a simple reason for observed experimental differences. Without reconstitution and additional purification steps the protein is usually neither fully loaded with metal ions nor fully reduced when isolated from biological sources. A different starting point obviously would affect the determination of stability constants. (167) For a complicated system such as MT that binds seven Zn(II) ions to 20 sulfur donors, the determination of affinities is not a trivial matter, and it is critically important to employ highly sensitive methods that allow an investigation of affinities over a wide range. It is generally not achievable when using just one reagent because the signal resulting from Zn(II) binding/dissociation changes only 1 to 2 orders of magnitude in standard spectroscopic titrations at a constant pH. Highly sensitive fluorescent zinc probes made it possible to differentiate affinities for Zn(II) ions in MT2 for the first time. (73) UV-monitored pH titrations of MT do not have enough sensitivity for the resolution of stepwise changes of Zn(II) ion affinities. They are sensitive enough, however, in the case of Cd(II) ion affinities that are grouped according to the formation of the clusters and with log K differences reaching a value of 3. The application of electrospray ionization mass spectrometry (ESI-MS) to analyze the metal-to-thionein and metal-to-thionein + apo-carbonic anhydrase (apo-CA) systems led to the conclusion that all Zn(II) ions are bound tightly in human MT1a with very similar affinities (log K from 11.4 to 12.5, Figure 19, right panel). (172) In this approach, relative intensities of signals are treated quantitatively under the assumption that they reflect equilibria in solution when in fact protein species are measured as ions in the gas phase. The assumption of identical equilibria in both phases is inappropriate for several reasons. Similar to the spectroscopic studies the application of only one competitor for the analysis of the MT system and the resulting accuracy in the determination of signal intensity can overestimate affinities. When using apo-CA as a competitor, association/dissociation rates of Zn(II) between MT and CA are vastly different and can be modified in the gas phase. (172) The last but not least reason is a limitation inherent to ESI-MS-based determinations of affinities, namely, that Zn(II)-peptide complexes undergo metal deposition or supermetalation under conditions of ESI-MS. (177,178) At present, a comparison of the two models (Figure 19, middle and right panels) is not possible for severals reasons. Two different human MTs were used (MT1a and MT2). There could be an intrinsic difference between the two forms, or a difference could be due to the fact that the recombinant MT1a protein used had an additional 11 amino acids (72 instead of 61) as a result of the cloning and expression strategy. Given the pH dependence of thiolate coordination (Figure 17), the difference in pH of the MT solutions (pH 6.8 for the ESI-MS investigations and pH 7.4 for the fluorescence investigations) is expected to have a significant effect on affinities and speciation. Above all, there is no definition of pH in the gas phase, and the salts used in ESI-MS are usually not buffers in the physiological range.
As discussed above, analyses of tissues and cell lines indicated the presence of significant amounts of the apoprotein. Because of the low resolution of the differential labeling methods used for the detection of T/(T+MT) ratios, it was not possible to address partially metal-saturated species. Zn6MT, Zn5MT, and Zn4MT species are readily formed when aliquots of fully saturated MT (Zn7MT) and the metal-free form (T) are mixed and equilibrate. Depending on the ratios of reactants, different metal-saturated species form. For instance, the highest concentration of the Zn6MT species is formed when thionein over total protein is ∼14%, while Zn5MT and Zn4MT species predominate when the molar ratio reaches ∼29 and 43%, respectively. The species distribution matches exactly the results obtained from ABD-F differential labeling, indicating that partially metalated species of MT indeed form in vivo under normal physiological conditions. It is worth noting that an application of the differential labeling method for the determination of T/(MT+T) ratios provides information on freely available Zn(II) in tissues and cells. The biological significance became apparent only when independent measurements of free Zn(II) concentrations with fluorescent chelating agents became possible and established their range. One needs to keep in mind that the differential labeling measures all MTs present, whereas the above speciation is based on the properties of only one form, human MT2. Thus, the different MTs and the different amounts of MTs present in cells contribute to buffering and, if affinities of MTs other than MT2 for Zn(II) are different, the buffering range in addition to the buffering capacity will differ. Adding the number of inducers of differential MT gene expression, gene silencing, the redox and pH effects on the proteins, ligand interactions, and protein turnover, one begins to appreciate the complexity, subtlety, and possible fine-tuning of buffering cellular and subcellular Zn(II) and controlling signaling Zn(II) ions.
3.3.2. Structures of Partially Metalated MT Species
Metal-binding properties of MTs have been investigated extensively for several decades using a wide range of physicochemical methods. (73,179−182) Originally, the most informative ones were spectroscopic methods such as UV–vis absorption spectroscopy and spectropolarimetry (circular dichroism (CD)) to examine the number of metal ions that bind to MTs, for conformational studies that are associated with metal binding, kinetics of complex formation, and dissociation in the presence of numerous competing reagents and proteins. From the very beginning of the field, two processes were investigated: metal association with the apoprotein (T) and metal dissociation from the holo-protein (MT). Since Zn(II) has no signatures in most spectroscopic methods, other metal ions such as Cd(II) or Co(II) were used as probes for the Zn(II) binding processes and coordination modes. (183) Cd(II) complexes with Cys-rich proteins have conspicuous ligand-to-metal charge transfer bands with lower energy when compared to Zn(II), facilitating the interpretation of conformational changes. (184−186) Spectra of Co(II) complexes allow a direct observation of d-d bands and conclusions about coordination environment and geometries of the metal ions in partially and fully saturated MT species. (133,187) Fluorimetric methods were applied for structural studies of MT with attached FRET probes and to examine the metal association and dissociation and the determination of metal affinities. (73,151,154,188,189)113Cd and 111Cd-NMR spectroscopy was critical for the elucidation of the cluster formation in the α- and β-domains, cluster dynamics, and the kinetics of Cd(II) transfer. (190−193) However, there is a caveat: the Cd(II) metalation process differs from that for Zn(II), and despite the chemical similarity of the two cations information obtained for one cannot be transferred readily to the other. (98,154,163) Important steps in the characterization of MTs were the determination of NMR structures of isolated domains of human and rabbit MT2 as well as the solution of the crystal structure of rat MT2. (44,46−48) The MT structures characterized are the ones for the fully metal-saturated proteins. 3D structural investigations on partially metalated species were never performed. Metalation and demetalation pathways were investigated with various spectroscopies. However, because of the limited resolution of the physicochemical techniques, the absence of well-defined secondary structures, and significant coordination dynamics, structural details about partially saturated MT species remained elusive.
Advances in the structural characterization of dynamic and multiconformer, metal-depleted MT species came with the use of high-resolution mass spectrometric techniques. The pioneer in the application of mass spectrometry for the investigation of MT was Catherine Fenselau and her group. With ESI-MS as a high-resolution technique in the 1990s, it became possible to analyze MT alkylation with pharmaceuticals such as melphalan and chlorambucil, (194,195) metal ions in native and reconstituted MTs, and reactions with chelators. (196,197) In the latter experiments, numerous partially metal-saturated species were identified by using a direct infusion of solutions that contained either T with variable metal ion equivalents or MT with different reagents. ESI-MS became rather popular due to the ease of sample preparation and the rapid acquisition of a wealth of data. It is widely used for the determination of metal-to-protein stoichiometries as well as the characterization of the metalation processes in both mammalian and other MTs. (198−201) Different spectrometric profiles observed at various metal-to-apo-form molar ratios and intensities of signals that correspond to particular MxMT species provide information on the association of metal ions with the protein. While this information is rather useful for a protein characterization or a comparison of various metal ions or proteins, it is critical to understand its limitation. In particular, it is assumed that speciation in the aqueous, nonbuffered solution injected into the mass spectrometer corresponds quantitatively to that in the gas phase as represented by relative intensities of peaks in the mass spectrum. Under well-controlled conditions, native-like conformations of proteins can be retained during the ESI process. However, it does not apply readily to metal centers that underly their own rules in the ESI process. (202,203) In the case of MT, the structure of species is based almost entirely on metal networks and the intrinsically disordered character of the apoprotein, and the coupling of the folding and metalation processes must be considered. The dynamic character of the different metal binding sites may produce various profiles in mass spectra and is not exclusively linked to the thermodynamic stability of the binding sites. Several intriguing conclusions about MT metalation have been drawn from investigations of ESI-MS profiles of the whole protein and its isolated α- and β-domains. (138,163,204,205) One limitation is that the properties of the isolated domains are not fully additive in describing the whole protein. With such an approach, it has been proposed that Zn(II) and Cd(II) differ in the formation of clustered and independent sites at various pH values, notwithstanding that there is no definition of pH in the gas phase. (204,206) It is impossible to gain insights into the metalation mechanisms based on only relative changes of ESI-MS profiles. Other approaches with higher resolution must be used. For example, N-ethylmaleimide (NEM) is highly effective for a differential modification of free and Cd(II)-bound cysteine residues. (98,157) When combined with protein fragmentation or in-solution proteolytic digestion it can be employed to distinguish coordinated and free Cys residues and accurate and precise measurements of Cd(II) metalation, for example, to demonstrate that the Cd4Cys11 cluster is exlusively formed in the α-domain prior to a metalation of the β-domain. Likewise, iodoacetamide (IAM) was employed for the differential labeling of Cys residues in MT when Zn7MT was equilibrated with Zn(II)-depleted sorbitol dehydrogenase (apo-SDH). (153) With a subsequent top-down and bottom-up approach enhanced by a matrix-assisted laser desorption/ionization (MALDI) analysis, the importance of Cys21 in both Zn(II) association to and dissociation from the weakest Zn(II) binding site in the β-domain has been demonstrated. (153) The application of NEM and IAM together in a dual labeling method allowed further resolution of the Zn(II) metalation process. (98,207) It demonstrated that the first four Zn(II) ions bind to both α- and β-domains as suggested from the quenching of the reactivity of the thiols with Zn(II) ions and the analysis of ESI-MS profiles. (72,73,162,204)
MS-based investigations of MTs either confirmed conclusions drawn from previously performed spectroscopic investigations or further characterized the metalation-coupled protein folding. MS techniques are promising for those metal ions that do not form clusters cooperatively but sequentially, such as physiologically important Zn(II). The presence of multiple conformers in metal-depleted species with partially clustered and independent binding sites is especially challenging for a structure determination with mass spectrometry as well as analytical methods that are crucial for the obtainment of valuable solution-concurring freeze frames. One of the newer MS techniques adding a new dimension to MT research is ion mobility MS (IM-MS). Like ESI-MS it is based on characteristic m/z ratios, but in addition it can interrogate the possible heterogeneity of peptide/protein ions and resolve populations of conformers in an assembly. (155,208,209) Thus, MT conformations were first demonstrated for apo-MT2 and Cd7MT species. (210) It allows a distinguishing of final products, differently metalated states, and intermediates in the metalation reactions. (156) Experimental collision cross sections (CCS) can be correlated with those simulated by molecular dynamics (MD) to obtain information on the conformation of particular protein species. (136,211−214) The CCS profile analysis is also used to probe differences in gas-phase stabilities of partially metalated species upon collision-induced unfolding. (158,215)
Very recently a mass spectrometric investigation supported by molecular dynamics calculations characterized the coordination environments in all seven metalated species of human MT2 and demonstrated how the coordination changes upon successive metal association/dissociation. (216) Remarkably, the binding of Zn(II) is different from that of Cd(II). Thus, Zn(II)- and Cd(II)-MTs are isostructural only in the species with seven metal ions, limiting the use of Cd(II) for investigations of MTs in zinc metabolism. The work provides the highest resolution yet in terms of structure and reactivity of the protein with metal ions and sulfhydryl modifying agents. An important difference is that the first two Zn(II) and Cd(II) ions select different coordination sites and begin with mononuclear tetrathiolate coordination in the α-domain (Figure 20). The third and fourth metal ions then coordinate in the α-domain only for Cd(II) while they begin to populate the β-domain in the case of Zn(II), rendering the formation of clusters but one aspect of the coordination. In these metalation processes, the protein adapts many conformations depending on the metal load and the type of metal ion affecting the folding and generates a huge conformational space (“structural landscape”) to shape the functions of the protein for reactivity and interactions. The different binding properties of copper further expand this conformational space. The metalation process in the entire protein is different from that in the individual domain peptides because metalation in one domain affects sulfhydryl reactivity in the other. The investigation also provided evidence for the hyperreactivity of Cys21 and Cys29 in the β-domain of the zinc but not the cadmium protein. In the crystal structure of rat Cd5Zn2MT2, these sulfur atoms show S···HN hydrogen bonding to the peptide backbone. (47) How the suppression of such a reactivity by Cd(II) reflects on the biological function remains to be determined. With significance to the biological context, the investigation demonstrates that the structures and reactivities of MTs change depending on the availability of metal ions and illustrate the importance of other MT structures in addition to the only one determined with seven divalent metal ions by X-ray crystallography and NMR spectroscopy. This aspect of how the biological context dictates structures of MTs and how it relates to functions of structurally highly flexible MTs in metal buffering and interactions will be discussed further in section 4.
Figure 20
Figure 20. Order of the stepwise Zn(II) dissociation for the unfolding pathway for (A) Zn7MT2 and (B) Cd7MT2 obtained by constant-speed steered molecular dynamics simulations that involve Zn7MT2 and Cd7MT2 with the C-termini fixed. (216) The bars show the binding/unbinding of the metal ion for each metal-loaded species. Representative conformations of the protein when six, five, and four Zn(II) ions (C) or Cd(II) ions (D) are bound. Zn(II) and Cd(II) are represented by gray spheres, and the sulfur atoms are shown in yellow. (57) The figure is adopted from ref (216) with permission of the American Chemical Society.
3.4. Copper MT
Some copper was found in the first-ever preparation of MT from horse kidney. (2) A copper protein isolated from the liver of newborn calves and localized in heavy lysosomes showed an amino acid composition similar to that of MT. (7) Likewise, a major Cu(I)-binding protein from human fetal liver was identified as MT. (8) The Cu(I)-rich form is in an insoluble fraction, whereas the Zn(II)-rich form is in a soluble fraction. (217) When fetal and neonatal human livers were investigated, a linear correlation between cytosolic MT and Zn(II) but not Cu(I)/Cu(II) was noted, and an involvement of MT in copper and zinc metabolism was suggested. (218)
The variable metal content of MTs isolated from native tissues is summarized in at least two published tables. (181,219) The data indicate that native mammalian MTs contain Cu(I) in all instances when MT is not induced by Zn(II). The isolation of MT from tissues where its content is rather high, such as the liver and kidney, introduces a certain bias toward metal metabolism in these organs. In the eye, the stoichiometry of MT was found to be Zn6CuMT, which changed to Zn7MT after the induction of the protein with Zn(II). (220) The situation is different for the Cd(II) induction, where some Cu(I) can be found in mouse MT. (221) In Cd(II)-exposed rats, Cu(I) is found in plasma MT. (222) These observations suggest that Cd(II) competes with Cu(I) in copper proteins and that the displaced Cu(I) binds to MT. However, an indirect mechanism is also conceivable. Exposure to Cd(II) elicits an oxidative stress. Thus, the reactive (oxygen) species could release Cu(I) from its binding sites. The harsh procedures employed to isolate MT from tissues raise the issue of whether the presence of some Cu(I) in MT is an artifact of the isolation, namely, that MT, due to its high affinity for Cu(I), binds some Cu(I) that may become available when the tissue is homogenized. The substantial amounts of Cu(I) in MT3 and MT4 from natural sources seem to indicate that their Cu(I) content is not an artifact of isolation and that these proteins have a role in copper metabolism. MT4 was classified as a copper MT based on metal-binding properties and comparison with other MTs, although it binds Zn(II) as well. (223) It is not clear whether the amount of copper in MT is simply determined by the specifics of copper metabolism in a particular tissue. Yet another consideration is that subcellular differences in the metal metabolism may contribute to the different metal loads of MTs. For instance, in the rapidly growing liver of the bank vole, cytosolic ZnMT disappears while nuclear CuMT appears, suggesting the replacement of Zn(II) by Cu(I). (224) In cyanobacteria, it has been observed that the location where a protein folds influences its metal content. (225) A location-specific metalation of MT would lead to a mixed-metal composition of isolated MTs and add yet another level of complexity to the elemental speciation of MTs in vivo.
3.4.1. Cu(I) Binding Affinity of MT
When Cu(I)─in most cases as [Cu(MeCN)4]+─is titrated into Zn7MT, in which the Zn(II)-thiolate clusters determine its protein structure, different structures are obtained compared to when Cu(I) is titrated into T. (53,226) The stoichiometry of seven divalent metal ions does not apply to monovalent ions. Rabbit liver Zn(II)-MT2 binds 12 Cu(I) ions in one Cu6S9 and one Cu6S11 cluster. (53) Two distinct Cu(I)4-thiolate clusters are formed with 12–14 cysteine residues when T─prepared from rabbit liver─binds Cu(I) ions. (226) Cu8MT is a stable intermediate, as the phosphorescence titration demonstrates a breakpoint after the addition of eight metal ions before the binding of an additional four Cu(I) ions occurs. A Cu4S8–9 cluster instead of the typical Zn3S9 cluster has also been observed in human MT3. (227) A remarkable spectroscopic property is being exploited in the investigations of Cu(I) binding, namely, the luminescence of Cu(I) in MT, which is due to efficient MLCT in the lowest excited states and results in a thermally activated delayed fluorescence (TADF) combined with a short-life phosphorescence. Accordingly, the emission, depending on temperature, consists of two decay paths at variable relative intensities: direct phosphorescence and TADF-only. (228) Cu4S6 and Cu6S9 clusters in the β-domain were observed in titrations (followed with ESI-MS) before Cu4S6 and Cu7Sx clusters in the α-domain are formed with specific emission and CD properties. (229) There seems to be a domain preference for Cu(I) binding with the β-domain having a marginally higher affinity for Cu(I). (229,230) A stoichiometry of up to 20 Cu(I) ions can be reached as in the case with Hg(II) and Ag(I). A caveat is, however, that a human MT1a protein with 72 instead of the 61 amino acids of native MT1a was used in these investigations. Such “supermetalated/hypermetalated” species are expected not to exist under normal physiological conditions, as the cellular availability of Zn(II) and Cu(I) is strictly controlled and the synthesis of more MT would be induced to cope with a surplus of metal ions. Supermetalation can be a result of the nature of the mass spectrometric investigation. At this juncture, a reminder is necessary that the exact structures of the Cu(I)-thiolate clusters in mammalian MTs have not been determined.
Gaps in our knowledge about Cu(I) interactions with MTs remain for several reasons. One reason is related to the redox properties of copper. When Cu(II) reacts with MTs, Cu(II) is reduced to Cu(I), and the thiolate sulfurs in the protein are oxidized (the redox chemistry of Cu(I)/Cu(II) in MT is discussed in section 3.4.2). Investigations of Cu(I) interactions with T or MT require anaerobic conditions. Another reason is related to the coordination geometry of Cu(I), which forms diagonal, trigonal, and occasionally tetrahedral binding sites with cysteinyl sulfur. Because of the high number of sulfur donors in the MT clusters, complexes with various stoichiometries are formed, making Cu(I)/MT interactions difficult to investigate. The last but not least reason is the thermodynamics of the interactions of Cu(I)/Cu(II) with MTs. Cu(I) forms extremely tight complexes with the cysteinyl sulfurs in MTs resulting in stability constants that cannot be determined directly. To investigate the interaction, competing ligands, the choice for which has been much more limited compared to those for Zn(II) until recently, are required. (231−233) All approaches involve the determination of stability constants based on a reference to a competing reagent. In addition to the very high Cu(I)-MT stability, the instability toward disproportionation further complicates the measurements. Therefore, the Cu(I) ion needs to be stabilized by a low-affinity ligand complexation such as in the complex with acetonitrile. (234)
The first investigations of the affinity of Cu(I) to MT led to a formation constant (K) of at least 2 × 1016 M–1. (141) It is not an absolute value determined by any particular method but one estimated based on the known affinity of Zn(II) and Cd(II) to MT and the fact that Cu(I) displaces both metal ions from their MT complexes and, hence, must have a higher affinity. Taking into account that Zn(II) and Cd(II) bind to MTs with various affinities, the estimated value was corrected for the weaker Zn(II) and tighter Cd(II) sites in MT. Subsequently, the stability of Cu(I)-containing MTs was investigated with chromophoric chelating probes with a known Cu(I) affinity. These probes change their characteristic electronic absorption when Cu(I) binds, and they report the fractions of Cu(I) being in the complex and in the MT species. The application of bicinchoninate (Bca), a bidentate ligand with log β12 = 17.2, (235) demonstrated that the average Cu(I) affinity (Kd) in MTs is less than 10–17 M. (170) The application of another bidentate ligand, bathocuproine disulfonate (Bcs), with a higher Cu(I) affinity (log β12 = 19.8) (235) allowed the determination of the average stability constants of human MT2 and MT3 with Cu(I) as Kd = 4.3 × 10–19 M and Kd = 5.6 × 10–20 M, respectively, at pH 7.4 (25 mM Tris, 50 mM NaCl). (170) Using the stability constant of the Cu(Bcs)2 complex recently determined by cyclic voltammetry (log β12 = 20.81) and the above average Kd values of MT2 and MT3, one recalculates values of Kd = 4.2 × 10–20 M for MT2 and Kd = 5.5 × 10–21 M for MT3. (170,233) When correcting for pH 7.0, at which the constants for Bcs were redetermined, one may expect slightly lower Kd values of Cu(I) in MT2 and MT3. Importantly, in these competition experiments MT2 and MT3 species that are not fully Cu(I)-saturated were characterized, namely, Cu(I)4Zn4MT2 and Cu(I)4Zn4MT3 species that were obtained in redox reactions between Cu(II) and Zn7MTs. The presence of disulfide bonds due to redox reactions with Cu(II) and resulting different CuxSy clusters may change the average Cu(I) affinity for the β-cluster when compared to the protein with fully reduced cysteinyl sulfurs.
As with the spectroscopic approaches, competition with a ligand of known Cu(I) affinity is critical and serves as a reference for affinity when mass spectrometry (ESI-MS) is employed. Common ligands for competition are reduced glutathione (GSH), DL-dithiothreitol (DTT), or diethyldithiocarbamic acid (DETC). The literature on the affinity of Cu(I) for metalloproteins is replete with false assumptions, resulting in constants that are over- or underestimated. For instance, the average dissociation constant of the rabbit Cu10MT2 species characterized by ESI-MS and competition with DETC is reported as 4.1 × 10–16 M. (236) However, the Kd value of the reference Cu(I)-DETC complex had been determined in competition with DTT as 1.4 × 10–14 M under the assumption that the Kd of the Cu(I)-DTT species is in the range of 10–12 M (236) when in fact it is much lower, that is, ∼10–16 M. (237) When a different reference Kd value for Cu(I)-DTT (5.0 × 10–16 M, pH 7.3) based on spectroscopic competition with Bcs was used, the average Kd value for Cu(I)10MT2 turned out to be 2.6 × 10–20 M. (235,238) The use of the subsequently updated Cu(I)(Bcs)2 affinity (obtained from cyclic voltammetry) gave an average Kd value of 2.5 × 10–21 M for rabbit Cu(I)10MT2 (pH 7.0). It is expected to be slightly lower at pH 7.4. (233) Currently, it is unknown whether minor differences in the affinities of Cu(I) in Cu4Zn4MT2 (human) and in Cu10MT2 (rabbit) originate from methodological issues or are indeed related to the origin of the protein or the stoichiometry of the particular species. Similar values for both proteins indicate that Cu(I) binds, on average, several orders of magnitude more tightly than Zn(II) or Cd(II). More information was garnered from an investigation of human MT1a. (205) When apo-MT1a, that is, thionein (T), was titrated with [Cu(MeCN)4]PF6 and the reaction was monitored by ESI-MS, multiple Cu(I)1–20MT1a complexes with various relative signal intensities were detected. With the average Kd value of rabbit Cu(I)10MT2 (see above) as an affinity standard, average Kd values of particular Cu(I)1–20MT1a species varied from 3.3 × 10–21 M for Cu(I)4MT1a to 7.1 × 10–18 M for Cu(I)20MT1a. (205) When this analysis was applied to the α- and β-domain peptides of human MT1a and the same affinity standard was used, a much wider range of dissociation constants for Cu(I)20–xMT1a species was found. (54) It was suggested that only some of the CuxMT1a species have very high affinities. For instance, average Kd values for the stable complexes Cu(I)4MT1a, Cu(I)6MT1a, and Cu(I)10MT1a are 6.3 × 10–23, 2.5 × 10–21, and 2.5 × 10–20 M, respectively. A rather moderate affinity (Kd = 1.3 × 10–11 M) was found for the Cu(I)13MT species, while the affinities for other weak complexes vary between 10–4 and 10–10 M. (54) Bearing in mind that ESI-MS has some major limitations in a quantitative analysis and for the determination of equilibrium constants, these investigations show that the affinities of Cu(I) sites in MT vary by several orders of magnitude.
3.4.2. Cu(II) Interactions with MT
Currently, the exact mechanism of the interaction of MTs with Cu(II) is unknown. The reaction of one thiol with one Cu(II) ion results in the reduction to Cu(I) and the formation of a thiyl radical. When Cu(II) is added to Zn7MT2 or Zn7MT3, a cooperative formation of a Cu4Sx cluster in the β-domain together with dissociation of three Zn(II) ions and the formation of two disulfides was proposed; however, this was confirmed only by a shift of molecular mass in mass spectrometry. (239) The exact localization of the disulfide bridges is unknown and so is the potential interaction of the sulfur atoms in the disulfides with Cu(I). (240−242) This four-electron reaction occurs in situ and is believed to be part of the protection of the cell against any extracellular Cu(II). The addition of another equivalent of Cu(II) to Cu4MT2ox or Cu4MT3ox in vitro causes a similar oxidative dissociation of four Zn(II) from the α-domain and formation of the second Cu4Sy cluster and two disulfides. (239)
The redox chemistry and biology of copper MT is twofold. T or MT are reductants for Cu(II), but whether this reaction is ever important inside cells is unknown, because the main form of copper in the cell is Cu(I), and any Cu(II) would be readily reduced by glutathione and then sequestered by MT. Thiyl radicals can be formed in the one-electron oxidation of the thiolate(s). (242,243) MT3, as isolated, has the composition Cu4Zn3–4MT3. The Cu(I)4-thiolate cluster is in the β-domain and is stable against air oxidation. (227) However, a Cu4Zn4MT3 prepared in vitro has two homometallic clusters, a Cu4 thiolate cluster in the β-domain and a Zn4 thiolate cluster in the α-domain. The latter is air-sensitive, and one Zn(II) ion dissociates concomitant with the thiolate oxidation, resulting in a Zn(II)3-thiolate cluster in the α-domain. (223,244,245) Thus, both sulfur and copper redox chemistry occur in MTs. MTs can inhibit copper redox cycling, but they can also be the target of oxidants and electrophiles for a copper dissociation for yet to be defined purposes or, under an oxidative stress, to initiate cellular damage. (126)
In contrast to Cu(I), the chances are higher for any Cu(II) to escape from proteins because of its lower affinity (see the discussion in section 4.4), unless specific kinetic barriers evolved in proteins to prevent the dissociation of Cu(II) in the cell. Outside cells, Cu(II) can be scavenged and reduced by extracellular MT3. (246,247) Zn7MT3 suppresses the Cu(I)-catalyzed formation of hydroxyl radicals in the presence of ascorbate. (239) The finding could be interpreted as Zn(II)-MT controlling the redox activity of free Cu(II) ions. The growth inhibitory function of MT3, which is supported by both Zn7MT3 and native Cu4Zn3–4MT3, has not been explained in molecular terms, and thus, it is not clear whether it relates to copper metabolism. Because Cu(II) is needed for cell proliferation, the sequestration of Cu(II) as Cu(I) could be a function of Zn7MT3 with the native copper protein being the product of such a function.
Cu(II) is present extracellularly, not only in pathological states but also under normal conditions. Any MT secreted from cells is expected to encounter Cu(II). Where exactly and which MT protein interacts with Cu(II) and whether the same species as in vitro form is not known. The isolation of Cu(I)/Zn(II)-MTs demonstrates that species with less than four Cu(I) can form in vivo. These forms are likely partially oxidized (CuyZn7–xTo) and contain variable amounts of Zn(II). The location of Cu(I), Zn(II), and the disulfides in such forms are not known. The interaction of several Cu(II) complexes with MT has been investigated. (246,248,249) Although some of the Cu(II) ligands form stable complexes they react the same way as inorganic Cu(II) salts resulting in an oxidative formation of Cu(I)-thiolate clusters. However, complexes with a very high affinity for either Cu(I) or Cu(II) (with log K > 20) do not react with MT, demonstrating an important limitation of MT as a cellular Cu(I) acceptor and reducing agent: The competing molecule (protein or low-molecular-weight ligand) must bind Cu(I) more tightly than log K > 20 in order not to undergo a Cu(I) transfer or, in the case of Cu(II) complexes, in order not to be reduced to Cu(I) complexes. (249) The implication is that only copper proteins with these features can function independently and be resistant to competition with MTs. Proteins that do not fulfill these conditions are prone to react with MTs.
From investigations of the affinity of isolated MTs one could conclude that Cu(I)-MTs cannot coexist with Zn(II)-MTs in cells. One factor that allows both to occur in cells is the difference in subcellular localization: Cu(I)-MT in lysosomes, in particular, under conditions of high Cu(I) concentrations, and Zn(II)-MT in the cytosol. The higher chemical stability of the Cu(I)–S(thiolate) bond compared to the Zn(II)–S(thiolate) bond allows Cu(I)-MT to exist under the acidic conditions in lysosomes.
3.5. Cadmium MT
The discovery of MT as a cadmium protein from horse kidney as a result of searching for a function of Cd(II) in biology set a separate line of research into motion, namely, the putative role of MT in the detoxification of some metal ions. The discussion of cadmium MT here has another reason: Cd(II), either when used for an induction of MT in animals or when added during a heterologous expression of recombinant MT proteins, confers a higher stability on the protein. Therefore, and because its isotopes lend themselves for NMR investigations, it became the metal of choice for many biophysical investigations and consequently the basis for our knowledge about structure. The preoccupation with Cd(II)-MTs for decades has a very important educational consequence. It distracted from a focus on the physiological functions of these mammalian proteins in zinc and copper metabolism. Without doubt, it is one of the reasons why insights into the functions of MTs were delayed so much.
Cd(II) accumulates in the α-cluster, in contrast to Cu(I), for which a thermodynamic and kinetic preference for the β-domain was noted. (230) It is also different from Zn(II), which does not bind cooperatively to the α-domain and does not have a preference for either one of the domains. (98) Heterometallic Cd5Zn2MT can be formed in vivo upon induction with Cd(II), and five ions are the maximum number of Cd(II) to be incorporated. Homometallic Cd7MT can be produced only in vitro or in heterologous expression systems. The binding of an additional two Cd(II) ions per monomer induces dimerization. It is accompanied by a major rearrangement in the β-cluster, as only the four peaks of 113Cd in the α-cluster are conserved in NMR spectra. (250) Phosphate is involved in the dimerization. Two hydrogen phosphate anions (HPO42–) per dimer bind noncovalently with a binding constant of Kd = 23 μM at pH 8.0. (251)
The binding affinity of Cd(II) to its sites in MT is ∼3 orders of magnitude tighter than the strongest binding of Zn(II). It is typical for this group in the periodic system of the elements (PSE) that the kinetic lability decreases in the order Hg(II) > Cd(II) > Zn(II). Thus, intracluster exchange rates of Cd(II) are t1/2 = 0.5 s for the β-cluster (at 35 °C) as determined with 111Cd(II) saturation transfer NMR and 16 min for the α-cluster as determined with the radioisotope 109Cd(II). (58,252) However, the exchange rates of the metal ions in the β-cluster are different with half-lives differing by a factor of 4 and metal site 4 (Figure 5) with sulfur donors from Cys5, Cys7, Cys21, and Cys24 exchanging more slowly. Site 4 was identified recently as the site for the most weakly bound Zn(II) ion in human MT2 thus showing the highest kinetic lability. (153) The mechanism of this intramolecular self-exchange reaction is an intermolecular transfer with Cd(II) sharing sulfur donors from both β-domains in a dimeric intermediate. (252) An interdomain exchange is also possible and has been shown recently for Zn(II) in partially metalated species. (98,153) Thus, at least three types of dimers can form: noncovalent dimers during metal exchange or with additional Cd(II) ions and a phosphate ion bound and covalent dimers bridged by disulfides. (192) The cadmium protein was essential for structural work, especially for the determination of the cluster structures with 113Cd NMR (35) but also for the NMR solution and X-ray crystal structures. Protein dynamics also plays a role, as the Cd(II) exchange in the β-cluster of Cd7MT1 is faster than in Cd7MT2. (253) Cluster volumes are ∼20% higher for Cd(II) than for Zn(II). (48)
111Cd perturbed angular correlation of γ-rays (PAC) spectroscopy of rabbit liver MT provided evidence for Cd(II) in two different coordination environments. (254) A remarkable fact is that seven resonances are well-resolved for the seven sites in the 113Cd NMR spectra of MT (Figure 5). It signifies small differences in coordination environments despite each metal ion being in a tetrahedral geometry with four sulfur donors from cysteines.
Kinetics and thermodynamics determine the metal exchange. Ag(I) and Cu(I) have a preference for the β-domain, while Zn(II) and Cd(II) prefer the α-domain. Metal ions in one domain do not influence the reactivity of metal ions in the other domain. The mixing of Cd7MT and Zn7MT leads to a thermodynamics-based distribution in the clusters. With inorganic clusters, the mixing of homometallic clusters always results in a statistical distribution of metal ions, but in MT the formation of heterometallic clusters demonstrates “an appreciable selectivity in metal partitioning among and within the clusters” as afforded by the protein. (255) From a structural point of view, there is an interplay between coordination dynamics of the clusters and protein dynamics. In biological tissues, the availability of metal ions such as Cu(I) or Cd(II) and the occupancy of any Zn(II)-MT already existing will affect binding preferences. The different behavior of the two domains has led to the suggestion that the α-domain and β-domain have functions in storage or distribution of metal ions, respectively.
3.6. Metal Ion Selectivity
MTs bind the following ions: monovalent Cu(I), Ag(I), Au(I); divalent Zn(II), Cd(II), Co(II), Ni(II), Fe(II), Hg(II), Pt(II), Pb(II); trivalent In(III), Sb(III), Bi(III), As(III). (39) Even a derivative of rabbit MT1 containing 6 equiv of the TcO3+ ion has been prepared. (256) There are three issues with regard to physiological significance. First, the binding of many metal ions can be readily investigated in vitro. There is no a priori reason why any metal ion with a sufficient thiophilicity should not bind. The investigation of such binding can be a purely academic interest if the metal ion is never present in the organism. When a particular nonessential metal ion becomes available under occasions of environmental exposure, the question is how it will affect the function of MT in zinc (and copper) metabolism and whether it will be permanently sequestered or redistributed to harm other biomolecules. Second, in vivo foreign metal ions will never reach the high stoichiometries measured in vitro, for example, 20 Hg(II) bound to 20 cysteine residues. And third, the questions remain as to which relative concentrations of essential metal ions determine the native metal composition of MTs and whether the different MTs present in one cell have the same or different metal compositions. The translation of in vitro investigations to significance in biology faces several additional uncertainties: Which metal ions are available when T is made on the ribosome? How does metal composition and distribution change when the gene expression of MTs is induced, the redox state or reactive species changes, or when MTs translocate to subcellular compartments where metal concentrations differ? And, do other metal ions bind to the free coordination sites of MTs that are not fully saturated with Zn(II), and, if so, what are the effects on Zn(II) or Cu(I) availability in the cell?
Investigations of the interaction of iron with MTs are limited to only a few physicochemical reports. Fe(II) has a high similarity to other divalent metal ions in terms of binding to mammalian MTs with the same stoichiometry. The saturation of the T requires a higher excess of Fe(II) over the protein due to its corresponding lower affinity in the Irving-Williams series. (257) At a neutral pH, Fe(II)7MT is yellow and extremely air-sensitive, and it turns to a wine-red color when it is exposed to air. The characterization of Fe(II)-MT complexes by electronic absorption and magnetic circular dichroism spectra indicates that seven Fe(II) ions are bound to the protein in tetrahedral coordination environments. The absorption spectra show a 5E → 5T2 d-d transition in the near-infrared region at ∼1850 nm and a broad charge-transfer absorption in the UV region with a shoulder at 314 nm. Spectroscopic titrations demonstrate that the coordination environment of the first four equivalents is similar to that of Fe(II)/tetrathiolate in rubredoxin. The addition of more Fe(II) causes a red-shift of the electronic absorption until metal occupancy is reached with formation of Fe(II)-thiolate clusters that have either an S = 0 or S = 2 ground state as a result of antiferromagnetic coupling between high-spin Fe(II) ions. The clusters with S = 0 and S = 2 are attributed to tetranuclear and trinuclear centers, respectively. (258) Electron paramagnetic resonance (EPR) spectra of Fe(II)-MT complexes show a broad resonance. Their intensity increases up to 4 equiv, remaining almost constant between 4 and 6 equiv, and undergo a marked decrease between 6 and 7 equiv. Mössbauer spectroscopy shows the presence of paramagnetic and diamagnetic subspectra in the ratio of 3:4. (259) A diamagnetic component has been assigned to a four-metal cluster with antiferromagnetic coupling between high-spin Fe(II) ions. Magnetic susceptibility measurements show an average magnetic moment of 8.5 μB for the three Fe(II) ions responsible for the paramagnetic component. This value is slightly lower than for three completely uncoupled Fe(II) ions and suggests a three-metal cluster with a weak exchange coupling between adjacent Fe(II) ions. An analysis of the data demonstrated a pseudoplanar geometry rather than a chairlike geometry in the Fe3S9 cluster, indicating a significant difference to the β-cluster formed by Zn(II) and Cd(II).
Given the extensive Fe–S chemistry, one wonders why interactions of iron with MT in vivo have not been reported. Even though Fe(II)-MT complexes form in vitro, the occurrence of such a complex in vivo has not been documented. Yet, the reactions of Zn(II)-MT with iron complexes cannot be ruled out, especially under conditions of iron overload that may provide enough Fe(II) for a competition with Zn(II) or under conditions of low saturation of MT with Zn(II). Zn7MT induces iron release from the iron-storage protein ferritin, where iron is stored in the Fe(III) oxidation state. (260) Perhaps there is a thiolate/disulfide-based redox reaction involved in electron transfer through the ferritin shell. MT itself is too large to penetrate the ferritin channels for a direct contact with Fe(III). In another potentially biological significant interaction, MT may protect lysosomes by an iron sequestration. (261) Fe(II) does not displace Zn(II) from proteins due to its lower affinity compared to that of Zn(II). However, the addition of Fe(II) together with NO results in the formation of iron nitrosyl thiolates with a stoichiometry of Fe(NO)2(SR)2 as confirmed by EPR signals with g values of 2.013 and 2.039. (262) The same product was observed when Fe(II) and NO were added to apo-MT. In another investigation addressing reactivity, four Fe(II) ions were incorporated into an α-domain peptide of MT. The tetranuclear cluster was found to participate in one-electron and multielectron reductions of biological and artificial substrates. (263)
4. Relating Metallothionein Thermodynamics and Kinetics to Control of Cellular Zn(II) and Cu(I)
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4.1. Buffers for the Most Competitive Metal Ions
The Irving-Williams series describes the trend of affinities of the first d-series of divalent metal ions. (264,265) The late RJP Williams also suggested that the series is an overarching principle for the control of metal ions in biology. (266) The higher the affinity of the metal ion for the same coordination environment the lower is its availability, endowing the most competitive metals ions, Zn(II) and Cu(II), with the lowest available free metal ion concentrations. (267) From the affinities of biological metal complexes, he estimated the free metal ion concentrations theoretically available. The series can be employed to understand the metalation of metalloproteins. (268,269) Metalloproteins are in equilibrium with a pool of LMW ligands that remains largely undefined in terms of affinity, capacity, and coordination chemistry. Thus, for Zn(II), there is not a single equilibrium between only bound and unbound metal ions as in the original description of the series. Instead, one needs to consider the contribution of a LMW ligand pool of Zn(II):
Zn(II) metalloproteins (including MT) ⇌ Zn(II) LMW ligand pool ⇌ Zn(II) complexes such as aquo complexes and complexes with simple inorganic anions
In contrast, the Cu(I) transfer is thought to be mediated exclusively by metallchaperones without the intermediacy of an LMW ligand pool.
However, glutathione has been discussed in the binding of Cu(I) in the cell and in participating in the transfer of this ion to MT. (102)
The Irving-Williams series predicts the free concentrations of divalent metal ions from the set of the same ligand(s). The situation is different in biology, since the metal ions are in different coordination environments, and a myriad of additional equilibria pertain. One aspect that modifies the trend in d-block elements somewhat is the increase in thiophilicity of the metal ions and the decreases in the oxophilicity from left to right in the periodic table. (270) The employment of sulfur as a ligand for Cu(I) amplifies the affinity for the most competitive metal ions. (271) It emphasizes the significance of MTs by using sulfur ligand donors only. The series is a thermodynamic concept. Kinetic factors play a role as well. Kinetic trapping mechanisms are not considered in this equilibrium situation. The dissociation rates of metal ions in some proteins are so slow that, for equilibrium considerations, they are quasi irreversible. For example, in carbonic anhydrase t1/2 for the off-rate of Zn(II) has been estimated to be in the order of three years (4 °C, neutral pH). (272,273) In the series, there is a reversal for the most competitive metal ions: Ni(II) < Cu(II) > Zn(II), indicating that Zn(II) has a lower affinity than Cu(II) for the same ligand. If this would be the only determinant of metal distribution, then MT should always contain copper. Several additional factors explain how biology circumvented this restriction in the need for buffering in a specific range to avoid an overlap of functions. First, under the reducing condition in the cell, most of the copper is in the Cu(I) state, not the Cu(II) state considered in the series. The transition from Cu(II) to Cu(I) increases the affinity for copper in a sulfur environment even further. Second, while the Cu(II)/Zn(II) ratio outside cells is ∼1:1, there is ∼10 times more Zn(II) than Cu(I)/Cu(II) in the cell, and certain ratios of the total metal ions must be maintained. Third, membrane transporters participate in the control of cellular metal ions. They contribute a kinetic component to the buffering, which is called muffling. (274) Thus, the entire system of homeostatic control for the two essential metals zinc and copper must be considered in order to understand how Zn(II)-MT can exist in the presence of Cu(I)/Cu(II) in the cell. The phenomenon has been expressed with the term “the cell rules”, namely, that metalloproteins such as MTs do not have the ability for an absolute discrimination of these metal ions and that their metal preference is in part determined by other components of the metal buffering and muffling systems of cells. (275) Fourth, protein metallochaperones safeguard the trafficking of Cu(I) to a limited number of copper proteins, but apparently none exist for the distribution of Zn(II) to a few thousand proteins. Metallochaperones apparently obtain Cu(I) directly from its transporter, which itself safeguards Cu(I) as a pseudochaperone, minimizing the chance for Zn(II) instead of Cu(I) being bound to the metallochaperone.
Other, nonessential thiophilic metal ions challenge the system and compete on the basis of affinity, because there is no homeostatic system to control them. They piggyback on proteins for essential metal ions. Thus, the functions of MTs will depend on the burden of nonessential metals such as Cd(II), Hg(II), Au(I), Ag(I), and Bi(III), all of which have a higher affinity for MT than Zn(II). MT, as the name already foreshadowed, is different from other metalloenzymes that become metalated with the specific metal ion they need for catalytic function. MT is more promiscuous in this regard, arguing against a role of just one metal ion in the protein.
MTs participate in the control of the most competitive metal ions Zn(II) and Cu(I). With their affinities commensurate with the availability of free Zn(II) or Cu(I), which is so tightly bound that there is essentially no free Cu(I), they control potent effects that these metal ions have on proteins, and they control their cytotoxicity. Zn(II) in a copper site will not support any redox activity that copper has in an enzyme and inactivate it. The two competitive metal ions must be regulated in a way that Zn(II) does not end up in copper sites and Cu(I) does not end up in zinc sites. One way of avoiding a mismetalation is through a regulation of the availability of metal ions. (276) Zn(II) can end up in copper sites in heterologous expression systems, for example, when azurin is expressed in E. coli. (277) Cu(I), however, does not appear to end up in zinc sites in vivo, though it can bind in them, for example, copper alcohol dehydrogenase. (278) There is the possibility that MT has a role in keeping Zn(II) out of copper sites and also, perhaps, in fetal liver in keeping Cu(I) out of zinc sites and, of course, to ascertain that any Cd(II) present is not ending up in either zinc or copper sites. In addition, MT may remove directly the wrong metal from mismetalated proteins. This aspect is discussed further in section 4.7.2.
Zn(II) is buffered to picomolar, while Cu(I) is buffered to zeptomolar concentrations─a relatively new and very challenging area for bioanalytical chemistry (Figure 21). On the basis of the affinity to MT, cellular cadmium buffering occurs in a window between Zn(II) and Cu(I) (Figure 21). Any in vitro investigations of the effects of these metal ions on proteins, therefore, must consider these concentrations in the cellular context in order to have physiological relevance.
Figure 21
Figure 21. Free metal ion concentrations and buffering of the most competitive essential metal ions Zn(II) and Cu(I) and nonessential and toxic Cd(II). Estimated steady-state concentrations for Zn(II) and Cu(I) are picomolar and zeptomolar, respectively. Cd(II) interacts with the metabolism of both. Very low concentrations require stochastics when the volume limits the definition of concentrations. (279) Physiological and pathophysiological redox changes and other triggering events can increase the concentrations (arrows). Zn(II) fluctuations are used in cellular signaling at concentrations lower than those in calcium signaling. Recent work suggests a dynamic copper pool that is involved in copper signaling as further discussed in section 4.5.
4.2. Zinc Buffering
The exceptional scope of cellular zinc biochemistry should be recognized when possible roles of MTs are discussed. In parallel with the history of the MT field, the zinc field developed over the second half of the last century from a single zinc enzyme to the recognition that there are over 3000 human zinc proteins. (280−282) To satisfy these requirements of a huge number of proteins for Zn(II), the total Zn(II) concentration in cells is at least 1 order of magnitude higher than the one in blood. Two families of 24 zinc transporters altogether coordinate cellular uptake, extrusion, and supply to organelles. (283) The affinities of mammalian zinc proteins vary over a wide range from nanomolar for extracellular zinc sites to femtomolar for some intracellular sites (Table 1).
Table 1. Dissociation Constants (−log Kd) or Inhibitory Constants (Ki) of Zinc Proteins
| protein/protein domain | binding amino acid residues | zinc site function | –log Kd or Ki | conditionsa (method of determination) | ref |
|---|---|---|---|---|---|
| Aminopeptidase-B (Rattus norvegicus) | HHE | Catalytic | 12.4 | 50 mM Tris pH 7.4, 75 mM NaCl (EAMB) | (284) |
| Angiotensin converting enzyme (Oryctolagus cuniculus - rabbit) | HHE | Catalytic | 8.2 | 50 mM HEPES pH 7.5, 300 mM NaCl (EADT) | (285) |
| Carbonic anhydrase (Homo sapiens) | HHH | Catalytic | 12.0 | 15 mM MOPS pH 7.0 (EAMB) | (286,287) |
| 11.4 | |||||
| 15 mM phosphate pH 7 | |||||
| Carboxypeptidase A (Bos taurus) | HEH | Catalytic | 10.5 | 50 mM Tris-HCl pH 8, 1 M NaCl (EQD) | (288) |
| Dipeptidyl peptidase III (Rattus norvegicus) | HHE | Catalytic | 12.3 | 50 mM phosphate pH 7.4 (EAMB) | (289) |
| Erythrocyte glyoxalase I (Homo sapiens) | QEHE | Catalytic | 10.6 | 100 mM Tris-HCl pH 8.5, 100 mM NaCl (EAMB) | (290) |
| Leucine aminopeptidase (Bos taurus) | Binuclear | Catalytic | 9–11 | 100 mM Tris-HCl pH 7.5, 1 M KCl (EQD) | (291) |
| Porphobilinogen synthase (Homo sapiens) | CCCC | Catalytic | 1.6 pM | pH 7.2 I = 0.1 M (EAC) | (292) |
| Human sonic hedgehog (Homo sapiens) | HDH | <10 | 100 mM HEPES pH 7.5, 150 mM NaCl (FLDT) | (293) | |
| Sorbitol dehydrogenase (Ovis aries) | HEC | Catalytic | 11.2 | 50 mM HEPES pH 7.4, 100 mM KNO3 | (294) |
| Human serum albumin (Homo sapiens) | HDHD | Transport | 7.5 | 30 mM HEPES pH 7.0, 250 mM NaCl (EQD) | (295) |
| Bovine serum albumin (Bos taurus) | HDHD | Transport | 7.3 | 30 mM HEPES pH 7.0, 250 mM NaCl (EQD) | (295) |
| DNA-binding domains (DBD) of nuclear hormone receptors (Homo sapiens) | 2 sites: CCCC | Structural | 9.3, 10.0 | 100 mM bis-Tris pH 7.4 (RT) | (296) |
| hERα-DBD | 9.5, 9.7 | ||||
| GR-DBD | |||||
| Keap1 (Mus musculus) | CCCC | Structural | 11.0 | (PAR) | (297) |
| Mammalian serum retinol-binding protein (Sus domesticus) | HHH | Structural | 11.7 | 20 mM HEPES pH 7.5, 50 mM K2SO4 (EQD) | (298) |
| MTF-1 (Mus musculus) | 6 sites: CCHH | Structural | 10.5 (average value) | 100 mM HEPES, pH 7.0, 50 mM NaCl (RT) | (299,300) |
| first zinc finger | |||||
| 50 mM HEPES, pH 7.0, 100 mM NaClO4 (CDC) | |||||
| 11.6 | |||||
| PDZ and LIM domain protein 1 – LIM domain (Homo sapiens) | 2 sites: CCHC, CCCH | Structural | 14.5 (average value) | 50 mM Tris pH 7.4, 150 mM NaCl (CDC) | (301) |
| Rad50 protein (Homo sapiens) | CC+CC | Structural | ∼19b | 50 mM HEPES pH 7.4, 150 mM NaCl (FLC) | (302) |
| Superoxide dismutase (Homo sapiens) | HHHD | Structural | 13.4 | 100 mM phosphate pH, 7.4 (PAR) | (303) |
| Transcription factor Sp1 (Homo sapiens) | CCHH (third zinc finger) | Structural | 9.2 | 50 mM pH 7.0, NaCl (RT) | (302,304) |
| 12.7 | 50 mM pH 7.0, NaClO4 (CDC) | ||||
| Tristetraprolin (Mus musculus) | 2 sites: CCCH | Structural | 10.2 | 200 mM HEPES pH, 100 mM NaCl (RT) | (305) |
| Xeroderma pigmentosum group A complementing protein XPAzf (Homo sapiens) | CCCC | Structural | 9.8 | 50 mM phosphate pH 7.4 (RT) | (306) |
| CD4-Lck complex, zinc clasp (Homo sapiens) | CC+CC (interprotein site, heterodimer) | Structural, regulatory | 18.6b | 50 mM HEPES pH 7.4, 100 mM KNO3 (FLC) | (307,308) |
| Ca2+ ATPase (Homo sapiens) | N.D. | Regulatory | 80 pM | 20 mM HEPES-Tris pH 7.4 (EAMB) | (309) |
| Caspase 3 | KEH | Regulatory | 6.9 nM | 100 mM HEPES pH 7.5, 100 mM NaCl (EAMB) | (310) |
| Caspase 6 | 2.6 nM | ||||
| Caspase 7 | 76 nM | ||||
| Caspase 8 (Homo sapiens) | 4.3 nM | ||||
| Cathepsins | HC for cathepsin S | Regulatory | IC50 ∼160 nM | (311) | |
| Dimethylarginine dimethylaminohydrolase-1 (Bos taurus) | HC | Regulatory | 4.2 nM | 25 mM HEPES pH 7.4, 50 mM NaCl (EAMB) | (312) |
| Kallikreins | HH or HE | Regulatory | 10 nM-10 μM | (313) | |
| NMDA receptor | HHED | Regulatory | 10 nM | 10 mM tricine pH 7.3 (EAMB) | (314) |
| Phosphoglucomutase (rabbit) | SDDD | Regulatory | 11.6 | 25 mM histidine-Tris pH 7.5, 1.5 mM Mg2+ (EAMB) | (315) |
| Protein tyrosine phosphatase 1B (Homo sapiens) | N.D. | Regulatory | 7.8 | 50 mM HEPES pH 7.4, 100 mM KNO3 (EAMB) | (294) |
| Receptor protein tyrosine phosphatase β (Homo sapiens) | N.D. | Regulatory | 21 pM | 50 mM HEPES pH 7.4 (EAMB) | (316) |
a
EQD – equilibrium dialysis, EADT – enzyme activity: direct titration, EAMB – enzyme activity in metal buffers, EAC – enzyme activity measured in the cells, PAR – competition with PAR [4-(2-pyridylazo)resorcinol], CDC – CD-monitored competition with chelating agents, FLDT- fluorescence monitored direct titration, FLC – fluorescence monitored competition with chelating agents, RT – reverse titration.
b
Dissociation constant of the homo- or heterodimeric interprotein Zn(II) complex–constant is defined by [A][B][Zn(II)]/[ZnAB], where A and B are components of interprotein binding sites. The constant has the unit M2.
An important conclusion is that the buffering discussed for MTs (Figure 19, middle panel) covers this range of affinities. Therefore, MTs can have a dynamic buffering role in cellular Zn(II) redistribution, in controlling the metalation (“zincation”) of zinc proteins, and in reprogramming the system in such a way that cells in different states adjust to different pZn (−log[Zn(II)]free) values─the zinc potential─and zinc buffering capacities. (89) Zinc buffering by MTs is reduced when MTF-1, the zinc-sensing transcription factor, is ablated and the sensitivity of the non-MTF1-controlled Zn(II)-responsive transcriptome increases. (317) Adjustments of the “MT/T system” to various pZn values have been observed in the human HT-29 colon cancer cell line. (89) Cells incubated with an increasing concentration of ZnSO4 (0–200 μM) demonstrated a decreased pZn value from 9.1 to 8.3, corresponding to the range from 0.8 pM to 5.2 nM free Zn(II) concentrations. Simultaneously, the zinc buffering capacity, the surplus of cellular ligands binding Zn(II) tightly, and the MT/T molar ratio changed from 65 to 219 μM, from 28.2 to 22.6 μM, and from 2.3 to 3.6, respectively. Under this paradigm of increased total cellular zinc, free Zn(II) and the concentrations of MTs increase, while the zinc buffering properties of the cell decrease. (89,318) In addition to serving as a steady-state buffer to maintain homeostasis, the dynamic regulation of MTs allows a resetting of the zinc buffering to satisfy Zn(II) requirements when the state of the cell changes, for example, in differentiation. (89) When mouse mammary epithelial cells differentiate into secretory cells zinc homeostasis is remodelled by an induction of MT1 and MT2 through the glucocorticoid receptor pathway, concomitant with an increase in cytosolic free Zn(II) to maintain the lactating phenotype. (319) Increases of free Zn(II) modulate gene expression of proteins that are important for specific processes, for example, neurite expansion and synaptic growth. (320)
4.3. Zinc Signaling
A remarkable development, which followed the advent of the synthesis of fluorescent Zn(II)-chelating probes and sensors similar to the ones employed in the calcium signaling field, was the recognition of intra- and extracellular zinc signals with important effector roles. (117) In contrast to micromolar to nanomolar changes for Ca(II) ions, the nanomolar to picomolar changes of Zn(II) ions and the coordination chemistry make investigations more challenging. The occurrence of signaling Zn(II) ion transients that variously have been referred to zinc waves, sparks, or spikes means that Zn(II) ions need to be buffered not only at a steady state but temporal changes in their availability also need to be controlled. Thus, buffering must allow zinc signals to occur, reach their targets, and then restore concentrations to steady-state levels. On the one hand, MT is a source of signaling Zn(II) ions when it transduces redox signals into zinc signals or possibly as result of local pH changes due to an inherent pH dependence of the Zn(II) affinity. (86,321) T, on the other hand, removes Zn(II) ions from sites that are targets of the signaling Zn(II) ions.
4.4. Copper Buffering
In contrast to the few thousand zinc proteins, copper usage is much more limited. Estimates are that the human copper proteome consists of only 54 human copper proteins. (322) The reducing environment of the cell comes at a cost. Cu(I) is even more potent than Fe(II) in catalyzing Fenton chemistry, and it can disproportionate into Cu0 and Cu(II). MTs sequester Cu(I) in such a way that Cu(I) is no longer solvent-accessible once it is bound and that there is basically no free Cu(I). Since the volume of a typical cell is in the range of a few picoliters (10–12 L), femtomolar concentrations of free Cu(I) (Figure 21) amount to only approximately a single ion per cell, thus essentially solving the issue of how Fenton chemistry can be avoided. In MT3, Cu(I) is redox-stable in the presence of oxygen. (227) However, it does not mean that Cu(I)-MT does not participate in redox reactions. In vitro, Cu(I)-MT reacts with cytochrome c, and a role in a redox chain was proposed. (323) Furthermore, copper chaperones safeguard Cu(I) in direct transfer to the limited number of proteins. These proteins and copper transporters use sulfur donors in their coordination environments. The copper transfer from the copper chaperone of superoxide dismutase (CCS) to the active site of superoxide dismutase (SOD) involves the trigonal sulfur coordination of Cu(I) in CCS and redox reactions in a sequence of events that has turned out to be remarkably complex and recently shown to even involve the copper transporter Ctr1. (324,325) Such sulfur chemistry could apply to a Cu(I) transfer from MT. A “catalyzed” transfer is necessary. With diffusion-limited association rates, copper dissociation from sites with attomolar binding constants would take years. The use of metallochaperones for copper would seem to make a copper buffer unnecessary. Indeed, in many accounts of copper metabolism, MTs do not have a central role and are suggested to function in storage or salvage only. One model for cellular copper homeostasis is based on the observation that significant amounts of Cu(I) have been postulated to be in the glutathione pool (102,326) and that this high-capacity pool has a role in copper buffering. Glutathione forms preferentially a tetranuclear [Cu4(GS)6]2– complex. Since such a complex forms superoxide, it is likely not the copper buffering species. On the basis of its affinity of ∼1016 M–1 the actually free Cu(I) concentration is sub-femtomolar, that is, attomolar. (327) With a ratiometric two-photon probe, crips-17 with a Kd of 8 aM, it was possible to demonstrate that Cu(I) indeed is buffered to ∼1 aM free Cu(I), in agreement with dissociation constants of copper chaperones. (145) A role of glutathione in copper metabolism is further supported by the fact that induced redox changes in the glutathione pool affect free Cu(I) concentrations as measured with the ratiometric FRET probe FCP-1. (328) A possible conclusion from these studies is that ZnMT is needed as a high-affinity and low-capacity pool for scavenging Cu(I) in order to avoid the Fenton chemistry of Cu(I) buffered by the glutathione pool.
In order to discuss a role of MTs in copper metabolism, one must examine how their affinities and binding mechanisms are related to those of copper proteins. Copper enzymes are sometimes described as Cu(II) enzymes, for example, copper superoxide dismutase, though the major valence state of cellular copper is Cu(I). The estimated Cu(I) binding constant for yeast copper superoxide dismutase (1020 M–1) is 5 orders of magnitude higher than the one for Cu(II) (∼1015 M–1). (329) Murine S-adenosylhomocysteine hydrolase has a K of 3.8 × 1014 M–1 for Cu(II) in the same range. (330) Thus, on the basis of equilibrium concentrations only, Cu(II) is more likely to dissociate than Cu(I) from ligand environments with nitrogen or oxygen donors, rendering cellular concentrations of free Cu(II) potentially significantly higher than those of Cu(I) with implications not only for competition with Zn(II) but also redox biology, where any free Cu(II) in the cell is expected to be reduced to Cu(I) immediately. Thus, free Cu(II) in the cell is unlikely to be of physiological relevance. Its contribution to pathophysiological processes is a different matter, though.
With the exception of MT3, MT4, and MTs in neonatal liver, highly Cu(I)-loaded MT species in human cells have not been reported, and all the forms described contain Zn(II) as well. (181,219) However, there are large variations of metal content in different species. For example, variable Cu/Zn ratios from 0.1 to 20 were found in calf livers, with a total metal content from 7 to 15 g-atoms/mol of protein. (331) We anticipate that the formation of Cu(I)-MT species may occur by different mechanisms. One mechanism involves a direct Cu(I) binding to Zn(II)-depleted species present under conditions of low free Zn(II) concentrations (eq 8) and occurring without Zn(II) dissociation. A reorganization of metals sites is expected to take place. In this case, ZnxMT may have role as a “copper sponge”, and the CuyZnxMT species formed could serve as a Cu(I) acceptor and donor similar to the Zn(II) buffering process. In such a mechanism Zn(II) and Cu(I) buffering would occur independently; that is, Zn(II) metabolism is not affected.
(8)
Alternatively, Cu(I)/Zn(II)MT species could form via a metal ion exchange when Cu(I) with its higher affinity for MT causes a Zn(II) dissociation (eq 9). The equilibrium constant describing this exchange constant (Kex) is different from the one describing the process in eq 8, and it will be yet different if other species such as Zn6MT are involved (eq 10).
(9)
(10)
In this process, Cu(I) binding impacts the Zn(II) availability significantly, and the relative concentrations of both metal ions are linked. Increases in Cu(I) concentrations would increase the Cu(I) load of MT, while free Zn(II) concentrations would increase simultaneously. Likewise the cellular Zn(II) influx could cause a Cu(I) dissociation from MT and increase free Cu(I) concentrations. This scenario is potentially very important, since the buffering of both Zn(II) and Cu(I) converge in the MT molecule, which could serve as a rheostat for the ratio and indicate a metabolic link between the two essential metal ions. The actual affinities of both metal ions in mixed CuyZn7–xMT species remain unknown. They likely differ from those in the binary systems. Eq 11, rearranged from eq 10, expresses how MT species may control the concentrations of both essential metal ions simultaneously.
(11)
Figure 22 summarizes the two described hypothetical modes of action for CuyZnxMT in an independent or linked buffering of the two metal ions.
Figure 22
Figure 22. Two modes of action in which MT buffers Cu(I) either independent of Zn(II) or dependent on Zn(II).
Another variable in this buffering is that mixed Cu(I)/Zn(II) MT complexes may be oxidized either during their formation involving Cu(II) or in reactions with oxidizing agents (Figure 23). Sulfur oxidation in CuyZn7–xMT will change the buffering properties of the system due to either a dissociation of one metal ion or a change of affinity toward a particular metal ion.
Figure 23
Figure 23. Pathways for the formation of mixed CuyZn7–xMT in reduced and oxidized forms.
4.5. Copper Signaling
Evidence for copper signaling accumulated recently, not only in the brain for olfaction and circadian rhythm but also generally for proliferation, autophagy, and fat metabolism. (328,332) Like in the case of zinc, it appears to involve both copper secretion from cells by exocytosis (extracellular signaling) and the release of labile Cu(I) ions in the cell (intracellular signaling). Besides Zn(II)-containing vesicles (zincosomes), copper storage vesicles (CSV) have been identified in the brain and in other tissues. (333−336) The copper concentration in these vesicles is estimated to be 100 mM. (333) The CSV are considered a dynamic copper pool that is regulated for signaling with labile copper ions. Targets are, for example, phosphodiesterase 3B (PDE3B), which is involved in cAMP-dependent lipolysis, (337) or β-secretase-1 (BACE), (338) an enzyme that cleaves amyloid precursor protein (APP) to generate amyloidβ (Aβ). Copper signaling requires a control of the transients in time and space and the state of cupric and cuprous ions, and neither the chemistry nor the biology is presently known. The chemical nature of the labile copper pool remains to be defined. Because free Cu(I) is virtually absent in cells─and needs to be extremely low due to its toxicity─it remains unknown how copper signaling can operate. The cellular biology where in the cell such labile copper pools exist also needs to be addressed. Major unresolved issues are how the dynamic pools of the two most competitive ions are regulated separately─or whether they interact through the linked buffering discussed for MT─what the exact role of glutathione is and how the released ions can target specific proteins in signaling. And then there is the overarching question of why signaling with both metal ions became advantageous in evolution.
4.6. Controlling Zn(II)/Cu(I) Ratios
It is an unproductive state of affairs that scientific communities became so specialized that they address either zinc or copper biology but rarely both. Focusing on a single metal ion will provide only incomplete answers. Regrettably, it also applies to research on MT and glutathione, both of which are discussed in either zinc or copper metabolism. With the exception of MT3, mixed Cu(I)/Zn(II) species have received almost no attention. The capacity of the MT/T and GSH/GSSG systems to buffer essential metal ions can be challenged by nonessential metal ions such as Cd(II). Metal buffering in these thiolate coordination environments further depends on the redox state of the system, relating it to redox buffering. How the glutathione and metallothionein redox systems buffer Zn(II) and Cu(I), assisted by kinetic muffling by transporters and dynamic regulation at multiple levels, how kinetic lability is incorporated into thermodynamic stability, and how the two systems interact are most remarkable aspects of biological inorganic chemistry. Answers about the physiological function will not be found by investigations only in vitro. Needed are determinations of the in situ metal compositions of these pools and correlations with total metal concentrations and respective zinc or copper metalloproteins in the same cellular compartment.
In the absence of bona fide metallochaperones for Zn(II), MT can have an active role in cellular Zn(II) redistribution, which will be discussed now for metal transfer reactions.
4.7. Metal Transfer and Exchange Reactions
In addition to metal exchange between MTs and between MTs and proteins, there is metal transfer. Metal transfer reactions have been discussed as presumed function of MTs. How and when a protein acquires Zn(II) for function remains an unresolved question in biochemistry. Metal transfer reactions have been investigated in both directions: MT as a Zn(II) donor to the apoforms of zinc proteins and T as a Zn(II) acceptor from zinc proteins. While the investigations to be discussed show metal transfer in principle, it must be acknowledged that the conditions under which the experiments were performed do not reflect the conditions in vivo. With the knowledge we have now about partially metal-saturated MT species, neither the fully Zn(II)-loaded protein (Zn7MT) nor the Zn(II)-free protein (T) are expected to exist under normal conditions, and hence the investigations must be reinterpreted by considering ZnxMT species under various cellular conditions.
4.7.1. Metal Transfer from MT to Proteins
Zn(II)-MT can deliver Zn(II) to several apoforms of zinc enzymes restoring their enzymatic activity. (339) Zn(II)-MT (log K7.4 from 8 to 12) is a much better Zn(II) donor than ZnEDTA (apparent log K7.4 = 13.6) to apo-carbonic anhydrase (CA) (log K7.0 = 12). (287,340) Accordingly, it was suggested already 40 years ago that metal transfer by an associative mechanism is the physiological function of MT. (341) For the interaction of EDTA with zinc finger peptides it was shown that an associative ligand exchange mechanism gives a 6 orders of magnitude faster Zn(II) transfer compared to a dissociative mechanism, emphasizing the power of kinetic mechanisms in controlling metal transfer. (273) However, MT is not a genuine metal transferase because metal transfer occurs at stoichiometric amounts and there is no evidence for catalysis, making MT a carrier protein similar to metallochaperones in this context.
Not only the apoforms of enzymes but also those of zinc finger peptides are acceptors of Zn(II) from MT. The preferential transfer of a single Zn(II) ion from Zn7MT to a zinc finger peptide requires interprotein contact. (135,342) The absence of metal transfer when the proteins are separated by a dialysis membrane supports an associative mechanism. When the Zn(II) transfer potential of the chemically synthesized individual MT domain peptides was examined, the N-terminal β-domain was the better Zn(II) donor to apo-SDH, but the cumulative properties of the two individual domains do not describe the properties of the whole protein. (75) This result is consistent with a weaker Zn(II) binding in the β-domain and observations that investigations with the individual domains do not recapitulate all the properties of intact MT.
An associative process does not necessarily mean a ligand-exchange mechanism with the ligand donors in the active site. MT may simply bind to the surface of the Zn(II) acceptor protein, and the interaction then triggers metal transfer. Some active sites are buried and not readily accessible for chelating agents, but there can be pathways for the migration of the metal ion from a binding site on the surface to the active site. (343) A direct interaction of MT with a protein is not the only mechanism of transfer. The GSH/GSSG couple mediates transfer, suggesting that the state of specific redox couples is a driving force for the Zn(II) distribution. (92) With apo-SDH as a Zn(II) acceptor, GSH was found to inhibit the transfer in the absence of GSSG but to enhance it in its presence. In the absence of either GSH or GSSG a stoichiometry of 1:1 (MT/apo-SDH) restored the activity, indicating that one Zn(II) ion was transferred. In order to determine whether the transfer is kinetically or thermodynamically controlled, carbonic anhydrase was also examined. It did not serve as a Zn(II) donor to apo-SDH (log K = 11.2). (294) Since the prevailing opinion at that time was that MT has a Kd of 1.4 × 10–13 M (pH 7) for Zn(II) while CA has a Kd of 1 × 10–12 M (pH 7) for Zn(II), it was concluded that the transfer from MT must be kinetically controlled. Observations of metal transfer from an apparently stronger to a weaker Zn(II) binding site were reconciled by postulating a kinetic lability in thermodynamically stable Zn(II)/thiolate clusters in MT. The results must now be re-evaluated in view of the different affinities of MT for Zn(II), which make thermodynamically controlled transfer possible. Indeed, different molar MT/T ratios modulate the activity of apo-SDH. Below a midpoint ratio of 0.4 [T/(T+MT)] Zn(II) transfer occurs, and the enzyme is activated. Above this ratio, Zn(II) transfer does not occur. (294)
Using stable isotope-labeled 67Zn3Cd4MT2 to examine metal transfer to apo-CA, it was noted that, in the absence of GSH or GSSG, both Zn(II) and Cd(II) are transferred to apo-CA and that, in the presence of GSH, the transfer of Zn(II) was increased while that of Cd(II) was reduced. Thus, GSH protects against a Cd(II) mismetalation. (344) A metal transfer by an exchange of different metal ions has been proposed as a mechanism of rescuing proteins that inadvertently have been metalated with Cd(II). Thus, the higher affinity of MT for Cd(II) compared to Zn(II) allows Zn(II)-MT to bind Cd(II) from a protein mismetalated with Cd(II) and then to donate Zn(II) to form the native zinc protein. (345) It suggests a function of MTs as a “clean sweeper in metalloprotein quality control.”
4.7.2. Metal Transfer from Proteins to Thionein
In vitro, T removes Zn(II) from Zn(II) binding sites in proteins in a process mediated by other Zn(II) chelating agents such as GSH. (72) With the multiple arrangements of its cysteines and their flexibility, T is a chelating agent par excellence and has the potential to remove Zn(II) from numerous, structurally different sites. However, on the basis of affinity, it would not be able to remove Zn(II) from sites in proteins where Zn(II) binds with femtomolar affinities. Clearly, the presence of such a strong chelating agent would be detrimental to many Zn(II)-dependent cellular processes. The present view delineated above, therefore, is that T does not exist in the cell. Upon induction, it will bind any surplus of readily available Zn(II) and re-equilibrate with any existing MTs. Nevertheless, the induction of T will increase the chelating capacity in the cell and adjust it to affinities needed to restore steady-state concentrations of free Zn(II) ions once zinc transients (signals) have been induced and have targeted regulatory Zn(II)-binding sites, which are a third category of binding sites in addition to catalytic and structural sites in proteins (Table 1). For example, a Zn(II)-binding site is present in mitochondrial aconitase, inhibiting its activity. (346) When heart extracts of MT-null mice were incubated with radioisotope-labeled 65Zn(II)-MT, Zn(II) transfer to m-aconitase but not c-aconitase was demonstrated. (347) The affinities of Zn(II) for regulatory zinc sites in proteins are often lower than those for catalytic or structural sites (Table 1). The regulation of proteins by reversible Zn(II) binding is not widely acknowledged but sufficiently documented. (348) Once inhibited, the proteins likely need to be activated again at some point in cellular time. T removes Zn(II) from Zn(II)-inhibited enzymes. (349) Other examples are protein tyrosine phosphatases, enzymes that are not zinc enzymes when they are active, but they are tightly inhibited by Zn(II) ions. (316,350) The MT/T ratio also modulates their inhibition. Protein tyrosine phosphatase 1B with a log K value of 7.8 for Zn(II) binding has a midpoint ratio [T/(T+MT)] of 0.08 for inhibition. (294) These results demonstrate a role of the MT/T system in regulating enzymatic and other protein activities via controlling Zn(II) availability. Also, MT/T would remove any Zn(II) bound adventitiously on the surface of proteins. If loosely bound Zn(II) on proteins were to contribute to buffering, the control of Zn(II) transients for signaling would not be possible. Zn(II) in catalytic and many structural sites in zinc proteins is kinetically trapped and not in fast exchange and does not contribute to buffering. These boundary conditions determine the range for MT/T buffering and zinc regulation.
The presence of a second chelator can accelerate the rate of metal transfer from a metalloenzyme to T. T removes Zn(II) from the active site of carboxypeptidase A ∼400-fold faster in the presence of D-penicillamine (β,β′-dimethyl-d-cysteine). (351,352) The effect is because T has a higher affinity for Zn(II) than D-penicillamine and consequently by transferring Zn(II) from D-penicillamine to T the removal of Zn(II) from the enzyme becomes quasi-irreversible. The process has been referred to as catalytic chelation and could well present a way of how an LMW ligand such as glutathione kinetically controls metal transfer involving the MT system.
T also abolishes DNA binding of the zinc finger transcription factor Sp1. (353) Likewise, Zn(II) is transferred from the zinc finger transcription factor TFIIIA to T. (354) Another system investigated is the estrogen receptor, the DNA binding domain of which requires two structural Zn(II) ions in tetrathiolate coordination environments. T abrogates and MT restores estrogen receptor binding to DNA. (355) The observations of MT transferring Zn(II) to the apo-forms of zinc transcription factors and the latter transferring Zn(II) to T suggest that the MT/T ratio could also modulate gene expression, including the induction or repression of MT genes. (356) With the exception of MTF-1, knowledge about the zinc regulation of Zn(II)-binding domains of transcription factors is sparse. There are many types of transcription factors with different zinc finger domains and different Zn(II) affinities. (135,357) Some of these Zn(II)-binding domains are involved directly in DNA interaction while others are not. (358,359) Thus, whether MT/T regulates Zn(II)-dependent transcription factors requires further investigations with knowledge of which ZnxMT species are present in the nucleus and with a consideration of different Zn(II) affinities and Zn(II) coordination environments of the transcription factors.
To address the issue of metal transfer with a more “realistic” experiment, a zinc protein (SDH) or a Zn(II)-inhibited protein (PTP1B) were mixed with different MT/T ratios, more closely mimicking the distribution of ZnxMT species. The result of these experiments shows that the MT/T ratio determines the Zn(II) availability, (294) indicating that the MT system is indeed a determinant of biological activity. (78)The important point is that regulation with Zn(II) ions would not be possible if they were freely available. MT as a zinc homeostatic gene product is a regulated zinc buffer that serves as a rheostat for the availability of Zn(II) ions and their role as cellular signaling ions.
4.7.3. A Role of ZnATP
In addition to MT/glutathione interactions, there is an interaction of MT with nucleotides. On the basis of an earlier observation of GTP binding to MT, (360) the binding of ATP and GTP was investigated in more detail. (361) For ATP, a 1:1 complex with a Kd = 176 μM at pH 7.4 was observed for rabbit Cd,ZnMT2. ATP enhances the Zn(II) transfer from human Zn7MT2 to apo-SDH and the reaction of MT with DTNB. These phenomena can now be explained by ATP initially binding to MT and then binding Zn(II) to form a ZnATP complex. The removal of Zn(II) will increase the reactivity with DTNB as free thiols become available. With the poorly hydrolyzable analogue adenosine 5′-[β,y-imido]triphosphate it was shown that the hydrolysis of ATP is not involved. GTP elicited virtually identical effects, but adenosine diphosphate (ADP) had no effect on the Zn(II) transfer. One group of investigators found no evidence for MT binding ATP. (362) Subsequently, the binding was confirmed with a Kd value of 310 μM at pH 7.6 when a kinetic method was employed to follow the reaction of MT with DTNB. (363) In this assay, rabbit MT1, MT2, and MT3 had similar effects. ADP affects the reactivity of the thiols in MT, but adenosine monophosphate (AMP) does not. It was then shown that chloride binds to MT, allowing 35Cl NMR to be employed in a competition assay with ATP to demonstrate the interaction of MT with ATP. (57) The estimated binding constant is 993 μM at pH 7.4 for Cd7MT. Furthermore, scanning tunneling microscopy, which was earlier employed to visualize the shape of single MT molecules, (56) demonstrated a change in the shape of MT upon interacting with ATP, as further corroborated by changes of the N- and C-terminal amino acids in 1H NMR total correlation spectroscopy (TOCSY) spectra. (57) The change of shape is due to either the binding of ATP or the removal of Zn(II). Given the typical ATP concentrations in a cell, MT is expected to interact with ATP.
ZnATP rather than MgATP is the preferred substrate for some kinases involved in cofactor biosynthesis: pyridoxal kinase, riboflavin kinase, and NAD kinase. (364−367) X-ray structures of the first two enzymes indeed show the interaction with a ZnATP complex. (368,369) Noteworthy, MT activates pyridoxal kinase. (370) An equilibrium dialysis showed that ATP removes Zn(II) from MT. This observation was confirmed in competition experiments with the Zn(II) probe FluoZin-3. (73) These experiments provide a unifying explanation for the observed MT/ATP interaction, namely, binding of ATP to MT followed by the removal of Zn(II) and the formation of a ZnATP complex that activates pyridoxal kinase. ATP forms a stronger complex with Zn(II) than ADP, and it has a stronger tendency to form ternary complexes with other LMW ligands. (371) The MT/ZnATP interaction could channel Zn(II) into specific reactions that require ZnATP as a cofactor. The very low free Zn(II) concentrations would not be sufficient to form a ZnATP complex. Therefore, Zn(II)-MT is thought to be the limiting molecule in the activation of pyridoxal kinase and probably other specific kinases that ulilize ZnATP as the substrate. It is a case of metal transfer to a cofactor rather than an enzyme and possibly a fundamental metabolic link between energy metabolism and Zn(II)-dependent cofactor biosynthesis (Figure 24).
Figure 24
Figure 24. Interaction of pyridoxal kinase with ZnATP (PDB code: 1LHR). The crystal structure of pyridoxal kinase (PK) shows complexation with ZnATP. (368) There is not enough free Zn(II) available to form the ZnATP needed to activate PK directly. However, the interaction of Zn(II)-MT with ATP could provide the ZnATP to activate PK. (370)
5. Regulation of Metallothionein in Biological Space and Time
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5.1. Multiplicity of MT Genes and Their Regulation
Molecular and cellular biology and molecular genetics provided new powerful tools for MT research. However, while they added important biological context, they did not answer the question about the function(s) of MTs either, primarily because the state of the protein is not addressed. The advent of the polymerase chain reaction (PCR) and rapid nucleic acid sequencing generated a wealth of information on sequences of additional MTs and their expression in many cells, previously not amenable to otherwise laborious MT isolation. It demonstrated a rather ubiquitous expression of MT1/2, cell-specific expression patterns of MTs that change in many diseases as investigated widely in cancer, (372) and rather restricted expression of MT3 in the brain and MT4 in squamous epithelia. With regard to transcription profiling, there is a caveat, however. The correspondence between mRNA and protein levels is generally poor for MTs. (373) Multiple mutations in MT genes were detected, and their associations with different health conditions widened the functional implications of MTs even further. (374)
The human MT gene cluster on chromosome 16q13 was defined. (19,375) With the cloning of six additional genes and MT3 and MT4, the number of expressed human proteins is at least 11. (182,376) There are yet additional genes; some are likely genuine pseudogenes, but for others the translation into stable or “functional” proteins has not been demonstrated yet. Referring to the different gene products as isoproteins implies similar structures and functions─an assumption of perceived redundancy rather than a fact. The variation of a significant number of amino acids in different MT1 proteins and their differential regulation underscores qualitative as well as quantitative differences.
The HUGO Gene Nomenclature Committee (HGNC) (www.genenames.org) has listed symbols and names for human MT genes (Table 2). Among these 19 genes, 7 are pseudogenes, leaving 12 genes for possible expression of MTs. Remarkably, one gene, MT1HL1─inferred from homology to be related to MT1h─has been retrotransposed to chromosome 1. The classification as a “pseudogene” is based on structural faults or atypical amino acids. It is a supposition that MTs with atypical amino acids are not expressed, as such amino acids have been observed in MTs from other species. MT1HL1 and MT1b are not thought to be pseudogenes. The putative proteins have 18 and 21 cysteine residues, respectively. In MT1HL1, each domain has one cysteine residue less. In addition, it has a tyrosine. The incorporation of the additional Cys into human MT2 at the corresponding position found in MT1b leads to a functional MT. (189) The expression of MT4 and MT1b is very restricted to a specific tissue or condition.
Table 2. Human MT Genes and Pseudogenes in Alphabetical Order Based on HGNC (HUGO Gene Nomenclature Committee)
| HGNC ID (gene) | approved symbol | approved name | previous symbols | aliases | chromosome |
|---|---|---|---|---|---|
| HGNC:7393 | MT1A | metallothionein 1A | MT1, MT1S | 16q13 | |
| HGNC:7394 | MT1B | metallothionein 1B | MT1, MT1Q | 16q13 | |
| HGNC:7395 | MT1CP | metallothionein 1C, pseudogene | 16q13 | ||
| HGNC:7396 | MT1DP | metallothionein 1D, pseudogene | MTM | 16q13 | |
| HGNC:7397 | MT1E | metallothionein 1E | MT1 | MTD | 16q13 |
| HGNC:7398 | MT1F | metallothionein 1F | MT1 | 16q13 | |
| HGNC:7399 | MT1G | metallothionein 1G | MT1 | MT1K | 16q13 |
| HGNC:7400 | MT1H | metallothionein 1H | MT1 | 16q13 | |
| HGNC:31864 | MT1HL1 | metallothionein 1H like 1 | MT1P2 | 1q43 | |
| HGNC:7401 | MT1IP | metallothionein 1I, pseudogene | MT1, MT1I | MTE | 16q13 |
| HGNC:7402 | MT1JP | metallothionein 1J, pseudogene | MT1, MT1NP, MT1J | MTB | 16q13 |
| HGNC:7404 | MT1L | metallothionein 1L, pseudogene | MT1 | MTF, MT1R | 16q13 |
| HGNC:14296 | MT1M | metallothionein 1M | MT1, MT1K | 16q13 | |
| HGNC:23681 | MT1P1 | metallothionein 1 pseudogene 1 | bA435O5.3 | 9q22.32 | |
| HGNC:16120 | MT1P3 | metallothionein 1 pseudogene 3 | C20orf127, MTL4 | dJ614O4.6 | 20q11.22 |
| HGNC:7405 | MT1X | metallothionein 1X | MT1 | MT1l | 16q13 |
| HGNC:7406 | MT2A | metallothionein 2A | MT2 | 16q13 | |
| HGNC:7408 | MT3 | metallothionein 3 | GIF | 16q13 | |
| HGNC:18705 | MT4 | metallothionein 4 | MTIV | 16q13 |
In addition to defining the extent of the MT family, the second most important contribution of molecular biology was insights into the regulation of basal and induced MT gene expression. It linked MTs and the cellular signal transduction pathways and networks. Induction is remarkably complex and dynamic and includes many environmental factors and hormones: heavy metal ions (essential ones, Zn(II) and Cu(I)/Cu(II), and toxic ones, Cd(II) and others), glucocorticoids, bacterial endotoxins (lipopolysaccharides, LPS), physical and chemical stress, high-energy radiation (UV, X-rays, and γ-rays), heat shock, tissue damage, infection, and inflammation (pro-inflammatory cytokines: IL1β, IL6, TNFα), that is, factors that demonstrate a specific role in the acute phase response, chemotherapeutic and other drugs, changes in oxygen concentration, that is, hypoxia and reoxygenation, interferons related to viral infections, phorbol 12-myristate 13-acetate (PMA)─an activator of protein kinase C, a key signaling enzyme, cold induction, and even psychological stress, such as physical restraint or social isolation. Overall, a major area of MT function is the biology of stress, defense, and inflammation. Protein kinase C has also been shown to interact with MT2a. (377) Moreover, the enzyme phosphorylates rat MT1 at Ser32, a highly conserved residue, and affects the Zn(II) regulation of gene expression. (378)
The many pathways of induction give the impression of pleiotropic actions of MT. However, the question remains: Are they all part of one primary molecular function, or do they reflect different functions of MTs? Some of the pathways are linked to zinc metabolism, but for others the link is either not obvious or investigations on how the inducers affect distribution or redistribution of cellular Zn(II) or Cu(I) under either changes of the cellular state or exposure to stress have not been reported.
In a way, the generally investigated bioinorganic chemistry of MT can be seen with the metaphor of the protein being the tip of the iceberg. One needs to look at what lies underneath: how the regulatory network of the expression of MT genes relates to the functions of the protein in health and disease. The complex control of the gene expression of MTs by a slew of inducers and the differential expression of the MT genes is an essential biological feature and contains crucial and yet to be fully delineated information about the specific function(s) of the proteins. The regulation of gene expression through multiple and pivotal signal transduction cascades targeting cis-regulatory elements (CREs) has left the impression that MTs are multifunctional and multipurpose proteins. However, molecular biology does not address protein chemistry or function directly. We suggest that the diverse conditions of induction, most of which are Zn(II)-dependent themselves, are a reflection of the many facets of zinc metabolism and that they converge into a major function in buffering and redistributing Zn(II). The critical observation that the Zn(II)-sensing transcription factor MTF-1, which binds at multiple MREs and requires Zn(II) for activation, is required for both basal and induced expression of MT1/2 genes in combination with coactivators or other transcription factors, and that other proteins are repressors of gene expression, supports this concept. Such control is well-fitting to adjust the buffering capacity for Zn(II) at the gene level with further control of the pZn of buffering afforded by redox reactions and other interactions at the protein level. The pathways leading to gene expression of MTs pinpoint the different scenarios where Zn(II) is involved. Understanding what MT does requires understanding the roles of its induction in specific aspects of zinc biology, the knowledge of which is still evolving and was not contemporaneous with the discovery of the pathways for MT induction, which started over 40 years ago. An interpretation of how these scientific fields inform each other has much to offer. With the knowledge about the inorganic biochemistry of MTs available now, it becomes possible to attempt a synthesis of the myriad of observations and integrate MTs in the cellular signal transduction and metal homeostatic networks. A synthesis underscores the complexity of functions of MTs in multiple and principal cellular processes. When leaving out the rest of the iceberg, the protein itself can be investigated ad infinitum without ever defining biological functions.
An even more copious literature discusses the involvement of MTs in diseases. However, without a functional context that links the gene expression and the state of the protein an interpretation is tenuous or impossible. Generally, investigations are limited to changes in gene expression of MTs, and the findings are advanced by an emphasis on the potential for a diagnosis and prognosis. Mutations in MTs also have been linked to specific diseases. (374) With a focus on transcriptomics only, pursued widely because of a lack of specific antibodies for the different gene products or metamorphic forms of MT, a molecular mechanism of the protein is seldom addressed, and therefore the exact roles of MTs in the diseases remain enigmatic. MT induction is often seen as generating an “antioxidant” rather than reflecting on the implications for changes in metal metabolism. In our opinion, statements that try to relate the protein to general observations of redox changes are superficial without identifying targets and should address changes in metal metabolism and resolving the ambiguity of whether the induced protein restricts metal ions or makes them available as would be the case in an antioxidant function when the protein thiols are oxidized. We will discuss general pathways that underlie many diseases and not discuss in detail noncommunicable diseases such as cancer, diabetes, and neurodegeneration, for which a lot of work regarding a positive or negative effect of MTs and a role of MT3 in the brain has been published as apparent from the cited reviews. (372,379−383) With specific reference to the healthy, diseased, or injured brain, MT3 is mainly in neurons, while MT1/2 are mainly in astrocytes. (384) Various forms of stress, inflammation, hypoxia, bacterial and viral infections, and aspects of cell fate (cell cycle/proliferation, differentiation/development, cell death/apoptosis) and cell adhesion/migration are such general processes linked to pathways of MT induction. Translating phenomenology into teleology, that is, what the protein does specifically, will reveal the implications for metal metabolism. Overall, such consequences of changed metal metabolism have not caught the interest of the wider biomedical community despite the prospect that knowledge about specific functions of MTs would open the field for therapeutic interventions. Thus, not surprisingly, not a single protein in the family has been approached for drug discovery so far.
We will pursue the discussion of induction through CREs, including the link with Zn(II) when known, but the discussion is not comprehensive, as there are additional inducers that transactivate the gene expression of MTs with roles yet to be established. MT3 is also different in terms of its induction from MT1/2. Hypoxia induces MT3 in adipocytes. (385) Only a modest induction by metal ions was noted in human neuronal cells. (386) In macrophages the interleukins IL4 and IL13 induce MT3. (387)
An in silico analysis of the complex promoters of MT genes provides information on the different binding sites of transcription factors and the differential expression of the MT genes. (388) In many cases, transcription factors heterodimerize and establish crosstalk between several pathways in gene expression. Pro-inflammatory cytokines are inducers with specific response elements for interleukin 6 (IL-6RE), interferons (ISREs), and signal tranducers and activators of transcription (STAT) transcription factors. However, MTs also have anti-inflammatory effects, probably mediated through ARE alone or in combination with an upstream stimulatory factor/major late transcription factor (USF/MLTF). Specific inductions of particular MTs and cell-specific inductions then need to be related to the specific properties of the proteins, once they become known, and to the coordinate expression of zinc transporters or other Zn(II)-dependent proteins.
MRE: Metal response element (RE). Among several proteins discussed in the metal-dependent induction of MTs, MRE-binding transcription factor-1 (MTF-1) is a key transcription factor. (389,390) It regulates the expression of MT1/2 genes. The induction requires Zn(II). The induction of MTF-1-mediated MT transcription by Cd(II) or Cu(I) occurs through the displacement of Zn(II) by these metals. (391) The responsiveness of MTs differs, as there are between one and five MRE sites in the promoters of different MT genes. When Zn(II) concentrations are elevated, MTF-1 induces T to bind Zn(II). It also induces ZnT1, the major cellular zinc exporter. It is certainly a mechanism to deal with increased metal concentrations. But MTF-1 can also serve as a transcriptional repressor, for example, for the zinc importer Zip10, suggesting an additional regulatory loop to control Zn(II) import. In this latter case, the MRE is downstream of the transcription start site, and the interaction with MTF-1 prevents the RNA polymerase from initiating the transcription. (392) While MTF-1 is a key factor in increasing T, that is, increasing the chelating capacity for excess concentrations of Zn(II), the question remains as to what happens under a zinc deficiency. In a mouse fibroblast cell line that has adapted to an extreme zinc deprivation, an amplification of the MT gene locus was noted, suggesting a role of MT in scavenging Zn(II) from the environment under these conditions. (393)
GRE: Glucocorticoid RE. It was the first specific induction pathway found for MT and is related to the response to various forms of stress that produce steroid hormones. (394) There is also a response to other ligands targeting the transcription factors of the nuclear hormone receptor family to which the glucocorticoid receptor, a zinc protein, belongs, for example, the hormones vitamin D and retinoic acid formed from vitamin A, but also ligands of the arylhydrocarbon receptor (AhR).
HRE: Hypoxia RE. Human MT2A mRNA increases under hypoxia, and evidence for an HRE was found, thus identifying this particular MT as an oxygen-regulated protein. (395) The transcription factor involved is hypoxia-inducible factor Hif1α. Hif1α interacts with MTF-1, (396) is stabilized by MT, (397) activated by phosphorylation, and interacts also with Hif1β (aryl hydrocarbon receptor nuclear translocator (ARNT)). Hypoxia responsiveness links MT and the important process of oxygen regulation, inflammation and angiogenesis, the formation of blood vessels under normal conditions but also critically involved in cancer metastasis, kidney, lung and heart disease, diabetes and acute respiratory syndrome as experienced in the Covid-19 pandemic due to SARS-CoV-2 infection.
XRE: Xenobiotic RE. The only directly ligand-activated transcription factor AhR, which heterodimerizes with ARNT, binds at this cis element. Xenobiotics such as halogenated aromatic hydrocarbons activate it, but in contrast to what the name indicates, also many endogenous substances important for stem cell maintenance and differentiation are activators. AhR interacts with the GR to activate human MT2A. (398) Furthermore, the induction of MT gene expression by 2,3,7,8-tetrachlorodibenzodioxin (TCDD, an AhR ligand) and the presence of MREs in close proximity of XREs has been observed and further discussed. (399)
ARE: Antioxidant RE. This response element was first described for the mouse MT1 gene (400) and, either alone or together with the USF/MLTF, is responsible for the induction of MT by oxidative stress. Oxidative stress can also activate MTF-1, presumably through Zn(II) released during oxidative stress. Like XRE, the ARE is activated by many substances such as electrophiles that pose a potential threat to the cell, and it is a major factor in the transcriptional response of MT to so many substances. The transcription factor is nuclear factor erythroid 2-related factor 2 (nrf2), which is not known to be a zinc protein. However, it interacts with Kelch-like ECH-associated protein1 (Keap1), which is a zinc protein, in which electrophiles react with the Zn(II)/thiolates and eject Zn(II). (297)
In addition, MT modulates other important signal transduction pathways for which no CREs on MTs have been identified. One is the tumor necrosis factor alpha (TNFα) pathway in inflammation. The transcription factor is nuclear factor kappaB (NF-κB), a dimer of p50/p65. p50 has been suggested to interact with MT (401) while MTF-1 interacts with p65 under conditions of hypoxia. (402) Altogether, at least 24 zinc sites in proteins involved in the pathway have been identified. (403) Controversies about the role of MT in this pathway have been reviewed. (404)
The other transcription factor discussed in interactions with MT is p53. It is the product of the TP53 gene and is called the guardian of the genome. Mutations in this gene are responsible for approximately half of cancers. p53 is a zinc protein. The zinc site has a structural function and is essential for its transcriptional activity, as the protein will misfold if it is devoid of Zn(II), for example, mutation in the zinc site (R175H), leading to a 1000-fold weaker Zn(II) binding. A very short exposure to Zn(II) (<30 min) rescues the protein and leads to cancer cell death, adducing to the unique concept that zinc metallochaperone therapy is a viable drug approach to rescue mutant p53. (405) It has been suggested that MT modulates Zn(II) binding to p53 and hence the transcriptional activity of p53 and that the two proteins interact. (406,407)
Numerous reviews have discussed the regulation of MT genes. The effects are often cell-, metal-, and MT-specific. Some MT1 and MT2 genes are constitutively expressed in most tissues, and their expression can be induced, in particular, by metal ions. In contrast, MT3 and MT4 are expressed only in some specific tissues and are genererally not directly induced by metal ions. Some of the transcription factors and their DNA recognition elements are specific for individual MT genes. The information has not been fully exploited to provide comprehensive insights into how specific signal transduction pathways and metal and redox metabolism are linked. In summary, instead of looking at MTs in isolation one needs to look at both levels of regulation, the processes upstream in its induction─the input─and those downstream in its activities in metal and redox metabolism─the output─to understand how the MT system works in cells (Figure 25).
Figure 25
Figure 25. Combined gene and protein regulation of the MT system. (left) Many signal transduction pathways converge at the MT promoters to induce the expression of at least 11 human MT genes. Various steps in these pathways depend on Zn(II). A Zn(II)-sensing transcription factor (MTF-1) controls the metal-dependent transcription at metal regulatory elements. Among the other inducers are interferons, glucocorticoids, and redox signals acting on the respective regulatory elements (ISRE, GRE, HRE, XRE, and ARE). DNA methylation can silence the effects of these cis-acting factors. (right) Newly synthesized thioneins bind metal ions to form metallothioneins. The availability of metal ions from MT proteins is linked to redox changes and redox signaling. Protein modifications, interactions with low molecular ligands and other proteins, and protein degradation afford additional layers of regulation as discussed in the main text. With regard to the induction of gene expression, it is a generalized scheme, as not all the respective regulator elements are present in a given MT gene.
When the MT protein is looked at in isolation, an important aspect is missed, namely, that Zn(II) coordination is part of some of the transcription factors for a constitutive and induced expression of MTs and that additional steps in the signal transduction pathways leading to expression are Zn(II)-regulated. The zinc transcription factors include proteins with coordination of the CCCC type, CCHH finger type (Sp1, retinoic acid receptor, Ikaros), and other types such as in Churchill, a neural inducing factor with at least three zinc sites, including a thiolate sulfur μ2-bridge (Figure 26)
Figure 26
Figure 26. Structures of mammalian proteins containing Znx(Cys/His)y clusters. (top left and right) Human embryonic neural inducing factor churchill (2JOX) (408) and dimerization domain (1RMD) (409) of mouse V(D)J recombination-activating protein RAG1, respectively. In both structures unique Zn2HisCys6 clusters with one bridging thiolate sulfur are present. (bottom) Structure of human euchromatic histone methyltransferase 2 containing a Zn3Cys6 cluster. (410)
There is virtually no information on a possible regulation of Zn(II)-containing transcription factors via their Zn(II) content, with the exception of the in vitro work of T removing Zn(II) from transcription factors.
In contrast to the induction of the MT gene expression, negative regulators have received comparatively less attention. (411) A negative regulation includes the silencing of genes by a DNA methylation on their cis-elements, that is, the DNA sequences recognized by transcription factors, and several transcription factors that are repressors. Among those, zinc finger transcription factors feature as well, for example, PZ120 with a CCHH zinc finger (POZ) motif. (412)
Epigenetic effects include histone modifications that affect chromatin structure and hence transcription factor access to DNA. Posttranscriptional control at the mRNA level of MTs has also been observed. (413,414)
Regarding proteolysis, mainly the lysosomal enzymes cathepsin B and L degrade MT with cell-, metal-, and MT-specific differences. (62) While cathepsins are not zinc enzymes, cathepsin B is strongly Zn(II)-inhibited with a half-maximal inhibitory concentration (IC50) of 150 nM (pH 7.4). (127) A Zn(II) ion has been found in the active site of cathepsin L. (415) The interaction of Zn(II) with cathepsins indicates a role of Zn(II) in controlling MT degradation by these enzymes in addition to the effect of the metal load of MT on degradation. It is generally accepted that T is more susceptible to degradation than MT. Further investigations are now warranted to determine whether the partially metalated MT species have different rates of degradation.
5.2. Spatiotemporal Distribution of MT in Cells
After having discussed the dynamics of the protein in terms of reactivity and metal coordination and the exquisite regulation of their genes, yet another significant biological aspect of dynamics is the distribution of the protein in cellular space and the changes in biological time. It involves additional interactions with proteins and important activities of MTs in the redistribution of metal ions and controlling metal metabolism in subcellular organelles. Investigations have addressed translocations to mitochondria, lysosomes, and the nucleus.
Despite lacking a mitochondrial targeting sequence, Zn7MT1/MT2/MT3 are translocated into the intermembrane space of liver, but not heart, mitochondria. Translocation results in a dissociation of Zn(II), which inhibits mitochondrial respiration. (416) Vice versa, T binds Zn(II) from the inhibitory site and activates respiration. The effect seems to be specific for the β-domain, indicating either a Zn(II) dissociation from this domain or a disulfide formation with proteins of the mitochondrial import machinery. Modifying the lysines of MT abolishes translocation. Investigations with Cu(I)-containing MTs regarding mitochondrial biology have not been reported.
MTs do not possess a nuclear localization sequence either. In the developing, but not adult, rat liver and kidney, MT was detected in the nucleus and cytoplasm. (417) In cultured primary hepatocytes of adult rats, however, MT was found in nuclei, but only in the early S phase and not in the G0 or G1 phases of the cell cycle, indicating that the localization is linked to proliferation and the cell cycle. (418) Nuclear MT was found in many tumors and suggested as a marker of the proliferative capacity of cells. (419) With a monoclonal antibody raised against monomeric rat liver MT1, cell cycle-specific nuclear localization of MT was shown in proliferating human HT-29 colon cancer cells. (420) A transient nuclear localization was noted early in the cell cycle when cells differentiated and were shown to depend on mitogenic signaling cascades. (421) Several attributes of the nucleocytoplasmic shuttling have been reported: requirement for energy (ATP) and dependence on phosphorylation signaling, the oxidation of a cytosolic factor, and the state of the cell. A perinuclear localization of the MT1 mRNA and association with the cytoskeleton mediated by the 3′ untranslated region is deemed necessary for nuclear import. (422) The translocation to the nucleus was also suggested to require the small GTPase Ran, an interaction of MT with a cytosolic protein that must be oxidized for nuclear translocation, and dependence on energy. (423−425) Nitric oxide was also proposed to be an essential signal for nuclear translocation and Zn(II) dissociation in the nucleus. (120) The observation that the decrease of cytoplasmic Zn(II)MT correlates with an increase of Cu(I)MT in the nucleus complicates matters further, as it could indicate that a metal exchange process occurs in the cytoplasm and then Cu(I)-MT translocates to the nucleus. (224) The function of MT in the nucleus is presently unknown.
While MTs are found in lysosomes, not much information is available on the translocation to lysosomes. A case has been made for functions of MT3 in lysosomes. MT3 is thought to participate in the process of bringing Zn(II) into lysosomes, controlling the lysosomal pH and activities of proteins, resulting in a role in autophagy and cell death in cortical astrocytes. (426)
The implications of these findings for investigating the functions of MT are huge, as they suggest that MTs need to be investigated with regard to subcellular localization as a function of specific cellular stages in addition to their identity and metal load.
5.3. Extracellular MT
In addition to intracellular redistribution, MTs have been found outside cells. The transfer of Cd(II)MT from liver to kidney requires secretion and uptake. (427−429) Under Cd(II) exposure, extracellular MT contains mainly Cd(II) and copper. (222) Extracellular MT has been determined in blood, urine, or bile. In blood, it is a zinc, copper, or cadmium protein, and degradation products of the protein were also found. (430) Reference ranges of 32 ± 16 ng/mL in plasma and 10 ± 6 ng per micromole creatinine in urine were determined with antibodies specific against human MT1. (431) After surgery, a significant increase in blood was noted, suggesting that MT is an acute phase protein. Since Zn(II) decreases in blood while Cu(II) increases during the acute phase response, it remains a pressing issue to determine whether the increase of MT is linked to both metal ions or only one of them. (432) There is a general lack of knowledge about whether specific MT forms are extracellular or all forms can be found in the extracellular fluid and what the metal load and oxidation state are in the extracellular environment, which is much more oxidizing than the intracellular environment. MT3 is slightly different in this regard. MT3 was identified originally in an assay investigating its effect on inhibiting neuronal growth when added extracellularly. (13) The growth inhibitory factor activity is maintained with Zn(II)-MT3 or Zn(II)/Cu(I)-MT3, the native form, but not other MTs. MT3 appears to be involved in clearing the synaptic cleft from Zn(II), which is a neurotransmitter/neuromodulator of specific neurons, and the reloading of Zn(II) into their synaptic vesicles. (433)
Neither uptake nor secretion mechanisms of MTs are known in any particular detail. For uptake, there is some information, namely, that it occurs through endocytosis via the lipoprotein receptor megalin/LRP2. MT1/2 secreted from astrocytes is found in the extracellular fluid of the injured brain and taken up by a megalin receptor on neurons where it affects axon regeneration. (434) The MT binding on human astrocytes was characterized with a Kd value of 0.84 nM and a Bmax of 99.82 fmol/mg protein to an unidentified binding site. (435) In proximal tubule cells, the multiligand receptor complex megalin: cubulin: amnionless is involved in uptake, but the 24p3/NGAL/lipocalin-2 (LCN2) receptor (SLC22A17) has a 1000-fold higher affinity for MT. (436,437) Human hepatocytes (HepG2 cell line) take up MT by a lipid-raft-dependent endocytosis. (311) MT colocalizes with albumin but not transferrin, also supporting a pathway of nonclassical endocytosis. After uptake in liver cells, MT ends up in an endosomal cellular compartment, from which the metal is released. These experiments were not yet performed with copper MT. For secretion, there is no known mechanism. MT does not have sequence motifs for export or any glycosylation sites. MT secretion is not due to leakage from damaged cells, though. It was described in the intestine, (438) white adipose tissue, (439) astrocytes, (434) and immune cells. (440) Of particular interest is that MT2 concentrations were found to be rather high in exosomes from mesenchymal stromal cells and are required to suppress the inflammatory response in the colon. (441) Taken together, MTs are part of intraluminal vesicles in multivesicular bodies (endosomes), and there seems to be a role for them in metal uptake and in biological communication, in particular, between immune cells. (440)
5.4. Interaction of MT with Proteins
In addition to the membrane receptors involved in cellular uptake of MT numerous interactions with cellular and extracellular proteins have been reported. (442) However, none of these protein–protein interactions (PPIs) has been investigated with structural methods that would reveal the molecular features of the presumed interactions, and none of them fulfill the criteria set out for firmly establishing stable PPIs by employing different techniques. Since MTs are relatively small and metamorphic proteins, nonspecific interactions through covalent or ionic interactions can occur. With its 20 cysteine residues MT is prone to form mixed disulfides. Also conspicuous for interactions are the eight lysine residues, which provide positive charges on the surface of the protein. Transient or stable interactions between MTs and proteins could also be mediated by metal ions. For example, PPIs of other proteins are mediated by Zn(II). Such interactions will depend on pZn and thus on MTs. (171) Implications about functions of interactions are also tenuous. Most of the interactions do not seem to have any obvious or direct connection to zinc, copper, or redox metabolism. The exceptions are some metal transfer reactions, which were found not to occur when proteins were separated by a dialysis membrane, indicating that a direct interaction is necessary for transfer.
With ZnT3, the zinc exporter that loads secretory vesicles with Zn(II), MT3 is part of a synaptic vesicle cycle. MT3 interacts with the small GTPase Rab3a. (433) Further support for such a role of MT3 comes from additional interactions with many other proteins participating in the synaptic vesicle cycle. (443) It was pointed out that such a specific role of MT3 would be different from a general one as a metal buffer.
An assumption in all these PPIs is that we know what the structure of MT is in vivo─by no means a foregone conclusion. Interactions have been examined with metal-reconstituted MTs that may not be relevant physiologically. When antibodies are employed for MT detection, their specificity is fraught with uncertainty, including which MT they detect. Future work has to identify the particular forms of MT involved in any putative interaction. Interactions have focused on MT only, and hence another unresolved issue remains whether there are PPIs with T. With these caveats, any information gleaned from the databases dealing with interactomes and reactomes deserves much scepticism.
6. From Metallothionein Structure to Functions, Roles, and Purposes
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6.1. MTs in Other Organisms
On the basis of sequence information, MTs were divided into three classes: Class I MTs related to mammalian MTs, class II MTs with no or only a seemingly distant relationship to mammalian MTs, and class III MTs as nontranslationally synthesized polypeptides that bind metal ions with cysteine thiolates. (18) These definitions were made by committees of scientists who convened at international meetings on metallothioneins. The problem with these definitions is arbitrariness. For class II, a criterion is lacking how the degree of similarity should be defined, while for class III MTs there is no reason why these peptides should be considered MTs in the first place.
Phylogenetic trees were constructed when many sequences of MTs became available. MTs were divided into 15 families. (444) However, it was noted that these trees are unrooted, suggesting that MTs did not evolve from a common ancestor. The heterogeneity of MTs lumped in class II increased continuously. Thousands of MTs bear no evolutionary relationship to class I MTs, again emphasizing their polyphyletic origin. (445) Phylogenetic and synteny analyses of vertebrate MTs identified ortholog and paralog genes, established a root for the fish clade, and support the functional divergence of MT3 and MT4 relative to MT1&2. (446) The enormous diversity of MTs may have evolved for different functions in different organisms. Class II MTs bind different metal ions (Zn(II), Cu(I), Cd(II)), and indeed some are Cu(I)- and others Cd(II)-binding proteins. They can have only one or even three domains, mononuclear zinc finger-type of structures or binuclear Zn2Cys6 clusters; they can be shorter, for example, fungi, Neurospora crassa MT with 25 amino acids including 7 Cys, or much longer, for example, flatworm MT with 227 amino acids with 34 Cys, than mammalian MTs, and they have atypical amino acids such as tyrosine residues. Histidine residues are also quite common in some and participate in metal binding. There are 91 Cys in Trichomonas vaginalis MT. Tetrahymena thermophile MTT1 has several CCC motifs. Cys motifs are not unique for MTs, nor are Zn(II)/thiolate clusters. A Zn3Cys9 cluster as in the β-domain of class I MTs was found in fungal and fly proteins and in human histone methyltransferases (Figure 26). (41,42,410) This variability demonstrates that all the definitions set out initially for equine renal cortex MT, (18) the prototype “MT”, do not hold when considering class II MTs: low molecular weight proteins, high metal content, characteristic amino acid composition (high cysteine, no aromatic amino acids or histidine), unique amino acid sequence, and a characteristic distribution of tetrahedral metal–thiolate complexes and metal–thiolate clusters.
What is a metallothionein then? The name for the protein was coined at a time when the sulfur binding of metal ions in biology was a curiosity. Looking at the heterogeneity of proteins and peptides classified as MTs, the use of the word “MT” does not mean much beyond the statement inherent in the name that it defines a protein that binds metals with the sulfur donor atoms from cysteines. It describes a property that is not unique to MT and is therefore trite and trivial. Taking it ad absurdum, the definition would apply to some zinc finger type of proteins or even iron–sulfur proteins. Nevertheless, the word MT is constantly used to indicate function. It is a most unsatisfactory situation, as the word cannot be used both generically and specifically at the same time as discussed here for the specific meaning related to the structures and functions of mammalian MTs. Even for mammalian MTs many additional investigations are required to explain sequence variations in terms of metal usage and metabolism in each species. The consequence of these uncertainties is that MTs are being called multifunctional and multipurpose proteins. In the worst-case scenario, it can become a pretense for investigations under an assumption of function. A clear understanding of what the word MT means hinders progress and gives a license for unfounded conclusions. For each MT, a possible function needs to be investigated in the biological context of zinc and copper metabolism, redox metabolism, and the environmental exposure of the organism to essential and nonessential elements. Chemical studies alone are not sufficient. To overcome this conundrum, the word MT should be understood as a generic term only when it is applied to proteins that do not bear any relationship to mammalian MTs, and further conclusions about function need a combination of chemical and biological investigations and not an a priori assumption of a perceived classification as an MT. To paraphrase the problem of epistemology with a quote from Voltaire (François-Marie Arouet): "Everything that is not clear is not right." Unclear terminology does not advance science.
6.2. The Dilemma of MT’s Biological Functions
In chemistry, the concept of “function” is relatively straightforward. Functional groups determine reactivity. Pure chemistry gains functions in applications, for example, the combination of Zn(s)/Zn(II) and Cus/Cu(II) in the Daniell cell─a battery. In biology, functions are restricted to the chemistry that is possible in the biological milieu─the very foundation of biological inorganic chemistry as opposed to the entire field of bioinorganic chemistry, which also addresses the chemistry of biomolecules regardless of physiological significance. As functional groups, the thiols of the cysteines in MT are involved in metal binding and in the reactivity with oxidants or electrophiles, providing a chemical basis for the functional potential of MT. While the interaction of the zinc and copper electrodes and their respective electrolytes defines a battery, one molecule alone does not define biological function but rather its interaction(s) with other biomolecules.
We believe that a general discussion of the meaning of function in biology will be helpful in order to recognize how chemical structure relates to chemical reactivity in biology. Specifically, we follow a history of philosophical and pragmatic discussions in the context of gene ontology, where the concept of biological function is “not as straightforward as it might seem” because function is divided into causal role function and selected effect function. (447) A causal role is connected to how a part has a role in a system, that is, an “activity” that is part of a more complex “mechanism”. A selected effect gives the ultimate answer why a part exists in the first place. It is linked to teleology: Why was the part selected in evolution? The necessity to pursue investigations about the origin of MTs has been pointed out. (448) An answer to this question imposes constraints on the myriad of causal functions and distinguishes a proper function from an accidental or adventitious function, an issue that is critically important when analyzing the many functions proposed for MTs. Molecular biologists conceive of function as specific, coordinated activities that serve a purpose. But it remains a candidate function until the evolved function has been related to past survival and reproduction. “Function” has two distinct aspects. The first is how the protein works at the mechanistic, molecular level, and the second is the functional role at the mechanistic, cellular level. It is this second aspect that purely chemical studies of MTs fail to address, as it requires relating the molecular properties to cellular biology. These definitions and the hierarchy of functions from molecules to cells to tissues to organisms needs to inform and direct any discussion about the functions of MTs. Working toward a molecular mechanism can be seen as a reductionist approach. But a holistic approach in the context of the operation of a cell is needed as well.
Gene ontology (GO) terms are categorized under Molecular Function, Cellular Component, Biological Process (www.geneontology.org). Gene ontology states for the molecular function of human MT2a (www.genecards.org): protein, drug, Zn(II) ion, metal ion binding. It is listing properties, not necessarily biological functions. The entry for biological process is left blank for the protein. Attempts to define the overall function of MT based on its chemical properties and existing data largely failed because the biological context was not considered. Likewise, defining functions solely on its biology also failed because the chemical properties were not considered.
The inducers of gene expression of MTs are seen as making “metallothionein” when in fact it is “thionein” that is synthesized, perhaps the central misrepresentation leading to confusion. What the induced protein does and where it gets its metal from and whether or not it is induced for additional functions, such as reducing another substrate, is largely left unanswered. One issue persisting in the field is that there is no name for the uniquely biological principle that MT embodies. Without a name, the biological principle is difficult to fathom and to communicate. One could coin a new name, but we will refrain from doing so, because the term “buffer” with the appropriate qualifications, we believe, may suffice for the time being. There are also semantic issues because the name metallothionein is used for many types of other proteins that are sulfur- and metal-rich and with reference to a mammalian protein with a given stoichiometry of seven divalent metal ions. After more than 60 years, it is time to get at the gist of the matter and discuss what the words used to describe or indicate functions of MTs mean, that is, buffer, scavenger, storage/reservoir, homeostatic protein, chaperone, detoxification. Chaperone means a specific protein to deliver Zn(II) or Cu(I) by direct protein–protein interactions. The word is fitting for genuine copper metallochaperones. For Zn(II), the situation is different, as there is free Zn(II) and no bona fide metallochaperone. A dozen MTs are unlikely to serve a role as specific chaperones to provide Zn(II) to ∼3000 proteins by direct interactions. For Cu(I), the term scavenger seems to be appropriate, as MTs sequester Cu(I) when the concentrations are high. For Zn(II), the opposite proposition was made, namely, that MT is a scavenger of Zn(II) at low Zn(II) concentrations, (393) serving as a temporary storage and reservoir. For Zn(II), we prefer the term buffer, as the protein is not saturated and the “MT/T” ratio is related to the free Zn(II) concentrations like a metal buffer, albeit with the notion that there are interactions with other biomolecules and mechanism for accepting and donating metal ions in specific processes. When discussing MTs as metal buffers, the first issue again is a semantic one, namely, naming the protein only metallothionein and not naming it after the ligand, that is, thionein. A metal buffer exists in a metal-free and a metal-bound form. Hence, the buffer would be MT/T. Neither term specifies the degree of saturation with metal ions, which is needed to define the different pKd values for buffering. In chemistry, applications of metal buffers are relatively rare, but in biology metal buffering is essential. Metals need to be homeostatically controlled in order not to interfere with other biological functions of the cell, AND each metal ion needs to be controlled relative to the other metal ions. The circumstances of chemical buffering are specific for the biological context. The biological metal buffer is dynamic and changes its buffering capacity through changes in gene expression of the protein AND its ratio to effect changes in pM (−log[Mn+]free). The protein does not buffer at one constant pM, and it remains to be shown whether the different MTs differ in their buffering properties. Buffering by MTs is affected by changes of pH and operates on specific biological time scales. And, importantly, MT is not inert─a property for which a chemical buffer is usually chosen─because it interacts with the system, being modified by reactions that change metal composition, thiols, and perhaps other groups. Last but not least, its metal buffering is linked to redox buffering. Thus, when MT is widely discussed as an antioxidant and as a cytoprotective agent without reference to the linked properties as a metal buffer, it must be understood that the reactivity of the thiol(ate)s with oxidants and electrophiles affects metal equilibria and that the ensuing changes of metal ion concentrations can have either a pro-antioxidant or a pro-oxidant effect. A role in detoxification after intoxication also relates to buffering essential metals and functions in redox biology. A longstanding issue is whether or not mammals possess a detoxification system for Cd(II). The late professor H.A.O. Hill asked a critical question in a discussion section during one of the earlier International Metallothionein Symposia: If it is detoxification, why do we not get rid of it? Subsequent research weakened the argument that a primary function of MT is the detoxification of Cd(II). Cd(II) is not an inducer of MTF-1 controlled gene transcription but, rather, displaces Zn(II) in MT, which is the actual inducer. (391) Cd(II) is not safely sequestered in MT, as the exchange rates are higher than those of Zn(II) and it can dissociate by a reaction of MT with NO. (125) The argument in favor of a role in detoxification is based on higher amounts of MT protecting against cadmium toxicity, MT1/MT2 null mice being hypersensitive to Cd(II), and genetic polymorphisms of MTs modulating cadmium toxicity. (449) All these observations indicate that MT has a role when exposed to Cd(II), but they do not refute a primary function in zinc and copper metabolism as supported by the complexity of the system, its regulation, and its integration into cellular signaling networks. The question remains what the capacity of the MT system is to accumulate Cd(II) before interfering with its functions in zinc or copper metabolism.
Chemical approaches to characterize the structure of the protein have advanced, but ultimately the function of the protein is a biological issue, which will not be resolved by purely chemical approaches that do not consider the biological context of the expression of MT genes controlled by so many factors. Combining knowledge from different scientific disciplines provides untapped opportunities for determining functions. Defining a structure of MT has been problematic, as it depends on the type of metal, metal load, and redox conditions, resulting in some purely chemical observations of a biological macromolecule with limited relevance to biology. Comprehensive attempts to combine the information about the gene expression of MTs and the cellular redistribution of metal ions or changes in redox potential are largely missing. Primarily, we need to look at the reason for the induction of T as a metal acceptor to restrict metal ions and, secondarily, at a donor of metal ions from the produced MT when metal ions are needed. The time scales are different: gene induction is slow; protein chemistry is fast.
With Zn(II) and Cu(I) as cargo, MT functions are inevitably linked to the numerous global functions of Zn(II) and the more specific functions of Cu(I). Compared to zinc MT, the literature is even less clear about the functions of copper MT. (450) Further investigations are necessary to clarify whether MT is involved in the storage, scavenging, or buffering of Cu(I). Either donating or accepting Zn(II) can activate or inhibit biological processes. An understanding is necessary how Zn(II) is redistributed to where it is required for function. Both actions have been described: MT delivering Zn(II) and T removing Zn(II). In the acquisition of Zn(II), there can be a huge number of protein–protein interactions, as T is an intrinsically disordered protein─maybe exactly what is required, namely, having a vast conformational space for serving as a versatile and multidentate chelating agent. Having one molecule performing the function of an otherwise large number of single chelating agents would again be a uniquely biological activity. Likewise, one can argue that the metamorphic states of MT with different metal load and oxidation state provide a vast conformational space for the recognition of Zn(II) acceptor proteins and for interactions with LMW ligands.
The discussion of MT function provides unparalleled lessons for protein science and biology, in general, and for control of metal metabolism, in particular. Why have chemical, biochemical, and genetic approaches not answered the question of MTs’ primary functions, and why has the situation persisted for 60 years? One reason is the limitations of each of the approaches and how the merger of them has been attempted with an insufficient integration of the different disciplines. Cysteine coordination chemistry became prominent in biochemistry only with the discovery of zinc finger and related proteins. Until that time MTs, with few exceptions, were rather unique with their sulfur coordination environments. Despite all the work, the redox connection was not considered as a major aspect of MT’s function in zinc metabolism until 40 years after its discovery, and all investigations were performed under the assumption of very tight binding of the metal ions, which was not challenged until 50 years after the discovery of MT. Tight binding suggested a role in storage, which was already inconsistent with the kinetic lability observed in metal transfer reactions. Also, on the basis of the tight binding of metal ions, a structural model based on seven divalent metal ions bound was considered THE metallothionein, and the structures of the metal/thiolate cluster, which turn out to be just one state of the metal coordination in the protein, preoccupied the imagination of many investigators.
Reasons for the failure to agree on the functions of MT thus are at least twofold, operational and a lack of considering the biological context. At least two reasons are operational. First, there is no method for probing the state of MT in the cell. Accordingly, the protein was isolated, and since heterogeneous either intrinsically or due to the procedures for isolation, metal ions were removed and the proteins were reconstituted with a chosen equivalent of metal ions. Structural investigations then led to one 3D structure. Second, determinations of metal affinities relied on methods that did not have a sufficient resolution to reveal differences. Thus, a critical insight later was that the metals do not bind with the same affinities and that there are weaker binding sites. Thus, the affinities of some sites are not stronger than those for other zinc or copper proteins, assigning MT a more dynamic function in metal metabolism. More robust methods for measuring affinities together with experiments showing the presence of T challenged the dogma that it is a protein saturated with seven divalent metal ions. As to the biological context, retrospectively, the failure to define a function has yet other reasons. The biological information on how metal ions are controlled was inadequate or even nonexistent, and therefore it was not understood how the chemical properties of MT could play a role in biology. But equally important, coming to the overarching question of the role in zinc and copper metabolism, insight into the quantitative aspects of how cellular Zn(II) and Cu(I) ions need to be buffered and are controlled and how these metal ions can become regulatory and signaling ions did not exist. Hence, it was not clear why a molecule such as MT would be needed. Therefore, its chemical activities could not be converted to biological activities, and the chemical properties did not match a biological need for them. Accordingly, no specific functional tests could have been applied to investigate the phenotype of the genetically modified animals, in which MT1/2 were ablated. Retrospectively, one can understand the confusion about role, function, and purpose in the many discussions about functions that are the result of its primary functions.
Some scientists had the vision and steadfastness to emphasize the importance of MTs for zinc metabolism. “Although a single essential function of MT has not been demonstrated, MT of higher eukaryotes evolved as a mechanism to regulate zinc levels and distribution within cells and organisms.” (emphasis added). (451) Or with regard to the role of MT in intermolecular metal transfer: “It may represent the mechanistic basis for the chaperoning function that MT was suggested to have in channeling and regulating the flow of essential metals, in particular of zinc, to and from their many sites of action”. (36) MT is the “keystone that has been endowed with all the necessary attributes (emphasis added) to safeguard and regulate zinc homeostasis.” (452) Such and similar statements have been made repeatedly, and they were in essence correct. However, the characterization of MT was incomplete to put the function on a firm molecular basis. Only now it seems possible to relate the uniquely biological principles of MTs described here to a central role in the control of the homeostasis of two essential metal ions. It provides a new and different perspective how the molecular functions and activities of MTs translate into biological activities: MT has arolein zinc, copper, and redox metabolism with afunctionof buffering these metals ions for thepurposeof controlling their availability for control of biological processes.
Since a primary function had not been assigned to MT unambiguously, some investigators called it a multifunctional or multipurpose protein because of the pleiotropic effects observed. It blurred the focus on its mechanism of action and perpetuated a conundrum, in which functions, roles, purposes, properties, and chemical and biological activities became mixed up. Without a clear understanding of what MT does work remains phenomenological, and past work begs for an interpretation.
Starting with chemistry, the major functional group in the protein is the bifunctional sulfhydryl group: it binds metal ions, and it is redox-active. The complexity increases when the MT protein is considered. Two metal clusters in two domains are important for its function and behave differently. At least the three-metal cluster is not unique for MTs, though. The properties indicate two additional functions in the molecule─one for “storage” of metal ions in the C-terminal domain and one for “delivery” of metal ions in the N-terminal domain. The properties of the clusters are the sum of the different affinities for metal ions─and likely different redox activities─of the 20 cysteines. Multifunctionality starts with these chemical activities. We then need to ask how these chemical properties become biological properties. Here, an understanding of the biological context is necessary, as biology controls metal concentrations and redox potentials and, therefore, limits the number of molecular states in which MT can exist. In other words, not all chemical properties are necessarily biological properties. Chemistry focused on the structure to solve the issue of function, but even structure underlies the constraints of biology. Biology focused on function with insufficient reference to structure and reactivity. Moving on to biology, several purely biological factors need to be added to the chemical properties, in particular, the biological systems that regulate expression of MTs and control the proteins. In biology, it is not just MT but the whole system and its control under different conditions. Do the different MT proteins have different functions or purposes or is there redundancy such that they have the same functions or purposes under different circumstances? The multitude of inducers of gene expression of MTs could be the basis for different purposes of MT but not necessarily different functions. For this aspect of the MT system, a plausibility argument was put forward: “proteins whose synthesis is subject to such complex and fine regulation as MTs are likely to occupy a central role in cellular metabolism”. (66) This role was then related to normal metabolism and to conditions of stress. However, the function of MTs under normal physiological conditions was considered elusive based on genetic experiments that did not provide evidence for an essential function in the reproduction, development, and maintenance of mice. They epitomize the difficulties of functional genomics. (453) The interpretation of the knockout (ko) experiments is based on an operational failure at the time, namely, a limited examination of the phenotype. The ko mice, which were engineered by two independent groups and in which only MT1 and MT2 were ablated, were examined for cadmium toxicity, which was enhanced. (64,64) Originally, the mice were not subjected to meaningful tests that would have examined specific roles of Zn(II). Later, these mice showed deficiencies under many experimental challenges such as being sensitive to high and low Zn(II). Crosses of a murine model of Menkes disease (ATP7a mutation) and Mt1/Mt2 ko mice support a physiological “function” of MT in ameliorating copper toxicity through Cu(I) sequestration. (454) MT is also involved in the Cu(I)-dependent trafficking of ATP7a from the trans Golgi to the plasma membrane. (455) These findings indeed show a biological function in copper metabolism. There is an important implication, namely, that the Zn(II) induction of MT restricts the Cu(I) availability, thus demonstrating a role of zinc in copper metabolism. Changes in the expression of MT have a multitude of effects, and the lack of a phenotype under laboratory conditions is apparent only.
The functioning of MT in zinc and copper metabolism obviously affects the many functions that these metal ions have. It appears that the roles of MTs in zinc and copper metabolism for various purposes can be reduced to a definite primary function, that is, its molecular mechanism. To examine this hypothesis, we discuss the relationship of its primary function in a hierarchy of roles and purposes at four levels (Table 3). The number of levels could be increased, of course, but we apply Ockham’s razor.
Table 3. Levels of Biological “Functions” of MTs
| Primary Level | Secondary Level | Tertiary Level | Quaternary Level |
|---|---|---|---|
| Function & Purpose | Function & Purpose | Role | Role |
| Metal metabolism | Redox metabolism | Protection, natural compounds, and radiation | Protection, man-made compounds, and radiation |
| Metal donation and chelation | Drug resistance cytotoxic agents, based on thiol/thiolate reactivity only | ||
| Zinc “buffer”? Redox control in Zn(II) and Cu(I)/Cu(II) metabolism | Redox “buffer”? Zn(II) control in redox metabolism | Scavenger: Cd(II), other metal ions/metalloids, free radicals |
Specifically, we posit:
-Primary (physiological) function. It is based on the chemical activity of a biologically controlled and highly dynamic metal buffer that controls the availability and biological activitites of Zn(II) and Cu(I) ions. MT ascertains the right balance between these two most competitive metal ions. As a biological metal buffer, MT has at least three additional biological features. Its molecular state depends on the redox state, it is regulated by many factors and interactions, and it is translocated to where it functions inside and outside cells, making it highly dynamic in biological time and space. The biological terms chaperone, storage, transport, and receptor binding are all additional roles but remain linked to its primary function. Whether it is a genuine metallochaperone for Zn(II) or Cu(I) will depend on the demonstration of specific mechanisms of metal loading and unloading in interactions with specific targets. There may be some specialization in terms of roles for some MTs such as MT3 and MT4, perhaps being more involved in copper metabolism. With regard to purpose, we see an involvement in cell proliferation, differentiation, and apoptosis under normal conditions but also under specific types of stress such as in the acute phase response and viral defense. Under all these conditions the characteristics of the buffer changes so that the availability of Zn(II) ions is increased or decreased. The functions of MTs seem to become essential under conditions of Zn(II) deficiency and overload. It reflects exactly the behavior of a buffer, where larger fluctuations are observed if the buffer capacity is diminished.
-Secondary (physiological) functions. There is the possibility that MT/T is a redox buffer only, and one that is metal-controlled, but the evidence is much weaker. Many studies that consider its antioxidant role do not address the targets and functions of the dissociated metal ions. Unless T has redox functions in the absence of metal ions, there is no need to postulate a secondary function.
-Tertiary functions. MT provides some protection against naturally occurring toxins such as toxic metal ions under conditions of exposure, for example, Cd(II), and overproduction of reactive species/radicals in oxidative distress and physical and psychological stress. It can have two consequences, namely, that the Zn(II) made available is needed for cytoprotection, clearly a purpose, or that the agents interfere with the functions of Zn(II) or Cu(I) when they overwhelm the buffering capacity. Again, these tertiary functions are linked to the primary (or secondary) function. Protection against radiation is also a tertiary function, which is linked to protective functions of Zn(II) and its involvement in cellular and molecular repair processes. These functions clearly have a role in pathology and toxicology.
-Quaternary functions. It includes the role in quenching natural and man-made chemicals that are sulfhydryl-reactive, hence lowering their toxicity or leading to resistance in the case of drugs. It is not a purpose but a role.
For the field to advance, a clear understanding is necessary of what defines a protein as a metallothionein based on a molecular mechanism and on biological functions. How these attributes are modified in other organisms and correspond to modifications in zinc and copper metabolism remains to be defined.
6.3. Challenges for Future Research
A major impediment for progress is that investigations have not addressed─notwithstanding the difficulties in analytical chemistry─the metal occupancy of MTs in vivo and correlated it with specific parameters of zinc, copper, and redox metabolism. This aim should be pursued with a speciation of the different MT proteins. Such analytics will be very challenging, as they would need to include the oxidation state of the cysteinyl sulfur in the proteins and any other modifications, such as phosphorylation, that may have occurred. Once such high-resolution and sensitive methods have been developed and verified, the changes over time and in cellular space need to be addressed to understand the dynamics of the protein. Since the type of metal ion and occupancy determine the protein’s structure, such investigations are crucial when both direct and metal-mediated transient or stable protein–protein interactions are addressed. There was no incentive to look at mixed-metal compositions because it was thought to be either Zn(II) or Cu(I) but not both. Insights about the intricacies of zinc and copper metabolism in terms of their regulation, interaction, and regulatory functions such as dynamic pools involved in signaling is still developing. There was also no incentive to look at properties of MT1 isoforms, because the name “iso” is often understood to indicate that they are the same proteins. The differential gene regulation and changes of up to 10 amino acid residues in the protein structure of MT1 “isoforms” certainly are expected to endow these proteins with different properties. Thus,
| 1. | What is the metal composition and metal load in vivo for all the different MT proteins present in a tissue? It requires new techniques that address proteins, redox state, metal saturation, and modifications. For cell biological investigations there is the need for specific antibodies to the different gene products, although the availability of such antibodies would not yet solve the analytical challenge of addressing the state of the protein. Presently, most investigations focus on mRNA levels only, for which the correlation with the corresponding proteins is uncertain. | ||||
| 2. | What are the structures of the proteins with different metal occupancies, including mixed-metal species and their physical properties, including different properties of the individual MT proteins? | ||||
| 3. | Extracellular MT, secretion and uptake pathways, including receptors, and mechanisms of intracellular translocation need to be characterized with the possibility that these processes are accompanied by changes in metalation. | ||||
| 4. | What are the dynamic changes of specific MTs in specific biological events such as proliferation, differentiation, and apoptosis and under conditions of various types of stress? | ||||
| 5. | Investigations should switch from a mere focus on toxic metals to how toxic metals affect zinc and copper metabolism. | ||||
| 6. | The interaction of MTs with other proteins needs to be characterized structurally, including investigations that address whether the metamorphic or multimorphic nature of MTs is a factor in the selection of binding partners. | ||||
| 7. | Once the analytical chemistry for determining specific MTs, their metal load, redox state, ligand interactions, and modifications is available, the dynamics of the proteins in the cell needs to be addressed. It is the most challenging part─and a gargantuan task─as it requires a subcellular resolution of the coordination dynamics and reactions of MTs and a methodology for simultaneously resolving metal ion fluctuations and signals spatiotemporally. | ||||
6.4. Conclusions
One of the mysteries in biochemistry for over 60 years has been the function of the gene family of MTs. The quest is sometimes a tale of red herrings and wild goose chases and illustrates the tantalizing difficulties of defining biological functions of proteins and metal ions. The pleiotropic effects of MTs observed with their gene expression under the control of so many pathways would seem to indicate a fundamentally important system. With reference to the immense structural variability of MTs in other organisms, it should be obvious why it did not make sense to treat all MTs in a review that tries to link structures and functions. The issues discussed here need to be the starting point and guiding principle for investigations of other proteins that are referred to as MTs. It is a huge task because one will need to understand zinc and copper metabolism in each organism and relate it to the variations in MT structures. An impediment for a wider understanding of function is the use of a generic name for many different types of MT proteins and the lack of a word that expresses the uniquely biological activities of MTs in metal and redox metabolism. A consensus about the relationship between the chemistry and biology of MTs is necessary to make progress in this important aspect of metallobiochemistry, which is at the center of how Zn(II) and Cu(I)─and Cu(II)─are controlled and what the consequences are for health if such control is perturbed. Biology needs a definition of molecular mechanisms of MTs that lead to biological functions, roles, and purposes rather than just an operational definition of these proteins being cysteine-rich and binding metal ions. After an exegesis of the literature, this article summarized and critically evaluated advances in the chemistry of MTs to provide interpretative guidance for ontology. Pivotal was the realization that MT is a highly dynamic multimorphic protein with coordination and protein dynamics as reflected in different metal loads, oxidation states, coordination environments, and protein conformations. It binds several Zn(II) and Cu(I) ions with different affinities dependent on the biological context. It will also bind nonessential, thiophilic metal ions, the binding of which interferes with its functions. The framework for the exclusive use of sulfur coordination chemistry in mammalian MTs is the redox chemistry, high discrimination afforded for metal ions, and the pH dependence of affinity for metal ions and reactivity. While this article focused primarily on affinities, the area of kinetics remains to be fully explored. The exquisite control of cellular zinc and copper homeostasis and the dynamic balance of these ions, buffering Zn(II) and Cu(I) to very low concentrations and yet serving as signaling ions in cellular regulation, suggests a role of MT as a regulated, uniquely biological, interacting buffer for these highly competitive metal ions. Such an interpretation was not possible until recently because critical insights about the molecular and cellular regulation of Zn(II) and Cu(I) were lacking. The establishment of a 3D structure was a landmark in MT research, but, unfortunately, it gave the wrong impression that a fully metal-loaded form is THE state of the protein. A myriad of publications are based on assumptions about both structure and function, describe phenomena such as changes in the proteins’ gene expression, and discuss no clear molecular relationship of the protein to either redox or metal metabolism. The theme in this article is how chemical activity informs biological roles and how biology informs chemistry by limiting what is possible in the biological milieu. It serves as a reminder that the overall problem is one of biology and not chemistry, though both are necessary, and that function(s) need(s) to be interpreted with a consideration of the availability of metal ions and the redox state in the biological system. MT affords some protection against Cd(II) and other toxic metal ions, metalloids, and chemicals, but it is a matter of natural or man-made exposure and cannot be the primary function of mammalian MTs; one must consider the extent to which these scavenging functions interfere with the primary function of MTs in metal metabolism. One would hope that refocusing on the essential questions and bridging the disjunct scientific communities that focus on Zn(II), Cu(I), toxic metal ions, or redox biology will lead to performing the right experiments to test specific hypotheses instead of continuing phenomenological research. The results of such research will have a tremendous impact on many disciplines and for the health and disease of living systems. Our attempt of a synthesis of the present knowledge, of course, does not exclude the possibility that yet additional functions, roles, and purposes of MTs may be discovered in the future.
Author Information
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- Wolfgang Maret - Departments of Biochemistry and Nutritional Sciences, School of Life Course Sciences, Faculty of Life Sciences and Medicine, King’s College London, London SE1 9NH, U.K.;
https://orcid.org/0000-0003-1458-8231;
- Artur Krężel - Department of Chemical Biology, Faculty of Biotechnology, University of Wrocław, Wrocław 50-383, Poland;https://orcid.org/0000-0001-9252-5784
Biographies
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Artur Krężel
Artur Krężel received his diploma in chemistry (master of science, 2000) and a PhD degree in bioinorganic chemistry (2004) on the topic of glutathione coordination chemistry from the University of Wrocław, Poland, under the supervision of Prof. Wojciech Bal. During his postdoctoral training (2004–2007) at the University of Texas Medical Branch in Galveston, he worked with Prof. Wolfgang Maret on mechanisms of cellular zinc homeostasis, which bore fruit in the discovery of metallothioneins as physiological zinc ion buffers. He then accepted a position as an assistant professor at the Faculty of Biotechnology of his alma mater. In 2011, he received a D.Sc. degree from his university. Currently, he is a full professor and head of the Department of Chemical Biology. His research work concentrates on several areas at the interfaces of inorganic biochemistry, biophysics, and chemical biology, in particular, an understanding of the molecular bases of zinc and copper metabolism. Examples of this endeavor are structure–function relationships, the stability of metalloproteins, protein folding and thermodynamics, and the development of new analytical methods, such as fluorescent probes for selective protein modifications and metal sensing.
Wolfgang Maret
Wolfgang Maret FRSC is the Professor of Metallomics in the Departments of Nutritional Sciences (research) and Biochemistry (teaching) at King’s College London, London, UK. He obtained his master of science (Dipl. Chem.) in chemistry and his PhD in Natural Sciences from Saarland University, Saarbrücken, Germany. His academic career includes postdoctoral research at The University of Chicago (Department of Biophysics & Theoretical Biology), assistant professor in the Center for Biochemical and Biophysical Sciences and Medicine at Harvard Medical School with an additional teaching appointment at The Bouvé College of Pharmacy, Northeastern University, Boston, MA, and associate professor at the University of Texas Medical Branch in Galveston, Texas (Departments of Preventive Medicine & Community Health and Anesthesiology). His research interests began with the catalytic mechanisms of metalloenzymes as investigated with spectroscopic and kinetic methods and continued with the molecular and cellular mechanisms of how metal ions control protein structure and function and how proteins control nutritionally essential elements and mitigate the effects of nonessential and toxic elements. His interest in the role of metal and redox biology in health and disease extends to the etiology of liver disease, diabetes, traumatic brain injury, skin disease, and arthritis. From 2012 to 2014, he served as the president of the International Society for Zinc Biology and from 2012 to 2016 as chair of the editorial board of the journal Metallomics of the Royal Society of Chemistry.
Acknowledgments
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We recognize the centennial of the birthday of Bert L. Vallee and the passing of Milan Vašák in 2019. Research in A.K.’s laboratory was supported by the National Science Center of Poland under Opus Grant No. 2018/31/B/NZ1/00567. Publication of this article was supported financially by the Excellence Initiative - Research University (IDUB) program for the University of Wrocław. W.M. thanks his colleagues at King’s College London, Professors C. Hogstrand and S. Sturzenbaum, Professor N. Bury, University of Suffolk, and Professor P. Kille, Cardiff University, for engaging discussions on all matters of metallothioneinology over the past decade.
| MT | metallothionein |
| T | thionein |
| DTNB | 5,5′-dithiobis(2-nitrobenzoic acid) |
| GSH | glutathione |
| GSSG | glutathione disulfide |
References
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This article references 455 other publications.
- 1Drinker, K. R.; Collier, E. S. The significance of zinc in the living organism. J. Industr. Hygiene 1926, 8, 257– 269Google ScholarThere is no corresponding record for this reference.
- 2Margoshes, M.; Vallee, B. L. A Cadmium protein from equine kidney cortex. J. Am. Chem. Soc. 1957, 79, 4813– 4814, DOI: 10.1021/ja01574a064Google Scholar2A cadmium protein from equine kidney cortexMargoshes, Marvin; Vallee, Bert L.Journal of the American Chemical Society (1957), 79 (), 4813-14CODEN: JACSAT; ISSN:0002-7863.cf. C.A. 50, 5817c. Fractionation of horse-kidney cortex with EtOH and (NH4)2SO4 gave a product contg. 20-5 mg./g. dry wt. of Cd in Cl3CCO2H-precipitable material in succesive fractionations. Ultracentrifugation in a synthetic-boundary cell showed the final products of 4 successive fractionations to be unidisperse with a sedimentation (uncor. for viscosity of diffusion) varying from 0.94 to 1.22 × 10-13. Paper electrophoresis at pH 8.5 of the product of 1 fractionation showed 3 components moving toward the cathode; the slowest comprised about 70% of the material. The Cd content rose 30-fold throughout the fractionation from the 1st ext. to the product. Cd was not removable by dialysis at pH 7, but was removed by treatment with hot Cl3CCO2H. With the exception of Zn, other metals initially present or introduced during fractionation were removed and occurred in low concn. in the final material. The possibility of isomorphism with Zn is discussed. The product contains 14% N, and reacts positively to ninhydrin and biuret. Hydrolysis and paper chromatography showed serine, glycine, aspartic and glutamic acids among other nonidentifiable amino acids. The last fraction contained about 1% hexosamine.
- 3Kägi, J. H. R.; Vallee, B. L. Metallothionein: a cadmium- and zinc-containing protein from equine renal cortex. J. Biol. Chem. 1960, 235, 3460– 3465, DOI: 10.1016/S0021-9258(18)64490-4Google Scholar3Metallothionein: a cadmium- and zinc-containing protein from equine renal cortexKagi, Jeremias H. R.; Vallee, Bert L.; Carlson, Janet M.Journal of Biological Chemistry (1960), 235 (), 3460-5CODEN: JBCHA3; ISSN:0021-9258.cf. CA 52, 7393c. Metallothionein, a protein of small mol. wt. isolated from equine renal cortex, contains 2.9% Cd, 0.6% Zn, and 4% S. The protein, homogeneous on ultracentrifugation but contg. minor impurities on electrophoresis, does not absorb radiation at or near 280 mμ; this finding indicates a very low aromatic amino acid content. A large no. of cysteine residues accounts for the high S content. The stoichiometry between titratable SH groups and metal atoms, the displacement of the metals by SH-specific agents, and the selective removal of Zn and Cd by H ions suggest isomorphous binding of the 2 metals to the protein through mercaptide linkages. Although the biol. function of metallothionein is not known thus far, the specific assocn. of Cd with this macromol. suggests a definite biol. role.
- 4Kägi, J. H. R.; Vallee, B. L.; Carlson, J. M. Metallothionein: a cadmium- and zinc-containing protein from equine renal cortex. II. Physicochemical properties. J. Biol. Chem. 1961, 236, 2435– 2442, DOI: 10.1016/S0021-9258(18)64017-7Google Scholar4Metallothionein: a cadmium and zinc-containing protein from equine renal cortex. II. Physicochemical propertiesKagi, Jeremias H. R.; Vallee, Bert L.Journal of Biological Chemistry (1961), 236 (), 2435-42CODEN: JBCHA3; ISSN:0021-9258.cf. Federation Proc. 19, 340(1960); CA 55, 7595e. Lyophilized metallothionein, prepd. from horse-kidney cortex by EtOH and salt fractionation (loc. cit.), was purified further by DEAE cellulose chromatography. The specific absorption of metallothionein at 250 mμ is a convenient monitor for its isolation. The purest prepn. of metallothionein contained 14.9% N and 8.5% S with 26 titratable mercapto groups accounting for more than 95% of the total S content. The mol. wt. of metallothionein is 10,000 ± 260, an av. of the values obtained from velocity sedimentation and diffusion detns. and the approach to equil. centrifugation. The small mol. wt. accounts for losses on dialysis through membranes of large pore size. The partial sp. vol., 0.648 ml./g., is unusually low, in part because of the high metal and S contents. Approx. 1 of 3 or 4 amino acids in this small protein mol. is a cysteine residue; proline, serine, and lysine residues come next in that order of abundance. Tryptophan and tyrosine are absent. The diffusion const., D20,w, is 12.42 ± 0.07 × 10-7, and the frictional ratio, f:f0, is 1.28. The most highly purified prepn., homogeneous by ultracentrifugation and electrophoresis, contains 5.9% Cd, 2.2% Zn, 0.2% Fe, and 0.1% Cu. Metallothionein is formed through the interaction of 1 atom of Cd or Zn with 3 sulfhydryl groups. The characteristic charge-transfer band, with max. absorption at 250 mμ, is due to the Cd mercaptide; it is absent in thionein, the metal-free protein. A similar band at 215 mμ is apparently due to the Zn mercaptide. The conformation of thionein, its component cysteine residues, and the resultant steric organization of the free sulfhydryl groups, disallow a large no. of possible structures of this protein and are thought to det. both the remarkable avidity for Cd and the preponderance of Cd over Zn.
- 5Kägi, J. H. R.; Himmelhoch, S. R.; Whanger, P. D.; Bethune, J. L.; Vallee, B. L. Equine hepatic and renal metallothioneins. J. Biol. Chem. 1974, 249, 3537– 3542, DOI: 10.1016/S0021-9258(19)42605-7Google Scholar5Equine hepatic and renal metallothioneins. Purification, molecular weight, amino acid composition, and metal contentKagi J H; Himmelhoch S R; Whanger P D; Bethune J L; Vallee B LThe Journal of biological chemistry (1974), 249 (11), 3537-42 ISSN:0021-9258.There is no expanded citation for this reference.
- 6Bühler, R. H. O.; Kägi, J. H. R. Human hepatic metallothioneins. FEBS Lett. 1974, 39, 229– 234, DOI: 10.1016/0014-5793(74)80057-8Google Scholar6Human hepatic metallothioneinsBuehler, Rolf H. O.; Kaegi, Jeremias H. R.FEBS Letters (1974), 39 (2), 229-34CODEN: FEBLAL; ISSN:0014-5793.Two Zn-contg. metallothioneins were isolated from human liver. Each had a min. mol. wt. of ∼6600, and based on this mol. wt. one contained 6 Zn atoms and the other 7. This was proportional to the cysteine and titratable SH groups of the proteins. The amino acid compns. were detd. No other metal were bound to these proteins.
- 7Porter, H. The particulate half-cysteine-rich copper protein of newborn liver. Relationship to metallothionein and subcellular localization in non-mitochondrial particles possibly representing heavy lysosomes. Biochem. Biophys. Res. Commun. 1974, 56, 661– 668, DOI: 10.1016/0006-291X(74)90656-1Google Scholar7Particulate half-cystine-rich copper protein of newborn liver. Relation to metallothionein and subcellular localization in nonmitochondrial particles possibly representing heavy lysosomesPorter, HuntingtonBiochemical and Biophysical Research Communications (1974), 56 (3), 661-8CODEN: BBRCA9; ISSN:0006-291X.The particulate half-cystine-rich Cu protein of newborn liver was partially purified by centrifugation of the heavy mitochondrial fraction through glycogen-sucrose or sucrose d. gradients. The resulting sediments contained ∼ 4% Cu, ∼ 20% half-cystine, and a 2-3-fold increase in β-glucuronidase specific activity. The Cu protein is not a true mitochondrial constituent and the data are consistent with its localization in a distinct population of heavy lysosomes. The amino acid composition of the polypeptide isolated from the crude insoluble Cu protein is strikingly similar to that of metallothionein, suggesting that the neonatal protein represents a Cu-rich form of metallothionein.
- 8Rydén, L.; Deutsch, H. F. Preparation and properties of the major copper─binding component in human fetal liver. J. Biol. Chem. 1978, 253, 519– 524, DOI: 10.1016/S0021-9258(17)38240-6Google Scholar8Preparation and properties of the major copper-binding component in human fetal liver. Its identification as metallothioneinRyden, Lars; Deutsch, Harold F.Journal of Biological Chemistry (1978), 253 (2), 519-24CODEN: JBCHA3; ISSN:0021-9258.Most of the relatively large amts. of Cu in human fetal liver is bound to a low-mol.-wt. protein. By a combination of gel filtrations and covalent chromatog. on thiopropyl-Sepharose, the monomeric form of the Cu-binding protein and some polymers of it were prepd. in good yield. The protein was identified as a Cu-thionein by the criteria of mol. wt., shape, amino acid compn., and amino acid sequence homol. Gel filtration in 6M guanidine-HCl showed that the mol. was a single peptide chain of 58 residues, corresponding to a mol. wt. of 6000. The mol. appears to be asym. with an approx. frictional ratio of 1.40. The protein contained 2.4 g atoms of Cu/mol and traces of Mn and Zn, but no Cd. The Cu was not paramagnetic. Possible roles of this Cu-binding protein in the transport and storage of Cu are discussed.
- 9Piscator, M. Om kadmium i normala människornjurar samt redogörelse för isolering av metallthionein ur lever från kadmiumexponderade kaniner. Nordisk Hygienisk Tidskrift 1964, 65, 76– 82Google ScholarThere is no corresponding record for this reference.
- 10Nordberg, G. F.; Nordberg, M.; Piscator, M.; Vesterberg, O. Separation of two forms of rabbit metallothionein by isoelectric focusing. Biochem. J. 1972, 126, 491– 498, DOI: 10.1042/bj1260491Google Scholar10Separation of two forms of rabbit metallothionein by isoelectric focussingNordberg, Gunnar F.; Nordberg, Monica; Piscator, Magnus; Vesterberg, OlofBiochemical Journal (1972), 126 (3), 491-8CODEN: BIJOAK; ISSN:0264-6021.Rabbits were given repeated injections of CdCl2. Cd- and Zn-contg. protein fractions were obtained from the livers of these animals by pptn. procedures and Sephadex G-75 chromatog. The protein thus obtained showed several characteristics similar to those of the earlier described protein metallothionein. Further sepn. by isoelec. focusing showed two main protein peaks with isoelec. points at 3.9 and 4.5, resp. Amino acid anal. of these 2 forms showed similar content of most amino acids [residues percent.: cysteine (28%), aspartate (8%), threonine (5-6), serine (12%), glycine (7%), alanine (13%), methionine (2%), isoleucine (2%)] but with a small difference in content of lysine (12 and 13%, resp.), proline (9 and 5%, resp.) and glutamate (2 and 4% resp.). The 2 forms of the protein both contained Cd, but only the one with pI 4.5 contained also significant amts. of Zn.
- 11Hunziker, P. E.; Kägi, J. H. R. Isolation and characterization of six human hepatic isometallothioneins. Biochem. J. 1985, 231, 375– 382, DOI: 10.1042/bj2310375Google Scholar11Isolation and characterization of six human hepatic isometallothioneinsHunziker, Peter E.; Kaegi, Jeremias H. R.Biochemical Journal (1985), 231 (2), 375-82CODEN: BIJOAK; ISSN:0264-6021.Human hepatic metallothionein (MT) was sepd. into 6 isoforms by using reversed-phase HPLC for anal. and prepn. By comparison with the HPLC elution profiles of the charge-separable species MT-1 and MT-2 isolated by earlier procedures, 5 of these isoproteins are identified as hitherto unresolved subforms of MT-1, and 1 is identical with MT-2. The 6 isoforms have distinct and reproducible retention times at neutral pH, where the metal remains bound to the protein, and at low pH, where the metal is removed. Their amino acid compns. display the high cysteine content and the lack of arom. amino acids and of histidine typical of mammalian metallothioneins, but they differ significantly with respect to all other amino acids. A survey of autopsy material indicates that in adult human liver, all 6 forms are usually expressed, albeit in somewhat variable relative proportions.
- 12Karin, M.; Richards, R. I. Human metallothionein genes – primary structure of the metallothionein-II gene and a related processed gene. Nature (London, U. K.) 1982, 299, 797– 802, DOI: 10.1038/299797a0Google Scholar12Human metallothionein genes. Primary structure of the metallothionein-II gene and a related processed geneKarin, Michael; Richards, Robert I.Nature (London, United Kingdom) (1982), 299 (5886), 797-802CODEN: NATUAS; ISSN:0028-0836.The complete nucleotide sequences of 2 of the human metallothionein gene family were elucidated and compared. One was a functional metallothionein-II gene; the other was a pseudogene, which lacked introns and which terminated in a poly(A) tail and was flanked by 2 direct repeats. A size polymorphism was detected which was assocd. with the processed gene in the population examd., and a region of apparent secondary structure homol. was obsd. between a 5' flanking region of the functional metallothionein-II gene and that of a mouse metallothionein-I gene.
- 13Uchida, Y.; Takio, K.; Titani, K.; Ihara, Y.; Tomonaga, M. The growth inhibitory factor that is deficient in the Alzheimer’s disease brain is a 68 amino acid metallothionein-like protein. Neuron 1991, 7, 337– 347, DOI: 10.1016/0896-6273(91)90272-2Google Scholar13The growth inhibitory factor that is deficient in the Alzheimer's disease brain is a 68 amino acid metallothionein-like proteinUchida, Yoko; Takio, Koji; Titani, Koiti; Ihara, Yasuo; Tomonaga, MasanoriNeuron (1991), 7 (2), 337-47CODEN: NERNET; ISSN:0896-6273.The authors purified and characterized the growth inhibitory factor (GIF) that is abundant in the normal human brain, but greatly reduced in the Alzheimer's disease (AD) brain. GIF inhibited survival and neurite formation of cortical neurons in vitro. Purified GIF is a 68 amino acid small protein, and its amino acid sequence is 70% identical to that of human metallothionein II with a 1 amino acid insert and a unique 6 amino acid insert in the NH2-terminal and the COOH-terminal portions, resp. The antibodies to the unique sequence of GIF revealed a distinct subset of astrocytes in the gray matter that appears to be closely assocd. with neuronal perikarya and dendrites. In the AD cortex, the no. of GIF-pos. astrocytes was drastically reduced, suggesting that GIF is down-regulated in the subset of astrocytes during AD.
- 14Sewell, A. K.; Jensen, L. T.; Erickson, J. C.; Palmiter, R. D.; Winge, D. R. Biocactivity of metallothionein-3 correlates with its novel β domain sequence rather than metal binding properties. Biochemistry 1995, 34, 4740– 4747, DOI: 10.1021/bi00014a031Google Scholar14Bioactivity of Metallothionein-3 Correlates with Its Novel β Domain Sequence Rather Than Metal Binding PropertiesSewell, Andrew K.; Jensen, Laran T.; Erickson, Jay C.; Palmiter, Richard D.; Winge, Dennis R.Biochemistry (1995), 34 (14), 4740-7CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)Human and mouse metallothionein-3 (MT-3) mols. exhibit the same metal binding stoichiometry with Zn(II), Cd(II), or Cu(I) as MT-1 or MT-2 mols., suggesting that MT-3 consists of two domains enfolding sep. polymetallic clusters. The kinetic reactivities of Zn(II) complexes of MT-3 with the chelator EDTA or the thiol reagent dithiobis(2-nitrobenzoic acid) (DTNB) resembles the reactivity of ZnMT-1. Furthermore, the candidate α and β domain peptides of human MT-3 are very similar to MT-1 domain peptides in the reactivity of Zn(II) complexes. Zn(II) complexes of human and mouse MT-3 inhibit the survival of rat cortical neurons cultured in the presence of an Alzheimer's disease brain ext. Inhibitory activity is unique to the MT-3 isoform and is a property of the N-terminal β domain. The inhibitory activity of the 32-residue MT-3 β domain is abolished by a double mutation within the β domain resulting in the conversion of the C-P-C-P sequence to either C-S-C-A or C-T-C-T. Thus, the bioactivity arises from a novel structure of the N-terminal β domain of MT-3 and not any unusual metal-binding properties.
- 15Palmiter, R. D.; Findley, S. D.; Whitmore, T. E.; Durnam, D. M. MT-III, a brain-specific member of the metallothionein gene family. Proc. Natl. Acad. Sci. U. S. A. 1992, 89, 6333– 6337, DOI: 10.1073/pnas.89.14.6333Google Scholar15MT-III, a brain-specific member of the metallothionein gene familyPalmiter, Richard D.; Findley, Seth D.; Whitmore, Theodore E.; Durnam, Diane M.Proceedings of the National Academy of Sciences of the United States of America (1992), 89 (14), 6333-7CODEN: PNASA6; ISSN:0027-8424.A third member of the metallothionein (MT) gene family, designated MT-III, was cloned by virtue of its homol. to a human protein that was shown previously to inhibit neuronal survival in culture and to be deficient in the brain of people with Alzheimer disease. Human and mouse brains MT-IIIs have two insertions relative to all other known mammalian MTs: a threonine after the fourth amino acid and a block of six amino acids near the carboxyl terminus. The genes encoding MT-III resemble all other mammalian MT genes in their small size and exon/intron organization. The MT-III genes are closely linked to the other functional MT genes on human chromosome 16 and mouse chromosome 8. Mouse MT-III gene expression appears to be restricted to brain; in addn., it fails to respond to zinc, cadmium, dexamethasone, or bacterial endotoxin in vivo, thereby distinguishing MT-III from other known MTs.
- 16Quaife, C. J.; Findley, S. D.; Erickson, J. C.; Froelick, G. J.; Kelly, E. J.; Zambrowicz, B. P.; Palmiter, R. D. Induction of a new metallothionein isoform (MT-IV) occurs during differentiation of stratified squamous epithelia. Biochemistry 1994, 33, 7250– 7259, DOI: 10.1021/bi00189a029Google Scholar16Induction of a New Metallothionein Isoform (MT-IV) Occurs during Differentiation of Stratified Squamous EpitheliaQuaife, Carol J.; Findley, Seth D.; Erickson, Jay C.; Froelick, Glenda J.; Kelly, Edward J.; Zambrowicz, Brian P.; Palmiter, Richard D.Biochemistry (1994), 33 (23), 7250-9CODEN: BICHAW; ISSN:0006-2960.A new member of the metallothionein (MT) gene family was discovered that lies about 20 kb 5' of the MT-III genes in both mouse and human. The MT-IV proteins are highly conserved in both species and have a glutamate insertion at position 5 relative to the classical MT-I and MT-II proteins. Murine MT-IV mRNA appears to be expressed exclusively in stratified squamous epithelia assocd. with oral epithelia, esophagus, upper stomach, tail, footpads, and neonatal skin. The MT derived from tongue epithelium contains both zinc and copper. Many of these epithelia develop parakaratosis during zinc deficiency in the rat. In situ hybridization reveals intense labeling of MT-IV mRNA in the differentiating spinous layer of cornified epithelia, whereas MT-I is expressed predominantly in the basal, proliferative layer; thus, there is a switch in MT isoform synthesis during differentiation of these epithelia. The authors suggest that MT-IV plays a special role in regulating zinc metab. during the differentiation of stratified epithelia.
- 17Kojima, Y.; Berger, C.; Vallee, B. L.; Kägi, J. H. R. Amino-acid sequence of equine renal metallothionein-1B. Proc. Natl. Acad. Sci. U. S. A. 1976, 73, 3413– 3417, DOI: 10.1073/pnas.73.10.3413Google Scholar17Amino-acid sequence of equine renal metallothionein-1BKojima, Yutaka; Berger, Christine; Vallee, Bert L.; Kaegi, Jeremias H. R.Proceedings of the National Academy of Sciences of the United States of America (1976), 73 (10), 3413-17CODEN: PNASA6; ISSN:0027-8424.The amino acid sequence of a metallothionein is reported. Metallothionein-1B is 1 of the 2 principal variants occurring in equine kidney cortex. The single-chain protein contains 61 amino acids. The N-terminal residue is N-acetylmethionine. The sequence shows distinct clustering of the 20 cysteinyl residues into 7 groups sepd. by stretches of ≥3 other residues. Within these groups the cysteines occur 7 times in alternating Cys-X-Cys sequences and 3 times each in Cys-Cys and Cys-X-X-Cys sequences where X is an amino acid other than cysteine. Another unique feature is the close assocn. of serine and of the basic amino acids with cysteine, as manifested by the occurrence of 7 Ser-Cys, 4 Cys-Lys, 7 Cys-Arg, and 3 Lys-Cys sequences. These findings are in agreement with the previous suggestion that metallothionein has structurally defined metal-binding sites, most of which contain 3 cysteinyl residues as the principal metal-binding ligands. The charge difference between the metal-free and the metal-contg. protein is consistent with the formation of neg. charged trimercaptide complexes with Cd2+ and(or) Zn2+. The possible addnl. involvement of serine and the basic amino acids in metal binding is discussed.
- 18Kojima, Y. Definitions and nomenclature of metallothioneins. Methods Enzymol. 1991, 205, 8– 10, DOI: 10.1016/0076-6879(91)05078-AGoogle Scholar18Definitions and nomenclature of metallothioneinsKojima, YutakaMethods in Enzymology (1991), 205 (Metallobiochem. Pt. B), 8-10CODEN: MENZAU; ISSN:0076-6879.Metallothioneins are defined and nomenclature of these proteins and the corresponding genes presented.
- 19Karin, M.; Eddy, R. L.; Henry, W. M.; Haley, L. L.; Byers, M. G.; Shows, T. B. Human metallothionein genes are clustered on chromosome 16. Proc. Natl. Acad. Sci. U. S. A. 1984, 81, 5494– 5498, DOI: 10.1073/pnas.81.17.5494Google Scholar19Human metallothionein genes are clustered on chromosome 16Karin, Michael; Eddy, Roger L.; Henry, W. Michael; Haley, Linda L.; Byers, Mary G.; Shows, Thomas B.Proceedings of the National Academy of Sciences of the United States of America (1984), 81 (17), 5494-8CODEN: PNASA6; ISSN:0027-8424.In man, the metallothioneins are encoded by ≥10-12 genes sepd. into 2 groups, MT-I and MT-II. To understand the genomic organization of these genes and their involvement in hereditary disorders of trace metal metab., their chromosomal location was detd. By using human-mouse cell hybrids and hybridization probes derived from clones and functional human MT1 and MT2 genes, it was shown that the functional human genes are clustered on human chromosome 16. Anal. of RNA from somatic cell hybrids indicated that hybrids which contained human chromosome 16 expressed both human MT1 and MT2 mRNA, and this expression is regulated by both heavy metal ions and glucocorticoid hormones.
- 20Katoh, K.; Standley, D. M. MAFFT multiple sequence alignment software version 7: Improvements in performance and usability. Mol. Biol. Evol. 2013, 30, 772– 780, DOI: 10.1093/molbev/mst010Google Scholar20MAFFT Multiple Sequence Alignment Software Version 7: Improvements in Performance and UsabilityKatoh, Kazutaka; Standley, Daron M.Molecular Biology and Evolution (2013), 30 (4), 772-780CODEN: MBEVEO; ISSN:0737-4038. (Oxford University Press)We report a major update of the MAFFT multiple sequence alignment program. This version has several new features, including options for adding unaligned sequences into an existing alignment, adjustment of direction in nucleotide alignment, constrained alignment and parallel processing, which were implemented after the previous major update. This report shows actual examples to explain how these features work, alone and in combination. Some examples incorrectly aligned by MAFFT are also shown to clarify its limitations. We discuss how to avoid misalignments, and our ongoing efforts to overcome such limitations.
- 21Troshin, P. V.; Procter, J. B.; Barton, G. J. Java bioinformatics analysis web services for multiple sequence alignment – JABAWS:MSA. Bioinformatics 2011, 27, 2001– 2002, DOI: 10.1093/bioinformatics/btr304Google Scholar21Java bioinformatics analysis web services for multiple sequence alignment-JABAWS:MSATroshin, Peter V.; Procter, James B.; Barton, Geoffrey J.Bioinformatics (2011), 27 (14), 2001-2002CODEN: BOINFP; ISSN:1367-4803. (Oxford University Press)Summary: JABAWS is a web services framework that simplifies the deployment of web services for bioinformatics. JABAWS:MSA provides services for five multiple sequence alignment (MSA) methods (Probcons, T-coffee, Muscle, Mafft and ClustalW), and is the system employed by the Jalview multiple sequence anal. workbench since version 2.6. A fully functional, easy to set up server is provided as a Virtual Appliance (VA), which can be run on most operating systems that support a virtualization environment such as VMware or Oracle VirtualBox. JABAWS is also distributed as a Web Application aRchive (WAR) and can be configured to run on a single computer and/or a cluster managed by Grid Engine, LSF or other queuing systems that support DRMAA. JABAWS:MSA provides clients full access to each application's parameters, allows administrators to specify named parameter preset combinations and execution limits for each application through simple configuration files. The JABAWS command-line client allows integration of JABAWS services into conventional scripts. Availability and Implementation: JABAWS is made freely available under the Apache 2 license and can be obtained from: http://www.compbio.dundee.ac.uk/jabaws. Contact: [email protected].
- 22Trinchella, F.; Esposito, M. G.; Scudiero, R. Metallothionein primary structure in amphibians: Insights from comparative evolutionary analysis in vertebrates. C. R. Biol. 2012, 335, 480– 487, DOI: 10.1016/j.crvi.2012.05.003Google Scholar22Metallothionein primary structure in amphibians: Insights from comparative evolutionary analysis in vertebratesTrinchella, Francesca; Esposito, Maria Grazia; Scudiero, RosariaComptes Rendus Biologies (2012), 335 (7), 480-487CODEN: CRBOCM; ISSN:1631-0691. (Elsevier Masson SAS)Metallothioneins are cysteine-rich, low-mol. wt. metal-binding proteins ubiquitously expressed in living organisms. In the last past years, the increasing amt. of vertebrate non-mammalian metallothionein sequences available have disclosed for these proteins differences in the primary structure that have not been supposed before. To provide a more up-to-date view of the metallothioneins in non-mammalian tetrapods, we decided to increase the still scarce knowledge concerning the primary structure and the evolution of metallothioneins in amphibians. Our data demonstrate an unexpected diversity of metallothionein sequences among amphibians, accompanied by remarkable features in their phylogeny. Phylogenetic anal. also reveals the complexity of vertebrate metallothionein evolution, made by both ancient and more recent events of gene duplication and loss.
- 23Madeira, F.; Park, Y. M.; Lee, J.; Buso, N.; Gur, T.; Madhusoodanan, N.; Basutkar, P.; Tivey, A.; Potter, S. C.; Finn, R. D.; Lopez, R. The EMBL-EBI search and sequence analysis tools APIs in 2019. Nucleic Acids Res. 2019, 47 (W1), W636– W641, DOI: 10.1093/nar/gkz268Google Scholar23The EMBL-EBI search and sequence analysis tools APIs in 2019Madeira, Fabio; Park, Young Mi; Lee, Joon; Buso, Nicola; Gur, Tamer; Madhusoodanan, Nandana; Basutkar, Prasad; Tivey, Adrian R. N.; Potter, Simon C.; Finn, Robert D.; Lopez, RodrigoNucleic Acids Research (2019), 47 (W1), W636-W641CODEN: NARHAD; ISSN:1362-4962. (Oxford University Press)The EMBL-EBI provides free access to popular bioinformatics sequence anal. applications as well as to a full-featured text search engine with powerful cross-referencing and data retrieval capabilities. Access to these services is provided via user-friendly web interfaces and via established RESTful and SOAP Web Services APIs (https://www.ebi.ac.uk/seqdb/confluence/display/JDSAT/EMBL-EBI+Web+Services+APIs+-+Data+Retrieval). Both systems have been developed with the same core principles that allow them to integrate an ever-increasing vol. of biol. data, making them an integral part of many popular data resources provided at the EMBL-EBI. Here, we describe the latest improvements made to the frameworks which enhance the interconnectivity between public EMBL-EBI resources and ultimately enhance biol. data discoverability, accessibility, interoperability and reusability.
- 24Letunic, I.; Bork, P. Interactive Tree Of Life (iTOL): an online tool for phylogenetic tree display and annotation. Bioinformatics 2007, 23, 127– 128, DOI: 10.1093/bioinformatics/btl529Google Scholar24Interactive Tree Of Life: an online tool for phylogenetic tree display and annotationLetunic, Ivica; Bork, PeerBioinformatics (2007), 23 (1), 127-128CODEN: BOINFP; ISSN:1367-4803. (Oxford University Press)Summary: Interactive Tree Of Life (iTOL) is a web-based tool for the display, manipulation and annotation of phylogenetic trees. Trees can be interactively pruned and re-rooted. Various types of data such as genome sizes or protein domain repertoires can be mapped onto the tree. Export to several bitmap and vector graphics formats is supported.
- 25McCormick, C. C.; Lin, L.-Y. Quantification and identification of metallothioneins by gel electrophoresis and silver staining. Methods Enzymol. 1991, 205, 71– 76, DOI: 10.1016/0076-6879(91)05087-CGoogle Scholar25Quantification and identification of metallothioneins by gel electrophoresis and silver stainingMcCormick, Charles C.; Lin, Lih YuanMethods in Enzymology (1991), 205 (Metallobiochem. Pt. B), 71-8, 2 platesCODEN: MENZAU; ISSN:0076-6879.A nondenaturing PAGE for isolating and identifying metallothioneins has been presented. The method employs gradient PAGE and extended electrophoresis of heat-treated tissue exts. (cytosol). The salient feature of the procedure is the use of Coomassie Blue stain as an initial treatment followed by silver stain enhancement. The latter process appears to specifically identify (enhance) MTs among other heat-stable proteins.
- 26Vašák, M. Standard isolation procedure for metallothionein. Methods Enzymol. 1991, 205, 41– 44, DOI: 10.1016/0076-6879(91)05082-7Google Scholar26Standard isolation procedure for metallothioneinVasak, MilanMethods in Enzymology (1991), 205 (Metallobiochem. Pt. B), 41-4CODEN: MENZAU; ISSN:0076-6879.Metallothionein (MT) isolation from rabbit liver included prepn. of crude fraction, gel-filtration chromatog., and ion-exchange chromatog. The purifn. of MT from human liver and horse liver and kidney is briefly described.
- 27Hunziker, P. E. Metal removal from mammalian metallothioneins. Methods Enzymol. 1991, 205, 451– 452, DOI: 10.1016/0076-6879(91)05129-JGoogle Scholar27Metal removal from mammalian metallothioneinsHunziker, Peter E.Methods in Enzymology (1991), 205 (Metallobiochem. Pt. B), 451-2CODEN: MENZAU; ISSN:0076-6879.Metal removal from metallothionein (MT) is a central step in the structural anal. of this protein. While zinc and cadmium are readily removed at low pH, copper remains partially bound and can be removed from MT only by using chelating agents. The methods for the prepn. of apo-MT that have been successfully used for the primary structure anal. of mammalian MTs are described.
- 28Vašák, M. Metal removal and substitution in vertebrate and invertebrate metallothioneins. Methods Enzymol. 1991, 205, 452– 458, DOI: 10.1016/0076-6879(91)05130-NGoogle Scholar28Metal removal and substitution in vertebrate and invertebrate metallothioneinsVasak, MilanMethods in Enzymology (1991), 205 (Metallobiochem. Pt. B), 452-8CODEN: MENZAU; ISSN:0076-6879.Prepn. of metal-free protein [mtallothionein(MT)] prepn. of metal-substituted MT, and examples of metal-substitution procedures are discussed.
- 29Winge, D. R.; Premakumar, R.; Rajagopalan, V. Metal-induced formation of metallothionein in rat liver. Arch. Biochem. Biophys. 1975, 170, 242– 252, DOI: 10.1016/0003-9861(75)90115-0Google Scholar29Metal-induced formation of metallothionein in rat liverWinge, Dennis R.; Premakumar, R.; Rajagopalan, K. V.Archives of Biochemistry and Biophysics (1975), 170 (1), 242-52CODEN: ABBIA4; ISSN:0003-9861.The low mol. wt. proteins induced in rats exposed to zinc [7440-66-6], mercury [7439-97-6], or silver [7440-22-4] were purified by the same procedure as was used for Cd-contg. metallothionein (Cd-thionein). In each case the thionein was resolved into the same two fractions on DEAE-cellulose. The two forms of each metalloprotein exhibited mobilities identical to those of the corresponding Cd-thionein on polyacrylamide gel electrophoresis. The amino acid compns. of the more anionic forms of Hg-thionein and Zn-thionein were quite similar to that of the corresponding Cd-thionein. Thus, the identity of the proteins induced in rats by zinc, mercury, and silver with the previously known metallothionein induced by cadmium [7440-43-9] has been established.
- 30Vašák, M.; Hawkes, G. E.; Nicholson, J. K.; Sadler, P. J. 113Cd NMR studies of reconstituted seven-cadmium metallothionein: evidence for structural flexibility. Biochemistry 1985, 24, 740– 747, DOI: 10.1021/bi00324a031Google Scholar30Cadmium-113 NMR studies of reconstituted seven-cadmium metallothionein: evidence for structural flexibilityVasak, Milan; Hawkes, Geoffrey E.; Nicholson, Jeremy K.; Sadler, Peter J.Biochemistry (1985), 24 (3), 740-7CODEN: BICHAW; ISSN:0006-2960.A reproducible method for the reconstitution of rabbit liver metallothionein (MT) contg. 7 Cd atoms/mol of protein is described. This protein was studied in detail by 113Cd NMR at 88-, 55-, and 44-MHz frequencies, including the effects of pH, temp., and ionic strength on the spectra. The results differ significantly from previous reports of 113Cd NMR on similar samples. Thus, the spectra of both chromatog. distinguishable isoforms MT1 and MT2 were not identical, and neither could be interpreted in terms of a unique static model with the 7 Cd2+ ions forming 2 independent clusters of 4 and 3 Cd2+ ions. Large differential shifts of 113Cd resonances were obsd. with changes in temp. over the range 277-320 K and ionic strength (0.02-0.5M). At low temp. a slow structural change (half-life of several minutes) was detected. The structure was more rigid at high ionic strength. The frequency dependence and 2-dimensional J-resolved spectra revealed that 113Cd resonances were composed of several overlapping peaks, complicating the interpretation of fine structure in 1-dimensional spectra. A new flexible model of the Cd cluster in metallothionein is proposed. This model incorporates dynamic thiolate exchange reactions and involves several configurational substrates of the protein. The possible relation of such flexibility to the function of metallothionein is discussed.
- 31Sutherland, D. E. K.; Stillman, M. J. The “magic numbers” of metallothionein. Metallomics 2011, 3, 444– 463, DOI: 10.1039/c0mt00102cGoogle Scholar31The "magic numbers" of metallothioneinSutherland, Duncan E. K.; Stillman, Martin J.Metallomics (2011), 3 (5), 444-463CODEN: METAJS; ISSN:1756-591X. (Royal Society of Chemistry)A review. Metallothioneins (MT) are a family of small cysteine rich proteins, which since their discovery in 1957, have been implicated in a range of roles including toxic metal detoxification, protection against oxidative stress, and as a metallochaperone involved in the homeostasis of both zinc and copper. The most well studied member of the family is the mammalian metallothionein, which consists of two domains: a β-domain with 9 cysteine residues, which sequesters 3 Cd2+ or Zn2+ or 6 Cu+ ions, and an α-domain with 11 cysteine residues and, which sequesters 4 Cd2+ or Zn2+ or 6 Cu+ ions. Despite over half a century of research, the exact functions of MT are still unknown. Much of current research aims to elucidate the mechanism of metal binding, as well as to isolate intermediates in metal exchange reactions; reactions necessary to maintain homeostatic equil. These studies further our understanding of the role(s) of this remarkable and ubiquitous protein. Recently, supermetallated forms of the protein, where supermetallation describes metalation in excess of traditional levels, have been reported. These species may potentially be the metal exchange intermediates necessary to maintain homeostatic equil. This review focuses on recent advances in the understanding of the mechanistic properties of metal binding, the implications for the metal induced protein folding reactions proposed for metallothionein metalation, the value of "magic nos.", which we informally define as the commonly detd. metal-to-protein stoichiometric ratios and the significance of the new supermetallated states of the protein and the possible interpretation of the structural properties of this new metalation status. Together we provide a commentary on current exptl. and theor. advances and frame our consideration in terms of the possible functions of MT.
- 32Palumaa, P.; Mackay, E. A.; Vašák, M. Nonoxidative cadmium-dependent dimerization of Cd7-metallothionein from rabbit liver. Biochemistry 1992, 31, 2181– 2186, DOI: 10.1021/bi00122a040Google Scholar32Nonoxidative cadmium-dependent dimerization of cadmium-metallothionein from rabbit liverPalumaa, Peep; Mackay, Elaine A.; Vasak, MilanBiochemistry (1992), 31 (7), 2181-6CODEN: BICHAW; ISSN:0006-2960.The effect of free Cd(II) ions on monomeric Cd7-metallothionein-2 (MT) from rabbit liver has been studied. Slow, concn.-dependent dimerization of this protein was obsd. by gel filtration chromatog. studies. The dimeric MT form, isolated by gel filtration, contains approx. two addnl. and more weakly bound Cd(II) ions per monomer. The incubation of MT dimers with complexing agents EDTA and 2-mercaptoethanol leads to the dissocn. of dimers to monomers. The results of CD and electronic absorption studies indicate that the slow dimerization process is preceded by an initial rapid Cd-induced rearrangement of the monomeric Cd7-MT structure. The 113Cd NMR spectrum of the MT dimer revealed only four 113Cd resonances at chem. shift positions similar to those obsd. for the Cd4 cluster of the well-characterized monomeric 113Cd7-MT. This result suggests that on dimer formation major structural changes occur in the original three-metal cluster domain of Cd7-MT.
- 33Vašák, M.; Kägi, J. H.; Hill, H. A. Zinc(II), cadmium(II), and mercury(II) thiolate transitions in metallothionein. Biochemistry 1981, 20, 2852– 2856, DOI: 10.1021/bi00513a022Google Scholar33Zinc(II), cadmium(II), and mercury(II) thiolate transitions in metallothioneinVasak, Milan; Kaegi, Jeremias H. R.; Hill, H. Allen O.Biochemistry (1981), 20 (10), 2852-6CODEN: BICHAW; ISSN:0006-2960.The metal-specific absorption envelopes of Zn-, Cd-, and Hg-metallothioneins and of complexes of these metal ions with 2-mercaptoethanol were analyzed in terms of Joergensen's electronegativity theory for charge-transfer excitations by using the spectra of Zn(II), Cd(II), and Hg(II) tetrahalides as refs. By Gaussian anal. the difference absorption spectra of the various forms of metallothionein vs. thionein and of the corresponding 2-mercaptoethanol complexes vs. 2-mercaptoethanol were resolved into 3 components. For each metal deriv. the location of the lowest energy band is in good agreement with the position of the first ligand-metal charge-transfer (LMCT) transition (type t2 → a1) predicted from the optical electronegativity difference of the thiolate ligands and of the central metal ion by assuming tetrahedral coordination. There is also a correspondence between the effects of the metal ion on the position of the first LMCT band and the binding energy of the 2p electrons of the S ligands as found by x-ray photoelectron spectroscopic measurements. Due to the lack of exact structural information, the assignment of the 2 other resolved metal-dependent bands remains conjectural, but it is likely that they include a second LMCT transition (type t2 → a1) analogous to that occurring in tetrahalide complexes of group-2B metal ions. The simplicity of the resolved thiolate spectra and their correspondence to those of tetrahedral models support the view that the various metal-binding sites of metallothionein are chem. similar and that the coordination environment of the metal ion has a symmetry related to that of a tetrahedron.
- 34Krebs, B.; Henkel, G. Transition-metal thiolates: From molecular fragments of sulfidic solids to models for active centers of biomolecules. Angew. Chem., Int. Ed. Engl. 1991, 30, 769– 788, DOI: 10.1002/anie.199107691Google ScholarThere is no corresponding record for this reference.
- 35Otvos, J. D.; Armitage, I. M. Structure of the metal clusters in rabbit liver metallothionein. Proc. Natl. Acad. Sci. U. S. A. 1980, 77, 7094– 7098, DOI: 10.1073/pnas.77.12.7094Google Scholar35Structure of the metal clusters in rabbit liver metallothioneinOtvos, James D.; Armitage, Ian M.Proceedings of the National Academy of Sciences of the United States of America (1980), 77 (12), 7094-8CODEN: PNASA6; ISSN:0027-8424.Cd-113 NMR was used to det. the structures of the multiple Cd-binding sites in the 2 major isoproteins of rabbit liver metallothionein. Isotopically labeled metallothionein was sepd. from the livers of rabbits that had been subjected to repeated injections of 113CdCl2. The native protein isolated from these livers contains an appreciable amt. of Zn in addn. to Cd: 2-3 mol/mol protein out of a total metal content of 7 mol/mol protein. The 113Cd NMR spectrum of Cd, Zn-contg. metallothionein is quite complex, reflecting the fact that the native protein is a heterogeneous mixt. of species contg. different relative amts. of Zn and Cd. Replacement of the native Zn with 113Cd in vitro gave a protein whose 113Cd NMR spectrum was much simpler, contg. 8 distinct multiplets with chem. shifts ranging from 611 to 670 ppm. The multiplet structures were due to 113Cd-113Cd scalar coupling arising from 2-bond interactions between 113Cd ions linked to one another by bridging cysteine thiolate ligands. The sizes and structures of the metal clusters in the protein were detd. by the application of selective homonuclear 113Cd decoupling techniques. Rabbit liver metallothionein contains 2 sep. metal clusters, one contg. 4 Cd2+ ions and the other contg. 3. These 2 clusters, whose structures are the same in both isoproteins, were designated cluster A and cluster B, resp. Structures for the clusters are proposed that account for the 113Cd spin-coupling data and for the participation of all 20 of the cysteine residues in metal ligation, 11 in cluster A and 9 in cluster B. The appearance in the spectrum of 8 multiplets rather than the 7 that would be expected on the basis of the no. of metal-binding sites in the protein is an indication of some residual heterogeneity in the 113Cd-labeled metallothionein sample. The origin of this heterogeneity is suggested to be the presence of a protein species that lacks metal ions at its cluster B binding sites.
- 36Kägi, J. H. R. Overview of metallothionein. Methods Enzymol. 1991, 205, 613– 626, DOI: 10.1016/0076-6879(91)05145-LGoogle Scholar36Overview of metallothioneinKagi J HMethods in enzymology (1991), 205 (), 613-26 ISSN:0076-6879.There is no expanded citation for this reference.
- 37Winge, D. R.; Miklossy, K. A. Domain nature of metallothionein. J. Biol. Chem. 1982, 257, 3471– 3476, DOI: 10.1016/S0021-9258(18)34802-6Google Scholar37Domain nature of metallothioneinWinge, Dennis R.; Miklossy, Kathy AnneJournal of Biological Chemistry (1982), 257 (7), 3471-6CODEN: JBCHA3; ISSN:0021-9258.Metallothionein purified from the livers of rats injected with CdCl2 was cleaved by proteolysis into a 32-residue polypeptide that contained 4 bound Cd ions. Appearance of this fragment designated α requires prior treatment of metallothionein with EDTA to remove Zn and destabilize the 3-metal cysteine cluster in the other domain. The half-mol. domain was not efficiently produced by proteolysis of native metallothionein. The Cd4-α fragment is asym. in shape, as is the parent mol. N-terminal sequence anal. revealed that the α fragment starts at lysine-30. Since the same amino acids are released from the C-terminus of intact thionein and the α fragment by carboxypeptidase Y, the α domain generated by digestion with subtilisin therefore comprises residues 30 through 61. The amino acid compn. of the α polypeptide is consistent with the structure of the 4-metal cysteine cluster proposed by J. D. Otvos and I. M. Armitage (1980). Metallothionein appears to consist of a 3-metal cysteine domain in the N-terminal half of the thionein mol. and the 4-metal cysteine domain in the C-terminal half.
- 38Boulanger, Y.; Armitage, I. M.; Miklossy, K. A.; Winge, D. R. 113Cd NMR study of a metallothionein fragment. Evidence for a two-domain structure. J. Biol. Chem. 1982, 257, 13717– 13719, DOI: 10.1016/S0021-9258(18)33506-3Google Scholar38Cadmium-113 NMR study of a metallothionein fragment. Evidence for a two-domain structureBoulanger, Yvan; Armitage, Ian M.; Miklossy, Kathy Anne; Winge, Dennis R.Journal of Biological Chemistry (1982), 257 (22), 13717-19CODEN: JBCHA3; ISSN:0021-9258.A 32-residue polypeptide fragment, designated αI, of rat liver metallothionein obtained by subtilisin digestion was studied by 113Cd NMR. The amino acid compn. of the fragment corresponded to residues 30-61 of the metallothionein primary structure, and it contained 3.4 g atoms of Cd2+/mol of αI-fragment. Four 113Cd resonances were obsd., 3 of which had identical chem. shifts to those assigned to the 4-metal cluster in human liver metallothionein-2 under the same pH and buffer conditions. The 5-ppm chem. shift difference between the remaining resonance assigned to the 4-metal cluster in the intact protein can be explained to result from the removal of the N-terminal polypeptide fragment contg. the 3-metal cluster. These results provide unambiguous evidence for the 2-domain structure of metallothionein, contg. a sep. 3- and a 4-metal cluster.
- 39Romero-Isart, N.; Vašák, M. Advances in the structure and chemistry of metallothioneins. J. Inorg. Biochem. 2002, 88, 388– 396, DOI: 10.1016/S0162-0134(01)00347-6Google Scholar39Advances in the structure and chemistry of metallothioneinsRomero-Isart, Nuria; Vasak, MilanJournal of Inorganic Biochemistry (2002), 88 (3-4), 388-396CODEN: JIBIDJ; ISSN:0162-0134. (Elsevier Science Inc.)A review. A low mol. wt. (6-7 kDa) class of metalloproteins, designated as metallothioneins (MTs), exhibit repeated sequence motifs of either CxC or CxxC through which mono or divalent d10 metal ions are bound in polymetallic-thiolate clusters. The preservation of metal-thiolate clusters in an increasing no. of three-dimensional structures of these proteins signifies the importance of this structural motif. This review focuses on the recent developments regarding the versatile and striking chem. reactivity of MTs as well as on the existence of conformational/configurational dynamics within their structure. Both properties and their interplay are likely to be essential for the still elusive biol. function of these proteins.
- 40Frey, M. H.; Wagner, G.; Vašák, M.; Soerensen, O. W.; Neuhaus, D.; Wörgötter, E.; Kägi, J. H. R.; Ernst, R. R.; Wüthrich, K. Polypeptide-metal cluster connectivities in metallothionein 2 by novel proton-cadmium-113 heteronuclear two-dimensional NMR experiments. J. Am. Chem. Soc. 1985, 107, 6847– 6851, DOI: 10.1021/ja00310a017Google Scholar40Polypeptide-metal cluster connectivities in metallothionein 2 by novel proton-cadmium-113 heteronuclear two-dimensional NMR experimentsFrey, Michael H.; Wagner, Gerhard; Vasak, Milan; Soerensen, Ole W.; Neuhaus, David; Woergoetter, Erich; Kaegi, Jeremias H. R.; Ernst, Richard R.; Wuethrich, KurtJournal of the American Chemical Society (1985), 107 (24), 6847-51CODEN: JACSAT; ISSN:0002-7863.Two-dimensional heteronuclear 1H-113Cd and homonuclear 113Cd-113Cd correlated NMR spectra were recorded for 113Cd-metallothionein 2 from rabbit liver. 1H detection was used for the heteronuclear expts. For 12 of the 20 cysteinyl residues, connectivities to one 113Cd could be identified; for the other 8 cysteinyl residues, connectivities to 2 113Cd nuclei were detected (bridging cysteines). In combination with the independently obtained sequence-specific NMR assignments of the cysteine 1H spin systems, the topol. and the locations of the metal-S clusters relative to the polypeptide structure could be established.
- 41Zhang, X.; Tamaru, H.; Khan, S. I.; Horton, J. R.; Keefe, L. J.; Selker, E. U.; Cheng, X. Structure of the Neurospora SET domain protein DIM-5, a histone H3 lysine methyltransferase. Cell 2002, 111, 117– 127, DOI: 10.1016/S0092-8674(02)00999-6Google Scholar41Structure of the Neurospora SET domain protein DIM-5, a histone H3 lysine methyltransferaseZhang, Xing; Tamaru, Hisashi; Khan, Seema I.; Horton, John R.; Keefe, Lisa J.; Selker, Eric U.; Cheng, XiaodongCell (Cambridge, MA, United States) (2002), 111 (1), 117-127CODEN: CELLB5; ISSN:0092-8674. (Cell Press)AdoMet-dependent methylation of histones is part of the "histone code" that can profoundly influence gene expression. We describe the crystal structure of Neurospora DIM-5, a histone H3 lysine 9 methyltransferase (HKMT), detd. at 1.98 Å resoln., as well as results of biochem. characterization and site-directed mutagenesis of key residues. This SET domain protein bears no structural similarity to previously characterized AdoMet-dependent methyltransferases but includes notable features such as a triangular Zn3Cys9 zinc cluster in the pre-SET domain and a AdoMet binding site in the SET domain essential for Me transfer. The structure suggests a mechanism for the methylation reaction and provides the structural basis for functional characterization of the HKMT family and the SET domain.
- 42Zheng, S.; Wang, J.; Feng, Y.; Wang, J.; Ye, K. Solution structure of MSL2 CXC domain reveals an unusual Zn3Cys9 cluster and similarity to Pre-SET domains of histone lysine methyltransferases. PLoS One 2012, 7, e45437 DOI: 10.1371/journal.pone.0045437Google Scholar42Solution structure of MSL2 CXC domain reveals an unusual Zn3Cys9 cluster and similarity to pre-SET domains of histone lysine methyltransferasesZheng, Sanduo; Wang, Jia; Feng, Yingang; Wang, Jinfeng; Ye, KeqiongPLoS One (2012), 7 (9), e45437CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)The dosage compensation complex (DCC) binds to single X chromosomes in Drosophila males and increases the transcription level of X-linked genes by approx. twofold. Male-specific lethal 2 (MSL2) together with MSL1 mediates the initial recruitment of the DCC to high-affinity sites in the X chromosome. MSL2 contains a DNA-binding cysteine-rich CXC domain that is important for X targeting. In this study, we detd. the soln. structure of MSL2 CXC domain by NMR spectroscopy. We identified three zinc ions in the CXC domain and detd. the metal-to-cysteine connectivities from 1H-113Cd correlation expts. The structure reveals an unusual zinc-cysteine cluster composed of three zinc ions coordinated by six terminal and three bridging cysteines. The CXC domain exhibits unexpected structural homol. to pre-SET motifs of histone lysine methyltransferases, expanding the distribution and structural diversity of the CXC domain superfamily. Our findings provide novel structural insight into the evolution and function of CXC domains.
- 43An, S.; Yeo, K. J.; Jeon, Y. H.; Song, J. J. Crystal structure of the human histone methyltransferase ASH1L catalytic domain and its implications for the regulatory mechanism. J. Biol. Chem. 2011, 286, 8369– 8374, DOI: 10.1074/jbc.M110.203380Google Scholar43Crystal Structure of the Human Histone Methyltransferase ASH1L Catalytic Domain and its Implications for the Regulatory MechanismAn, So-Jin; Yeo, Kwon-Joo; Jeon, Young-Ho; Song, Ji-JoonJournal of Biological Chemistry (2011), 286 (10), 8369-8374CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)Absent, small, or homeotic disk1 (Ash1) is a trithorax group histone methyltransferase that is involved in gene activation. Although there are many known histone methyltransferases, their regulatory mechanisms are poorly understood. Here, we present the crystal structure of the human ASH1L catalytic domain, showing its substrate binding pocket blocked by a loop from the post-SET domain. In this configuration, the loop limits substrate access to the active site. Mutagenesis of the loop stimulates ASH1L histone methyltransferase activity, suggesting that ASH1L activity may be regulated through the loop from the post-SET domain. In addn., we show that human ASH1L specifically methylates histone H3 Lys-36. Our data implicate that there may be a regulatory mechanism of ASH1L histone methyltransferases.
- 44Arseniev, A.; Schultze, P.; Wörgötter, E.; Braun, W.; Wagner, G.; Vašák, M.; Kägi, J. H.; Wüthrich, K. Three-dimensional structure of rabbit liver [Cd7]metallothionein-2a in aqueous solution determined by nuclear magnetic resonance. J. Mol. Biol. 1988, 201, 637– 657, DOI: 10.1016/0022-2836(88)90644-4Google Scholar44Three-dimensional structure of rabbit liver [Cd7]metallothionein-2a in aqueous solution determined by nuclear magnetic resonanceArseniev, Alexandre; Schultze, Peter; Woergoetter, Erich; Braun, Werner; Wagner, Gerhard; Vasak, Milan; Kaegi, Jeremias H. R.; Wuthrich, KurtJournal of Molecular Biology (1988), 201 (3), 637-57CODEN: JMOBAK; ISSN:0022-2836.In previous work the metal-polypeptide coordinative bonds in the major protein species of a reconstituted [113Cd7]metallothionein-2 prepn. from rabbit liver in aq. soln. were detd., the secondary polypeptide structure was found to contain several half-turns and 310-helical segments, and a preliminary characterization of the overall polypeptide backbone fold in the β-domain contg. the 3-metal cluster, and the α-domain contg. the 4-metal cluster, was obtained. Using a new, more extensive set of NMR data these earlier structures were improved by new structure calcns. The new exptl. data consist of distance constraints from measurements of nuclear Overhauser effects, and dihedral angle constraints derived from both coupling consts. and nuclear Overhauser effects. The structure calcns. were performed with the program DISMAN. Since no information on the orientation of the 2 domains relative to each other could be obtained, the structure calcns. were performed sep. for the α-domain and the β-domain. The av. of the pairwise root-mean-square distances among the 20 structures with the least residual violations of input constraints was 2.9 Å for the β-domain and 1.4 Å for the α-domain. The overall chirality of the polypeptide fold is right-handed for the β-domain and left-handed for the α-domain. For each of the 7 metal ions, the local chirality of the coordination of the 4 cysteinyl Sγ atoms is clearly defined. The improved structures of both domains show the previously noted differences relative to the recently published crystal structure of metallothionein-2a from rat liver.
- 45Schultze, P.; Wörgötter, E.; Braun, W.; Wagner, G.; Vašák, M.; Kägi, J. H. R.; Wüthrich, K. Conformation of [Cd7]-metallothionein-2 from rat liver in aqueous solution determined by nuclear magnetic resonance spectroscopy. J. Mol. Biol. 1988, 203, 251– 268, DOI: 10.1016/0022-2836(88)90106-4Google Scholar45Conformation of [Cd7]-metallothionein-2 from rat liver in aqueous solution determined by nuclear magnetic resonance spectroscopySchultze, Peter; Woergoetter, Erich; Braun, Werner; Wagner, Gerhard; Vasak, Milan; Kaegi, Jeremias H. R.; Wuethrich, KurtJournal of Molecular Biology (1988), 203 (1), 251-68CODEN: JMOBAK; ISSN:0022-2836.The 3-dimensional structure of [Cd7]-metallothionein-2 from rat liver was detd. in aq. soln., using NMR spectrometry and distance geometry calcns. The exptl. data provided proton-proton distance constraints from measurements of NOE, constraints on the geometry of the metal-cysteine clusters detd. by heteronuclear correlation spectroscopy, and dihedral angle constraints derived from both coupling consts. and NOE. The structure calcns. were performed with the program DISMAN. As in previous studies with rabbit liver metallothionein-2a, the structure calcns. were performed sep. for the α and β-domains contg. the 4 and 3-metal clusters, resp., since no interdomain constraints were found. For both domains, the global polypeptide fold, the location of polypeptide secondary structure elements, the architecture of the metal-S cluster, and the local chirality of the metal coordination are very similar to the soln. structure of rabbit metallothionein-2a, but show considerable difference relative to the crystal structure of rat metallothionein-2.
- 46Messerle, B. A.; Schäffer, A.; Vašák, M.; Kägi, J. H.; Wüthrich, K. Three-dimensional structure of human [113Cd7]metallothionein-2 in solution determined by nuclear magnetic resonance spectroscopy. J. Mol. Biol. 1990, 214, 765– 779, DOI: 10.1016/0022-2836(90)90291-SGoogle Scholar46Three-dimensional structure of human [113Cd7]metallothionein-2 in solution determined by nuclear magnetic resonance spectroscopyMesserle, Barbara A.; Schaeffer, Andreas; Vasak, Milan; Kaegi, Jeremias H. R.; Wuethrich, KurtJournal of Molecular Biology (1990), 214 (3), 765-79CODEN: JMOBAK; ISSN:0022-2836.The three-dimensional structure of human [113Cd7]metallothionein-2 was detd. by NMR spectroscopy in soln. Sequence-specific 1H resonance assignments were obtained using the sequential assignment method. The input for the structure calcns. consisted of the metal-cysteine coordinative bonds identified with heteronuclear correlation spectroscopy, 1H-1H distance constraints from nuclear Overhauser enhancement spectroscopy, and spin-spin coupling consts. 3JHNα and 3Jαβ. The mol. consists of 2 domains, the β-domain including amino acid residues 1-30 and 3 metal ions, and the α-domain including residues 31-61 and 4 metal ions. The NMR data present no evidence for a preferred relative orientation of the 2 domains. The polypeptide-to-metal coordinative bonds in human metallothionein-2 are identical to those in the previously detd. soln. structures of rat metallothionein-2 and rabbit metallothionein-2α, and the polypeptide conformations in the three proteins are also closely similar.
- 47Robbins, A. H.; McRhee, D. E.; Williamson, M.; Collett, S. A.; Xuong, N. H.; Furey, W. F.; Wang, B. C.; Stout, C. D. Refined Crystal Structure of Cd,Zn Metallothionein at 2.0 Å Resolution. J. Mol. Biol. 1991, 221, 1269– 1293, DOI: 10.1016/0022-2836(91)80126-FGoogle Scholar47Refined crystal structure of cadmium-zinc metallothionein at Å resolutionRobbins, A. H.; McRee, D. E.; Williamson, M.; Collett, S. A.; Xuong, N. H.; Furey, W. F.; Wang, B. C.; Stout, C. D.Journal of Molecular Biology (1991), 221 (4), 1269-93CODEN: JMOBAK; ISSN:0022-2836.The crystal structure of Cd5,Zn2-metallothionein from rat liver has been refined at 2.0 Å resoln. of a R-value of 0.176 for all obsd. data. The five Cd positions in the asym. unit of the crystal create a pseudo-centrosym. constellation about a crystallog. 2-fold axis. Consequently, the distribution of anomalous differences in almost ideally centrosym. Therefore, the previously reported metal positions and the protein model derived therefrom are incorrect. Direct methods were applied to the protein amplitudes to locate the Cd positions. The new positions were used to calc. a new electron d. map based on the Cd anomalous scattering and partial structure to model the metal clusters and the protein. Phases calcd. from this model predict the positions of three sites in a (NH4)2WS4 deriv. Single isomorphous replacement phases calcd. with these tungsten sites confirm the positions of the Cd sites from the new direct methods calcns. The refined metallothionein structure has a root-mean-square deviation of 0.016 Å from ideality of bonds and normal stereochem. of Φ, φ, and χ torsion angles. The metallothionein crystal structure is in agreement with the structures for the α and β domains in soln. derived by NMR methods. The overall chain folds and all metal to cysteine bonds are the same in the two structure detns. The handedness of a short helix in the α-domain (residues 41 to 45) is the same in both structures. The crystal structure provides information concerning the metal cluster geometry and cysteine solvent accessibility and side-chain stereochem. Short cysteine peptide sequences repeated in the structure adopt restricted conformations which favor the formation of amide to sulfur hydrogen bonds. The crystal packing reveals intimate assocn. of mols. about the diagonal 2-fold axes and trapped ions of crystn. (modeled as phosphate and sodium). Variation in the chem. and structural environments of the metal sites is in accord with data for metal exchange reactions in metallothioneins.
- 48Braun, W.; Vašák, M.; Robbins, A. H.; Stout, C. D.; Wagner, G.; Kägi, J. H. R.; Wüthrich, K. Comparison of the NMR solution and the x-ray crystal structure of rat metallothionein-2. Proc. Natl. Acad. Sci. U. S. A. 1992, 89, 10124– 10128, DOI: 10.1073/pnas.89.21.10124Google Scholar48Comparison of the NMR solution structure and the x-ray crystal structure of rat metallothionein-2Braun, W.; Vasak, M.; Robbins, A. H.; Stout, C. D.; Wagner, G.; Kaegi, J. H. R.; Wuethrich, K.Proceedings of the National Academy of Sciences of the United States of America (1992), 89 (21), 10124-8CODEN: PNASA6; ISSN:0027-8424.Metallothioneins are small cysteine-rich proteins capable of binding heavy metal ions such as Zn2+ and Cd2+. They are ubiquitous tissue components in higher organisms, which tentatively have been attributed both unspecific protective functions against toxic metal ions and highly specific roles in fundamental zinc-regulated cellular processes. In this paper a detailed comparison of the NMR soln. structure (Schultz, P., et al., 1988) and a recent x-ray crystal structure (Robbins, A. H., et al., 1991) of rat metallothionein-2 shows that the metal-lothionein structures in crystals and in soln. have identical mol. architectures. The structures obtained with both techniques now present a reliable basis for the discussions on structure-function correlations in this class of metalloproteins.
- 49Öz, G.; Zangger, K.; Armitage, I. M. Three-dimensional structure and dynamics of a brain specific growth inhibitory factor: Metallothionein 3. Biochemistry 2001, 40, 11433– 11441, DOI: 10.1021/bi010827lGoogle Scholar49Three-dimensional structure and dynamics of a brain specific growth inhibitory factor: metallothionein-3Oz G; Zangger K; Armitage I MBiochemistry (2001), 40 (38), 11433-41 ISSN:0006-2960.The brain specific member of the metallothionein (MT) family of proteins, metallothionein-3, inhibits the growth and survival of neurons, in contrast to the ubiquitous mammalian MT isoforms, MT-1 and MT-2, that are found in most tissues and are thought to function in metal ion homeostasis and detoxification. Solution NMR was utilized to determine the structural and dynamic differences of MT-3 from MT-1 and 2. The high-resolution solution structure of the C-terminal alpha-domain of recombinant mouse MT-3 revealed a tertiary fold very similar to MT-1 and 2, except for a loop that accommodates an acidic insertion relative to these isoforms. This loop was distinguished from the rest of the domain by dynamics of the backbone on the nano- to picosecond time-scale shown by (15)N relaxation studies and was identified as a possible interaction site with other proteins. The N-terminal beta-domain contains the region responsible for the growth inhibitory activity, a CPCP tetrapeptide close to the N-terminus. Because of exchange broadening of a large number of the NMR signals from this domain, homology modeling was utilized to calculate models for the beta-domain and suggested that while the backbone fold of the MT-3 beta-domain is identical to MT-1 and 2, the second proline responsible for the activity, Pro9, may show structural heterogeneity. (15)N relaxation analyses implied fast internal motions for the beta-domain. On the basis of these observations, we conclude that the growth inhibitory activity exhibited by MT-3 is a result of a combination of local structural differences and global dynamics in the beta-domain.
- 50Wang, H.; Zhang, Q.; Cai, B.; Li, H. Y.; Sze, K. H.; Huang, Z. X.; Wu, H. M.; Sun, H. Z. Solution structure and dynamics of human metallothionein-3 (MT-3). FEBS Lett. 2006, 580, 795– 800, DOI: 10.1016/j.febslet.2005.12.099Google Scholar50Solution structure and dynamics of human metallothionein-3 (MT-3)Wang, Hui; Zhang, Qi; Cai, Bin; Li, Hongyan; Sze, Kong-Hung; Huang, Zhong-Xian; Wu, Hou-Ming; Sun, HongzheFEBS Letters (2006), 580 (3), 795-800CODEN: FEBLAL; ISSN:0014-5793. (Elsevier B.V.)Alzheimer's disease is characterized by progressive loss of neurons accompanied by the formation of intraneural neurofibrillary tangles and extracellular amyloid plaques. Human neuronal growth inhibitory factor, classified as metallothionein-3 (MT-3), was found to be related to the neurotrophic activity promoting cortical neuron survival and dendrite outgrowth in the cell culture studies. We have detd. the soln. structure of the α-domain of human MT-3 (residues 32-68) by multinuclear and multidimensional NMR spectroscopy in combination with the mol. dynamic simulated annealing approach. The human MT-3 shows two metal-thiolate clusters, one in the N-terminus (β-domain) and one in the C-terminus (α-domain). The overall fold of the α-domain is similar to that of mouse MT-3. However, human MT-3 has a longer loop in the acidic hexapeptide insertion than that of mouse MT-3. Surprisingly, the backbone dynamics of the protein revealed that the β-domain exhibits similar internal motion to the α-domain, although the N-terminal residues are more flexible. Our results may provide useful information for understanding the structure-function relationship of human MT-3.
- 51Dolderer, B.; Echner, H.; Beck, A.; Hartmann, H.-J.; Weser, U.; Luchinat, C.; Del Bianco, C. Coordination of three and four Cu(I) to the α- and β-domain of vertebrate Zn-metallothionein-1, respectively, induces significant structural changes. FEBS J. 2007, 274, 2349– 2362, DOI: 10.1111/j.1742-4658.2007.05770.xGoogle Scholar51Coordination of three and four Cu(I) to the α- and β-domain of vertebrate Zn-metallothionein-1, respectively, induces significant structural changesDolderer, Benedikt; Echner, Hartmut; Beck, Alexander; Hartmann, Hans-Juergen; Weser, Ulrich; Luchinat, Claudio; Del Bianco, CristinaFEBS Journal (2007), 274 (9), 2349-2362CODEN: FJEOAC; ISSN:1742-464X. (Blackwell Publishing Ltd.)Vertebrate metallothioneins are found to contain Zn(II) and variable amts. of Cu(I), in vivo, and are believed to be important for d10-metal control. To date, structural information is available for the Zn(II) and Cd(II) forms, but not for the Cu(I) or mixed metal forms. Cu(I) binding to metallothionein-1 has been investigated by CD, luminescence and 1H NMR using two synthetic fragments representing the α- and the β-domain. The 1H NMR data and thus the structures of Zn4α metallothionein (MT)-1 and Zn3βMT-1 were essentially the same as those already published for the corresponding domains of native Cd7MT-1. Cu(I) titrn. of the Zn(II)-reconstituted domains provided clear evidence of stable polypeptide folds of the three Cu(I)-contg. α- and the four Cu(I)-contg. β-domains. The soln. structures of these two species are grossly different from the structures of the starting Zn(II) complexes. Further addn. of Cu(I) to the two single domains led to the loss of defined domain structures. Upon mixing of the sep. prepd. aq. three and four Cu(I) loaded α- and β-domains, no interaction was seen between the two species. There was neither any indication for a net transfer of Cu(I) between the two domains nor for the formation of one large single Cu(I) cluster involving both domains.
- 52Rupp, H.; Weser, U. Conversion of metallothionein into Cu-thionein, the possible low molecular weight form of neonatal hepatic mitochondrocuprein. FEBS Lett. 1974, 44, 293– 297, DOI: 10.1016/0014-5793(74)81161-0Google Scholar52Conversion of metallothionein into copper-thionein, the possible low molecular weight form of neonatal hepatic mitochondrocupreinRupp, Heinz; Weser, UlrichFEBS Letters (1974), 44 (3), 293-7CODEN: FEBLAL; ISSN:0014-5793.When chicken metallothionein was titrated using Cu(CH3CN)4ClO4, absorbance did not occur in the visible region. A plot of the concn. of added Cu vs. absorbance at const. wavelengths revealed that at 250 μ the linear response decreased and at 280, 300, and 320 μ it increased. All curves showed a sharp bending at the same point corresponding to 15.2 g atoms of Cu/12,000 g of protein. Increased Cu concns. resulted in 2 new Cotton effects with pos. and neg. extremes at 359 and 302 μ, resp. The neg. Cotton effect at 238 μ leveled off. The 2 new Cotton effects were assigned to electronic transitions of Cu chelates where the free functional groups of lysine, histidine, and cysteine participated as ligands. Displacement of Zn2+ and Cd2+ by H+, using partially loaded Cu-thionein, gave a highly polymeric form which was similar to neonatal hepatic mitochondrocuprein. The binding energy of the S core electrons of this polymer, 162.5 eV, was between that of the resp. S-binding energies of Cu-thionein and the fully oxidized cystine-thionein. Polymeric Cu-thionen is probably composed of either Cu-thionein or cysteine-thionein residues.
- 53Presta, A.; Green, A. R.; Zelazowski, A.; Stillman, M. J. Formation of a continuum of copper(I) thiolate stoichiometric species. Eur. J. Biochem. 1995, 227, 226– 240, DOI: 10.1111/j.1432-1033.1995.tb20380.xGoogle Scholar53Copper binding to rabbit liver metallothionein. Formation of a continuum of copper(I)-thiolate stoichiometric speciesPresta, Anthony; Green, Anna Rae; Zelazowski, Andrzej; Stillman, Martin J.European Journal of Biochemistry (1995), 227 (1/2), 226-40CODEN: EJBCAI; ISSN:0014-2956. (Springer)CD and UV absorption spectral data have been used to probe the binding mechanism for formation and the structure of the copper(I)-thiolate binding clusters in rabbit liver metallothionein during addn. of Cu+ to aq. solns. of Zn7-metallothionein 2 and Cd5Zn2-metallothionein 2. Mammalian metallothionein binds metals in two binding sites, namely the α and β domains. Spectral data which probe the distribution of Cu(I) between the two binding domains clearly show that both the site of binding (α or β), and the structures of the specific metal-thiolate clusters formed, are dependent on temp. and on the nature of the starting protein (either Zn7-metallothionein or Cd5Zn2-metallothionein). CD spectra acquired during the addn. of Cu+ to Zn7-metallothionein show that Cu+ replace the bound Zn(II) in a domain-distributed manner with complete removal of the Zn(II) after addn. of 12 Cu+. Spectral and metal analyses prove that a series of Cu(I)-metallothionein species are formed by a non-cooperative metal-binding mechanism with a continuum of Cu(I):metallothionein stoichiometries. Observation of a series of spectral satn. points signal the formation of distinct optically active Cu(I)-thiolate structures for the Cu9Zn2-metallothionein, Cu12-metallothionein, and the Cu15-metallothionein species. These data very clearly show that for Cu(I) binding to Zn7-metallothionein, there are several key Cu(I):metallothionein stoichiometric ratios, and not just the single value of 12. The CD spectra up to the Cu12-metallothionein species are defined by bands located at 255(+) nm and 280(-) nm. Interpretation of the changes in the CD and UV absorption spectral data recorded between 3 °C and 52 °C as Cu+ is added to Zn-metallothionein show that copper-thiolate cluster formation is strongly temp. dependent. These changes in spectral properties are interpreted in terms of kinetic vs. thermodn. control of the metal-binding pathways as Cu+ binds to the protein. At low temps. (3°C and 10°C) the spectral data indicate a kinetically controlled mechanism whereby an activation barrier inhibits formation of ordered copper-thiolate structures until formation of Cu12-metallothionein. At higher temps. (>30°C) the activation barrier is overcome, allowing formation of new Cu(I)-thiolate clusters with unique spectral properties, esp. at the Cu9Zn2-metallothionein point. The CD spectra also show that a Cu15-metallothionein species with a well-defined, three-dimensional structure forms at all temps., characterized by a band near 335 nm, indicating the presence of digonal Cu(I). Complicated CD spectral changes are obsd. when Cu+ is added to Cd5Zn2-metallothionein. The spectral data are interpreted in terms of domain-distributed binding followed by rearrangement to form the domain-specific product. In the Cu6Cd4-metallothionein species, the Cu+ are ultimately bound specifically to the β domain of the protein. This complex is characterized by the CD spectrum of the Cd4S'Cys'11 in the α domain. The domain-specific product arises from the result of two interdependent driving forces, leading to formation of the Cu6S'Cys'9, β-domain cluster and the Cd4S'Cys'11 α-domain cluster. These findings imply physiol. roles for the individual domains of this protein. Further Cu+ addn. yields the mixed Cu12Cd4-metallothionein species which exhibits a unique CD spectrum with bands at 240, 268, 293 and 332 nm. Mol. modeling calcns. were used to create a structure for the Cu12-metallothionein 2 species, based on domain stoichiometries identified by the spectroscopic data of Cu6S'Cys'11 (α domain) and Cu6S'Cys'9 (β domain). In accord with the CD spectral data, this structure involves exclusive trigonal coordination of all 12 bound Cu+ to the 20 cysteinyl thiolates. All cysteinyl thiolates in the β domain adopt bridging geometry, while cysteinyl thiolates in the α domain adopt both bridging and terminal geometries.
- 54Melenbacher, A.; Korkola, N. C.; Stillman, M. J. The pathways and domain specificity of Cu(I) binding to human metallothionein 1A. Metallomics 2020, 12, 1951– 1964, DOI: 10.1039/D0MT00215AGoogle Scholar54The pathways and domain specificity of Cu(I) binding to human metallothionein 1AMelenbacher, Adyn; Korkola, Natalie C.; Stillman, Martin J.Metallomics (2020), 12 (12), 1951-1964CODEN: METAJS; ISSN:1756-591X. (Royal Society of Chemistry)Copper is an essential element, but as a result of numerous adverse reactions, it is also a cellular toxin. Nature protects itself from these toxic reactions by binding cuprous copper to chaperones and other metalloproteins. Metallothionein has been proposed as a storage location for Cu(I) and potentially as the donor of Cu(I) to copper-dependent enzymes. We report that the addn. of Cu(I) to apo recombinant human metallothionein 1a cooperatively forms a sequential series of Cu(I)-cysteinyl thiolate complexes that have specific Cu(I) : MT stoichiometries of 6 : 1, 10 : 1, and finally 13 : 1. The individual domain Cu : SCys stoichiometries were detd. as Cu6S9 (for 6 : 1), Cu6S9 + Cu4S6 (for 10 : 1), and Cu6S9 + Cu7S9 (for 13 : 1) based on the no. of modified free cysteines not involved in Cu(I) binding. The stoichiometries are assocd. with Cu-SCys cluster formation involving bridging thiols in the manner similar to the clusters formed with Cd(II) and Zn(II). The locations of these clustered species within the 20 cysteine full protein were detd. from the unique speciation profiles of Cu(I) binding to the β and α domain fragments of recombinant human metallothionein 1a with 9 and 11 cysteines, resp. Competition reactions using these domain fragments challenged Cu(I) metalation of the βα protein, allowing the sequence of cluster formation in the full protein to be detd. Relative binding consts. for each Cu(I) bound are reported. The emission spectra of the Cu4S6, Cu6S9, and Cu7S9 clusters have unique λmax and phosphorescent lifetime properties. These phosphorescent data provide unambiguous supporting evidence for the presence of solvent shielded clusters reported concurrently by ESI-MS. Simulated emission spectra based on the cluster specific emission profiles matched the exptl. spectra and are used to confirm that the relative concns. seen by ESI-MS are representative of the soln. Our results suggest that the availability of a series of sequential Cu(I)-thiolate clusters provides flexibility as a means of protecting the cell from toxicity while still allowing for homeostatic control of the total copper content in the cell. This mechanism provides a dynamic and reactive method of reducing the cellular free copper concns.
- 55Calderone, V.; Dolderer, B.; Hartmann, H.-J.; Echner, H.; Luchinat, C.; Del Bianco, C.; Mangani, S.; Weser, U. The crystal structure of yeast copper thionein: The solution of a long-lasting enigma. Proc. Natl. Acad. Sci. U. S. A. 2005, 102, 51– 56, DOI: 10.1073/pnas.0408254101Google Scholar55The crystal structure of yeast copper thionein: The solution of a long-lasting enigmaCalderone, Vito; Dolderer, Benedikt; Hartmann, Hans-Juergen; Echner, Hartmut; Luchinat, Claudio; Del Bianco, Cristina; Mangani, Stefano; Weser, UlrichProceedings of the National Academy of Sciences of the United States of America (2005), 102 (1), 51-56CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)We report here the crystal structure of yeast copper thionein (Cu-MT), detd. at 1.44-Å resoln. The Cu-MT structure shows the largest known oligonuclear Cu(I) thiolate cluster in biol., consisting of six trigonally and two digonally coordinated Cu(I) ions. This is at variance with the results from previous spectroscopic detns., which were performed on MT samples contg. seven rather than eight metal ions. The protein backbone has a random coil structure with the loops enfolding the copper cluster, which is located in a cleft where it is bound to 10 cysteine residues. The protein structure is somewhat different from that of Ag7-MT and similar, but not identical, to that of Cu7-MT. Besides the different structure of the metal cluster, the main differences lie in the cysteine topol. and in the conformation of some portions of the backbone. The present structure suggests that Cu-MT, in addn. to its role as a safe depository for copper ions in the cell, may play an active role in the delivery of copper to metal-free chaperones.
- 56Davis, J. J.; Hill, H. A. O.; Kurz, A.; Jacob, C.; Maret, W.; Vallee, B. L. A scanning tunnelling microscopy study of rabbit metallothionein. PhysChemComm 1998, 1, 12– 22, DOI: 10.1039/a806057fGoogle ScholarThere is no corresponding record for this reference.
- 57Maret, W.; Heffron, G.; Hill, H. A. O.; Djuricic, D.; Jiang, L.-J.; Vallee, B. L. The ATP/metallothionein interaction: NMR and STM. Biochemistry 2002, 41, 1689– 1694, DOI: 10.1021/bi0116083Google Scholar57The ATP/Metallothionein Interaction: NMR and STMMaret, Wolfgang; Heffron, Gregory; Hill, H. Allen O.; Djuricic, Dejana; Jiang, Li-Juan; Vallee, Bert L.Biochemistry (2002), 41 (5), 1689-1694CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)We have previously established that ATP binds to mammalian metallothionein-2 (MT). The interaction between ATP and MT and the assocd. conformational change of the protein affect the sulfhydryl reactivity and zinc transfer potential of MT [Jiang, L.-J., Maret, W., and Vallee, B. L. (1998) The ATP-metallothionein complex. Proc. Natl. Acad. Sci. U.S.A. 95, 9146-9149]. NMR spectroscopic investigations have now provided further evidence for the interaction. 35Cl NMR spectroscopy has further identified chloride as an addnl. biol. MT ligand, which can interfere with the interaction of ATP with MT. 1H NMR/TOCSY spectra demonstrate that ATP binding affects the N- and C-terminal amino acids of the MT mol. Scanning tunneling microscopy recorded images of single MT mols. in buffered solns. Moreover, this technique demonstrates that the otherwise nearly linear MT mol. bends by about 20° at its central hinge region between the domains in the presence of ATP. These results may bear on the development of mild obesity in MT null mice and the role of MT in the regulation of energy balance. The interaction suggests a mechanism for the cellular translocation, retention, and reactivity of the ATP·MT complex in the mitochondrial intermembrane space. Both MT and ATP are localized there, and MT and thionein alternately bind and release zinc, thereby affecting mitochondrial respiration.
- 58Otvos, J. D.; Liu, X.; Li, H.; Shen, G.; Basti, M. Dynamic aspects of metallothionein structure. In Metallothionein III; Suzuki, K. T., Imura, N., Kimura, M., Eds.; Birkhäuser: Basel, Switzerland, 1993; pp 57– 74.Google ScholarThere is no corresponding record for this reference.
- 59Maret, W.; Larsen, K. S.; Vallee, B. L. Coordination dynamics of biological zinc “clusters” in metallothioneins and in the DNA-binding domain of the transcription factor Gal4. Proc. Natl. Acad. Sci. U. S. A. 1997, 94, 2233– 2237, DOI: 10.1073/pnas.94.6.2233Google Scholar59Coordination dynamics of biological zinc "clusters" in metallothioneins and in the DNA-binding domain of the transcription factor Gal4Maret, Wolfgang; Larsen, Kjeld S.; Vallee, Bert L.Proceedings of the National Academy of Sciences of the United States of America (1997), 94 (6), 2233-2237CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)The almost universal appreciation for the importance of zinc in metab. has been offset by the considerable uncertainty regarding the proteins that store and distribute cellular zinc. We propose that some zinc proteins with so-called zinc cluster motifs have a central role in zinc distribution, since they exhibit the rather exquisite properties of binding zinc tightly while remaining remarkably reactive as zinc donors. We have used zinc isotope exchange both to probe the coordination dynamics of zinc clusters in metallothionein, the small protein that has the highest known zinc content, and to investigate the potential function of zinc clusters in cellular zinc distribution. When mixed and incubated, metallothionein isoproteins-1 and -2 rapidly exchange zinc, as demonstrated by fast chromatog. sepn. and radiometric anal. Exchange kinetics exhibit two distinct phases (kfast ≃ 5000 min-1·M-1; kslow ≃ 200 min-1·M-1, pH 8.6, 25°C) that are thought to reflect exchange between the three-zinc clusters and between the four-zinc clusters, resp. Moreover, we have obsd. and examd. zinc exchange between metallothionein-2 and the Gal4 protein (k ≃ 800 min-1·M-1, pH 8.0, 25°C), which is a prototype of transcription factors with a two-zinc cluster. This reaction constitutes the first exptl. example of intermol. zinc exchange between heterologous proteins. Such kinetic reactivity distinguishes zinc in biol. clusters from zinc in the coordination environment of zinc enzymes, where the metal does not exchange over several days with free zinc in soln. The mol. organization of these clusters allows zinc exchange to proceed through a ligand exchange mechanism, involving mol. contact between the reactants.
- 60Hamer, D. H. Metallothionein. Annu. Rev. Biochem. 1986, 55, 913– 951, DOI: 10.1146/annurev.bi.55.070186.004405Google Scholar60MetallothioneinHamer, Dean H.Annual Review of Biochemistry (1986), 55 (), 913-51CODEN: ARBOAW; ISSN:0066-4154.A review, with 217 refs., on metallothionein. Nomenclature, occurrence, and detection of the proteins are described, and their structure and metal-binding properties are reviewed. In addn., consideration is given to the genetics of metallothioneins and to the regulation of expression of metallothionein genes. Possible functions of metallothioneins are also discussed.
- 61Westin, G.; Schaffner, W. A zinc-responsive factor interacts with a metal-regulated enhancer element (MRE) of the mouse metallothionein-I gene. EMBO J. 1988, 7, 3763– 3770, DOI: 10.1002/j.1460-2075.1988.tb03260.xGoogle Scholar61A zinc-responsive factor interacts with a metal-regulated enhancer element (MRE) of the mouse metallothionein-I geneWestin, Gunnar; Schaffner, WalterEMBO Journal (1988), 7 (12), 3763-70CODEN: EMJODG; ISSN:0261-4189.Heavy metal ions are effective inducers of metallothionein gene transcription. The metal response is dependent on short DNA motifs, so-called MREs (metal responsive elements) that occur in multiple copies in the promoter region of these genes. An MRE of the mouse metallothionein-I gene (MREd) was analyzed, and it was demonstrated that this can function over long distances as a bona fide metal ion-inducible enhancer. The transcription factor Sp1 and a zinc-inducible factor, designated MTF-1, bind to the MREd enhancer in vitro. The combined use of MREd mutants in a transient assay in HeLa cells and a competition band shift assay show that the zinc-inducible formation of the MTF-1/DNA complex in vitro correlates with zinc-inducible transcription in vivo. A chem. methylation interference assay revealed remarkably similar but non-identical guanine interference patterns for the MTF-1 and Sp1 complexes, which may mean that MTF-1 is related to the Sp1 factor.
- 62Miles, A. T.; Hawksworth, G. M.; Beattie, J.; Rodilla, V. Induction, regulation, degradation, and biological significance of mammalian metallothioneins. Crit. Rev. Biochem. Mol. Biol. 2000, 35, 35– 70, DOI: 10.1080/10409230091169168Google Scholar62Induction, regulation, degradation, and biological significance of mammalian metallothioneinsMiles, A. T.; Hawksworth, G. M.; Beattie, J. H.; Rodilla, V.Critical Reviews in Biochemistry and Molecular Biology (2000), 35 (1), 35-70CODEN: CRBBEJ; ISSN:1040-9238. (CRC Press LLC)A review with 328 refs. Metallothioneins (MTs) are small cysteine-rich metal-binding proteins found in many species and, although there are differences between them, it is of note that they have a great deal of sequence and structural homol. Mammalian MTs are 61 or 62 amino acid polypeptides contg. 20 conserved cysteine residues that underpin the binding of metals. The existence of MT across species is indicative of its biol. demand, while the conservation of cysteines indicates that these are undoubtedly central to the function of this protein. Four MT isoforms have been found so far, MT-1, MT-2, MT-3, and MT-4, but these also have subtypes with 17 MT genes identified in man, of which 10 are known to be functional. Different cells express different MT isoforms with varying levels of expression perhaps as a result of the different function of each isoform. Even different metals induce and bind to MTs to different extents. Over 40 yr of research into MT have yielded much information on this protein, but have failed to assign to it a definitive biol. role. The fact that multiple MT isoforms exist, and the great variety of substances and agents that act as inducers, further complicates the search for the biol. role of MTs. This article reviews the current knowledge on the biochem., induction, regulation, and degrdn. of this protein in mammals, with a particular emphasis on human MTs. It also considers the possible biol. roles of this protein, which include participation in cell proliferation and apoptosis, homeostasis of essential metals, cellular free radical scavenging, and metal detoxification.
- 63Beyersmann, D.; Haase, H. Functions of zinc in signaling, proliferation and differentiatioin of mammalian cells. BioMetals 2001, 14, 331– 341, DOI: 10.1023/A:1012905406548Google Scholar63Functions of zinc in signaling, proliferation and differentiation of mammalian cellsBeyersmann, Detmar; Haase, HajoBioMetals (2001), 14 (3-4), 331-341CODEN: BOMEEH; ISSN:0966-0844. (Kluwer Academic Publishers)A review and discussion with 88 refs. Zn is essential for cell proliferation and differentiation, esp. for the regulation of DNA synthesis and mitosis. On the mol. level, it is a structural constituent of a great no. of proteins, including enzymes of cellular signaling pathways and transcription factors. Zn homeostasis in eukaryotic cells is controlled on the levels of uptake, intracellular sequestration in Zn-storing vesicles ("zincosomes"), nucleocytoplasmic distribution, and elimination. These processes involve the major Zn-binding protein, metallothionein, as a tool for the regulation of the cellular Zn level and the nuclear translocation of Zn in the course of the cell cycle and differentiation. In addn., there is also increasing evidence for a direct signaling function for Zn on all levels of signal transduction. Zn can modulate cellular signal recognition, 2nd messenger metab., protein kinase and protein phosphatase activities, and it may stimulate or inhibit activities of transcription factors, depending on the exptl. systems studied. Zn has been shown to modify specifically the metab. of cGMP, the activities of protein kinase C and MAP kinases, and the activity of transcription factor MTF-1 which controls the transcription of the genes for metallothionein and Zn transporter ZnT-1. As a conclusion of these observations, new hypotheses regarding regulatory functions of Zn2+ ions in cellular signaling pathways are proposed.
- 64Masters, B. A.; Kelly, E. J.; Quaife, C. J.; Brinster, R. L.; Palmiter, R. D. Targeted disruption of MTI and MTII genes increases sensitivity to cadmium. Proc. Natl. Acad. Sci. U. S. A. 1994, 91, 584– 588, DOI: 10.1073/pnas.91.2.584Google Scholar64Targeted disruption of metallothionein I and II genes increases sensitivity to cadmiumMasters, Brian A.; Kelly, Edward J.; Quaife, Carol J.; Brinster, Ralph L.; Palmiter, Richard D.Proceedings of the National Academy of Sciences of the United States of America (1994), 91 (2), 584-8CODEN: PNASA6; ISSN:0027-8424.The authors inactivated the mouse metallothionein (MT)-I and MT-II genes in embryonic stem cells and generated mice homozygous for these mutant alleles. These mice were viable and reproduced normally when reared under normal lab. conditions. They were, however, more susceptible to hepatic poisoning by cadmium. This proves that these widely expressed MTs are not essential for development but that they do protect against cadmium toxicity. These mice provide a means for testing other proposed functions of MT in vivo.
- 65Michalska, A. E.; Choo, A. K. H. Targeting and germ-line transmission of a null mutation at the metallothionein I and II loci in mouse. Proc. Natl. Acad. Sci. U. S. A. 1993, 90, 8088– 8092, DOI: 10.1073/pnas.90.17.8088Google Scholar65Targeting and germ-line transmission of a null mutation at the metallothionein I and II loci in mouseMichalska, Anna E.; Choo, K. H. AndyProceedings of the National Academy of Sciences of the United States of America (1993), 90 (17), 8088-92CODEN: PNASA6; ISSN:0027-8424.The authors report the generation of transgenic mice deficient in the metallothionein MT-I and MT-II genes. The mutations were introduced into embryonic stem cells by homologous recombination. Chimeric mice resulting from the targeted embryonic stem cells transmitted the disrupted alleles through their germ line. Homozygous animals were born alive and appeared phenotypically normal and fertile. Absence of MT proteins was confirmed by direct measurement in liver exts. Challenging the mutant animals with moderate levels of CdSO4 indicated their greater susceptibility to cadmium toxicity than wild-type animals. These mice should provide a useful model to allow detailed study of the physiol. roles of MT-I and MT-II.
- 66Karin, M. Metallothioneins: Proteins in search of function. Cell 1985, 41, 9– 10, DOI: 10.1016/0092-8674(85)90051-0Google Scholar66Metallothioneins: proteins in search of functionKarin, MichaelCell (Cambridge, MA, United States) (1985), 41 (1), 9-10CODEN: CELLB5; ISSN:0092-8674.A review, with 20 refs., of induction of metallothioneins (MT) by heavy metal ions and protection against toxicity, regulation of MT by hormones and MT expression in development, MT and metab. regulation, MT and differentiation and proliferation regulation, MT and free radicals, and MT and the UV response.
- 67Thornalley, P. J.; Vašák, M. Possible role for metallothionein in protection against radiation-induced oxidative stress. Kinetics and mechanism of its reaction with superoxide and hydroxyl radicals. Biochim. Biophys. Acta, Protein Struct. Mol. Enzymol. 1985, 827, 36– 44, DOI: 10.1016/0167-4838(85)90098-6Google Scholar67Possible role for metallothionein in protection against radiation-induced oxidative stress. Kinetics and mechanism of its reaction with superoxide and hydroxyl radicalsThornalley, Paul J.; Vasak, MilanBiochimica et Biophysica Acta, Protein Structure and Molecular Enzymology (1985), 827 (1), 36-44CODEN: BBAEDZ; ISSN:0167-4838.Rabbit liver metallothionein-1 (MT) [mol. wt. (Mr) 6500], which contains Zn and(or) Cd ions, apparently scavenges free •OH and O-2 produced by the xanthine/xanthine oxidase reaction much more effectively than the control, bovine serum albumin (Mr 65,000). Kinetic competition studies between MT and either a spin trap for •OH or ferricytochrome c for O2- gave bimol. rate consts. on the order of k•OH/MT ≈1012 M-1/s and kO2-/MT ≈5 × 105 M-1/s, resp. The former value suggests that all 20 cysteine S atoms are involved in this quenching process and that they all act in the diffusion control limit. Aerobic radiolysis of an aq. soln. of MT, which generated O2- and •OH, induced metal ion loss and thiolate oxidn. These effects were reversed by incubation of the irradiated protein with GSH and the appropriate divalent metal ion. MT is apparently an extraordinarily efficient •OH scavenger, even compared with proteins 10-50-fold its mol. wt. •OH damage to MT evidently occurs at the metal ion-thiolate clusters, which may be repaired in the cell by GSH. MT has characteristics of a sacrificial but renewable cellular target for •OH-mediated cellular damage.
- 68Sato, M.; Bremner, I. Oxygen free radicals and metallothionein. Free Radical Biol. Med. 1993, 14, 325– 337, DOI: 10.1016/0891-5849(93)90029-TGoogle Scholar68Oxygen free radicals and metallothioneinSato, Masao; Bremner, IanFree Radical Biology & Medicine (1993), 14 (3), 325-37CODEN: FRBMEH; ISSN:0891-5849.A review, with 128 refs., on the evidence supporting a physiol. role as a free radical scavenger and describing induction of metallothionein synthesis by oxidative stress, possible mediators for this induction, and the radical scavenging capability of metallothionein in tissues and cells. The relationship between metallothionein and other antioxidant defense systems and the medical implications of the free radical scavenging properties of metallothionein are also discussed.
- 69Fliss, H.; Ménard, M. Oxidant-induced mobilization of zinc from metallothionein. Arch. Biochem. Biophys. 1992, 293, 195– 199, DOI: 10.1016/0003-9861(92)90384-9Google Scholar69Oxidant-induced mobilization of zinc from metallothioneinFliss, Henry; Menard, MichelArchives of Biochemistry and Biophysics (1992), 293 (1), 195-9CODEN: ABBIA4; ISSN:0003-9861.Neutrophils which accumulate at sites of inflammation secrete a no. of injurious oxidants which are highly reactive with protein sulfhydryls. The present study examd. the possibility that this reactivity with thiols may cause protein damage by mobilizing zinc from cellular metalloproteins in which the metal is bound to cysteine. The ability of the 3 principal neutrophil oxidants, HOCl, O2-, and H2O2, to cleave thiolate bonds and mobilize complexed zinc was compared using 2 model compds. (2,3-dimercaptopropanol and metallothionein peptide fragment 56-61), as well as metallothionein. With all compds., 50 μM HOCl caused high rates of Zn2+ mobilization as measured spectrophotometrically with the metallochromic indicator 4-(2-pyridylazo)resorcinol. Xanthine (500 μM) plus xanthine oxidase (30 mU), which produced a similar concn. of O2-, also effected a rapid rate of Zn2+ mobilization which was inhibited by superoxide dismutase but not catalase, indicating that O2- is also highly reactive with thiolate bonds. In contrast, H2O2 alone was much less reactive at comparable concns. Thus, HOCl and O2- can cause damage to cellular metalloproteins through the mobilization of complexed zinc. In view of the essential role played by zinc in numerous cellular processes, Zn2+ mobilization by neutrophil oxidants may cause significant cellular injury at sites of inflammation.
- 70Otvos, J. D.; Petering, D. H.; Shaw, C. F. Structure-reactivity relationships of metallothionein, a unique metal-binding protein. Comments Inorg. Chem. 1989, 9, 1– 35, DOI: 10.1080/02603598908035801Google Scholar70Structure-reactivity relationships of metallothionein, a unique metal-binding proteinOtvos, James D.; Petering, David H.; Shaw, C. FrankComments on Inorganic Chemistry (1989), 9 (1), 1-35CODEN: COICDZ; ISSN:0260-3594.A review, with 89 refs., on the structure of metallothioneins and relating the structure to its metal-binding properties and kinetics. Particular emphasis is placed on its binding of Cd, Au, Pt, and Zn.
- 71Li, T. Y.; Minkel, D. T.; Shaw, C. F., 3rd; Petering, D. H. On the reactivity of metallothioneins with 5,5′-dithiobis(2-nitrobenzoic acid). Biochem. J. 1981, 193, 441– 446, DOI: 10.1042/bj1930441Google Scholar71On the reactivity of metallothioneins with 5,5'-dithiobis-(2-nitrobenzoic acid)Li, Ta-Yuen; Minkel, Daniel T.; Shaw, C. Frank, III; Petering, David H.Biochemical Journal (1981), 193 (2), 441-6CODEN: BIJOAK; ISSN:0264-6021.Rat liver and horse kidney metallothioneins reacted with 5,5'-dithiobis(2-nitrobenzoic acid) (I), releasing 5-thio-2-nitrobenzoate and metal ions. The reactions were slow and showed biphasic kinetics, each process having an empirical rate law of similar form. The pseudo-1st-order kinetics were insensitive to pH, but were modified in guanidine-HCl soln. Rat liver metallothioneins of variable Zn, Cu, and Cd compn. reacted similarly, giving observable thiol/total metal ratios in good agreement with stoichiometries of SH/(Cd + Zn) and SH/Cu of 3 and 1, resp. A model complex, Cd-2,3-dimercaptopropanol, resembled the protein in its reaction with I.
- 72Jacob, C.; Maret, W.; Vallee, B. L. Control of zinc transfer between thionein, metallothionein and zinc proteins. Proc. Natl. Acad. Sci. U. S. A. 1998, 95, 3489– 3494, DOI: 10.1073/pnas.95.7.3489Google Scholar72Control of zinc transfer between thionein, metallothionein, and zinc proteinsJacob, Claus; Maret, Wolfgang; Vallee, Bert L.Proceedings of the National Academy of Sciences of the United States of America (1998), 95 (7), 3489-3494CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Metallothionein (MT), despite its high metal binding const. (KZn = 3.2 × 1013 M-1 at pH 7.4), can transfer zinc to the apoforms of zinc enzymes that have inherently lower stability consts. To gain insight into this paradox, we have studied zinc transfer between zinc enzymes and MT. Zinc can be transferred in both directions-i.e., from the enzymes to thionein (the apoform of MT) and from MT to the apoenzymes. Agents that mediate or enhance zinc transfer have been identified that provide kinetic pathways in either direction. MT does not transfer all of its seven zinc atoms to an apoenzyme, but apparently contains at least one that is more prone to transfer than the others. Modification of thiol ligands in MT zinc clusters increases the total no. of zinc ions released and, hence, the extent of transfer. Aside from disulfide reagents, we show that selenium compds. are potential cellular enhancers of zinc transfer from MT to apoenzymes. Zinc transfer from zinc enzymes to thionein, on the other hand, is mediated by zinc-chelating agents such as Tris buffer, citrate, or glutathione. Redox agents are asym. involved in both directions of zinc transfer. For example, reduced glutathione mediates zinc transfer from enzymes to thionein, whereas glutathione disulfide oxidizes MT with enhanced release of zinc and transfer of zinc to apoenzymes. Therefore, the cellular redox state as well as the concn. of other biol. chelating agents might well det. the direction of zinc transfer and ultimately affect zinc distribution.
- 73Krężel, A.; Maret, W. Dual nanomolar and picomolar Zn(II) binding properties of metallothionein. J. Am. Chem. Soc. 2007, 129, 10911– 10921, DOI: 10.1021/ja071979sGoogle Scholar73Dual Nanomolar and Picomolar Zn(II) Binding Properties of MetallothioneinKrezel, Artur; Maret, WolfgangJournal of the American Chemical Society (2007), 129 (35), 10911-10921CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Each of the seven Zn(II) ions in the Zn3S9 and Zn4S11 clusters of human metallothionein is in a tetrathiolate coordination environment. Yet anal. of Zn(II) assocn. with thionein, the apoprotein, and anal. of Zn(II) dissocn. from metallothionein using the fluorescent chelating agents FluoZin-3 and RhodZin-3 reveal at least three classes of sites with affinities that differ by 4 orders of magnitude. Four Zn(II) ions are bound with an apparent av. log K of 11.8, and with the methods employed, their binding is indistinguishable. This binding property makes thionein a strong chelating agent. One Zn(II) ion is relatively weakly bound, with a log K of 7.7, making metallothionein a zinc donor in the absence of thionein. The binding data demonstrate that Zn(II) binds with at least four species: Zn4T, Zn5T, Zn6T, and Zn7T. Zn5T and Zn6T bind Zn(II) with a log K of ∼10 and are the predominant species at micromolar concns. of metallothionein in cells. Central to the function of the protein is the reactivity of its cysteine side chains in the absence and presence of Zn(II). Chelating agents, such as physiol. ligands with moderate affinities for Zn(II), cause dissocn. of Zn(II) ions from metallothionein at pH 7.4 (Zn7T (symbol) Zn7-nT + nZn2+), thereby affecting the reactivity of its thiols. Thus, the rate of thiol oxidn. increases in the presence of Zn(II) acceptors but decreases if more free Zn(II) becomes available. Thionein is such an acceptor. It regulates the reactivity and availability of free Zn(II) from metallothionein. At thionein/metallothionein ratios > 0.75, free Zn(II) ions are below a pZn (-log[Zn2+]free) of 11.8, and at ratios < 0.75, relatively large fluctuations of free Zn(II) ions are possible (pZn between 7 and 11). These chem. characteristics match cellular requirements for Zn(II) and suggest how the mol. structures and redox chemistries of metallothionein and thionein det. Zn(II) availability for biol. processes.
- 74Savas, M. M.; Petering, D. H.; Shaw, C. F., III On the rapid, monophasic reaction of rabbit liver metallothionein α-domain with 5,5′-dithiobis(2-nitrobenzoic acid)(DTNB). Inorg. Chem. 1991, 30, 581– 583, DOI: 10.1021/ic00003a049Google Scholar74On the rapid, monophasic reaction of the rabbit liver metallothionein α-domain with 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB)Savas, M. Meral; Petering, David H.; Shaw, C. Frank, IIIInorganic Chemistry (1991), 30 (3), 581-3CODEN: INOCAJ; ISSN:0020-1669.The first kinetic study of an isolated metallothionein domain, in order to obtain insight into the origin of the biphasic reactions of the holoprotein, is reported. The reactions of the α-Cd4 cluster from rabbit liver metallothionein-II with DTNB were monophasic and 1st-order in the presence of excess DTNB at pH 7.4 and 25°. This result also holds at pH 6.8 (25°) and at temps. 5 and 50° (pH 7.4). The obsd. rate consts. are linearly dependent upon DTNB concn. over the range studied, yielding a complex rate law, rate=k1+k2[DTNB]. For the DTNB-independent process, the 1st-order rate const. (intercept) is k1 = 6.4 × 10-4 s-1, and for the DTNB-dependent processes, the 2nd-order rate const. (slope) is k2 = 1.12 s-1 M-1. The significance of this result for the biphasic reaction of the holoprotein with DTNB is discussed briefly.
- 75Jiang, L.-J.; Vašák, M.; Vallee, B. L.; Maret, W. Zinc transfer potentials of the α- and β-clusters of metallothionein are affected by domain interactions in the whole molecule. Proc. Natl. Acad. Sci. U. S. A. 2000, 97, 2503– 2508, DOI: 10.1073/pnas.97.6.2503Google Scholar75Zinc transfer potentials of the α- and β-clusters of metallothionein are affected by domain interactions in the whole moleculeJiang, Li-Juan; Vasak, Milan; Vallee, Bert L.; Maret, WolfgangProceedings of the National Academy of Sciences of the United States of America (2000), 97 (6), 2503-2508CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)The α- and β-polypeptides of human metallothionein (isoform 2), obtained by chem. synthesis, were converted into their resp. zinc/thiolate clusters, and each domain was investigated sep. Proton titrn. data for the N-terminal β-domain fit a simple model with three ionizations of the same apparent pKa value of 4.9 and a collective binding const. for zinc of 5×10-12 M at pH 7.0. The zinc cluster in the C-terminal α-domain is more stable than that in the β-domain. Its pH titrn. is also more complex, indicating at least two classes of zinc sites with different affinities. The whole mol. is stabilized with regard to the individual domains. Chem. modification implicates lysine side chains in both the stabilization of the β-domain cluster and the mutual stabilization of the domains in the whole mol. The two zinc clusters also differ in the reactivity of their cysteine sulfurs and their potential to donate zinc to an acceptor mol. dependent on its type and characteristics. The isolated β-domain cluster reacts faster with Ellman's reagent and is a better zinc donor toward zinc-depleted sorbitol dehydrogenase than is the isolated α-domain cluster, whereas the reverse is obsd. when a chelating agent is the zinc acceptor. Thus, although each cluster assembles independently of the other, the cumulative properties of the individual domains do not suffice to describe metallothionein either structurally or functionally. The two-domain structure of the whole mol. is important for its interaction with ligands and for control of its reactivity and overall conformation.
- 76Savas, M. M.; Shaw, C. F., III; Petering, D. H. The oxidation of rabbit liver metallothionein-II by 5,5′-dithiobis(2-nitrobenzoic acid) and glutathione disulfide. J. Inorg. Biochem. 1993, 52, 235– 249, DOI: 10.1016/0162-0134(93)80028-8Google Scholar76The oxidation of rabbit liver metallothionein-II by 5,5'-dithiobis(2-nitrobenzoic acid) and glutathione disulfideSavas, M. Meral; Shaw, C. Frank, III; Petering, David H.Journal of Inorganic Biochemistry (1993), 52 (4), 235-49CODEN: JIBIDJ; ISSN:0162-0134.Because metallothionein (MT) may undergo thiol-disulfide or other redox reactions under certain cellular conditions, the partially and completely oxidized products of the reactions of Cd7MT-II with the electrophile 5,5'-dithiobis(2-nitrobenzoic acid), ESSE, and oxidized glutathione, GSSG, were characterized. Reaction with the stoichiometric quantity of ESSE (1 ESSE per MT thiolate) generates monomeric and polymeric MTs with three types of disulfide bonds: intra- and intermol. CyS-SCy linkages and a small no. (2-3/MT) of mixed disulfides, CyS-SE, involving thionitrobenzoate (ES-). Reaction with substoichiometric quantities of ESSE (0.02 or 0.1 per MT thiolate) causes the formation of intra- and intermol. CyS-SCy disulfides, but no mixed disulfides. In the latter reactions, two equiv. of ES- are released per mol of ESSE, but the release is described by a single first-order rate const. (k = 3.0 ± 0.5 s-1). Substantial amts. of cadmium remained bound to the MT monomers and polymers after reaction with the substoichiometric quantities. Despite the Cd bound to the MT after reaction with 0.1 ESSE per MT thiolate, no 111Cd NMR signals were detected, indicating rapid equilibration of the remaining metal ions among the disrupted binding sites. Large excesses of the endogenous aliph. disulfide, GSSG, displace Zn+2 from Zn7-MT slowly. The reaction is complete after 24 h with 5000 μM GSSG, but only 25% complete after 72 h with 250 μM GSSG. Approx. one Cd+2 is displaced rapidly from Cd7MT by 5000 μM GSSG and half as much by 250 μM GSSG, but no further reaction occurs. It is unlikely that GSSG oxidn. of MTs would be physiol. significant.
- 77Maret, W. Oxidative metal release from metallothionein via zinc-thiol/disulfide interchange. Proc. Natl. Acad. Sci. U. S. A. 1994, 91, 237– 241, DOI: 10.1073/pnas.91.1.237Google Scholar77Oxidative metal release from metallothionein via zinc-thiol/disulfide interchangeMaret, WolfgangProceedings of the National Academy of Sciences of the United States of America (1994), 91 (1), 237-41CODEN: PNASA6; ISSN:0027-8424.Mammalian metallothionein has been postulated to play a pivotal role in cellular zinc distribution. All seven of its metal atoms are bound with high thermodn. stability in two clusters buried deeply in the mol. If the protein is to function in metal delivery, there must be a biol. mechanism to facilitate metal release. One means to achieve this would be a labilization of the cluster by interaction of metallothionein with an appropriate cellular ligand. To search for such a mediator, the authors have designed a rapid radiochromatog. method that can detect changes in the zinc content of 65Zn-labeled metallothionein in response to other biomols. Using this methodol., the authors have established that rabbit liver metallothionein 2 interacts with glutathione disulfide with concomitant release of zinc. Under conditions of pseudo-first-order kinetics, the monophasic reaction depends linearly on the concn. of glutathione disulfide in the range from 5 to 30 mM with a second-order rate const. k = 4.9 × 10-3 s-1·M-1 (pH 8.6; 25°C). Apparently, zinc release does not involve direct access of glutathione disulfide to the inner coordination sphere of the metals. Rather it appears that the solvent-accessible zinc-bound thiolates in two clefts of each domain of metallothionein [Robbins, A. H., McRee, D. E., Williamson, M., Collett, S. A., Xuong, N. H., Furey, W. F. Wang, B. C. & Stout, C. D. (1991) J. Mol. Biol. 221, 1269-1293] participate in a thiol/disulfide interchange with glutathione disulfide. This rate-limiting initial S-thiolation, which occurs with indistinguishable rates in both clusters, then causes the clusters to collapse and release their zinc. Such a mechanism of metal release would link the control of the metal content of metallothionein to the cellular glutathione redox status and raises important questions about the physiol. implications of this observation with regard to a role of glutathione in zinc metab. and in making zinc available for other biomols.
- 78Maret, W.; Krężel, A. Cellular zinc and redox buffering capacity of metallothionein/thionein in health and disease. Mol. Med. 2007, 13, 371– 375, DOI: 10.2119/2007-00036.MaretGoogle Scholar78Cellular zinc and redox buffering capacity of metallothionein/thionein in health and diseaseMaret, Wolfgang; Krezel, ArturMolecular Medicine (Manhasset, NY, United States) (2007), 13 (7-8), 371-375CODEN: MOMEF3; ISSN:1076-1551. (Feinstein Institute for Medical Research)A review. Zinc is involved in virtually all aspects of cellular and mol. biol. as a catalytic, structural, and regulatory cofactor in over 1000 proteins. Zinc binding to proteins requires an adequate supply of zinc and intact mol. mechanisms for redistributing zinc ions to make them available at the right time and location. Several dozen gene products participate in this process, in which interactions between zinc and sulfur donors det. the mobility of zinc and establish coupling between cellular redox state and zinc availability. Specifically, the redox properties of metallothionein and its apoprotein thionein are crit. for buffering zinc ions and for controlling fluctuations in the range of picomolar concns. of "free" zinc ions in cellular signaling. Metallothionein and other proteins with sulfur coordination environments are sensitive to redox perturbations and can render cells susceptible to injury when oxidative stress compromises the cellular redox and zinc buffering capacity in chronic diseases. The implications of these fundamental principles for zinc metab. in type 2 diabetes are briefly discussed.
- 79Maret, W.; Vallee, B. L. Thiolate ligands in metallothionein confer redox activity on zinc clusters. Proc. Natl. Acad. Sci. U. S. A. 1998, 95, 3478– 3482, DOI: 10.1073/pnas.95.7.3478Google Scholar79Thiolate ligands in metallothionein confer redox activity on zinc clustersMaret, Wolfgang; Vallee, Bert L.Proceedings of the National Academy of Sciences of the United States of America (1998), 95 (7), 3478-3482CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)We postulate a novel and general mechanism in which the redox-active sulfur donor group of cyst(e)ine confers oxidoreductive characteristics on stable zinc sites in proteins. Thus, the present, an earlier, and accompanying manuscripts [Maret, W., Larsen, K. S. & Vallee, B. L. (1997) Proc. Natl. Acad. Sci. USA 94, 2233-2237; Jiang, L.-J., Maret, W. & Vallee, B. L. (1998) Proc. Natl. Acad. Sci. USA 95, 3483-3488; and Jacob, C., Maret, W. & Vallee, B. L. (1998) Proc. Natl. Acad. Sci. USA 95, 3489-3494] demonstrate that the interactive network featuring multiple zinc/sulfur bonds as found in the clusters of metallothionein (MT) constitutes a coordination unit crit. for the concurrent oxidn. of cysteine ligands and the ensuing release of zinc. The low position of MT (<-366 mV) on a scale of redox reagents allows its effective oxidn. by relatively mild cellular oxidants, in particular disulfides. When MT is exposed to an excess of dithiodipyridine, all of its 20 cysteines are oxidized within 1 h with the concomitant release of all 7 zinc atoms; similarly, the thiol/disulfide oxidoreductase DsbA reacts stoichiometrically with MT to release zinc. Zinc and sulfur ligands in the clusters are in a spatial arrangement that seemingly favors disulfide bond formation. Jointly, this and the above-mentioned manuscripts conclude that the control of cellular zinc distribution as a function of the energy state of the cell is the long sought role of MT. This specific MT function renders dubious the widely held belief that MT primarily scavenges radicals or detoxifies metals and is consistent with the frequent use of cysteine as a zinc ligand in proteins as a means of both tight and weak zinc binding of thiols and disulfides, resp. Thus, we relate changes in the reducing power of the cell to the stability of the zinc/sulfur network in MT and the relative mobility of zinc and its control.
- 80Maret, W. The function of zinc metallothionein: A link between cellular zinc and redox state. J. Nutr. 2000, 130, 1455S– 1458S, DOI: 10.1093/jn/130.5.1455SGoogle Scholar80The function of zinc metallothionein: a link between cellular zinc and redox stateMaret, WolfgangJournal of Nutrition (2000), 130 (5S), 1455S-1458SCODEN: JONUAI; ISSN:0022-3166. (American Society for Nutritional Sciences)A review with 42 refs. A chem. and biochem. mechanism of action of the metallothionein (MT)/thionein (T) couple has been proposed. The mechanism emphasizes the importance of zinc/sulfur cluster bonding in MT and the significance of the two cluster networks as redox units that confer mobility on otherwise tightly bound and redox-inert zinc in MT. In this article, it is further explored how this redox mechanism controls the metabolically active cellular zinc pool. The low redox potential of the sulfur donor atoms in the clusters readily allows oxidn. by mild cellular oxidants with concomitant release of zinc. Such a release by oxidants and the preservation of zinc binding by antioxidants place MT under the control of the cellular redox state and, consequently, energy metab. The binding of effectors, e.g., ATP, elicits conformational changes and alters zinc binding in MT. The glutathione/glutathione disulfide redox couple as well as selenium compds. effect zinc delivery from MT to the apoforms of zinc enzymes. This novel action of selenium on zinc/sulfur coordination sites has significant implications for the interaction between these essential elements. Tight binding and kinetic lability, modulation of MT by cellular ligands and the redox state, control of MT gene expression by zinc and many other inducers all support a crit. function of the MT/T system in cellular homeostasis and distribution of zinc.
- 81Maret, W. Cellular zinc and redox stress converge in the metallothionein/thionein pair. J. Nutr. 2003, 133, 1460S– 1462S, DOI: 10.1093/jn/133.5.1460SGoogle Scholar81Cellular zinc and redox states converge in the metallothionein/thionein pairMaret, WolfgangJournal of Nutrition (2003), 133 (5S-1), 1460S-1462SCODEN: JONUAI; ISSN:0022-3166. (American Society for Nutritional Sciences)A review. The paramount importance of zinc for a wide range of biol. functions is based on its occurrence in thousands of known zinc proteins. To regulate the availability of zinc dynamically, eukaryotes have compartmentalized zinc and the metallothionein/thionein pair, which controls the pico- to nanomolar concns. of metabolically active cellular zinc. Interactions of zinc with sulfur ligands of cysteines turn out to be crit. both for tight binding and creation of a redox-active coordination environment from which the redox-inert zinc can be distributed. Biol. oxidants such as disulfides and S-nitrosothiols oxidize the zinc/thiolate clusters in metallothionein with concomitant zinc release. In addn., selenium compds. that have the capacity to form selenol(ate)s catalytically couple with the glutathione/glutathione disulfide and metallothionein/thionein redox pairs to either release or bind zinc. In this pathway, selenium expresses its antioxidant effects through redox catalysis in zinc metab. Selenium affects the redox state of thionein, an endogenous chelating agent. With its 20 cysteines, thionein contributes significantly to the zinc- and thiol-redox-buffering capacity of the cell. Thus, hitherto unknown interactions between the essential micronutrients zinc and selenium on the one hand and zinc and redox metab. on the other are key features of the cellular homeostatic zinc system.
- 82IUPAC. Compendium of Chemical Terminology, 2nd ed.; McNaught, A. D., Wilkinson, A., Eds.; Blackwell Scientific Publications: Oxford, England,1997. DOI: 10.1351/goldbook .Google ScholarThere is no corresponding record for this reference.
- 83Maret, W. Zinc and sulfur: A critical biological partnership. Biochemistry 2004, 43, 3301– 3309, DOI: 10.1021/bi036340pGoogle Scholar83Zinc and Sulfur: A Critical Biological PartnershipMaret, WolfgangBiochemistry (2004), 43 (12), 3301-3309CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)A review. The Zn-S interaction offers specific mechanisms for enzyme catalysis, establishes reactivities of zinc sites that hitherto were believed to have only a structural role, allows zinc to be tightly bound and yet to be available, and, importantly, generates redox-active coordination environments for the redox-inert zinc ion. These activities are critically involved in the regulation of protein structure and function, and in mobility, transfer, redistribution, and sensing of cellular zinc, as is becoming evident from the continuing exploration of the protein metallothionein and other proteins involved in cellular zinc homeostasis.
- 84Lee, S.-H.; Maret, W. Redox control of zinc finger proteins: Mechanisms and role in gene regulation. Antioxid. Redox Signaling 2001, 3, 531– 534, DOI: 10.1089/15230860152542907Google Scholar84Redox control of zinc finger proteins: mechanism and role in gene regulationLee, Suk-Hee; Maret, WolfgangAntioxidants & Redox Signaling (2001), 3 (4), 531-534CODEN: ARSIF2; ISSN:1523-0864. (Mary Ann Liebert, Inc.)A review, with 10 refs., on the redox control of zinc finger proteins (ZFPs) and its effect on gene expression and DNA metab.
- 85Maret, W. Metallothionein/disulfide interactions, oxidative stress, and the mobilization of cellular zinc. Neurochem. Int. 1995, 27, 111– 117, DOI: 10.1016/0197-0186(94)00173-RGoogle Scholar85Metallothionein/disulfide interactions, oxidative stress, and the mobilization of cellular zincMaret, WolfgangNeurochemistry International (1995), 27 (1), 111-17CODEN: NEUIDS; ISSN:0197-0186. (Elsevier)Glutathione disulfide, the major cell. disulfide, releases zinc from metallothionein (MT). Here, the interaction of rabbit liver MT-II with other selected biol. disulfides (CoA/glutathione mixed disulfide, CoA disulfide, and cystamine) was investigated by measuring concomitant release of radioactive 65-zinc from MT. These disulfides react more rapidly than glutathione disulfide, thus underscoring the reactivity of zinc sulfur bonds in the clusters of MT and the importance of the MT/disulfide interactions as a chem. mechanism for mobilizing zinc from a thermodynamically stable zinc complex. Two implications of these in vitro findings are discussed. (I) Apparently, in the case of zinc which is redox inert, Nature has availed itself of the redox activity of the cysteine ligand to mobilize the metal, and, presumably to permit redox-control of cellular zinc distribution. The mobilization of zinc from MT suggests a possible function of MT as a physiol. zinc donor. (Ii) A shift of the glutathione redox balance under conditions of oxidative stress will accelerate metal release from MT. Such a disturbance of metal metab. has important consequences for the progression of diseases such as Alzheimer's and Parkinson's disease where oxidative stress occurs in affected brain tissue.
- 86Maret, W. Zinc coordination environments in proteins as redox sensors and signal transducers. Antioxid. Redox Signaling 2006, 8, 1419– 1441, DOI: 10.1089/ars.2006.8.1419Google Scholar86Zinc coordination environments in proteins as redox sensors and signal transducersMaret, WolfgangAntioxidants & Redox Signaling (2006), 8 (9 & 10), 1419-1441CODEN: ARSIF2; ISSN:1523-0864. (Mary Ann Liebert, Inc.)A review. Zn/Cys coordination environments in proteins are redox-active. Oxidn. of the S ligands mobilizes Zn, while redn. of the oxidized ligands enhances Zn binding, providing redox control over the availability of Zn2+ ions. Some Zn-proteins are redox sensors, in which Zn release is coupled to conformational changes that control varied functions such as enzymic activity, binding interactions, and mol. chaperone activity. Whereas the released Zn2+ ion in redox sensors has no known function, the redox signal is transduced to specific and sensitive Zn signals in redox transducers. Released Zn can bind to sites on other proteins and modulate signal transduction, generation of metabolic energy, mitochondrial function, and gene expression. The paradigm of such redox transducers is the Zn-protein, metallothionein, which, together with its apoprotein, thionein, functions at a central node in cellular signaling by redistributing cellular Zn, presiding over the availability of Zn, and interconverting redox and Zn signals. In this regard, the transduction of NO signals into Zn signals by metallothionein has received particular attention. It appears that redox-inert Zn has been chosen to control some aspects of cellular thiol/disulfide redox metab. Tight control of Zn is essential for redox homeostasis because both increases and decreases of cellular Zn elicit oxidative stress. Depending on its availability, Zn can be cytoprotective as a pro-antioxidant or cytotoxic as a pro-oxidant. Any condition with acute or chronic oxidative stress is expected to perturb Zn homeostasis.
- 87Pattanaik, A.; Shaw, C. F., III; Petering, D. H.; Garvey, J.; Kraker, A. J. Basal metallothionein in tumours: Widespread presence of apoprotein. J. Inorg. Biochem. 1994, 54, 91– 105, DOI: 10.1016/0162-0134(94)80023-5Google Scholar87Basal metallothionein in tumors: widespread presence of apoproteinPattanaik, Asima; Shaw, C. Frank; Petering, David H.; Garvey, Justine; Kraker, Alan J.Journal of Inorganic Biochemistry (1994), 54 (2), 91-105CODEN: JIBIDJ; ISSN:0162-0134. (Elsevier)A survey has been conducted of solid and ascites tumors from mice and solid tumors in rats for the presence of metallothionein or metallothionein-like protein. In most tumors, a pos. identification was made on the basis of Sephadex G-75 and HPLC-DEAE chromatog. followed by competitive RIA for metallothionein. Apometallothionein was revealed in a no. of tumor for the first time by comparing in Sephadex G-75 chromatog. profiles of Zn in native cytosol and Cd in cytosol incubated briefly with CdCl2 to sat. free binding sites on the protein before Sephadex G-75 chromatog. In two cases unsatn. of metallothionein was correlated with a lack of zinc in the ascites fluid which supplies the tumor with zinc.
- 88Yang, Y.; Maret, W.; Vallee, B. L. Differential fluorescence labeling of cysteinyl clusters uncovers high tissue levels of thionein. Proc. Natl. Acad. Sci. U. S. A. 2001, 98, 5556– 5559, DOI: 10.1073/pnas.101123298Google Scholar88Differential fluorescence labeling of cysteinyl clusters uncovers high tissue levels of thioneinYang, Yi; Maret, Wolfgang; Vallee, Bert L.Proceedings of the National Academy of Sciences of the United States of America (2001), 98 (10), 5556-5559CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)The isolation of thionein (T) from tissues has not been reported heretofore. T contains 20 cysteinyl residues that react with 7-fluorobenz-2-oxa-1,3-diazole-4-sulfonamide to form fluorescent adducts. In metallothionein (MT), the cysteinyl residues, which are bound to zinc, do not react. However, they do react in the presence of a chelating agent such as EDTA. The resultant difference in chem. reactivity provides a means to measure T in the absence of EDTA, (MT + T) in its presence, and, of course, MT by difference. The 7-fluorobenz-2-oxa-1,3-diazole-4-sulfonamide deriv. of T can be isolated from tissue homogenates by HPLC and quantified fluorimetrically with a detection limit in the femtomolar range and a linear response over 3 orders of magnitude. Anal. of liver, kidney, and brain of rats reveals almost as much T as MT. Moreover, in contrast to earlier views, MT in tissue exts. appears to be less stable than T. The existence of T in tissues under normal physiol. conditions has important implications for its function both in zinc metab. and the redox balance of the cell.
- 89Krężel, A.; Maret, W. Zinc-buffering capacity of a eukaryotic cell at physiological pZn. JBIC, J. Biol. Inorg. Chem. 2006, 11, 1049– 1062, DOI: 10.1007/s00775-006-0150-5Google Scholar89Zinc-buffering capacity of a eukaryotic cell at physiological pZnKrezel, Artur; Maret, WolfgangJBIC, Journal of Biological Inorganic Chemistry (2006), 11 (8), 1049-1062CODEN: JJBCFA; ISSN:0949-8257. (Springer GmbH)In spite of the paramount importance of zinc in biol., dynamic aspects of cellular zinc metab. remain poorly defined at the mol. level. Investigations with human colon cancer (HT-29) cells establish a total cellular zinc concn. of 264 μM. Remarkably, about 10% of the potential high-affinity zinc-binding sites are not occupied by zinc, resulting in a surplus of 28 μM ligand (av. Kcd = 83 pM) that ascertain cellular zinc-buffering capacity and maintain the "free" zinc concn. in proliferating cells at picomolar levels (784 pM, pZn = 9.1). This zinc-buffering capacity allows zinc to fluctuate only with relatively small amplitudes (ΔpZn = 0.3; below 1 nM) without significantly perturbing physiol. pZn. Thus, the "free" zinc concns. in resting and differentiated HT-29 cells are 614 pM and 1.25 nM, resp. The calcn. of these "free" zinc concns. is based on measurements at different concns. of the fluorogenic zinc-chelating agent and extrapolation to a zero concn. of the agent. It depends on the state of the cell, its buffering capacity, and the zinc dissocn. const. of the chelating agent. Zinc induction of thionein (apometallothionein) ensures a surplus of unbound ligands, increases zinc-buffering capacity and the availability of zinc (ΔpZn = 0.8), but preserves the zinc-buffering capacity of the unoccupied high-affinity zinc-binding sites, perhaps for crucial physiol. functions. Jointly, metallothionein and thionein function as the major zinc buffer under conditions of increased cellular zinc.
- 90Krężel, A.; Maret, W. Different redox states of metallothionein/thionein in biological tissue. Biochem. J. 2007, 402, 551– 558, DOI: 10.1042/BJ20061044Google Scholar90Different redox states of metallothionein/thionein in biological tissueKrezel, Artur; Maret, WolfgangBiochemical Journal (2007), 402 (3), 551-558CODEN: BIJOAK; ISSN:0264-6021. (Portland Press Ltd.)Mammalian metallothioneins are redox-active metalloproteins. In the case of zinc metallothioneins, the redox activity resides in the cysteine sulfur ligands of zinc. Oxidn. releases zinc, whereas redn. re-generates zinc-binding capacity. Attempts to demonstrate the presence of the apoprotein (thionein) and the oxidized protein (thionin) in tissues posed tremendous anal. challenges. One emerging strategy is differential chem. modification of cysteine residues in the protein. Chem. modification distinguishes three states of the cysteine ligands (reduced, oxidized and metal-bound) based on (i) quenched reactivity of the thiolates when bound to metal ions and restoration of thiol reactivity in the presence of metal-ion-chelating agents, and (ii) modification of free thiols with alkylating agents and subsequent redn. of disulfides to yield reactive thiols. Under normal physiol. conditions, metallothionein exists in three states in rat liver and in cell lines. Ras-mediated oncogenic transformation of normal HOSE (human ovarian surface epithelial) cells induces oxidative stress and increases the amt. of thionin and the availability of cellular zinc. These expts. support the notion that metallothionein is a dynamic protein in terms of its redox state and metal content and functions at a juncture of redox and zinc metab. Thus redox control of zinc availability from this protein establishes multiple methods of zinc-dependent cellular regulation, while the presence of both oxidized and reduced states of the apoprotein suggest that they serve as a redox couple, the generation of which is controlled by metal ion release from metallothionein.
- 91Haase, H.; Maret, W. A differential assay for metallothionein and the reduced and oxidized states of thionein. Anal. Biochem. 2004, 333, 19– 26, DOI: 10.1016/j.ab.2004.04.039Google Scholar91A differential assay for the reduced and oxidized states of metallothionein and thioneinHaase, Hajo; Maret, WolfgangAnalytical Biochemistry (2004), 333 (1), 19-26CODEN: ANBCA2; ISSN:0003-2697. (Elsevier)In the cellular environment, the sulfur ligands in zinc/thiolate coordination sites of proteins can be oxidized with concomitant mobilization of zinc. The characterization of such "redox zinc switches" requires the detn. of three species, i.e., the zinc-contg. complex and the zinc-free complex with the thiolate ligands either reduced or oxidized. Differential chem. modification of thiol groups in the presence and absence of either reducing or chelating agents allows the anal. speciation of such systems as demonstrated here for the characterization of the redox and metal-binding states of mammalian metallothionein. Thiol derivatization with 6-iodoacetamidofluorescein in the presence and absence of the reducing agent tris(2-carboxyethyl)phosphine, high-performance liq. chromatog. sepn., and photometric detection are employed to det. the reduced and oxidized protein. Because the holoprotein reacts only in the presence of a chelating agent such as ethylenediaminetetraacetate (EDTA) its amt. can be detd. as the difference between measurements in the presence and the absence of EDTA. This method is applied to the study of the chem. and enzymic oxidn. of metallothionein/thionein. It should also greatly facilitate the characterization of the redox and metal-binding properties of zinc/thiolate coordination environments of other proteins such as zinc finger proteins.
- 92Feng, W.; Benz, F. W.; Cai, J.; Pierce, W. M.; Kang, Y. J. Metallothionein disulfides are present in metallothionein-overexpressing transgenic mouse heart and increase under conditions of oxidative stress. J. Biol. Chem. 2006, 281, 681– 687, DOI: 10.1074/jbc.M506956200Google Scholar92Metallothionein Disulfides Are Present in Metallothionein-overexpressing Transgenic Mouse Heart and Increase under Conditions of Oxidative StressFeng, Wenke; Benz, Frederick W.; Cai, Jian; Pierce, William M.; Kang, Y. JamesJournal of Biological Chemistry (2006), 281 (2), 681-687CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)Metallothionein (MT) releases zinc under oxidative stress conditions in cultured cells. The change in the MT mol. after zinc release in vivo is unknown although in vitro studies have identified MT disulfide bond formation. The present study was undertaken to test the hypothesis that MT disulfide bond formation occurs in vivo. A cardiac-specific MT-overexpressing transgenic mouse model was used. Mice were administered saline as a control or doxorubicin (20 mg/kg), which is an effective anticancer drug but with severe cardiac toxicity at least partially because of the generation of reactive oxygen species. A differential alkylation of cysteine residues in MT of the heart exts. was performed. Free and metal-bound cysteines were first trapped by N-ethylmaleimide and the disulfide bonds were reduced by dithiothreitol followed by alkylation with radiolabeled iodoacetamide. Analyses of the differentially alkylated MTs in the heart ext. by high performance liq. chromatog., SDS-PAGE, Western blot, and mass spectrometry revealed that disulfide bonds were present in MT in vivo under both physiol. and oxidative stress conditions. More disulfide bonds were found in MT under the oxidative stress conditions. The MT disulfide bonds were likely intramol. and both α- and β-domains were involved in the disulfide bond formation, although the α-domain appeared to be more easily oxidized than the β-domain. The results suggest that under physiol. conditions, the formation of MT disulfide bonds is involved in the regulation of zinc homeostasis. Addnl. zinc release from MT under oxidative stress conditions is accompanied by more MT disulfide bond formation.
- 93Hathout, Y.; Fabris, D.; Fenselau, C. Stoichiometry in zinc ion transfer from metallothionein to zinc finger peptides. Int. J. Mass Spectrom. 2001, 204, 1– 6, DOI: 10.1016/S1387-3806(00)00343-2Google Scholar93Stoichiometry in zinc ion transfer from metallothionein to zinc finger peptidesHathout, Y.; Fabris, D.; Fenselau, C.International Journal of Mass Spectrometry (2001), 204 (1-3), 1-6CODEN: IMSPF8; ISSN:1387-3806. (Elsevier Science B.V.)Electrospray and nanospray ionizations are used to study the transfer of zinc ions between Zn7-metallothionein and apo peptides that are models for several kinds of zinc finger proteins. A membrane expt. is reported here which demonstrates that interprotein contact is required for these transfers. Anal. on a quadrupole ion trap has allowed all reactants and all products to be monitored simultaneously. Evidence is provided for the preferential transfer of a single zinc ion from each Zn7-metallothionein complex.
- 94Haase, H.; Maret, W. Partial oxidation and oxidative polymerization of metallothionein. Electrophoresis 2008, 29, 4169– 4176, DOI: 10.1002/elps.200700922Google Scholar94Partial oxidation and oxidative polymerization of metallothioneinHaase, Hajo; Maret, WolfgangElectrophoresis (2008), 29 (20), 4169-4176CODEN: ELCTDN; ISSN:0173-0835. (Wiley-VCH Verlag GmbH & Co. KGaA)One mechanism for regulation of metal binding to metallothionein (MT) involves the non-enzymic or enzymic oxidn. of its thiols to disulfides. Formation and speciation of oxidized MT have not been investigated in detail despite the biol. significance of this redox biochem. While metal ion-bound thiols in MT are rather resistant towards oxidn., free thiols are readily oxidized. MT can be partially oxidized to a state in which some of its thiols remain reduced and bound to metal ions. Anal. of the oxidn. products with SDS-PAGE and a thiol-specific labeling technique, employing eosin-5-iodoacetamide, demonstrates higher-order aggregates of MT with intermol. disulfide linkages. The polymn. follows either non-enzymic or enzymic oxidn., indicating that it is a general property of oxidized MT. Supramol. assemblies of MT add new perspectives to the complex redox and metal equil. of this protein.
- 95Hou, T.; An, Y.; Ru, B.; Bi, R.; Xu, X. Cysteine-independent polymerization of metallothioneins in solutions and in crystals. Protein Sci. 2000, 9, 2302– 2312, DOI: 10.1110/ps.9.12.2302Google Scholar95Cysteine-independent polymerization of metallothioneins in solutions and in crystalsHou, Tingjun; An, Yu; Ru, Binggen; Bi, Ruchang; Xu, XiaojieProtein Science (2000), 9 (12), 2302-2312CODEN: PRCIEI; ISSN:0961-8368. (Cambridge University Press)Polymn. of metallothioneins complicates the research of metallothioneins' structure and function. Our work focuses on the cysteine-independent polymn. of metallothionein monomers in different milieus. After the purifn. of metallothionein monomers, a dynamic light-scattering technique is used to detect the polymd. states of rabbit liver metallothionein I and II in different buffers. This is the first systematical detection of polymd. states of metallothioneins in solns. The effects of buffer compn. are discussed in detail. Steric complementarity, hydrophobic, and electrostatic interaction characteristics are studied, following the modeling of monomers and relevant polymers of rat metallothionein II, rabbit liver metallothionein I and II. These theor. calcns. are the first complete computer simulations on different factors affecting metallothioneins' polymn. A mol. recognition mechanism of metallothioneins' polymn. in solns. is proposed on the bases of exptl. results and theor. calcns. Preliminary X-ray studies of two crystal forms of rabbit liver metallothionein II are compared with the crystal structure of rat metallothionein II, and the polymd. states in crystal packing are discussed with the knowledge of polymn. of metallothioneins in solns. The hypothesis, which is consistent with theor. calcns. and exptl. results, is expected to construct a connection between the biochem. characteristics and physiol. functions of metallothioneins, and this research may give some enlightenment to the topics of protein polymns.
- 96Vašák, M. Large-scale preparation of metallothionein: Biological sources. Methods Enzymol. 1991, 205, 39– 41, DOI: 10.1016/0076-6879(91)05081-6Google Scholar96Large-scale preparation of metallothionein: biological sourcesVasak, MilanMethods in Enzymology (1991), 205 (Metallobiochem. Pt. B), 39-41CODEN: MENZAU; ISSN:0076-6879.The title process included metallothionein induction by Cd and Zn (the latter metal preferred). Some comments are included related to large-scale protein isolation.
- 97Hong, S.-H.; Toyama, M.; Maret, W.; Murooka, Y. High yield expression and single step purification of human thionein/metallothionein. Protein Expression Purif. 2001, 21, 243– 250, DOI: 10.1006/prep.2000.1372Google Scholar97High Yield Expression and Single Step Purification of Human Thionein/MetallothioneinHong, Sung-Hye; Toyama, Mitsutoshi; Maret, Wolfgang; Murooka, YoshikatsuProtein Expression and Purification (2001), 21 (1), 243-250CODEN: PEXPEJ; ISSN:1046-5928. (Academic Press)Human metallothionein (MT), isoform 2, was expressed in Escherichia coli as an intein (protein splicing element) fusion protein in the absence of added metals and purified by intein-mediated purifn. with an affinity chitin-binding tag (IMPACT system). This procedure constitutes a novel and simple strategy to prep. thionein (T), the metal-free form, or MT when reconstituting T with metals in vitro. The yield was 8 mg of T or 6 mg of pure Cd7- or Zn7-MT from a 1-L culture, significantly higher than yields from any other expression system. Purified recombinant protein is indistinguishable from the native protein on the basis of its metal-binding ability, titrn. of its sulfhydryls, and UV and CD spectra. The MALDI-TOF mass spectrum is consistent with that of T with a free N-terminus. (c) 2001 Academic Press.
- 98Peris-Díaz, M. D.; Guran, R.; Zitka, O.; Adam, V.; Krężel, A. Metal- and affinity-specific dual labeling of cysteine-rich proteins for identification of metal-binding sites. Anal. Chem. 2020, 92, 12950– 12958, DOI: 10.1021/acs.analchem.0c01604Google Scholar98Metal- and Affinity-Specific Dual Labeling of Cysteine-Rich Proteins for Identification of Metal-Binding SitesPeris-Diaz, Manuel David; Guran, Roman; Zitka, Ondrej; Adam, Vojtech; Krezel, ArturAnalytical Chemistry (Washington, DC, United States) (2020), 92 (19), 12950-12958CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)Here, using human metallothionein (MT2) as an example, the authors describe an improved strategy based on differential alkylation coupled to MS, assisted by zinc probe monitoring, for identification of cysteine-rich binding sites with nanomolar and picomolar metal affinity using iodoacetamide (IAM) and N-ethylmaleimide reagents. An SN2 reaction provided by IAM is more suitable to label free Cys residues, avoiding nonspecific metal dissocn. Afterward, metal-bound Cys can be easily labeled in a nucleophilic addn. reaction after sepn. by reverse-phase C18 at acidic pH. Finally, the authors evaluated the efficiency of the method by mapping metal-binding sites of Zn7-xMT species using a bottom-up MS approach with respect to metal-to-protein affinity and element(al) resoln. The methodol. presented might be applied not only for MT2 but to identify metal-binding sites in other Cys-contg. proteins.
- 99Jiang, L.-J.; Maret, W.; Vallee, B. L. The glutathione redox couple modulates zinc transfer from metallothionein to zinc-depleted sorbitol dehydrogenase. Proc. Natl. Acad. Sci. U. S. A. 1998, 95, 3483– 3488, DOI: 10.1073/pnas.95.7.3483Google Scholar99The glutathione redox couple modulates zinc transfer from metallothionein to zinc-depleted sorbitol dehydrogenaseJiang, Li-Juan; Maret, Wolfgang; Vallee, Bert L.Proceedings of the National Academy of Sciences of the United States of America (1998), 95 (7), 3483-3488CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)The release and transfer of zinc from metallothionein (MT) to zinc-depleted sorbitol dehydrogenase (EC 1.1.1.14) in vitro has been used to explore the role of MT in cellular zinc distribution. A 1:1 molar ratio of MT to sorbitol dehydrogenase is required for full reactivation, indicating that only one of the seven zinc atoms of MT is transferred in this process. Reduced glutathione (GSH) and glutathione disulfide (GSSG) are crit. modulators of both the rate of zinc transfer and the ultimate no. of zinc atoms transferred. GSSG increases the rate of zinc transfer 3-fold, and its concn. is the major determinant for efficient zinc transfer. GSH has a dual function. In the absence of GSSG, it inhibits zinc transfer from MT, indicating that MT is in a latent state under the relatively high cellular concns. of GSH. In addn., it primes MT for the reaction with GSSG by enhancing the rate of zinc transfer 10-fold and by increasing the no. of zinc atoms transferred to four. 65Zn-labeling expts. confirm the release of one zinc from MT in the absence of glutathione and the more effective release of zinc in the presence of GSH and GSSG. In vivo, MT may keep the cellular concns. of free zinc very low and, acting as a temporary cellular reservoir, release zinc in a process that is dynamically controlled by its interactions with both GSH and GSSG. These results suggest that a change of the redox state of the cell could serve as a driving force and signal for zinc distribution from MT.
- 100Brouwer, M.; Brouwer-Hoexum, T.; Cashon, R. Crustaceans as models for metal metabolism. III. Interaction of lobster and mammalian metallothionein with glutathione. Mar. Environ. Res. 1993, 35, 13– 17, DOI: 10.1016/0141-1136(93)90006-LGoogle ScholarThere is no corresponding record for this reference.
- 101Brouwer, M.; Hoexum-Brouwer, T.; Cashon, R. E. A putative glutathione-binding site in CdZn metallothionein identified by equilibrium binding and molecular-modelling studies. Biochem. J. 1993, 294, 219– 225, DOI: 10.1042/bj2940219Google Scholar101A putative glutathione-binding site in cadmium-zinc-metallothionein identified by equilibrium binding and molecular-modeling studiesBrouwer, Marius; Hoexum-Brouwer, Thea; Cashon, Robert E.Biochemical Journal (1993), 294 (1), 219-25CODEN: BIJOAK; ISSN:0264-6021.Glutathione (GSH) has been found to form a complex with both vertebrate and invertebrate copper-metallothionein (CuMT) [Freedman, Ciriolo and Peisach (1989) J. Biol. Chem. 264, 5598-5605; Brouwer and Brouwer-Hoexum (1991) Arch. Biochem. Biophys. 290, 207-213]. In this paper the authors report on the interaction of GSH with CdZnMT-I and CdZnMT-II from rabbit liver and with CdMT-I from Blue crab hepatopancreas. Ultrafiltration expts. showed that all three MTs combined with GSH. The measured binding data for the three MTs could be described by a single binding isotherm. The GSH/MT stoichiometry was 1.4 ± 0.3 and Kdiss. = 14 ± 6 μM. Partially Zn-depleted MT does not significantly bind GSH, indicating that the GSH-binding site is located on MT's Zn-contg. N-terminal domain. The putative GSH-binding site on rabbit liver MT was investigated using mol.-graphics anal. A cleft on the MTs N-terminal domain, which has the labile Zn-2 at its base, could easily accommodate GSH. Cysteine-ligand exchange between the terminal (non-bridging) Cys-26, bound to Zn-2, and the cysteine in GSH is stereochem. possible. Based on these considerations a model of MT-GSH was built in which GSH's cysteine replaces Cys-26 as a terminal Zn-2 ligand. This complex was energy-minimized by mol.-mechanics calcns., taking into account computed partial electrostatic charges on all atoms, including Cd and Zn. These calcns. showed that the MT-GSH complex was thermodynamically more stable than MT, due to favorable non-bonded, electrostatic and van der Waals interactions. Six hydrogen bonds can form between GSH and MT. The av. pairwise root-mean-square deviations (RMSD) of the metals in energy-minimized MT and MT-GSH, compared with the metals in the crystal structure, were 0.0087 ± 0.0028 nm (0.087 ± 0.028 Å) and 0.0168 ± 0.0087 nm (0.168 ± 0.087 Å) resp. The RMSD values for the polypeptide-backbone α carbons were 0.0136 ± 0.0060 nm (0.136 ± 0.060 Å) and 0.0491 ± 0.0380 nm (0.491 ± 0.380 Å) resp. No other docking sites for GSH were found. The energy-minimized structure of an MT-2-mercaptoethanol complex was somewhat less stable than the native MT domain, attesting to the specificity of the MT-GSH interaction. The possible physiol. significance of the MT-GSH interaction is discussed.
- 102Freedman, J. H.; Ciriolo, M.; Peisach, J. The role of glutathione in copper metabolism and toxicity. J. Biol. Chem. 1989, 264, 5598– 5605, DOI: 10.1016/S0021-9258(18)83589-XGoogle Scholar102The role of glutathione in copper metabolism and toxicityFreedman, Jonathan H.; Ciriolo, Maria Rosa; Peisach, JackJournal of Biological Chemistry (1989), 264 (10), 5598-605CODEN: JBCHA3; ISSN:0021-9258.Cellular copper metab. and the mechanism of resistance to copper toxicity were investigated using a wild type hepatoma cell line (HAC) and a copper-resistant cell line (HAC600) that accumulates copper and has a highly elevated level of metallothionein (MT). Of the enzymes involved in reactive oxygen metab., only glutathionine peroxidase was elevated (3-4-fold) in resistant cells, suggestive of an increase in the cellular flux of hydrogen peroxide. A majority of the cytoplasmic copper (>60%) was isolated from both cell lines as a GSH complex. Kinetic studies of 67Cu uptake showed that GSH bound 67Cu before the metal was complexed by MT. Depletion of cellular GSH with buthionine sulfoximine inhibited the incorporation of 67Cu into MT by >50%. Apparently, copper is complexed by GSH soon after entering the cell. The complexed metal is then transferred to MT where it is stored. This study also indicates that resistance to metal toxicity in copper-resistant hepatoma cells is due to increases in both cellular GSH and MT. Furthermore, it is suggested that elevated levels of GSH peroxidase allows cells to more efficiently accommodate an increased cellular hydrogen peroxide flux that may occur as a consequence of elevated levels of cytoplasmic copper.
- 103Krężel, A.; Wójcik, J.; Maciejczyk, M.; Bal, W. May GSH and L-His contribute to intracellular binding of zinc? Thermodynamic and solution structural study of a ternary complex. Chem. Commun. 2003, 704– 705, DOI: 10.1039/b300632hGoogle Scholar103May GSH and L-His contribute to intracellular binding of zinc? Thermodynamic and solution structural study of a ternary complexKrezel, Artur; Wojcik, Jacek; Maciejczyk, Maciej; Bal, WojciechChemical Communications (Cambridge, United Kingdom) (2003), (6), 704-705CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)GSH and L-His are abundant biomols. and likely biol. ligands for Zn(II) under certain conditions. Potentiometric titrns. provide evidence of formation of ternary Zn(II) complexes with GSH and L-His or D-His with slight stereoselectivity in favor of L-His (ca. 1 log unit of stability const.). The soln. structure of the ZnH(GSH)(L-His)(H2O) complex at pH 6.8, detd. by NMR, includes tridentate L-His, monodentate (sulfur) GSH, and weak interligand interactions. Calcns. of competitiveness of this complex for Zn(II) binding at pH 7.4 indicate that it is likely to be formed in vivo under conditions of GSH depletion. Otherwise, GSH alone emerges as a likely Zn(I) carrier.
- 104Krężel, A.; Bal, W. Studies of zinc(II) and nickel(II) complexes of GSH, GSSG and their analogs shed more light on their biological relevance. Bioinorg. Chem. Appl. 2004, 2, 293– 305, DOI: 10.1155/S1565363304000172Google Scholar104Studies of zinc(II) and nickel(II) complexes of GSH, GSSG and their analogs shed more light on their biological relevanceKrezel, Artur; Bal, WojciechBioinorganic Chemistry and Applications (2004), 2 (3-4), 293-305CODEN: BCAIAH; ISSN:1565-3633. (Freund Publishing House)A review. Glutathione, γ-Glu-Cys-Gly, is one of the most abundant small mols. in biosphere. Its main form is the reduced monomer (GSH), serving to detoxicate xenobiotics and heavy metals, reduce protein thiols, maintain cellular membranes and deactivate free radicals. Its oxidized dimer (GSSG) controls metal content of metallothionein. The results presented provided a quant. and structural description of Zn(II)-glutathione complexes, including a novel ternary Zn(II)-GSH-His complex. A soln. structure for this complex was obtained using 2D-NMR. The complexes studied may contribute to both zinc and glutathione physiol. In the case of Ni(II) complexes an interesting dependence of coordination modes on the ratios of reactants was found. At high GSH excess a Ni(GSH)2 complex is formed, with Ni(II) bonded through S and N and/or O donor atoms. This complex may exist as a high- or low-spin species. Another goal of the studies presented was to describe the catalytic properties of Ni(II) ions towards GSH oxidn., which appeared to be an important step in nickel carcinogenesis. The pH dependence of oxidn. rates allowed to det. the Ni(GSH)2 complex as the most active among the toxicol. relevant species. Protonation and oxidn. of metal-free GSH and its analogs were also studied in detail. The monoprotonated form HL2- of GSH is the one most susceptible to oxidn., due to a salt bridge between S- and NH3+ groups, which activates the thiol.
- 105Casadei, M.; Persichini, T.; Polticelli, F.; Musci, G.; Colasanti, M. S-glutathionylation of metallothioneins by nitrosative/oxidative stress. Exp. Gerontol. 2008, 43, 415– 422, DOI: 10.1016/j.exger.2007.11.004Google Scholar105S-Glutathionylation of metallothioneins by nitrosative/oxidative stressCasadei, Manuela; Persichini, Tiziana; Polticelli, Fabio; Musci, Giovanni; Colasanti, MarcoExperimental Gerontology (2008), 43 (5), 415-422CODEN: EXGEAB; ISSN:0531-5565. (Elsevier)Cysteine residues within metallothionein (MT) structure have been shown to be particularly prone to S-nitrosylation. The objective of this study was to examine the possibility that MTs undergo S-glutathionylation under nitrosative/oxidative stress. MT from rabbit liver was treated with different concns. of GSNO, diamide plus GSH or H2O2 plus GSH. Parallel sets of samples were treated with 10 mM DTT for 30 min at 37 °C to reduce mixed disulfides. Incubations were then processed for Western blot or dot-immunobinding assay. Western blot with anti-MT or anti-GSH were also performed on peripheral blood mononuclear cell exts. Structural aspects of S-glutathionylation of MTs were also examd. Treatment with GSNO, diamide/GSH or H2O2/GSH induced a dose-dependent increase in the levels of MT S-glutathionylation. This effect was completely reversed by treatment with the reducing agent DTT, indicating that S-glutathionylation of MT protein was related to formation of protein-mixed disulfides. Structural anal. of rat MT indicated that Cys residues located in the N-terminal domain of the protein are the likely targets for S-glutathionylation, both for their solvent accessibility and electrostatics induced reactivity. S-Glutathionylation of MT, given its reversibility, would provide protection from irreversible oxidn. of Cys residues, thus representing a mechanism of high potential biol. relevance.
- 106Zangger, K.; Öz, G.; Haslinger, E.; Kunert, O.; Armitage, I. M. Nitric oxide selectively releases metals from the amino-terminal domain of metallothioneins: potential role at inflammatory sites. FASEB J. 2001, 15, 1303– 1305, DOI: 10.1096/fj.00-0641fjeGoogle Scholar106Nitric oxide selectively releases metals from the N-terminal domain of metallothioneins: potential role at inflammatory sitesZangger, Klaus; Oz, Guilin; Haslinger, Ernst; Kunert, Olaf; Armitage, Ian M.FASEB Journal (2001), 15 (7), 1303-1305, 10.1096/fj/00-0641/fjeCODEN: FAJOEC; ISSN:0892-6638. (Federation of American Societies for Experimental Biology)Metallothioneins (MTs) and various other metal binding proteins release metals when exposed to nitric oxide (NO). We investigated the structural consequences of the interaction between MTs and NO by using 1H- and 113Cd-NMR spectroscopy and found that only the three metals from the N-terminal β-domain were selectively released whereas the C-terminal α-domain remains intact. Since it has been proposed that the β-domain is responsible for the postulated role of MTs in zinc homeostasis, whereas the tight binding of metals in the α-domain appears to play a role in heavy metal detoxification, our results suggest a potential regulatory role of NO in zinc distribution. Specifically, we present a mechanism whereby MT counteracts the cytotoxic effects of NO at inflammatory sites.
- 107Barbato, J. C.; Catanescu, O.; Murray, K.; DiBello, P. M.; Jacobsen, D. W. Targeting of metallothionein by L-homocysteine. A novel mechanism for disruption of zinc and redox homeostasis. Arterioscler., Thromb., Vasc. Biol. 2007, 27, 49– 54, DOI: 10.1161/01.ATV.0000251536.49581.8aGoogle Scholar107Targeting of metallothionein by L-homocysteine: a novel mechanism for disruption of zinc and redox homeostasisBarbato John C; Catanescu Otilia; Murray Kelsey; DiBello Patricia M; Jacobsen Donald WArteriosclerosis, thrombosis, and vascular biology (2007), 27 (1), 49-54 ISSN:.OBJECTIVE: L-homocysteine and/or L-homocystine interact in vivo with albumin and other extracellular proteins by forming mixed-disulfide conjugates. Because of its extremely rich cysteine content, we hypothesized that metallothionein, a ubiquitous intracellular zinc-chaperone and superoxide anion radical scavenger, reacts with L-homocysteine and that homocysteinylated-metallothionein suffers loss of function. METHODS AND RESULTS: 35S-homocysteinylated-metallothionein was resolved in lysates of cultured human aortic endothelial cells in the absence and presence of reduced glutathione by SDS-PAGE and identified by Western blotting and phosphorimaging. Using zinc-Sepharose chromatography, L-homocysteine was shown to impair the zinc-binding capacity of metallothionein even in the presence of reduced glutathione. L-Homocysteine induced a dose-dependent increase in intracellular free zinc in zinquin-loaded human aortic endothelial cells within 30 minutes, followed by the appearance of early growth response protein-1 within 60 minutes. In addition, intracellular reactive oxygen species dramatically increased 6 hours after L-homocysteine treatment. In vitro studies demonstrated that L-homocysteine is a potent inhibitor of the superoxide anion radical scavenging ability of metallothionein. CONCLUSIONS: These studies provide the first evidence that L-homocysteine targets intracellular metallothionein by forming a mixed-disulfide conjugate and that loss of function occurs after homocysteinylation. The data support a novel mechanism for disruption of zinc and redox homeostasis.
- 108Aizenman, E.; Stout, A. K.; Hartnett, K. A.; Dineley, K. E.; McLaughlin, B.; Reynolds, I. J. Induction of neuronal apoptosis by thiol oxidation: Putative role of intracellular zinc release. J. Neurochem. 2000, 75, 1878– 1888, DOI: 10.1046/j.1471-4159.2000.0751878.xGoogle Scholar108Induction of neuronal apoptosis by thiol oxidation: putative role of intracellular zinc releaseAizenman, Elias; Stout, Amy K.; Hartnett, Karen A.; Dineley, Kirk E.; McLaughlin, BethAnn; Reynolds, Ian J.Journal of Neurochemistry (2000), 75 (5), 1878-1888CODEN: JONRA9; ISSN:0022-3042. (Lippincott Williams & Wilkins)The membrane-permeant oxidizing agent 2,2'-dithiodipyridine (DTDP) can induce Zn2+ release from metalloproteins in cell-free systems. Here, we report that brief exposure to DTDP triggers apoptotic cell death in cultured neurons, detected by the presence of both DNA laddering and asym. chromatin formation. Neuronal death was blocked by increased extracellular potassium levels, by tetraethylammonium, and by the broad-spectrum cysteine protease inhibitor butoxy-carbonyl-aspartate-fluoromethylketone. N,N,N',N'-Tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN) and other cell-permeant metal chelators also effectively blocked DTDP-induced toxicity in neurons. Cell death, however, was not abolished by the NMDA receptor blocker MK-801, by the intracellular calcium release antagonist dantrolene, or by high concns. of ryanodine. DTDP generated increases in fluorescence signals in cultured neurons loaded with the zinc-selective dye Newport Green. The fluorescence signals following DTDP treatment also increased in fura-2- and magfura-2-loaded neurons. These responses were completely reversed by TPEN, consistent with a DTDP-mediated increase in intracellular free Zn2+ concns. Our studies suggest that under conditions of oxidative stress, Zn2+ released from intracellular stores may contribute to the initiation of neuronal apoptosis.
- 109Fass, D.; Thorpe, C. Chemistry and enzymology of disulfide cross-linking in proteins. Chem. Rev. 2018, 118, 1169– 1198, DOI: 10.1021/acs.chemrev.7b00123Google Scholar109Chemistry and Enzymology of Disulfide Cross-Linking in ProteinsFass, Deborah; Thorpe, ColinChemical Reviews (Washington, DC, United States) (2018), 118 (3), 1169-1198CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)A review. Cysteine thiols are among the most reactive functional groups in proteins, and their pairing in disulfide linkages is a common post-translational modification in proteins entering the secretory pathway. This modest amino acid alteration, the mere removal of a pair of hydrogen atoms from juxtaposed cysteine residues, contrasts with the substantial changes that characterize most other post-translational reactions. However, the wide variety of proteins that contain disulfides, the profound impact of crosslinking on the behavior of the protein polymer, the numerous and diverse players in intracellular pathways for disulfide formation, and the distinct biol. settings in which disulfide bond formation can take place belie the simplicity of the process. Here we lay the groundwork for appreciating the mechanisms and consequences of disulfide bond formation in vivo by reviewing chem. principles underlying cysteine pairing and oxidn. We then show how enzymes tune redox-active cofactors and recruit oxidants to improve the specificity and efficiency of disulfide formation. Finally, we discuss disulfide bond formation in a cellular context and identify important principles that contribute to productive thiol oxidn. in complex, crowded, dynamic environments.
- 110Hu, H. Y.; Cheng, H. Q.; Li, Q.; Zou, Y. S.; Xu, G. J. Study of the redox properties of metallothionein in vitro by reacting with DsbA protein. J. Protein Chem. 1999, 18, 665– 670, DOI: 10.1023/A:1020654206878Google Scholar110Study of the redox properties of metallothionein in vitro by reacting with DsbA proteinHu, H. Y.; Cheng, H. Q.; Li, Q.; Zou, Y. S.; Xu, G. J.Journal of Protein Chemistry (1999), 18 (6), 665-670CODEN: JPCHD2; ISSN:0277-8033. (Kluwer Academic/Plenum Publishers)Mammalian metallothionein (MT) contains 20 cysteine residues involved in the two metal clusters without a disulfide bond. The redox reaction of the Cys thiols was proposed to be assocd. with the metal distribution of MT. The E. coli DsbA protein is extremely active in facilitating thiol/disulfide exchange both in vivo and in vitro. To further investigate the redox properties of MT, reaction between MT and DsbA was carried out in vitro by fluorescence detection. Equil. characterization indicates that the reaction is stoichiometric (1:1) under certain conditions. Kinetic study gives a rate const. of the redox reaction of 4.42 × 105 sec-1 M-1, which is 103-fold larger than that of glutathione reacting with DsbA. Metal-free MT (apo-MT) shows a higher equil. redn. potential than MT, but exhibits an indistinguishable kinetic rate. Oxidn. of MT by DsbA leads to metal release from the clusters. The characteristic fluorescence increase during redn. of DsbA may provide a sensitive probe for exploring the redox properties of some reductants of biol. interest. The result also implies that oxidn. of Cys thiols may influence the metal release or delivery from MT.
- 111Jacob, C.; Maret, W.; Vallee, B. L. Selenium redox biochemistry of zinc/sulfur coordination sites in proteins and enzymes. Proc. Natl. Acad. Sci. U. S. A. 1999, 96, 1910– 1914, DOI: 10.1073/pnas.96.5.1910Google Scholar111Selenium redox biochemistry of zinc-sulfur coordination sites in proteins and enzymesJacob, Claus; Maret, Wolfgang; Vallee, Bert L.Proceedings of the National Academy of Sciences of the United States of America (1999), 96 (5), 1910-1914CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Selenium has been increasingly recognized as an essential element in biol. and medicine. Its biochem. resembles that of sulfur, yet differs from it by virtue of both redox potentials and stabilities of its oxidn. states. Selenium can substitute for the more ubiquitous sulfur of cysteine and as such plays an important role in more than a dozen selenoproteins. We have chosen to examine zinc-sulfur centers as possible targets of selenium redox biochem. Selenium compds. release zinc from zinc/thiolate-coordination environments, thereby affecting the cellular thiol redox state and the distribution of zinc and likely of other metal ions. Arom. selenium compds. are excellent spectroscopic probes of the otherwise relatively unstable functional selenium groups. Zinc-coordinated thiolates, e.g., metallothionein (MT), and uncoordinated thiolates, e.g., glutathione, react with benzeneseleninic acid (oxidn. state +2), benzeneselenenyl chloride (oxidn. state 0) and selenocystamine (oxidn. state -1). Benzeneseleninic acid and benzeneselenenyl chloride react very rapidly with MT and titrate substoichiometrically and with a 1:1 stoichiometry, resp. Selenium compds. also catalyze the release of zinc from MT in peroxidn. and thiol/disulfide-interchange reactions. The selenoenzyme glutathione peroxidase catalytically oxidizes MT and releases zinc in the presence of t-Bu hydroperoxide, suggesting that this type of redox chem. may be employed in biol. for the control of metal metab. Moreover, selenium compds. are likely targets for zinc/thiolate coordination centers in vivo, because the reactions are only partially suppressed by excess glutathione. This specificity and the potential to undergo catalytic reactions at low concns. suggests that zinc release is a significant aspect of the therapeutic antioxidant actions of selenium compds. in antiinflammatory and anticarcinogenic agents.
- 112Chen, Y.; Maret, W. Catalytic oxidation of zinc/sulfur coordination sites in proteins by selenium compounds. Antioxid. Redox Signaling 2001, 3, 651– 656, DOI: 10.1089/15230860152542998Google Scholar112Catalytic oxidation of zinc/sulfur coordination sites in proteins by selenium compoundsChen, Yu; Maret, WolfgangAntioxidants & Redox Signaling (2001), 3 (4), 651-656CODEN: ARSIF2; ISSN:1523-0864. (Mary Ann Liebert, Inc.)Zinc/thiolate (cysteine) coordination occurs in a very large no. of proteins. These coordination sites are thermodynamically quite stable. Yet the redox chem. of thiolate ligands confers extraordinary reactivities on these sites. The significance of such ligand-centered reactions is that they affect the binding and release of zinc, thus helping to distribute zinc, and perhaps controlling zinc-dependent cellular events. One new aspect focuses on the thiolate ligands of zinc as targets for the redox action of selenium compds. A distinctive feature of this chem. is the capacity of selenols to catalyze the oxidn. of zinc/thiolate sites. We here use a chromophoric compd., 2-nitrophenylselenocyanate, to investigate its reaction mechanism with the zinc/thiolate clusters of metallothionein, a protein that is a cellular reservoir for zinc and together with its apoprotein, thionein, is involved in zinc distribution as a zinc donor/acceptor pair. The reaction is particularly revealing as it occurs in two steps. A selenenylsulfide intermediate is formed in the fast oxidative step, followed by the generation of 2-nitrophenylselenol that initiates the second, catalytic step. The findings demonstrate the high reactivity of selenium compds. with zinc/thiolate coordination sites and the potent catalytic roles that selenoproteins and selenium redox drugs may have in affecting gene expression via modulation of the zinc content of zinc finger proteins.
- 113Chen, Y.; Maret, W. Catalytic selenols couple the redox cycles of metallothionein and glutathione. Eur. J. Biochem. 2001, 268, 3346– 3353, DOI: 10.1046/j.1432-1327.2001.02250.xGoogle Scholar113Catalytic selenols couple the redox cycles of metallothionein and glutathioneChen, Yu; Maret, WolfgangEuropean Journal of Biochemistry (2001), 268 (11), 3346-3353CODEN: EJBCAI; ISSN:0014-2956. (Blackwell Science Ltd.)Co-ordination of zinc to the thiol group of cysteine allows mobilization of zinc through oxidn. of its ligand. This mol. property links the binding and release of zinc in metallothionein (MT) to the cellular redox state. Biol. disulfides such as glutathione disulfide (GSSG) oxidize MT with concomitant release of zinc, while glutathione (GSH) reduces the oxidized protein to thionein, which then binds to available zinc. Neither of these two redox processes is very efficient, even at high concns. of GSSG or GSH. However, the GSH/GSSG redox pair can efficiently couple with the MT/thionein system in the presence of a selenium compd. that has the capacity to form a catalytic selenol(ate). This coupling provides a very effective means of modulating oxidn. and redn. Remarkably, selenium compds. catalyze the oxidn. of MT even under overall reducing conditions such as those prevailing in the cytosol. In this manner, the binding and release of zinc from zinc-thiolate co-ordination sites is linked to redox catalysis by selenium compds., changes in the glutathione redox state, and the availability of either a zinc donor or a zinc acceptor. The results also suggest that the pharmacol. actions of selenium compds. in cancer prevention and other antiviral and anti-inflammatory therapeutic applications, as well as unknown functions of selenium-contg. proteins, may relate to coupling between the thiol redox state and the zinc state.
- 114Zhang, S.; Li, J.; Wang, C.-C.; Tsou, C.-L. Metal regulation of metallothionein participation in redox reactions. FEBS Lett. 1999, 462, 383– 386, DOI: 10.1016/S0014-5793(99)01562-8Google Scholar114Metal regulation of metallothionein participation in redox reactionsZhang, S.; Li, J.; Wang, C.-C.; Tsou, C.-L.FEBS Letters (1999), 462 (3), 383-386CODEN: FEBLAL; ISSN:0014-5793. (Elsevier Science B.V.)Like glutathione or dithiothreitol, metallothionein effects the formation of pancreatic RNase A from its S-sulfonated deriv. catalyzed by protein disulfide isomerase. EDTA increases the yield of RNase A activity recovery with metallothionein but does not affect the reaction with glutathione or dithiothreitol. EDTA also increases the reactivity of thiol groups in metallothionein with 5,5'-dithiobis-(2-nitrobenzoic acid) by chelation of zinc ions. It is suggested that the thiol groups in metallothionein form a part of the pool of cellular thiols in the regulation of cellular redox reactions and their availability is modulated by zinc chelation.
- 115Sagher, D.; Brunell, D.; Hejtmancik, J. F.; Kantorow, M.; Brot, N.; Weissbach, H. Thionein can serve as a reducing agent for the methionine sulfoxide reductases. Proc. Natl. Acad. Sci. U. S. A. 2006, 103, 8656– 8661, DOI: 10.1073/pnas.0602826103Google Scholar115Thionein can serve as a reducing agent for the methionine sulfoxide reductasesSagher, Daphna; Brunell, David; Hejtmancik, J. Fielding; Kantorow, Marc; Brot, Nathan; Weissbach, HerbertProceedings of the National Academy of Sciences of the United States of America (2006), 103 (23), 8656-8661CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)It has been generally accepted, primarily from studies on methionine sulfoxide reductase (Msr) A, that the biol. reducing agent for the members of the Msr family is reduced thioredoxin (Trx), although high levels of DTT can be used as the reductant in vitro. Preliminary expts. using both human recombinant MsrB2 (hMsrB2) and MsrB3 (hMsrB3) showed that although DTT can function in vitro as the reducing agent, Trx works very poorly, prompting a more careful comparison of the ability of DTT and Trx to function as reducing agents with the various members of the Msr family. Escherichia coli MsrA and MsrB and bovine MsrA efficiently use either Trx or DTT as reducing agents. In contrast, hMsrB2 and hMsrB3 show <10% of the activity with Trx as compared with DTT, raising the possibility that, in animal cells, Trx may not be the direct hydrogen donor or that there may be a Trx-independent reducing system required for MsrB2 and MsrB3 activity. A heat-stable protein has been detected in bovine liver that, in the presence of EDTA, can support the Msr reaction in the absence of either Trx or DTT. This protein has been identified as a zinc-contg. metallothionein (Zn-MT). The results indicate that thionein (T), which is formed when the zinc is removed from Zn-MT, can function as a reducing system for the Msr proteins because of its high content of cysteine residues and that Trx can reduce oxidized T.
- 116Sagher, D.; Brunell, D.; Brot, N.; Vallee, B. L.; Weissbach, H. Selenocompounds can serve as oxidoreductants with the methionine sulfoxide reductase enzymes. J. Biol. Chem. 2006, 281, 31184– 31187, DOI: 10.1074/jbc.M606962200Google Scholar116Selenocompounds Can Serve as Oxidoreductants with the Methionine Sulfoxide Reductase EnzymesSagher, Daphna; Brunell, David; Brot, Nathan; Vallee, Bert L.; Weissbach, HerbertJournal of Biological Chemistry (2006), 281 (42), 31184-31187CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)In a recent study on the reducing requirement for the methionine sulfoxide reductases (Msr) (Sagher, D., Brunell, D., Hejtmancik, J. F., Kantorow, M., Brot, N. & Weissbach, H. (2006) PROC: Natl. Acad. Sci. U. S. A. 103, 8656-8661), we have shown that thioredoxin, although an excellent reducing system for Escherichia coli MsrA and MsrB and bovine MsrA, is not an efficient reducing agent for either human MsrB2 (hMsrB2) or human MsrB3 (hMsrB3). In a search for another reducing agent for hMsrB2 and hMsrB3, it was recently found that thionein, the reduced, metal-free form of metallothionein, could function as a reducing system for hMsrB3, with weaker activity using hMsrB2. In the present study, we provide evidence that some selenium compds. are potent reducing agents for both hMsrB2 and hMsrB3.
- 117Maret, W. Regulation of cellular zinc ions and their signalling functions. In Zinc signalling; Fukada, T., Kambe, T., Eds.; 2019, Springer: Singapore; pp 5– 22.Google ScholarThere is no corresponding record for this reference.
- 118Kröncke, K. D.; Fehsel, K.; Schmidt, T.; Zenke, F. T.; Dasting, I.; Wesener, J. R.; Bettermann, H.; Breunig, K. D.; Kolb-Bachofen, V. Nitric oxide destroys zinc-sulfur clusters inducing zinc release from metallothionein and inhibition of the zinc-finger type yeast transcription activator LAC9. Biochem. Biophys. Res. Commun. 1994, 200, 1105– 1110, DOI: 10.1006/bbrc.1994.1564Google Scholar118Nitric oxide destroys zinc-sulfur clusters inducing zinc release from metallothionein and inhibition of the zinc finger-type yeast transcription activator LAC9Kroncke K D; Fehsel K; Schmidt T; Zenke F T; Dasting I; Wesener J R; Bettermann H; Breunig K D; Kolb-Bachofen VBiochemical and biophysical research communications (1994), 200 (2), 1105-10 ISSN:0006-291X.Nitric oxide, generated from S-nitrosocysteine or applied as gas mediates metal ion release from the Zn2+/Cd(2+)-complexing protein metallothionein via oxidation of SH-groups. Time-dependent S-nitrosylation and subsequent disulfide formation of metallothionein are demonstrated. Furthermore, nitric oxide inhibits DNA binding activity of the yeast transcription factor LAC9 containing a zinc finger like DNA binding domain. These results show that nitric oxide interacts with and destroys zinc-sulfur clusters in proteins.
- 119St Croix, C. M.; Wasserloos, K. J.; Dineley, K. E.; Reynolds, I. J.; Levitan, E. S.; Pitt, B. R. Nitric oxide-induced changes in intracellular zinc homeostasis are mediated by metallothionein/thionein. Am. J. Physiol. Lung Cell. Mol. Physiol. 2002, 282, L185– L192, DOI: 10.1152/ajplung.00267.2001Google Scholar119Nitric oxide-induced changes in intracellular zinc homeostasis are mediated by metallothionein/thioneinSt. Croix, Claudette M.; Wasserloos, K. J.; Dineley, K. E.; Reynolds, I. J.; Levitan, E. S.; Pitt, B. R.American Journal of Physiology (2002), 282 (2, Pt. 1), L185-L192CODEN: AJPHAP; ISSN:0002-9513. (American Physiological Society)We hypothesized that metallothionein (MT), a cysteine-rich protein with a strong affinity for Zn2+, plays a role in nitric oxide (NO) signaling events via sequestration or release of Zn2+ by the unique thiolate clusters of the protein. Exposing mouse lung fibroblasts (MLF) to the NO donor S-nitrosocysteine resulted in 20-30% increases in fluorescence of the Zn2+-specific fluorophore Zinquin that were rapidly reversed by the Zn2+ chelator N,N,N'N'-tetrakis-(2-pyridylmethyl)ethylenediamine. The absence of a NO-mediated increase in labile Zn2+ in MLF from MT knockouts and its restoration after MT complementation by adenoviral gene transfer inferred a crit. role for MT in the regulation of Zn2+ homeostasis by NO. Addnl. data obtained in sheep pulmonary artery endothelial cells suggested a role for the apo form of MT, thionein (T), as a Zn2+-binding protein in intact cells, as overexpression of MT caused inhibition of NO-induced changes in labile Zn2+ that were reversed by Zn2+ supplementation. Furthermore, fluorescence-resonance energy-transfer data showed that overexpression of green fluorescent protein-modified MT prevented NO-induced conformational changes, which are indicative of Zn2+ release from thiolate clusters. This effect was restored by Zn2+ supplementation. Collectively, these data show that MT mediates NO-induced changes in intracellular Zn2+ and suggest that the ratio of MT to T can regulate Zn2+ homeostasis in response to nitrosative stress.
- 120Spahl, D. U.; Berendji-Grün, D.; Suschek, C. V.; Kolb-Bachofen, V.; Kröncke, K. D. (2003) Regulation of zinc homeostasis by inducible NO synthase-derived NO: nuclear metallothionein translocation and intranuclear Zn2+ release. Proc. Natl. Acad. Sci. U. S. A. 2003, 100, 13952– 13957, DOI: 10.1073/pnas.2335190100Google Scholar120Regulation of zinc homeostasis by inducible NO synthase-derived NO: Nuclear metallothionein translocation and intranuclear Zn2+ releaseSpahl, Daniela U.; Berendji-gruen, Denise; Suschek, Christoph V.; Kolb-bachofen, Victoria; Kroencke, Klaus -D.Proceedings of the National Academy of Sciences of the United States of America (2003), 100 (24), 13952-13957CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Zn2+ is crit. for the functional and structural integrity of cells and contributes to a no. of important processes including gene expression. It has been shown that NO exogenously applied via NO donors resulting in nitrosative stress leads to cytoplasmic Zn2+ release from the zinc storing protein metallothionein (MT) and probably other proteins that complex Zn2+ via cysteine thiols. We show here that, in cytokine-activated murine aortic endothelial cells, NO derived from the inducible NO synthase (iNOS) induces a transient nuclear release of Zn2+. This nuclear Zn2+ release depends on the presence of MT as shown by the lack of this effect in activated endothelial cells from MT-deficient mice and temporally correlates with nuclear MT translocation. Data also show that NO is an essential but not sufficient signal for MT-mediated Zn2+ trafficking from the cytoplasm into the nucleus. In addn., we found that, endogenously via iNOS, synthesized NO increases the constitutive mRNA expression of both MT-1 and MT-2 genes and that nitrosative stress exogenously applied via an NO donor increases constitutive MT mRNA expression via intracellular Zn2+ release. In conclusion, we here provide evidence for a signaling mechanism based on iNOS-derived NO through the regulation of intracellular Zn2+ trafficking and homeostasis.
- 121Chen, Y.; Irie, Y.; Keung, W. M.; Maret, W. S-Nitrosothiols react preferentially with zinc thiolate clusters of metallothionein III through transnitrosation. Biochemistry 2002, 41, 8360– 8367, DOI: 10.1021/bi020030+Google Scholar121S-Nitrosothiols React Preferentially with Zinc Thiolate Clusters of Metallothionein III through TransnitrosationChen, Yu; Irie, Yoshifumi; Keung, Wing Ming; Maret, WolfgangBiochemistry (2002), 41 (26), 8360-8367CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)Metallothionein (MT) is a two-domain protein with zinc thiolate clusters that bind and release zinc depending on the redox states of the sulfur ligands. Since S-nitrosylation of cysteine is considered a prototypic cellular redox signaling mechanism, we here investigate the reactions of S-nitrosothiols with different isoforms of MT. MT-III is significantly more reactive than MT-I/II toward S-nitrosothiols, whereas the reactivity of all three isoforms toward reactive oxygen species is comparable. A cellular system, in which all three MTs are similarly effective in protecting rat embryonic cortical neurons in primary culture against hydrogen peroxide but where MT-III has a much more pronounced effect of protecting against S-nitrosothiols, confirms this finding. MT-III is the only isoform with consensus acid-base sequence motifs for S-nitrosylation in both domains. Studies with synthetic and zinc-reconstituted domain peptides demonstrate that S-nitrosothiols indeed release zinc from both the α- and the β-domain of MT-III. S-Nitrosylation occurs via transnitrosation, a mechanism that differs fundamentally from that of previous studies of reactions of MT with NO•. Our data demonstrate that zinc thiolate bonds are targets of S-nitrosothiol signaling and further indicate that MT-III is biol. specific in converting NO signals to zinc signals. This could bear importantly on the physiol. action of MT-III, whose biol. activity as a neuronal growth inhibitory factor is unique, and for brain diseases that have been related to oxidative or nitrosative stress.
- 122Zhang, L.-M.; Croix, C. S.; Cao, R.; Wasserloos, K.; Watkins, S. C.; Stevens, T.; Li, S.; Tyurin, V.; Kagan, V. E.; Pitt, B. R. Cell-surface protein disulfide isomerase is required for transnitrosation of metallothionein by S-nitroso-albumin in intact rat pulmonary vascular endothelial cells. Exp. Biol. Med. 2006, 231, 1507– 1515, DOI: 10.1177/153537020623100909Google Scholar122Cell-surface protein disulfide isomerase is required for transnitrosation of metallothionein by S-nitroso-albumin in intact rat pulmonary vascular endothelial cellsZhang, Li-Ming; St. Croix, Claudette; Cao, Rong; Wasserloos, Karla; Watkins, Simon C.; Stevens, Troy; Li, Song; Tyurin, Vladimir; Kagan, Valerian E.; Pitt, Bruce R.Experimental Biology and Medicine (Maywood, NJ, United States) (2006), 231 (9), 1507-1515CODEN: EBMMBE; ISSN:1535-3702. (Society for Experimental Biology and Medicine)S-nitrosation of the metal binding protein, metallothionein (MT) appears to be a crit. link in affecting endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS)-derived nitric oxide (NO)-induced changes in cytoplasmic and nuclear labile zinc, resp. Although low mol. wt. S-nitrosothiols also appear to affect this signaling system, less is known about the ability of extracellular protein nitrosothiols to transnitrosate MT. Accordingly, we synthesized fluorescently labeled S-nitroso-albumin (SNO-albumin, a major protein S-nitrosothiol in plasma) and detd., via confocal microscopy in fixed tissue, that it is transported into cultured rat pulmonary vascular endothelial cells in a temp. sensitive fashion. The cells were transfected with an expression vector that encodes human MT-IIa cDNA sandwiched between enhanced cyan (donor) and yellow (acceptor) fluorescent proteins (FRET-MT) that can detect conformational changes in MT through fluorescence resonance energy transfer (FRET). SNO-albumin and the membrane-permeant low mol. wt. S-nitroso-L-cysteine Et ester (L-SNCEE) caused a conformational change in FRET-MT as ascertained by full spectral laser scanning confocal microscopy in live rat pulmonary vascular endothelial cells, a result which is consistent with transnitrosation of the reporter mol. Transnitrosation of FRET-MT by SNO-albumin, but not L-SNCEE, was sensitive to antisense oligonucleotide-mediated inhibition of the expression of cell surface protein disulfide isomerase (csPDI). These results extend the original observations of Ramachandran et al. and suggest that csPDI-mediated denitrosation helps to regulate the ability of the major plasma NO carrier (SNO-albumin) to transnitrosate endothelial cell mol. targets (e.g. MT).
- 123Stoyanovsky, D. M.; Tyurina, Y. Y.; Tyurin, V. A.; Anand, D.; Mandavia, D. N.; Gius, D.; Ivanova, J.; Pitt, B.; Billiar, T. R.; Kagan, V. E. Thioredoxin and lipoic acid catalyse the denitrosation of low molecular weight and protein S-nitrosothiols. J. Am. Chem. Soc. 2005, 127, 15815– 15823, DOI: 10.1021/ja0529135Google Scholar123Thioredoxin and Lipoic Acid Catalyze the Denitrosation of Low Molecular Weight and Protein S-NitrosothiolsStoyanovsky, Detcho A.; Tyurina, Yulia Y.; Tyurin, Vladimir A.; Anand, Deepthi; Mandavia, Dhara N.; Gius, David; Ivanova, Juliana; Pitt, Bruce; Billiar, Timothy R.; Kagan, Valerian E.Journal of the American Chemical Society (2005), 127 (45), 15815-15823CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)The nitrosation of cellular thiols has attracted much interest as a regulatory mechanism that mediates some of the pathophysiol. effects of nitric oxide (NO). In cells, virtually all enzymes contain cysteine residues that can be subjected to S-nitrosation, whereby this process often acts as an activity switch. Nitrosation of biol. thiols is believed to be mediated by N2O3, metal-nitrosyl complexes, and peroxynitrite. To date, however, enzymic pathways for S-denitrosation of proteins have not been identified. Herein, we present exptl. evidence that two ubiquitous cellular dithiols, thioredoxin and dihydrolipoic acid, catalyze the denitrosation of S-nitrosoglutathione, S-nitrosocaspase 3, S-nitrosoalbumin, and S-nitrosometallothionenin to their reduced state with concomitant generation of nitroxyl (HNO), the one-electron redn. product of NO. In these reactions, formation of NO and HNO was assessed by ESR spectrometry, potentiometric measurements, and quantification of hydroxylamine and sodium nitrite as end reaction products. Nitrosation and denitrosation of caspase 3 was correlated with its proteolytic activity. We also report that thioredoxin-deficient HeLa cells with mutated thioredoxin reductase denitrosate S-nitrosothiols less efficiently. We conclude that both thioredoxin and dihydrolipoic acid may be involved in the regulation of cellular S-nitrosothiols.
- 124Aravindakumar, C. T.; Ceulemans, J.; De Ley, M. Nitric oxide induces Zn2+ release from metallothionein by destroying zinc-sulphur clusters without concomitant formation of S-nitrosothiol. Biochem. J. 1999, 344, 253– 258, DOI: 10.1042/bj3440253Google Scholar124Nitric oxide induces Zn2+ release from metallothionein by destroying zinc-sulphur clusters without concomitant formation of S-nitrosothiolAravindakumar, Charuvila T.; Ceulemans, Jan; De Ley, MarcBiochemical Journal (1999), 344 (1), 253-258CODEN: BIJOAK; ISSN:0264-6021. (Portland Press Ltd.)The reaction of nitric oxide (NO) with metallothionein (MT) has been investigated at neutral pH under strictly anaerobic conditions. It is obsd. that NO mediates zinc release from MT by destroying zinc-sulfur clusters, but that it does not by itself S-nitrosylate MT in contrast to common belief. Zinc release and loss of thiolate groups under anaerobic conditions is found to be much slower than under aerobic conditions. The obsd. percentage loss of Zn2+ and thiolate groups after 3 h of NO treatment are 62 and 39%, resp. The reaction of NO with cysteine is reinvestigated and it is found that cysteine is quant. converted to cystine after 5 min of NO treatment at pH 7.3. At lower pH, a much lower rate of conversion is obsd. confirming the base-catalyzed nature of the reaction of NO with thiols. On the basis of these results, a reaction mechanism involving electrophilic attack of NO on thiolate groups and subsequent formation of a nitrogen-centered radical, MTSNOH, as intermediate is proposed for the reaction of NO with MT that leads to zinc release.
- 125Misra, R. R.; Hochadel, J. F.; Smith, G. T.; Cook, J. C.; Waalkes, M. P.; Wink, D. A. Evidence that nitric oxide enhances cadmium toxicity by displacing the metal from metallothionein. Chem. Res. Toxicol. 1996, 9, 326– 332, DOI: 10.1021/tx950109yGoogle Scholar125Evidence That Nitric Oxide Enhances Cadmium Toxicity by Displacing the Metal from MetallothioneinMisra, R. Rita; Hochadel, James F.; Smith, George T.; Cook, John C.; Waalkes, Michael P.; Wink, David A.Chemical Research in Toxicology (1996), 9 (1), 326-32CODEN: CRTOEC; ISSN:0893-228X. (American Chemical Society)When growing Chinese hamster ovary cells were treated for 24 h with 0.5, 0.75, or 1.0 mM CdCl2 followed by a 1-h exposure to 1.0, 1.5, or 2.0 mM 1,1-diethyl-2-hydroxy-2-nitrosohydrazine (DEA/NO), an NO-generating sodium salt, NO enhanced Cd-induced inhibition of colony forming ability without affecting Cd-induced cytolethality. In expts. designed to det. whether NO acts by displacing Cd from cellular metallothionein (MT), cells treated with 2.0 mM CdCl2 followed by 1.5 or 3.0 mM DEA/NO exhibited 29 and 38% redns., resp., in the amt. of Cd bound to MT. When purified rat liver MT was used to further characterize NO-induced release of Cd from MT, dose-related increases in Cd displacement were obsd. at DEA/NO concns. between 0.1 and 0.5 mM, and a plateau was reached at 3 mol of Cd displaced/mol of MT at higher DEA/NO concns. Compared to cells exposed to Cd or DEA/NO alone, cells treated with Cd followed by DEA/NO also exhibited a transient 2-3-fold decrease in c-myc proto-oncogene expression. Taken together, our results support the hypothesis that NO mediates Cd release from MT in vivo and suggest that intracellular generation of free Cd may induce DNA damage and force cells into a period of growth arrest. Such findings may have particular relevance with regard to the etiol. of Cd-induced carcinogenesis in human populations.
- 126Fabisiak, J. P.; Tyurin, V. A.; Tyurina, Y. Y.; Borisenko, G. G.; Korotaeva, A.; Pitt, B. R.; Lazo, J. S.; Kagan, V. E. Redox regulation of copper-metallothionein. Arch. Biochem. Biophys. 1999, 363, 171– 181, DOI: 10.1006/abbi.1998.1077Google Scholar126Redox Regulation of Copper-MetallothioneinFabisiak, James P.; Tyurin, Vladimir A.; Tyurina, Yulia Y.; Borisenko, Grigory G.; Korotaeva, Alexandra; Pitt, Bruce R.; Lazo, John S.; Kagan, Valerian E.Archives of Biochemistry and Biophysics (1999), 363 (1), 171-181CODEN: ABBIA4; ISSN:0003-9861. (Academic Press)Copper (Cu) is an essential element whose localization within cells must be carefully controlled to avoid Cu-dependent redox cycling. Metallothioneins (MTs) are cysteine-rich metal-binding proteins that exert cytoprotective effects during metal exposure and oxidative stress. The specific role of MTs, however, in modulating Cu-dependent redox cycling remains unresolved. Our studies utilized a chem. defined model system to study MT modulation of Cu-dependent redox cycling under reducing (Cu/ascorbate) and mild oxidizing (Cu/ascorbate + H2O2) conditions. In the presence of Cu and ascorbate, MT blocked Cu-dependent lipid oxidn. and ascorbyl radical formation with a stoichiometry corresponding to Cu/MT ratios ≤12. In the presence of H2O2 the degree of protection by MT was less and biol. oxidns. and radical formation were inhibited only up to Cu/MT ratios of 6. Phys. interaction of MT and Cu was measured by using low-temp. EPR of free Cu2+ in soln. The maximal amt. of EPR-silent Cu1+ (presumably in complex with MT) corresponded to 12 molar equivalents of Cu/MT under reducing conditions, but only 9 in the presence of H2O2. H2O2 modulated the ability of MT to protect HL-60 cells from Cu-induced cell death in a manner that correlated with the ability of MT to mitigate Cu-redox cycling in cell-free systems. Thus, optimal binding of Cu to MT is achieved under reducing conditions; however, a portion of this Cu appears releasable under oxidizing conditions. Release of free Cu from MT during oxidative stress could enhance the formation of reactive oxygen species and potentiate cellular damage. (c) 1999 Academic Press.
- 127Hao, Q.; Maret, W. Aldehydes release zinc from proteins. A pathway from oxidative stress/lipid peroxidation to cellular functions of zinc. FEBS J. 2006, 273, 4300– 4310, DOI: 10.1111/j.1742-4658.2006.05428.xGoogle Scholar127Aldehydes release zinc from proteins. A pathway from oxidative stress/lipid peroxidation to cellular functions of zincHao, Qiang; Maret, WolfgangFEBS Journal (2006), 273 (18), 4300-4310CODEN: FJEOAC; ISSN:1742-464X. (Blackwell Publishing Ltd.)Oxidative stress, lipid peroxidn., hyperglycemia-induced glycations and environmental exposures increase the cellular concns. of aldehydes. A novel aspect of the mol. actions of aldehydes, e.g. acetaldehyde and acrolein, is their reaction with the cysteine ligands of zinc sites in proteins and concomitant zinc release. Stoichiometric amts. of acrolein release zinc from zinc-thiolate coordination sites in proteins such as metallothionein and alc. dehydrogenase. Aldehydes also release zinc intracellularly in cultured human hepatoma (HepG2) cells and interfere with zinc-dependent signaling processes such as gene expression and phosphorylation. Thus both acetaldehyde and acrolein induce the expression of metallothionein and modulate protein tyrosine phosphatase activity in a zinc-dependent way. Since minute changes in the availability of cellular zinc have potent effects, zinc release is a mechanism of amplification that may account for many of the biol. effects of aldehydes. The zinc-releasing activity of aldehydes establishes relationships among cellular zinc, the functions of endogenous and xenobiotic aldehydes, and redox stress, with implications for pathobiochem. and toxicol. mechanisms.
- 128Miyazaki, I.; Asanuma, M.; Hozumi, H.; Miyoshi, K.; Sogawa, N. Protective effects of metallothionein against dopamine quinone-induced dopaminergic neurotoxicity. FEBS Lett. 2007, 581, 5003– 5008, DOI: 10.1016/j.febslet.2007.09.046Google Scholar128Protective effects of metallothionein against dopamine quinone-induced dopaminergic neurotoxicityMiyazaki, Ikuko; Asanuma, Masato; Hozumi, Hiroaki; Miyoshi, Ko; Sogawa, NorioFEBS Letters (2007), 581 (25), 5003-5008CODEN: FEBLAL; ISSN:0014-5793. (Elsevier B.V.)Dopamine (DA) quinone as DA neuron-specific oxidative stress conjugates with cysteine residues in functional proteins to form quinoproteins. Here, we examd. the effects of cysteine-rich metal-binding proteins, metallothionein (MT)-1 and -2, on DA quinone-induced neurotoxicity. MT quenched DA semiquinones in vitro. In dopaminergic cells, DA exposure increased quinoproteins and decreased cell viability; these were ameliorated by pretreatment with MT-inducer zinc. Repeated L-DOPA administration markedly elevated striatal quinoprotein levels and reduced the DA nerve terminals specifically on the lesioned side in MT-knockout parkinsonian mice, but not in wild-type mice. Our results suggested that intrinsic MT protects against L-DOPA-induced DA quinone neurotoxicity in parkinsonian mice by its quinone-quenching property.
- 129Gauthier, M. A.; Eibl, J. K.; Crispo, J. A.; Ross, G. M. Covalent arylation of metallothionein by oxidized dopamine products: a possible mechanism for zinc-mediated enhancement of dopaminergic neuron survival. Neurotoxic. Res. 2008, 14, 317– 328, DOI: 10.1007/BF03033856Google Scholar129Covalent arylation of metallothionein by oxidized dopamine products: a possible mechanism for zinc-mediated enhancement of dopaminergic neuron survivalGauthier, Michelle A.; Eibl, Joseph K.; Crispo, James A. G.; Ross, Gregory M.Neurotoxicity Research (2008), 14 (4), 317-328CODEN: NURRFI; ISSN:1029-8428. (F. P. Graham Publishing Co.)Metallothioneins are a group of low mol. wt. proteins which can be induced upon exposure to metal ions, including Zn2+. These cysteine-rich proteins are believed to have anti-oxidant-like properties due to their ability to scavenge free radicals with their multiple sulfhydryl groups. Dopamine is a neurotransmitter that can form toxic quinone and semi-quinone products in an oxidative environment. While Zn2+ is known to be toxic to some neuron subtypes, here we report a beneficial effect of Zn2+ on dopaminergic neurons and identify a mechanism through which metallothionein may scavenge toxic dopamine oxidn. products. Cultured embryonic neurons were treated with Zn2+, and the no. of dopaminergic neurons surviving after two or three weeks in culture was detd. We demonstrate that under these conditions metallothionein is upregulated and is able to form covalent arylation products with dopamine and 6-hydroxydopamine both in vitro and in culture. These expts. suggest that Zn2+ enhances the survival of dopaminergic neurons, and we propose that as a mechanism, upregulated metallothioneins form covalent adducts with both dopamine and 6-hydroxydopamine, resulting in the obsd. neuroprotective effect of Zn2+ on these cells. As Zn2+ homeostasis and modulation of metallothionein expression are often markers of neurodegeneration, these studies may have significant implications for understanding the underlying basis of degenerative diseases involving dopaminergic neurons, including Parkinson's disease.
- 130Maret, W. Metallothionein redox biology in the cytoprotective and cytotoxic functions of zinc. Exp. Gerontol. 2008, 43, 363– 369, DOI: 10.1016/j.exger.2007.11.005Google Scholar130Metallothionein redox biology in the cytoprotective and cytotoxic functions of zincMaret, WolfgangExperimental Gerontology (2008), 43 (5), 363-369CODEN: EXGEAB; ISSN:0531-5565. (Elsevier)A review. A crit. aspect of cellular zinc metab. is the tight control of the picomolar concns. of free zinc ions and their fluctuations to balance folding and misfolding of proteins, supply of thousands of zinc-requiring proteins with zinc, and dual functions of zinc as either a pro-oxidant or a pro-antioxidant. Zinc/sulfur (cysteine) bonds in proteins have a key role in this control because they generate redox-active coordination environments. Metallothionein (MT) is such a redox-active zinc protein, which couples biochem. to the cellular redox state. The coordination dynamics and redox state of its zinc/thiolate clusters det. cellular zinc availability. A fraction of MT in tissues and cells contains free thiols and disulfides. Thus, MT with seven zinc ions and twenty reduced thiols as characterized by high-resoln. 3D structures does not represent its biol. active form. Redox stress affects the zinc and redox buffering capacity of MT and elicits fluctuations of zinc ions that are potent effectors of multiple metabolic and signaling pathways. We are beginning to appreciate the sensitivity of cellular zinc homeostasis to perturbations, the clin. importance of linked zinc and redox imbalances for aging and the development of chronic diseases, and the tangible benefits of preventive and therapeutic nutritional interventions.
- 131Xiao, Z.; Wedd, A. G. The challenges of determining metal-protein affinities. Nat. Prod. Rep. 2010, 27, 768– 789, DOI: 10.1039/b906690jGoogle Scholar131The challenges of determining metal-protein affinitiesXiao, Zhi-Guang; Wedd, Anthony G.Natural Product Reports (2010), 27 (5), 768-789CODEN: NPRRDF; ISSN:0265-0568. (Royal Society of Chemistry)A review. A key property of metallo-proteins and -enzymes is the affinity of metal ion M for protein ligand P as defined by the dissocn. const. KD = [M][P]/[MP]. Its accurate detn. is essential for a quant. understanding of metal selection and speciation. However, the surfaces of proteins are defined by the sidechains of amino acids and so abound in good metal ligands (e.g., imidazole of histidine, thiol of cysteine, carboxylate of aspartic and glutamic acids, etc.). Consequently, adventitious binding of metal ions to protein surfaces is common with KD values ≥ 10-6 M. On the other hand, transport proteins responsible for chaperoning' essential metals to their cellular destinations appear to bind the metal ions selectively (KD < 10-7 M), both for speciation and to minimize the toxic effects of free' metal ions. These ions are normally bound with still higher affinities at their ultimate destinations (the active sites of metallo-proteins and -enzymes). This review surveys possible approaches to estn. of these dissocn. consts. and pinpoints the various problems assocd. with each approach.
- 132Pearson, R. G. Hard and soft acids and bases. J. Am. Chem. Soc. 1963, 85, 3533– 3539, DOI: 10.1021/ja00905a001Google Scholar132Hard and soft acids and basesPearson, Ralph G.Journal of the American Chemical Society (1963), 85 (22), 3533-9CODEN: JACSAT; ISSN:0002-7863.A number of Lewis acids of diverse types are classified as (a) or (b) following the criterion of Ahrland, et al. (CA 53, 960c). Other, auxiliary criteria are proposed. Class (a) acids prefer to bind to "hard" or nonpolarizable bases. Class (b) acids prefer to bind to "soft" or polarizable bases. Since class (a) acids are themselves "hard" and since class (b) acids are "soft" a simple, useful rule is proposed: hard acids bind strongly to hard bases and soft acids bind strongly to soft bases. The explanations for such behavior include: (1) various degrees of ionic and covalent σ-bonding; (2) π-bonding; (3) electron correlation phenomena; (4) solvation effects.
- 133Ejnik, J.; Robinson, J.; Zhu, J.; Forsterling, H.; Shaw, C. F.; Petering, D. H. Folding pathway of apo-metallothionein induced by Zn2+, Cd2+ and Co2+. J. Inorg. Biochem. 2002, 88, 144– 152, DOI: 10.1016/S0162-0134(01)00393-2Google Scholar133Folding pathway of apo-metallothionein induced by Zn2+, Cd2+ and Co2+Ejnik, John; Robinson, James; Zhu, Jianyu; Forsterling, Holger; Shaw, C. Frank; Petering, David H.Journal of Inorganic Biochemistry (2002), 88 (2), 144-152CODEN: JIBIDJ; ISSN:0162-0134. (Elsevier Science Inc.)Metal ion binding to the sulfhydryl groups of apometallothionein (apo-MT) causes both the formation of native metal-thiolate clusters and the folding of the polypeptide chain of each domain. Cd2+ and Zn2+ react with apo-MT to form metal-thiolate bonds in reactions that are complete within milliseconds and which are pH-dependent. Dual mixing expts. were conducted that involve the initial reaction of metal ion and apo-MT followed by mixing with 5,5'-N-dithio-bis(2-nitrobenzoate) or EDTA after 26 ms. They showed that structures had formed within the brief reaction period which were resistant to rapid reaction with reagents that interact with sulfhydryl groups or metal ions, resp. It was concluded that native metallothionein domains had been constituted within this brief period. Apo-MT was also titrated with Co2+ to yield Con-MT (n=1-7). Initially, Co2+ bound to independent, tetrahedral thiolate sites. Spectrophotometric anal. of the titrn. suggested that the independent Co(II) sites began to coalesce into clusters at n=4 (pH 7.2) or n=5 (pH 8.4). Back titrn. of free sulfhydryl groups (S) in Con-MT (n=1-7) with iodoacetamide at pH 7.2 confirmed that clustering began at n=4. Upon conversion of these alkylated structures to the corresponding 113Cd2+ species 113Cd NMR spectroscopy established that the location of Co(II) in Con-MT (n=1-3) was non-specific and that at n=4, the only observable structure was Co(II)4S11. The results suggest possible kinetic pathways of folding that are conceptually similar to those hypothesized for other small proteins.
- 134Bertini, I.; Luchinat, C.; Messori, L.; Vašák, M. Proton NMR studies of the cobalt(II)-metallothionein system. J. Am. Chem. Soc. 1989, 111, 7296– 7300, DOI: 10.1021/ja00201a002Google Scholar134Proton NMR studies of the cobalt(II)-metallothionein systemBertini, Ivano; Luchinat, Claudio; Messori, Luigi; Vasak, MilanJournal of the American Chemical Society (1989), 111 (19), 7296-300CODEN: JACSAT; ISSN:0002-7863.The binding of Co2+ to metallothionein (MT) from rabbit liver was followed through 1H NMR spectroscopy of cysteine side-chain protons and magnetic susceptibility measurements at room temp. in soln. In the course of the titrn. of apo-MT with Co2+, no isotropically shifted 1H NMR signals are obsd. up to ∼3 equiv of metal. A few broad signals in the 300-50 ppm region are obsd. when 4 equiv are added, whereas >30 sharp signals in the same region are obsd. when ≥5 equiv are added, up to the full metal complement of 7 equiv. Parallel magnetic susceptibility measurements show that little magnetic coupling among metal ions occurs at ≤3 equiv. Strong coupling is obsd. when the 4th equiv. is added, and a sizeable coupling is maintained, although with a smaller redn. of susceptibility value per metal ion, at ≤7 equiv. The results are consistent with a metal binding scheme in which the 1st 3 metals do not share any cysteine S donor, 2 metals possibly occupying the four-metal site and 1 the three-metal site. The 4th metal binds in the four-metal site, giving rise to a strongly coupled Co3 cluster, and the 5th completes the four-metal cluster, yielding a well-resolved 1H NMR spectrum. The 6th and 7th equiv complete the three-metal cluster, where they are probably strongly coupled as well. It is proposed that the well-resolved 1H NMR spectrum of the Co7MT deriv. essentially arises from the four-metal cluster.
- 135Kluska, K.; Adamczyk, J.; Krężel, A. Metal binding properties, stability and reactivity of zinc fingers. Coord. Chem. Rev. 2018, 367, 18– 64, DOI: 10.1016/j.ccr.2018.04.009Google Scholar135Metal binding properties, stability and reactivity of zinc fingersKluska, Katarzyna; Adamczyk, Justyna; Krezel, ArturCoordination Chemistry Reviews (2018), 367 (), 18-64CODEN: CCHRAM; ISSN:0010-8545. (Elsevier B.V.)A review. Zinc fingers (ZFs) are among the most structurally diverse protein domains. They interact with nucleic acids, other proteins and lipids to facilitate a multitude of biol. processes. Currently, there are more than 10 known classes of ZFs, with various architectures, metal binding modes, functions and reactivity. The versatility, selectivity and stability of these short amino acid sequences is achieved mainly by (i) residues participating in Zn(II) coordination (mostly Cys and His), (ii) hydrophobic core and ZF structure formation, and (iii) variable residues responsible for inter- and intramol. interactions. Since their discovery, ZFs have been extensively studied in terms of their structure, stability and recognition targets by the application of various methodologies. Studies based on interactions with other metal ions and their complexes have contributed to the understanding of their chem. properties and the discovery of new types of ZF complexes, such as gold fingers or lead fingers. Moreover, due to the presence of nucleophilic thiolates, ZFs are targets for reactive oxygen and nitrogen species as well as alkylating agents. Interactions with many reactive mols. lead to disturb the native Zn(II) coordination site which further result in structural and functional damage of the ZFs. The post-translational modifications including phosphorylation, acetylation, methylation or nitrosylation frequently affect ZFs function via changes in the protein structure and dynamics. Even though the literature is replete with structural and stability data regarding classical (ββα) ZFs, there is still a huge gap in the knowledge on physicochem. properties and reactivity of other ZF types. In this review, metal binding properties of ZFs and stability factors that modulate their functions are reviewed. These include interactions of ZFs with biogenic and toxic metal ions as well as damage occurring upon reaction with reactive oxygen and nitrogen species, the methodol. used for ZFs characterization, and aspects related to coordination chem.
- 136Kluska, K.; Peris-Díaz, M. D.; Płonka, D.; Moysa, A.; Dadlez, M.; Deniaud, A.; Bal, W.; Krężel, A. Formation of highly stable multinuclear AgnSn clusters in zinc fingers disrupts their structure and function. Chem. Commun. (Cambridge, U. K.) 2020, 56, 1329– 1332, DOI: 10.1039/C9CC09418KGoogle Scholar136Formation of highly stable multinuclear AgnSn clusters in zinc fingers disrupts their structure and functionKluska, Katarzyna; Peris-Diaz, Manuel D.; Plonka, Dawid; Moysa, Alexander; Dadlez, Michal; Deniaud, Aurelien; Bal, Wojciech; Krezel, ArturChemical Communications (Cambridge, United Kingdom) (2020), 56 (9), 1329-1332CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)Silver (Ag(I)) binding to consensus zinc fingers (ZFs) causes Zn(II) release inducing a gradual disruption of the hydrophobic core, followed by an overall conformational change and formation of highly stable AgnSn clusters. A compact eight-membered Ag4S4 structure formed by a CCCC ZF is the first cluster example reported for a single biol. mol. Ag(I)-induced conformational changes of ZFs can, as a consequence, affect transcriptional regulation and other cellular processes.
- 137Carpenter, M. C.; Shami Shah, A.; DeSilva, S.; Gleaton, A.; Su, A.; Goundie, B.; Croteau, M. L.; Stevenson, M. J.; Wilcox, D. E.; Austin, R. N. Thermodynamics of Pb(II) and Zn(II) binding to MT-3, a neurologically important metallothionein. Metallomics 2016, 8, 605– 617, DOI: 10.1039/C5MT00209EGoogle Scholar137Thermodynamics of Pb(II) and Zn(II) binding to MT-3, a neurologically important metallothioneinCarpenter, M. C.; Shami Shah, A.; DeSilva, S.; Gleaton, A.; Su, A.; Goundie, B.; Croteau, M. L.; Stevenson, M. J.; Wilcox, D. E.; Austin, R. N.Metallomics (2016), 8 (6), 605-617CODEN: METAJS; ISSN:1756-591X. (Royal Society of Chemistry)Isothermal titrn. calorimetry (ITC) was used to quantify the thermodn. of Pb2+ and Zn2+ binding to metallothionein-3 (MT-3). Pb2+ binds to zinc-replete Zn7MT-3 displacing each zinc ion with a similar change in free energy (G) and enthalpy (H). EDTA chelation measurements of Zn7MT-3 and Pb7MT-3 reveal that both metal ions are extd. in a tri-phasic process, indicating that they bind to the protein in three populations with different binding thermodn. Metal binding is entropically favored, with an enthalpic penalty that reflects the enthalpic cost of cysteine deprotonation accompanying thiolate ligation of the metal ions. These data indicate that Pb2+ binding to both apo MT-3 and Zn7MT-3 is thermodynamically favorable, and implicate MT-3 in neuronal lead biochem.
- 138Melenbacher, A.; Korkola, N. C.; Stillman, M. J. The pathways and domain specificity of Cu(I) binding to human metallothionein 1A. Metallomics 2020, 12, 1951– 1964, DOI: 10.1039/D0MT00215AGoogle Scholar138The pathways and domain specificity of Cu(I) binding to human metallothionein 1AMelenbacher, Adyn; Korkola, Natalie C.; Stillman, Martin J.Metallomics (2020), 12 (12), 1951-1964CODEN: METAJS; ISSN:1756-591X. (Royal Society of Chemistry)Copper is an essential element, but as a result of numerous adverse reactions, it is also a cellular toxin. Nature protects itself from these toxic reactions by binding cuprous copper to chaperones and other metalloproteins. Metallothionein has been proposed as a storage location for Cu(I) and potentially as the donor of Cu(I) to copper-dependent enzymes. We report that the addn. of Cu(I) to apo recombinant human metallothionein 1a cooperatively forms a sequential series of Cu(I)-cysteinyl thiolate complexes that have specific Cu(I) : MT stoichiometries of 6 : 1, 10 : 1, and finally 13 : 1. The individual domain Cu : SCys stoichiometries were detd. as Cu6S9 (for 6 : 1), Cu6S9 + Cu4S6 (for 10 : 1), and Cu6S9 + Cu7S9 (for 13 : 1) based on the no. of modified free cysteines not involved in Cu(I) binding. The stoichiometries are assocd. with Cu-SCys cluster formation involving bridging thiols in the manner similar to the clusters formed with Cd(II) and Zn(II). The locations of these clustered species within the 20 cysteine full protein were detd. from the unique speciation profiles of Cu(I) binding to the β and α domain fragments of recombinant human metallothionein 1a with 9 and 11 cysteines, resp. Competition reactions using these domain fragments challenged Cu(I) metalation of the βα protein, allowing the sequence of cluster formation in the full protein to be detd. Relative binding consts. for each Cu(I) bound are reported. The emission spectra of the Cu4S6, Cu6S9, and Cu7S9 clusters have unique λmax and phosphorescent lifetime properties. These phosphorescent data provide unambiguous supporting evidence for the presence of solvent shielded clusters reported concurrently by ESI-MS. Simulated emission spectra based on the cluster specific emission profiles matched the exptl. spectra and are used to confirm that the relative concns. seen by ESI-MS are representative of the soln. Our results suggest that the availability of a series of sequential Cu(I)-thiolate clusters provides flexibility as a means of protecting the cell from toxicity while still allowing for homeostatic control of the total copper content in the cell. This mechanism provides a dynamic and reactive method of reducing the cellular free copper concns.
- 139Payne, J. C.; ter Horst, M. A.; Godwin, H. A. Lead fingers: Pb2+ binding to structural zinc-binding domains determined directly by monitoring lead-thiolate charge-transfer bands. J. Am. Chem. Soc. 1999, 121, 6850– 6855, DOI: 10.1021/ja990899oGoogle Scholar139Lead Fingers: Pb2+ Binding to Structural Zinc-Binding Domains Determined Directly by Monitoring Lead-Thiolate Charge-Transfer BandsPayne, John C.; Horst, Marc A. ter; Godwin, Hilary ArnoldJournal of the American Chemical Society (1999), 121 (29), 6850-6855CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Here, we report that lead-thiolate charge-transfer bands (250-400 nm) can be used to monitor lead binding to cysteine-rich sites in proteins and report the application of this technique to det. the thermodn. of lead binding to a series of structural zinc-binding domains. These studies reveal that Pb2+ binds tightly to structural zinc-binding domains with dissocn. consts. that range from KdPb = 10-9 to 10-14 M, depending on the no. of cysteine residues in the metal-binding site. Competition expts. with Zn2+ lead to two striking conclusions: first, the two metals rapidly equilibrate, and second, the ratio of Pb2+ to Zn2+ bound to a particular site is detd. by the relative affinities of the two metals for that site, rather than being under kinetic control. We conclude that Pb2+ should be able to compete effectively with Zn2+ for Cys4 sites under physiol. conditions. Despite the fact that Pb2+ binds tightly to cysteine-rich structural zinc sites, CD and 1H NMR studies reveal that Pb2+ does not stabilize the correct fold of the peptides.
- 140Mesterházy, E.; Lebrun, C.; Crouzy, S.; Jancsó, A.; Delangle, P. Short oligopeptides with three cysteine residues as models of sulphur-rich Cu(I)- and Hg(II)-binding sites in proteins. Metallomics 2018, 10, 1232– 1244, DOI: 10.1039/C8MT00113HGoogle Scholar140Short oligopeptides with three cysteine residues as models of sulphur-rich Cu(I)- and Hg(II)-binding sites in proteinsMesterhazy, Edit; Lebrun, Colette; Crouzy, Serge; Jancso, Attila; Delangle, PascaleMetallomics (2018), 10 (9), 1232-1244CODEN: METAJS; ISSN:1756-591X. (Royal Society of Chemistry)The essential Cu(I) and the toxic Hg(II) ions possess similar coordination properties, and therefore, similar cysteine rich proteins participate in the control of their intracellular concn. In this work we present the metal binding properties of linear and cyclic model peptides incorporating the three-cysteine motifs, CxCxxC or CxCxC, found in metallothioneins. Cu(I) binding to the series of peptides at physiol. pH revealed to be rather complicated, with the formation of mixts. of polymetallic species. In contrast, the Hg(II) complexes display well-defined structures with spectroscopic features characteristic for a HgS2 and HgS3 coordination mode at pH = 2.0 and 7.4, resp. Stability data reflect a ca. 20 orders of magnitude larger affinity of the peptides for Hg(II) (log βpH7.4HgP ≈41) than for Cu(I) (log βpH7.4CuP ≈ 18). The different behavior with the two metal ions demonstrates that the use of Hg(II) as a probe for Cu(I), coordinated by thiolate ligands in water, may not always be fully appropriate.
- 141Vašák, M.; Kägi, J. H. R. Metallothioneins. In Encyclopedia of Inorganic Chemistry; King, R. B., Ed.; John Wiley & Sons: New York, NY, 1994; pp 2229– 2241.Google ScholarThere is no corresponding record for this reference.
- 142Leiva-Presa, A.; Capdevila, M.; Gonzàlez-Duarte, P. Mercury(II) binding to metallothioneins. Variables governing the formation and structural features of the mammalian HgMT species. Eur. J. Biochem. 2004, 271, 4872– 4880, DOI: 10.1111/j.1432-1033.2004.04456.xGoogle Scholar142Mercury(II) binding to metallothioneins. Variables governing the formation and structural features of the mammalian Hg-MT speciesLeiva-Presa, Angels; Capdevila, Merce; Gonzalez-Duarte, PilarEuropean Journal of Biochemistry (2004), 271 (23/24), 4872-4880CODEN: EJBCAI; ISSN:0014-2956. (Blackwell Publishing Ltd.)With the aim of extending our knowledge on the reaction pathways of Zn-metallothionein (MT) and apo-MT species in the presence of Hg(II), we monitored the titrn. of Zn7-MT, Zn4-αMT and Zn3-βMT proteins, at pH 7 and 3, with either HgCl2 or Hg(ClO4)2 by CD and UV-vis spectroscopy. Detailed anal. of the optical data revealed that std. variables, such as the pH of the soln., the binding ability of the counterion (chloride or perchlorate), and the time elapsed between subsequent addns. of Hg(II) to the protein, play a determinant role in the stoichiometry, stereochem. and degree of folding of the Hg-MT species. Despite the fact that the effect of these variables is clear, it is difficult to generalize. Overall, it can be concluded that the reaction conditions [pH, time elapsed between subsequent addns. of Hg(II) to the protein] affect the structural properties more substantially than the stoichiometry of the Hg-MT species, and that the role of the counterion becomes particularly apparent on the structure of overloaded Hg-MT.
- 143Carter, K. P.; Young, A. M.; Palmer, A. E. Fluorescent sensors for measuring metal ions in living systems. Chem. Rev. 2014, 114, 4564– 4601, DOI: 10.1021/cr400546eGoogle Scholar143Fluorescent Sensors for Measuring Metal Ions in Living SystemsCarter, Kyle P.; Young, Alexandra M.; Palmer, Amy E.Chemical Reviews (Washington, DC, United States) (2014), 114 (8), 4564-4601CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)This review focuses on fluorescent sensors for transition metals commonly found in biol. organisms. The authors start this review by giving a basic overview of fluorescence imaging and sensor design, followed by a crit. anal. of parameters and properties to consider when using sensors in biol. systems. The authors then present a historical perspective of how the field has evolved. While this review focuses on transition metals, the authors discuss some of the key advances/milestones achieved in the development of fluorescent Ca2+ indicators as these helped lay the groundwork for much of the subsequent work developing sensors for transition metals. Finally, the authors highlight progress in sensor development for biol. metals, emphasizing recent advances, while including a discussion of the most widely used sensors. To demonstrate what kind of measurements can be made and what kind of information can be learned from using fluorescent sensors, the authors review several applications of sensors for defining metal homeostasis and dynamics in cells or organisms. The authors would also like to call readers' attention to several excellent prior reviews that focus on different aspects of sensor development.
- 144Hessels, A. M.; Merkx, M. Genetically-encoded FRET-based sensors for monitoring Zn2+ in living cells. Metallomics 2015, 7, 258– 266, DOI: 10.1039/C4MT00179FGoogle Scholar144Genetically-encoded FRET-based sensors for monitoring Zn2+ in living cellsHessels, Anne M.; Merkx, MaartenMetallomics (2015), 7 (2), 258-266CODEN: METAJS; ISSN:1756-591X. (Royal Society of Chemistry)A review. Genetically-encoded fluorescent sensor proteins are attractive tools for studying intracellular Zn2+ homeostasis and signaling. Here we provide an overview of recently developed sensors based on F.ovrddot.orster Resonance Energy Transfer (FRET). The pros and cons of the various sensors are discussed with respect to Zn2+ affinity, dynamic range, intracellular targeting and multicolor imaging. Recent applications of these sensors are described, as well as some of the challenges that remain to be addressed in future research.
- 145Morgan, M. T.; Bourassa, D.; Harankhedkar, S.; McCallum, A. M.; Zlatic, S. A.; Calvo, J. S.; Meloni, G.; Faundez, V.; Fahrni, C. J. Ratiometric two-photon microscopy reveals attomolar copper buffering in normal and Menkes mutant cells. Proc. Natl. Acad. Sci. U. S. A. 2019, 116, 12167– 12172, DOI: 10.1073/pnas.1900172116Google Scholar145Ratiometric two-photon microscopy reveals attomolar copper buffering in normal and Menkes mutant cellsMorgan, M. Thomas; Bourassa, Daisy; Harankhedkar, Shefali; McCallum, Adam M.; Zlatic, Stephanie A.; Calvo, Jenifer S.; Meloni, Gabriele; Faundez, Victor; Fahrni, Christoph J.Proceedings of the National Academy of Sciences of the United States of America (2019), 116 (25), 12167-12172CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Copper is controlled by a sophisticated network of transport and storage proteins within mammalian cells, yet its uptake and efflux occur with rapid kinetics. Present as Cu(I) within the reducing intracellular environment, the nature of this labile copper pool remains elusive. While glutathione is involved in copper homeostasis and has been assumed to buffer intracellular copper, the authors demonstrate with a ratiometric fluorescent indicator, crisp-17, that cytosolic Cu(I) levels are buffered to the vicinity of 1 aM, where negligible complexation by glutathione is expected. Enabled by the phosphine sulfide-stabilized phosphine (PSP) ligand design strategy, crisp-17 offers a Cu(I) dissocn. const. of 8 aM, thus exceeding the binding affinities of previous synthetic Cu(I) probes by four to six orders of magnitude. Two-photon excitation microscopy with crisp-17 revealed rapid, reversible increases in intracellular Cu(I) availability upon addn. of the ionophoric complex CuGTSM or the thiol-selective oxidant 2,2'-dithiodipyridine (DTDP). While the latter effect was dramatically enhanced in 3T3 cells grown in the presence of supplemental copper and in cultured Menkes mutant fibroblasts exhibiting impaired copper efflux, basal Cu(I) availability in these cells showed little difference from controls, despite large increases in total copper content. Intracellular copper is thus tightly buffered by endogenous thiol ligands with significantly higher affinity than glutathione. The dual utility of crisp-17 to detect normal intracellular buffered Cu(I) levels as well as to probe the depth of the labile copper pool in conjunction with DTDP provides a promising strategy to characterize perturbations of cellular copper homeostasis.
- 146Berg, J. M.; Merkle, D. L. On the metal ion specificity of zinc finger proteins. J. Am. Chem. Soc. 1989, 111, 3759– 3761, DOI: 10.1021/ja00192a050Google Scholar146On the metal ion specificity of zinc finger proteinsBerg, Jeremy M.; Merkle, Denise L.Journal of the American Chemical Society (1989), 111 (10), 3759-61CODEN: JACSAT; ISSN:0002-7863.The affinities of a "zinc finger" peptide, H-Pro-Phe-Pro-Cys-Lys-Glu-Glu-Gly-Cys-Glu-Lys-Gly-Phe-Thr-Ser-Leu-His-His-Leu-Thr-Arg-His-Ser-Leu-Thr-His-Thr-Gly-Glu-Lys-OH, for Co2+ and Zn2+ have been detd. by spectrophotometrically monitored titrns. The dissocn. consts. are 3.8 ± 0.5 × 10-6M and 2.8 ± 0.9 × 10-9M for Co2+ and Zn2+, resp. The preference for Zn2+ over Co2+ is interpreted in terms of changes in ligand field stabilization energy for Co2+ in leaving an octahedral environment in Co(OH2)62+ and entering a tetrahedral environment when bound by the peptide. This model predicts an enthalpy difference of -4.5 kcal/mol favoring Zn2+ over Co2+ for binding to the peptide. This compares favorably to the obsd. free energy difference of -4.3 kcal/mol. These results suggest that the loss in ligand field stabilization energy incumbent in the transition from an octahedral site in aq. soln. to a tetrahedral site in a protein may be responsible for the obsd. specificity of the "zinc finger" proteins (and other proteins with tetrahedral binding sites) for zinc.
- 147Lachenmann, M. J.; Ladbury, J. E.; Dong, J.; Huang, K.; Carey, P.; Weiss, M. A. Why zinc fingers prefer zinc: ligand-field symmetry and the hidden thermodynamics of metal ion selectivity. Biochemistry 2004, 43, 13910– 13925, DOI: 10.1021/bi0491999Google Scholar147Why Zinc Fingers Prefer Zinc: Ligand-Field Symmetry and the Hidden Thermodynamics of Metal Ion SelectivityLachenmann, Marcel J.; Ladbury, John E.; Dong, Jian; Huang, Kun; Carey, Paul; Weiss, Michael A.Biochemistry (2004), 43 (44), 13910-13925CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)The zinc finger, a motif of protein-nucleic acid recognition broadly conserved among eukaryotes, is a globular minidomain contg. a tetrahedral metal-binding site. Preferential coordination of Zn2+ (relative to Co2+) is proposed to reflect differences in ligand-field stabilization energies (LFSEs) due to complete or incomplete occupancy of d orbitals. LFSE predicts that the preference for Zn2+ should be purely enthalpic in accord with calorimetric studies of a high-affinity consensus peptide. Despite its elegance, the general predominance of LFSE is unclear as (i) the magnitude by which CP-1 prefers Zn2+ is greater than that expected and (ii) the analogous metal ion selectivity of a zinc metalloenzyme (carbonic anhydrase) is driven by changes in entropy rather than enthalpy. Because CP-1 was designed to optimize zinc binding, the authors have investigated the NMR structure and metal ion selectivity of a natural finger of lower stability derived from human tumor-suppressor protein WT1. Raman spectroscopy suggests that the structure of the WT1 domain is unaffected by interchange of Zn2+ and Co2+. As in CP-1, preferential binding of Zn2+ (relative to Co2+) is driven predominantly by differences in enthalpy, but in this case the enthalpic advantage is less than that predicted by LFSE. A theor. framework is presented to define the relationship between LFSE and other thermodn. factors, such as metal ion electroaffinities, enthalpies of hydration, and the topog. of the underlying folding landscape. The contribution of environmental coupling to entropy-enthalpy compensation is delineated in a formal thermodn. cycle. Together, these considerations indicate that LFSE provides an important but incomplete description of the stringency and thermodn. origin of metal-ion selectivity.
- 148Posewitz, M. C.; Wilcox, D. E. Properties of the Sp1 zinc finger 3 peptide: coordination chemistry, redox reactions, and metal binding competition with metallothionein. Chem. Res. Toxicol. 1995, 8, 1020– 1028, DOI: 10.1021/tx00050a005Google Scholar148Properties of the Sp1 Zinc Finger 3 Peptide: Coordination Chemistry, Redox Reactions, and Metal Binding Competition with MetallothioneinPosewitz, Matthew C.; Wilcox, Dean E.Chemical Research in Toxicology (1995), 8 (8), 1020-8CODEN: CRTOEC; ISSN:0893-228X. (American Chemical Society)Toxic and/or carcinogenic consequences may result from metal ion substitution for the Zn(II) in transcription factors contg. zinc fingers, and the small Cys-rich metal-binding protein metallothionein (MT) may play a role in this metal substitution. To begin to evaluate this hypothesis, with regard to the carcinogenic metal ion Ni(II), a peptide corresponding to the third finger of the transcription factor Sp1 (Sp1-3) has been synthesized and its metal binding and redox reactions have been studied. The peptide binds Zn(II), Co(II), and Ni(II), with spectroscopic data indicating a tetrahedral coordination for the latter two; metal ion affinities have been quantified (Kd = 6 × 10-10, 3 × 10-7, and 4 × 10-6, resp.) and less than those of an optimized zinc finger peptide (B. A. Krizek et al. (1993)) but greater than those of the second finger of transcription factor IIIA (J. M. Berg and D. L. Merkle (1989)). Reactions of the peptide and its metal-bound forms with dioxygen or hydrogen peroxide did not produce oxygen radical species; however, oxidn. of the two Sp1-3 cysteines was modulated by metal ions (Zn < Co = apo < Ni), suggesting a protective role for Zn(II) but an enhancing role for Ni(II). Metal binding competition between Sp1-3 and the α domain of human liver MT-2 (α-hMT2) indicates a similar affinity for Zn(II). However, α-hMT2 has a higher affinity for Ni(II), suggesting that MT may play a protective role by ensuring Zn(II), rather than Ni(II), coordination to zinc finger sequences of transcription factors.
- 149Romero-Isart, N.; Oliva, B.; Vašák, M. Influence of NH-Sγ bonding interactions on the structure and dynamics of metallothioneins. J. Mol. Model. 2010, 16, 387– 394, DOI: 10.1007/s00894-009-0542-xGoogle Scholar149Influence of NH-Sγ bonding interactions on the structure and dynamics of metallothioneinsRomero-Isart, Nuria; Oliva, Baldo; Vasak, MilanJournal of Molecular Modeling (2010), 16 (3), 387-394CODEN: JMMOFK; ISSN:0948-5023. (Springer GmbH)Mammalian metallothioneins (MII7MTs) show a clustered arrangement of the metal ions and a nonregular protein structure. The soln. structures of Cd3-thiolate cluster contg. β-domain of mouse β-MT-1 and rat β-MT-2 show high structural similarities, but widely differing structure dynamics. Mol. dynamics simulations revealed a substantially increased no. of NH-Sγ hydrogen bonds in β-MT-2, features likely responsible for the increased stability of the Cd3-thiolate cluster and the enfolding protein domain. Alterations in the NH-Sγ hydrogen-bonding network may provide a rationale for the differences in dynamic properties encountered in the β-domains of MT-1, -2, and -3 isoforms, believed to be essential for their different biol. function.
- 150Kochańczyk, T.; Nowakowski, M.; Wojewska, D.; Kocyła, A.; Ejchart, A.; Koźmiński, W.; Krężel, A. Metal-coupled folding as the driving force for the extreme stability of Rad50 zinc hook dimer assembly. Sci. Rep. 2016, 6, 36346, DOI: 10.1038/srep36346Google Scholar150Metal-coupled folding as the driving force for the extreme stability of Rad50 zinc hook dimer assemblyKochanczyk, Tomasz; Nowakowski, Michal; Wojewska, Dominika; Kocyla, Anna; Ejchart, Andrzej; Kozminski, Wiktor; Krezel, ArturScientific Reports (2016), 6 (), 36346CODEN: SRCEC3; ISSN:2045-2322. (Nature Publishing Group)The binding of metal ions at the interface of protein complexes presents a unique and poorly understood mechanism of mol. assembly. A remarkable example is the Rad50 zinc hook domain, which is highly conserved and facilitates the Zn2+-mediated homodimerization of Rad50 proteins. Here, we present a detailed anal. of the structural and thermodn. effects governing the formation and stability (logK12 = 20.74) of this evolutionarily conserved protein assembly. We have dissected the determinants of the stability contributed by the small β-hairpin of the domain surrounding the zinc binding motif and the coiled-coiled regions using peptides of various lengths from 4 to 45 amino acid residues, alanine substitutions and peptide bond-to-ester perturbations. In the studied series of peptides, an >650 000-fold increase of the formation const. of the dimeric complex arises from favorable enthalpy because of the increased acidity of the cysteine thiols in metal-free form and the structural properties of the dimer. The dependence of the enthalpy on the domain fragment length is partially compensated by the entropic penalty of domain folding, indicating enthalpy-entropy compensation. This study facilitates understanding of the metal-mediated protein-protein interactions in which the metal ion is crit. for the tight assocn. of protein subunits.
- 151Hong, S.-H.; Hao, Q.; Maret, W. Domain-specific fluorescence resonance energy transfer (FRET) sensors of metallothionein/thionein. Protein Eng., Des. Sel. 2005, 18, 255– 263, DOI: 10.1093/protein/gzi031Google Scholar151Domain-specific fluorescence resonance energy transfer (FRET) sensors of metallothionein/thioneinHong, S.-H.; Hao, Q.; Maret, W.Protein Engineering, Design & Selection (2005), 18 (6), 255-263CODEN: PEDSBR; ISSN:1741-0126. (Oxford University Press)Each of the two domains of mammalian metallothioneins contains a zinc-thiolate cluster. Employing site-directed mutagenesis and chem. modification, fluorescent probes were introduced into human metallothionein (isoform 2) with minimal perturbations of the structures of these clusters. The resulting FRET (fluorescence resonance energy transfer) sensors are specific for each domain. The design and construction of a sensor for the α-domain cluster is based on a FRET pair where a C-terminally added tryptophan serves as the donor for a fluorescence acceptor attached to a free cysteine in the linker region between the two domains. Mol. modeling studies and steady-state fluorescence polarization anisotropy measurements suggest unrestricted motion of the tryptophan donor, but limited motion of the AEDANS ({[(amino)ethyl]amino}naphthalene-1-sulfonic acid) acceptor, putting constraints on the use of the α-domain sensor with this FRET pair as a spectroscopic ruler. The fluorescent metallothioneins allow distance measurements during binding and removal of metals in the individual domains. The overall dimensions of the apoprotein, thionein, for which no structural information is available, do not seem to be significantly different from those of the holoprotein. The single- and double-labeled fluorescent metallothioneins overcome a longstanding impediment in studies of the function of this protein, namely its lack of intrinsic probe characteristics.
- 152Summers, K. L.; Mahrok, A. K.; Dryden, M. D. M; Stillman, M. J. Structural properties of metal-free apometallothioneins. Biochem. Biophys. Res. Commun. 2012, 425, 485– 492, DOI: 10.1016/j.bbrc.2012.07.141Google Scholar152Structural properties of metal-free apometallothioneinsSummers, Kelly L.; Mahrok, AnjanPreet K.; Dryden, Michael D. M.; Stillman, Martin J.Biochemical and Biophysical Research Communications (2012), 425 (2), 485-492CODEN: BBRCA9; ISSN:0006-291X. (Elsevier B.V.)The metalated forms of metallothionein (MT) are well studied (particularly Zn-MT, Cu-MT, and Cd-MT), but almost nothing is known about the chem. and structural properties of apo-MTs despite their importance in initial metalation and subsequent demetalation. Electrospray ionization mass spectrometry was used to provide a detailed view of the structural properties of the metal-free protein. Mass spectra of Zn7-MT and apo-MT at pH 7 exhibited the same charge state distribution, indicating that apo-MT was tightly folded like the metalated protein, whereas apo-MT at pH 3 exhibited a charge state spectrum assocd. with unfolding or denaturation. Benzoquinone was used to modify the Cys residues in the β-MT (9 Bq), and α-MT (11 Bq) fragments, and the full βα-MT (20 Bq) protein. ESI-MS showed that the overall vol. and, therefore, the extent of folding for the modified proteins was similar to that of Zn-MT. Mol. modeling using MM3-MD methods provided the vol. of each modified protein. The vols. of the partially modified proteins followed the same trend as the charge states, showing that ESI-MS is an excellent method with which to follow small changes in protein folding as a function of applied chem. stress. The data suggested that the structure of apo-βα-MT was more organized than previously considered.
- 153Drozd, A.; Wojewska, D.; Peris-Díaz, M. D.; Jakimowicz, P.; Krężel, A. Crosstalk of the structural and zinc buffering properties of mammalian metallothionein-2. Metallomics 2018, 10, 595– 613, DOI: 10.1039/C7MT00332CGoogle Scholar153Crosstalk of the structural and zinc buffering properties of mammalian metallothionein-2Drozd, Agnieszka; Wojewska, Dominika; Peris-Diaz, Manuel David; Jakimowicz, Piotr; Krezel, ArturMetallomics (2018), 10 (4), 595-613CODEN: METAJS; ISSN:1756-591X. (Royal Society of Chemistry)Metallothioneins (MTs), small cysteine-rich proteins, present in four major isoforms, are key proteins involved in zinc and copper homeostasis in mammals. To date, only one X-ray crystal structure of a MT has been solved. It demonstrates seven bivalent metal ions bound in two structurally independent domains with M4S11 (a) and M3S9 (β) clusters. Recent discoveries indicate that Zn(II) ions are bound with MT2 with the range from nano- to picomolar affinity, which dets. its cellular zinc buffering properties that are demonstrated by the presence of partially Zn(II)-depleted MT2 species. These forms serve as Zn(II) donors or acceptors and are formed under varying cellular free Zn(II) concns. Due to the lack of appropriate methods, knowledge regarding the structure of partially-depleted metallothionein is lacking. Here, we describe the Zn(II) binding mechanism in human MT2 with high resoln. with respect to particular Zn(II) binding sites, and provide structural insights into Zn(II)-depleted MT species. The results were obtained by the labeling of metal-free cysteine residues with iodoacetamide and subsequent top-down electrospray ionization anal., MALDI MS, bottom-up nanoLC-MALDI-MS/MS approaches and mol. dynamics (MD) simulations. The results show that the a-domain is formed sequentially in the first stages, followed by the formation of the β-domain, although both processes overlap, which is in contrast to the widely investigated cadmium MT. Independent ZnS4 cores are characteristic for early stages of domain formation and are clustered in later stages. However, Zn-S network rearrangement in the β-domain upon applying the seventh Zn(II) ion explains its lower affinity. Detailed anal. showed that the weakest Zn(II) ion assocs. with the β-domain by coordination to Cys21, which was also found to dissoc. first in the presence of the apo-form of sorbitol dehydrogenase. We found that Zn(II) binding to the isolated β-domain differs significantly from the whole protein, which explains its previously obsd. different Zn(II)-binding properties. MD results obtained for Zn(II) binding to the whole protein and isolated β-domain are highly convergent with mass spectrometry data. This study provides a comprehensive overview of the crosstalk of structural and zinc buffering related-to-thermodn. properties of partially metal-satd. mammalian MT2 and sheds more light on other MT proteins and zinc homeostasis.
- 154Wątły, J.; Łuczkowski, M.; Padjasek, M.; Krężel, A. Phytochelatins as a dynamic system for Cd(II) buffering from micro- to femtomolar range. Inorg. Chem. 2021, 60, 4657– 4675, DOI: 10.1021/acs.inorgchem.0c03639Google Scholar154Phytochelatins as a Dynamic System for Cd(II) Buffering from the Micro- to Femtomolar RangeWatly, Joanna; Luczkowski, Marek; Padjasek, Michal; Krezel, ArturInorganic Chemistry (2021), 60 (7), 4657-4675CODEN: INOCAJ; ISSN:0020-1669. (American Chemical Society)Phytochelatins (PCs) are short Cys-rich peptides with repeating γ-Glu-Cys motifs found in plants, algae, certain fungi, and worms. Their biosynthesis has been found to be induced by heavy metals-both biogenic and toxic. Among all metal inducers, Cd(II) has been the most explored from a biol. and chem. point of view. Although Cd(II)-induced PC biosynthesis has been widely examd., still little is known about the structure of Cd(II) complexes and their thermodn. stability. Here, we systematically investigated glutathione (GSH) and PC2-PC6 systems, with regard to their complex stoichiometries and spectroscopic and thermodn. properties. We paid special attention to the detn. of stability consts. using several complementary techniques. All peptides form CdL complexes, but CdL2 was found for GSH, PC2, and partially for PC3. Moreover, binuclear species CdxLy were identified for the series PC3-PC6 in an excess of Cd(II). Potentiometric and competition spectroscopic studies showed that the affinity of Cd(II) complexes increases from GSH to PC4 almost linearly from micromolar (log K7.4GSH = 5.93) to the femtomolar range (log K7.4PC4 = 13.39) and addnl. chain elongation does not increase the stability significantly. Data show that PCs form an efficient system which buffers free Cd(II) ions in the pico- to femtomolar range under cellular conditions, avoiding significant interference with Zn(II) complexes. Our study confirms that the favorable entropy change is the factor governing the elevation of phytochelatins' stability and illuminates the importance of the chelate effect in shifting the free Gibbs energy.
- 155Chen, S. H.; Russell, D. H. How closely related are conformations of protein ions sampled by IM-MS to native solution strcutures?. J. Am. Soc. Mass Spectrom. 2015, 26, 1433– 1443, DOI: 10.1007/s13361-015-1191-1Google Scholar155How Closely Related Are Conformations of Protein Ions Sampled by IM-MS to Native Solution Structures?Chen, Shu-Hua; Russell, David H.Journal of the American Society for Mass Spectrometry (2015), 26 (9), 1433-1443CODEN: JAMSEF; ISSN:1044-0305. (Springer)Here, we critically evaluate the effects of changes in the ion internal energy (Eint) on ion-neutral collision cross sections (CCS) of ions of two structurally diverse proteins, specifically the [M + 6H]6+ ion of ubiquitin (ubq6+), the [M + 5H]5+ ion of the intrinsically disordered protein (IDP) apo-metallothionein-2A (MT), and its partially- and fully-metalated isoform, the [CdiMT]5+ ion. The ion-neutral CCS for ions formed by "native-state" ESI show a strong dependence on Eint. Collisional activation is used to increase Eint prior to the ions entering and within the traveling wave (TW) ion mobility analyzer. Comparisons of exptl. CCSs with those generated by mol. dynamics (MD) simulation for soln.-phase ions and solvent-free ions as a function of temp. provide new insights about conformational preferences and retention of soln. conformations. The Eint-dependent CCSs, which reveal increased conformational diversity of the ion population, are discussed in terms of folding/unfolding of solvent-free ions. For example, ubiquitin ions that have low internal energies retain native-like conformations, whereas ions that are heated by collisional activation possess higher internal energies and yield a broader range of CCS owing to increased conformational diversity due to losses of secondary and tertiary structures. In contrast, the CCS profile for the IDP apoMT is consistent with kinetic trapping of an ion population composed of a wide range of conformers, and as the Eint is increased, these structurally labile conformers unfold to an elongated conformation.
- 156Chen, S. H.; Chen, L.; Russell, D. H. Metal-induced conformational changes of human metallothionien-2a: A combined theortical and experimental study of metal-free and partially metalated intermediates. J. Am. Chem. Soc. 2014, 136, 9499– 9508, DOI: 10.1021/ja5047878Google Scholar156Metal-Induced Conformational Changes of Human Metallothionein-2A: A Combined Theoretical and Experimental Study of Metal-Free and Partially Metalated IntermediatesChen, Shu-Hua; Chen, Liuxi; Russell, David H.Journal of the American Chemical Society (2014), 136 (26), 9499-9508CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Electrospray ionization ion mobility mass spectrometry (ESI IM-MS) and mol. dynamics (MD) simulations reveal new insights into metal-induced conformational changes and the mechanism for metalation of human metallothionein-2A (MT), an intrinsically disordered protein. ESI of solns. contg. apoMT yields multiple charge states of apoMT; following addn. of Cd2+ to the soln., ESI yields a range of CdiMT (i = 1-7) product ions. Ion mobility arrival-time distributions (ATDs) for the CdiMT (i = 0-7) ions reveal a diverse population of ion conformations. The ion mobility data clearly show that the conformational diversity for apoMT and partially metalated ions converges toward ordered, compact conformations as the no. of bound Cd2+ ions increase. MD simulations provide addnl. information on conformation candidates of CdiMT (i = 0-7) that supports the convergence of distinct conformational populations upon metal binding. Integrating the IM-MS and MD data provides a global view that shows stepwise conformational transition of an ensemble as a function of metal ion bound. ApoMT is comprised of a wide range of conformational states that populate between globular-like compact and coil-rich extended conformations. During the initial stepwise metal addn. (no. of metal ions bound i = 1-3), the metal ions bind to different sites to yield distinct conformations, whereas for i > 4, the conformational changes appear to be domain-specific, attributed to different degrees of disorder of the β domain; the β domain becomes more ordered as addnl. metal ions are added, promoting convergences to the dumbbell-shaped conformation.
- 157Chen, S. H.; Russell, W. K.; Russell, D. H. Combining chemical labeling, bottom-up and top-down ion-mobility mass spectrometry to identify metal-binding sites of partially metalated metallothionein. Anal. Chem. 2013, 85, 3229– 3237, DOI: 10.1021/ac303522hGoogle Scholar157Combining Chemical Labeling, Bottom-Up and Top-Down Ion-Mobility Mass Spectrometry To Identify Metal-Binding Sites of Partially Metalated MetallothioneinChen, Shu-Hua; Russell, William K.; Russell, David H.Analytical Chemistry (Washington, DC, United States) (2013), 85 (6), 3229-3237CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)Metalation and demetalation of human metallothionein-2A (MT) with Cd2+ is investigated by using chem. labeling and "bottom-up" and "top-down" proteomics approaches. Both metalation and demetalation of MT-2A by Cd2+ are shown to be domain specific and occur as two distinct processes. Metalation involves sequential addn. of Cd2+ to the α-domain resulting in formation of an intermediate, Cd4MT. Chem. labeling with N-ethylmaleimide (NEM) and tandem mass spectrometry expts. clearly show that the four metal ions are located in the α-domain. In the presence of excess Cd2+, the Cd4MT intermediate reacts to add Cd2+ to the β-domain to yield the fully metalated Cd7MT. Demetalation occurs in the reverse order, i.e., Cd2+ is removed (by EDTA) first from the β-domain followed by Cd2+ removal from the α-domain. Metalation of human MT-2A is shown to be metal ion specific by comparing relative metal ion binding consts. for Cd2+ and Zn2+.
- 158Dong, S.; Wagner, N. D.; Russell, D. H. Collision-induced unfolding of partially metalated metallothionein-2A: Tracking unfolding reactions of gas-phase ions. Anal. Chem. 2018, 90, 11856– 11862, DOI: 10.1021/acs.analchem.8b01622Google Scholar158Collision-Induced Unfolding of Partially Metalated Metallothionein-2A: Tracking Unfolding Reactions of Gas-Phase IonsDong, Shiyu; Wagner, Nicole D.; Russell, David H.Analytical Chemistry (Washington, DC, United States) (2018), 90 (20), 11856-11862CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)Metallothioneins (MTs) constitute a group of intrinsically disordered proteins that exhibit extreme diversity in structure, biol. functionality, and metal ion specificity. Structures of coordinatively satd. metalated MTs have been extensively studied, but very limited structural information for the partially metalated MTs exists. Here, the conformational preferences from partial metalation of rabbit metallothionein-2A (MT) by Cd2+, Zn2+, and Ag+ are studied using nanoelectrospray ionization ion mobility mass spectrometry. We also employ collision-induced unfolding to probe differences in the gas-phase stabilities of these partially metalated MTs. Our results show that despite their similar ion mobility profiles, Cd4-MT, Zn4-MT, Ag4-MT, and Ag6-MT differ dramatically in their gas-phase stabilities. Furthermore, the sequential addn. of each Cd2+ and Zn2+ ion results in the incremental stabilization of unique unfolding intermediates.
- 159Reddi, A. R.; Guzman, T. R.; Breece, R. M.; Tierney, D. L.; Gibney, B. R. Deducing the energetic cost of protein folding in zinc finger proteins using designed metallopeptides. J. Am. Chem. Soc. 2007, 129, 12815– 12827, DOI: 10.1021/ja073902+Google Scholar159Deducing the Energetic Cost of Protein Folding in Zinc Finger Proteins Using Designed MetallopeptidesReddi, Amit R.; Guzman, Tabitha R.; Breece, Robert M.; Tierney, David L.; Gibney, Brian R.Journal of the American Chemical Society (2007), 129 (42), 12815-12827CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Zinc finger transcription factors represent the largest single class of metalloproteins in the human genome. Binding of Zn(II) to their canonical Cys4, Cys3His1, or Cys2His2 sites results in metal-induced protein folding events required to achieve their proper structure for biol. activity. The thermodn. contribution of Zn(II) in each of these coordination spheres toward protein folding is poorly understood because of the coupled nature of the metal-ligand and protein-protein interactions. Using an unstructured peptide scaffold, GGG, we have employed fluorometry, potentiometry, and calorimetry to det. the thermodn. of Zn(II) binding to the Cys4, Cys3His1, and Cys2His2 ligand sets with minimal interference from protein folding effects. The data show that Zn(II) complexation is entropy driven and modulated by proton release. The formation consts. for Zn(II)-GGG with a Cys4, Cys3His1, or Cys2His2 site are 5.6×1016, 1.5×1015, or 2.5×1013 M-1, resp. Thus, the Zn(II)-Cys4, Zn(II)-Cys3His1, and Zn(II)-Cys2His2 interactions can provide up to 22.8, 20.7, and 18.3 kcal/mol, resp., in driving force for protein stabilization, folding, and/or assembly at pH values above the ligand pKa values. While the contributions from the three coordination motifs differ by 4.5 kcal/mol in Zn(II) affinity at pH 9.0, they are equiv. at physiol. pH, ΔG = -16.8 kcal/mol or a Ka = 2.0×1012 M-1. Calorimetric data show that this is due to proton-based enthalpy-entropy compensation between the favorable entropic term from proton release and the unfavorable enthalpic term due to thiol deprotonation. Since protein folding effects have been minimized in the GGG scaffold, these peptides possess nearly the tightest Zn(II) affinities possible for their coordination motifs. The Zn(II) affinities in each coordination motif are compared between the GGG scaffold and natural zinc finger proteins to det. the free energy required to fold the latter. Several proteins have identical Zn(II) affinities to GGG. That is, little, if any, of their Zn(II) binding energy is required to fold the protein, whereas some have affinities weakened by up to 5.7 kcal/mol; i.e., the Zn(II) binding energy is being used to fold the protein.
- 160Sénèque, O.; Latour, J. M. Coordination properties of zinc finger peptides revisited: ligand competition studies reveal higher affinities for zinc and cobalt. J. Am. Chem. Soc. 2010, 132, 17760– 17774, DOI: 10.1021/ja104992hGoogle Scholar160Coordination Properties of Zinc Finger Peptides Revisited: Ligand Competition Studies Reveal Higher Affinities for Zinc and CobaltSeneque, Olivier; Latour, Jean-MarcJournal of the American Chemical Society (2010), 132 (50), 17760-17774CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Zinc fingers are ubiquitous small protein domains which have a Zn(Cys)4-x(His)x site. They possess great diversity in their structure and amino acid compn. Using a family of six peptides, it was possible to assess the influence of hydrophobic amino acids on the metal-peptide affinities and on the rates of metal assocn. and dissocn. A model of a treble-clef zinc finger, a model of the zinc finger site of a redox-switch protein, and four variants of the classical ββα zinc finger were used. They differ in their coordination set, their sequence length, and their hydrophobic amino acid content. The speciation, metal binding consts., and structure of these peptides have been investigated as a function of pH. The zinc binding consts. of peptides, which adopt a well-defined structure, were found to be around 1015 at pH 7.0. The rates of zinc exchange between EDTA and the peptides were also assessed. We evidenced that the packing of hydrophobic amino acids into a well-defined hydrophobic core can have a drastic influence on both the binding const. and the kinetics of metal exchange. Notably, well-packed hydrophobic amino acids can increase the stability const. by 4 orders of magnitude. The half-life of zinc exchange was also seen to vary significantly depending on the sequence of the zinc finger. The possible causes for this behavior are discussed. This work will help in understanding the dynamics of zinc exchange in zinc-contg. proteins.
- 161Miłoch, A.; Krężel, A. Metal binding properties of the zinc finger metallome--insights into variations in stability. Metallomics 2014, 6, 2015– 2024, DOI: 10.1039/C4MT00149DGoogle Scholar161Metal binding properties of the zinc finger metallome - insights into variations in stabilityMiloch, Anna; Krezel, ArturMetallomics (2014), 6 (11), 2015-2024CODEN: METAJS; ISSN:1756-591X. (Royal Society of Chemistry)Zinc is one of the most widespread metal ions found in biol. systems. Of the expected 3000 zinc proteins in the human proteome, most contain zinc in structural sites. Among these structures, the most important are zinc fingers, which are well suited to facilitate interactions with DNA, RNA, proteins and lipid mols. Knowledge regarding their stability is a crit. issue in understanding the function of zinc fingers and their reactivity under fluxing cellular Zn(II) availability and different redox states. Zinc stability consts. that have been detd. using a variety of methods demonstrate wide diversity. Recent studies on the stability of consensus zinc fingers have demonstrated that the known metal-ion affinities for zinc fingers may have been underestimated by as much as three or more orders of magnitude. Here, using four natural ββα zinc fingers, we compare in detail several different methods that have been used for the detn. of zinc finger stability consts., such as common reverse-titrn., potentiometry, competition with metal chelators, and a new approach based on a three-step spectrophotometric titrn. We discuss why the stabilities of zinc fingers that are detd. spectrophotometrically are frequently underestimated due to the lack of effective equil. competition, which leads to large errors during the processing of the titrn. data. The literature stability consts. of many natural zinc fingers have been underestimated, and they are significantly lower when compared with the consensus peptides. Our data show that in the cell, some naturally occurring zinc fingers may potentially be unoccupied and are instead loaded transiently with Zn(II). Large variations in stability within the same class of zinc fingers have demonstrated that the thermodn. effects hidden in the sequence and structure are the key elements responsible for the differentiation of the stability of the zinc finger metallome.
- 162Alderighi, L.; Gans, P.; Ienco, A.; Peters, D.; Sabatini, A.; Vacca, A. Hyperquad simulation and speciation (HySS): a utility program for the investigation of equilibria involving soluble and partially soluble species. Coord. Chem. Rev. 1999, 184, 311– 318, DOI: 10.1016/S0010-8545(98)00260-4Google Scholar162Hyperquad simulation and speciation (HySS): a utility program for the investigation of equilibria involving soluble and partially soluble speciesAlderighi, Lucia; Gans, Peter; Ienco, Andrea; Peters, Daniel; Sabatini, Antonio; Vacca, AlbertoCoordination Chemistry Reviews (1999), 184 (), 311-318CODEN: CCHRAM; ISSN:0010-8545. (Elsevier Science S.A.)A review is presented with 23 refs. describing hyperquad simulation and speciation (HySS) that is a computer program written for the Windows operating system on personal computers which provides (a) a system for simulating titrn. curves and (b) a system for providing speciation diagrams. The calcns. relate to equil. in soln. and also include the possibility of formation of a partially sol. ppt. There are no restrictions on the no. of reagents that may be present or on the no. of complexes that may be formed.
- 163Irvine, G. W.; Pinter, T. B. J.; Stillman, M. J. Defining the metal binding pathways of human metallothionein 1a: balancing zinc availability and cadmium seclusion. Metallomics 2016, 8, 71– 81, DOI: 10.1039/C5MT00225GGoogle Scholar163Defining the metal binding pathways of human metallothionein 1a: balancing zinc availability and cadmium seclusionIrvine, Gordon W.; Pinter, Tyler B. J.; Stillman, Martin J.Metallomics (2016), 8 (1), 71-81CODEN: METAJS; ISSN:1756-591X. (Royal Society of Chemistry)Metallothioneins (MTs) are cysteine-rich, metal-binding proteins that are found throughout Nature. This ubiquity highlights their importance in essential metal regulation, heavy metal detoxification and cellular redox chem. Missing from the current description of MT function is the underlying mechanism by which MTs achieve their proposed biol. functions. To date, there have been conflicting reports on the mechanism of metal binding and the structures of the metal binding intermediates formed during metalation of apoMTs. The form of the metal-bound intermediates dictates the metal sequestering and metal-donating properties of the protein. Through a detailed anal. of spectral data from electrospray ionization mass spectrometric and CD methods we report that Zn(II) and Cd(II) metalation of the human MT1a takes place through two distinct pathways. The first pathway involves formation of beaded structures with up to five metals bound terminally to the 20 cysteines of the protein via a noncooperative mechanism. The second pathway is dominated by the formation of the four-metal domain cluster structure M4SCYS11via a cooperative mechanism. We report that there are different pathway preferences for Zn(II) and Cd(II) metalation of apo-hMT1a. Cd(II) binding follows the beaded pathway above pH 7.1 but beginning below pH 7.1 the clustered (Cd4Scys11) pathway begins to dominate. In contrast, Zn(II) binding follows the terminal, "beaded", pathway at all physiol. relevant pH (pH ≥ 5.2) only following the clustered pathway below pH 5.1. The results presented here allow us to reconcile the conflicting reports concerning the presence of different metalation intermediates of MTs. The conflict regarding cooperative vs. noncooperative binding mechanisms is also reconciled with the exptl. results described here. These two metal-specific pathways and the presence of radically different intermediate structures provide insight into the multi-functional nature of MT: binding Zn(II) terminally for donation to metalloenzymes and sequestering toxic Cd(II) in a cluster structure.
- 164Vazquez, F.; Vašák, M. Comparative 113Cd-n.m.r. studies on rabbit 113Cd7-, (Zn1,Cd6)- and partially metal-depleted 113Cd6-metallothionein-2a. Biochem. J. 1988, 253, 611– 614, DOI: 10.1042/bj2530611Google Scholar164Comparative cadmium-113 NMR studies on rabbit 113Cd7-, (Zn1,Cd6)- and partially metal-depleted 113Cd6-metallothionein-2aVazquez, Francisco; Vasak, MilanBiochemical Journal (1988), 253 (2), 611-14CODEN: BIJOAK; ISSN:0264-6021.Rabbit 113Cd7-metallothionein-2a (MT) contains 2 metal-thiolate clusters of 3 (cluster B) and 4 (cluster A) metal ions. The 113Cd NMR spectrum of 113Cd6-MT, isolated from 113Cd7-MT upon treatment with EDTA, is similar to that of 113Cd7-MT, but the cluster B resonances are lower in intensity, suggesting its cooperative metal depletion. Zn1,113Cd6-MT, formed by addn. of the Zn(II) ions to 113Cd6-MT, shows 113Cd NMR features characteristic of cluster B populations contg. both Cd(II) and Zn(II) ions. The overall intensity gain of the mixed cluster B resonances per Cd as to those in 113Cd6- and 113Cd7-MT suggests a stabilizing effect of the bound Zn(II) ions upon the previously established intramol. 113Cd exchange within this cluster.
- 165Li, T. Y.; Kraker, A. J.; Shaw, C. F., III; Petering, D. H. Ligand substitution reactions of metallothioneins with EDTA and apo-carbonic anhydrase. Proc. Natl. Acad. Sci. U. S. A. 1980, 77, 6334– 6338, DOI: 10.1073/pnas.77.11.6334Google Scholar165Ligand substitution reactions of metallothioneins with EDTA and apo-carbonic anhydraseLi, Ta-Yuen; Kraker, Alan J.; Shaw, C. Frank, III; Petering, David H.Proceedings of the National Academy of Sciences of the United States of America (1980), 77 (11), 6334-8CODEN: PNASA6; ISSN:0027-8424.The reactions of Zn-, Cd-, and Zn,Cd-thioneins with EDTA and apo-carbonic anhydrase were studied. The ligand substitution reaction of Zn with EDTA is multiphasic having both associative and dissociative components in the rate expression. The Cd sites are ∼2 orders of magnitude less reactive. In contrast, apo-carbonic anhydrase abstrs. Zn from Zn-thionein and Zn,Cd-thionein in 2nd-order processes that are 2-3 orders of magnitude more rapid than those involving EDTA and approach the rate for unligated Zn2+ with the apo-protein. In comparison with other Zn proteins, Zn-thionein contains unusually reactive metal sites, suggesting that this protein may be a physiol. Zn transfer protein, able to donate Zn to Zn-requiring apo macromols.
- 166Otvos, J. D.; Petering, D. H.; Shaw, C. F. Structure-reactivity relationships of metallothionein, a unique metal-binding protein. Comments Inorg. Chem. 1989, 9, 1– 35, DOI: 10.1080/02603598908035801Google Scholar166Structure-reactivity relationships of metallothionein, a unique metal-binding proteinOtvos, James D.; Petering, David H.; Shaw, C. FrankComments on Inorganic Chemistry (1989), 9 (1), 1-35CODEN: COICDZ; ISSN:0260-3594.A review, with 89 refs., on the structure of metallothioneins and relating the structure to its metal-binding properties and kinetics. Particular emphasis is placed on its binding of Cd, Au, Pt, and Zn.
- 167Namdarghanbari, M. A.; Meeusen, J.; Bachowski, G.; Giebel, N.; Johnson, J.; Petering, D. H. Reaction of the zinc sensor FluoZin-3 with Zn7-metallothionein. Inquiry into the existence of a proposed weak binding site. J. Inorg. Biochem. 2010, 104, 224– 231, DOI: 10.1016/j.jinorgbio.2009.11.003Google Scholar167Reaction of the zinc sensor FluoZin-3 with Zn7-metallothionein: Inquiry into the existence of a proposed weak binding siteNamdarghanbari, Mohammad Ali; Meeusen, Jeffrey; Bachowski, Gary; Giebel, Nicholas; Johnson, Jeremiah; Petering, David H.Journal of Inorganic Biochemistry (2010), 104 (3), 224-231CODEN: JIBIDJ; ISSN:0162-0134. (Elsevier B.V.)It has been reported that Zn7-metallothionein (MT), contains one weak binding site for Zn2+. To test this conclusion, rabbit liver MT isolated at pH 7 was reacted with chelating agents of modest affinity for Zn2+. Contrary to the previous study, no evidence was found for Zn2+ stoichiometrically bound to the protein with an apparent stability const. of about 108. Indeed, stability const. measurements based upon competition between Zn7-MT and ligands of known stability with Zn2+ showed that all of the protein bound Zn2+ displayed the same stability const. at pH 7.4 and 25 °C of (1.7 ± 0.6) × 1011. Brief reaction of Zn7-MT with strong acid converted it into MT* and upon re-neutralization into Zn7-MT*, which demonstrated reactivity of about 1 Zn2+/mol MT with competing ligands. Acid titrn. of Zn7-MT to pH 2 or below rapidly resulted in the formation of Zn7-MT* that displayed biphasic titrn. with base, revealing the re-binding of lower affinity Zn2+ between pH 5 and 7. Since MT is commonly acidified during prepn., care must be taken to document which form of the protein is present in subsequent expts. at pH 7.
- 168Hasler, D. W.; Jensen, L. T.; Zerbe, O.; Winge, D. R.; Vašák, M. Effect of the two conserved prolines of human growth inhibitory factor (metallothionein-3) on its biological activity and structure fluctuation: comparison with a mutant protein. Biochemistry 2000, 39, 14567– 14575, DOI: 10.1021/bi001569fGoogle Scholar168Effect of the Two Conserved Prolines of Human Growth Inhibitory Factor (Metallothionein-3) on Its Biological Activity and Structure Fluctuation: Comparison with a Mutant ProteinHasler, Daniel W.; Jensen, Laran T.; Zerbe, Oliver; Winge, Dennis R.; Vasak, MilanBiochemistry (2000), 39 (47), 14567-14575CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)Human neuronal growth inhibitory factor, a metalloprotein classified as metallothionein-3 (MT-3), impairs the survival and the neurite formation of cultured neurons. In these studies the double P7S/P9A mutant (mutMT-3) and single mutants P7S and P9A of human Zn7-MT-3 were generated, and their effects on the biol. activity and the structure of the protein were examd. The biol. results clearly established the necessity of both proline residues for the inhibitory activity, as even single mutants were found to be inactive. Using electronic absorption, CD (CD), magnetic CD (MCD), and 113Cd NMR spectroscopy, the structural features of the metal-thiolate clusters in the double mutant Cd7-mutMT-3 were investigated and compared with those of wild-type Cd7-MT-3 [Faller, P., Hasler, D. W., Zerbe, O., Klauser, S., Winge, D. R., and Vasak, M. (1999) Biochem. 38, 10158] and the well characterized Cd7-MT-2a from rabbit liver. Similarly to 113Cd7-MT-3 the 113Cd NMR spectrum of 113Cd7-mutMT-3 at 298 K revealed four major and three minor resonances (approx. 20% of the major ones) between 590 and 680 ppm, originating from a Cd4S11 cluster in the α-domain and a Cd3S9 cluster in the β-domain, resp. Due to the presence of dynamic processes in the structure of MT-3 and mutMT-3, all resonances showed the absence of resolved homonuclear [113Cd-113Cd] couplings and large apparent line widths (between 140 and 350 Hz). However, whereas in 113Cd7-mutMT-3 the temp. rise to 323 K resulted in a major recovery of the originally NMR nondetectable population of the Cd3S9 cluster resonances, no such temp. effect was obsd. in 113Cd7-MT-3. To account for the obsd. NMR features, a dynamic structural model for the β-domain is proposed, which involves a folded and a partially unfolded state. It is suggested that in the partially unfolded state a slow cis/trans isomerization of Cys-Pro(7) or Cys-Pro(9) amide bonds in 113Cd7-MT-3 takes place and that this process represents a rate-limiting step in a correct domain refolding. In addn., closely similar apparent stability consts. of human MT-3, mutMT-3, and rabbit MT-2a with Cd(II) and Zn(II) ions were found. These results suggest that specific structural features dictated by the repetitive (Cys-Pro)2 sequence in the β-domain of MT-3 and not its altered metal binding affinity compared to MT-1/MT-2 isoforms are responsible for the biol. activity of this protein.
- 169Peroza, E. A.; dos Santos Cabral, A.; Wan, X.; Freisinger, E. Metal ion release from metallothioneins: proteolysis as an alternative to oxidation. Metallomics 2013, 5, 1204– 1214, DOI: 10.1039/c3mt00079fGoogle Scholar169Metal ion release from metallothioneins: proteolysis as an alternative to oxidationPeroza, Estevao A.; dos Santos Cabral, Augusto; Wan, Xiaoqiong; Freisinger, EvaMetallomics (2013), 5 (9), 1204-1214CODEN: METAJS; ISSN:1756-591X. (Royal Society of Chemistry)Metallothioneins (MTs) are among others involved in the cellular regulation of essential ZnII and CuI ions. However, the high binding affinity of these proteins requires addnl. factors to promote metal ion release under physiol. conditions. The mechanisms and efficiencies of these processes leave many open questions. We report here a comprehensive anal. of the ZnII-release properties of various MTs with special focus on members of the four main subfamilies of plant MTs. ZnII competition expts. with the metal ion chelator 4-(2-pyridylazo)resorcinol (PAR) in the presence of the cellular redox pair glutathione (GSH)/glutathione disulfide (GSSG) show that plant MTs from the subfamilies MT1, MT2, and MT3 are remarkably more affected by oxidative stress than those from the Ec subfamily and the well-characterized human MT2 form. In addn., we evaluated proteolytic digestion with trypsin and proteinase K as an alternative mechanism for selective promotion of metal ion release from MTs. Also here the obsd. percentage of liberated metal ions depends strongly on the MT form evaluated. Closer evaluation of the data addnl. allowed deducing the thermodn. and kinetic properties of the ZnII release processes. The CuI-form of chickpea MT2 was used to exemplify that both oxidn. and proteolysis are also effective ways to increase the transfer of copper ions to other mols. ZnII release expts. with the individual metal-binding domains of Ec-1 from wheat grain reveal distinct differences from the full-length protein. This triggers the question about the roles of the long cysteine-free peptide stretches typical for plant MTs.
- 170Calvo, J. S.; Lopez, V. M.; Meloni, G. Non-coordinative metal selectivity bias in human metallothioneins metal-thiolate clusters. Metallomics 2018, 10, 1777– 1791, DOI: 10.1039/C8MT00264AGoogle Scholar170Non-coordinative metal selectivity bias in human metallothioneins metal-thiolate clustersCalvo, Jenifer S.; Lopez, Victor M.; Meloni, GabrieleMetallomics (2018), 10 (12), 1777-1791CODEN: METAJS; ISSN:1756-591X. (Royal Society of Chemistry)Mammalian metallothioneins (MT-1 through MT-4) are a class of metal binding proteins contg. two metal-thiolate clusters formed through the preferential coordination of d10 metals, Cu(I) and Zn(II), by 20 conserved cysteine residues located in two protein domains. MT metalation (homometallic or heterometallic Zn(II)/Cu(I) species) appears to be isoform specific and controlling zinc and copper concns. to perform specific and distinct biol. functions. Structural and functional relationships, and in vivo metalation studies, identified evolutionary features defining the metal-selectivity nature for MTs. Metallothionein-3 (MT-3) has been shown to possess the most pronounced Cu-thionein character forming Cu(I)-contg. species more favorably than metallothionein-2 (MT-2), which possesses the strongest Zn-thionein character. In this work, we identify isoform-specific determinants which control metal binding selectivity bias in different MTs isoforms. By studying the reactivity of Zn7MT-2, Zn7MT-3 and Zn7MT-3 mutants towards Cu(II) to form Cu(I)4Zn4MTs, we have identified isoform-specific key non-coordinating residues governing folding/outer sphere control of metal selectivity bias in MTs metal clusters. By mutating selected residues and motifs in MT-3 to the corresponding MT-2 amino acids, we dissected key roles in modulating cluster dynamic and metal exchange rates, in increasing the Cu(I)-affinity in MT-3 N-terminal β-domain and/or modulating the higher stability of the Zn(II)-thiolate cluster in MT-2 β-domain. We thus engineered MT-3 variants in which the copper-thionein character is converted into a zinc-thionein. These results provide new insights into the mol. determinants governing metal selectivity in metal-thiolate clusters.
- 171Kocyła, A.; Tran, J. B.; Krężel, A. Galvanization of protein-protein interactions in a dynamic zinc interactome. Trends Biochem. Sci. 2021, 46, 64– 79, DOI: 10.1016/j.tibs.2020.08.011Google Scholar171Galvanization of Protein-Protein Interactions in a Dynamic Zinc InteractomeKocyla, Anna; Tran, Jozef Ba; Krezel, ArturTrends in Biochemical Sciences (2021), 46 (1), 64-79CODEN: TBSCDB; ISSN:0968-0004. (Elsevier Ltd.)A review The presence of Zn2+ at protein-protein interfaces modulates complex function, stability, and introduces structural flexibility/complexity, chem. selectivity, and reversibility driven in a Zn2+-dependent manner. Recent studies have demonstrated that dynamically changing Zn2+ affects numerous cellular processes, including protein-protein communication and protein complex assembly. How Zn2+-involved protein-protein interactions (ZPPIs) are formed and dissoc. and how their stability and reactivity are driven in a zinc interactome remain poorly understood, mostly due to exptl. obstacles. Here, we review recent research advances on the role of Zn2+ in the formation of interprotein sites, their architecture, function, and stability. Moreover, we underline the importance of zinc networks in intersystemic communication and highlight bioinformatic and exptl. challenges required for the identification and investigation of ZPPIs.
- 172Pinter, T. B.; Stillman, M. J. The zinc balance: competitive zinc metalation of carbonic anhydrase and metallothionein 1A. Biochemistry 2014, 53, 6276– 6285, DOI: 10.1021/bi5008673Google Scholar172The Zinc Balance: Competitive Zinc Metalation of Carbonic Anhydrase and Metallothionein 1APinter, Tyler B. J.; Stillman, Martin J.Biochemistry (2014), 53 (39), 6276-6285CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)The small, cysteine-rich metallothionein family of proteins is currently considered to play a crit. role in the provision of metals to metalloenzymes. However, there is limited information available on the mechanisms of these fundamentally important interactions. We report on the competitive zinc metalation of apocarbonic anhydrase in the presence of apometallothionein 1A using electrospray-ionization mass spectrometry. These expts. revealed the relative affinities of zinc to all species in soln. The carbonic anhydrase is shown to compete efficiently only against Zn5-7MT. The calcd. equil. zinc binding consts. of each of the 7 zinc metallothionein 1A species ranged from a high of (log(KF)) 12.5 to a low of 11.8. The 8 equil. consts. connecting the 10 active species in competition for the zinc were modeled by fitting the KF values of the 8 competitive bimol. reactions to the ESI-mass spectral data. These modeled K values are shown to be exptl. connected to the metalation efficiency of the carbonic anhydrase. The series of 7 metallothionein binding affinities for zinc highlight the buffering role of zinc metallothioneins that permit simultaneously zinc storage and zinc sensing. Finally, the significance of the multiple zinc binding affinities of zinc metallothionein is discussed in relation to zinc homeostasis.
- 173Maret, W. Analyzing free zinc(II) ion concentrations in cell biology with fluorescent chelating molecules. Metallomics 2015, 7, 202– 211, DOI: 10.1039/C4MT00230JGoogle Scholar173Analyzing free zinc(ii) ion concentrations in cell biology with fluorescent chelating moleculesMaret, WolfgangMetallomics (2015), 7 (2), 202-211CODEN: METAJS; ISSN:1756-591X. (Royal Society of Chemistry)A review. Essential metal ions are tightly controlled in biol. systems. An understanding of metal metab. and homeostasis is being developed from quant. information of the sizes, concns., and dynamics of cellular and subcellular metal ion pools. In the case of human zinc metab., minimally 24 proteins of two zinc transporter families and a dozen metallothioneins participate in cellular uptake, extrusion, and re-distribution among cellular compartments. Significantly, zinc(II) ions are now considered signaling ions in intra- and intercellular communication. Such functions require transients of free zinc ions. It is exptl. quite challenging to distinguish zinc that is protein-bound from zinc that is not bound to proteins. Measurement of total zinc is relatively straightforward with anal. techniques such as at. absorption/emission spectroscopy or inductively coupled plasma mass spectrometry. Total zinc concns. of human cells are 200-300 μM. In contrast, the pool of non-protein bound zinc is mostly examd. with fluorescence microscopy/spectroscopy. There are two widely applied fluorescence approaches, one employing low mol. wt. chelating agents ("probes") and the other metal-binding proteins ("sensors"). The protein sensors, such as the CALWY, Zap/ZifCY, and carbonic anhydrase-based sensors, can be genetically encoded and have certain advantages in terms of controlling intracellular concn., localization, and calibration. When employed correctly, both probes and sensors can establish qual. differences in free zinc ion concns. However, when quant. information is sought, the assumptions underlying the applications of probes and sensors must be carefully examd. and even then measured pools of free zinc ions remain methodol. defined. A consensus is building that the steady-state free zinc ion concns. in the cytosol are in the picomolar range but there is no consensus on their concns. in subcellular compartments. Applying the extensive toolbox of available probes/sensors in biol. systems requires an understanding of the principles of cellular zinc homeostasis and the chem. biol. of the probes and sensors. Regardless of limitations in specificity (for a particular metal ion), selectivity (for a particular metal pool), and sensitivity (detection limit), the technol. is making remarkable contributions to imaging zinc with high spatiotemporal resoln. in single cells and to defining the biochem. functions of zinc ions in cellular regulation.
- 174Kochańczyk, T.; Drozd, A.; Krężel, A. Relationship between the architecture of zinc coordination and zinc binding affinity in proteins-insights into zinc regulation. Metallomics 2015, 7, 244– 257, DOI: 10.1039/C4MT00094CGoogle Scholar174Relationship between the architecture of zinc coordination and zinc binding affinity in proteins - insights into zinc regulationKochanczyk, Tomasz; Drozd, Agnieszka; Krezel, ArturMetallomics (2015), 7 (2), 244-257CODEN: METAJS; ISSN:1756-591X. (Royal Society of Chemistry)A review. Zinc proteins are an integral component of the proteome of all domains of life. Zn(II), one of the most widespread transition elements, serves multiple functions in proteins, such as a catalytic co-factor, a structural center and a signaling component. The mechanism by which proteins assoc. with and dissoc. from Zn(II) and the factors that modulate their affinity and stability remain incompletely understood. In this article, we aim to address how zinc binding sites present in proteins differ in their architecture and how their structural arrangement is assocd. with protein function, thermodn. and kinetic stability, reactivity, as well as zinc-dependent regulation. Here, we emphasize that the concn.-dependent functionality of the interprotein zinc binding site may serve as another factor regulating the relationship between cellular Zn(II) availability and protein function.
- 175Maret, W. Zinc in cellular regulation: The nature and significance of ″zinc signals″. Int. J. Mol. Sci. 2017, 18, 2285, DOI: 10.3390/ijms18112285Google Scholar175Zinc in cellular regulation: the nature and significance of "zinc signals"Maret, WolfgangInternational Journal of Molecular Sciences (2017), 18 (11), 2285/1-2285/12CODEN: IJMCFK; ISSN:1422-0067. (MDPI AG)In the last decade, we witnessed discoveries that established Zn2+ as a second major signalling metal ion in the transmission of information within cells and in communication between cells. Together with Ca2+ and Mg2+, Zn2+ covers biol. regulation with redox-inert metal ions over many orders of magnitude in concns. The regulatory functions of zinc ions, together with their functions as a cofactor in about three thousand zinc metalloproteins, impact virtually all aspects of cell biol. This article attempts to define the regulatory functions of zinc ions, and focuses on the nature of zinc signals and zinc signalling in pathways where zinc ions are either extracellular stimuli or intracellular messengers. These pathways interact with Ca2+, redox, and phosphorylation signalling. The regulatory functions of zinc require a complex system of precise homeostatic control for transients, subcellular distribution and traffic, organellar homeostasis, and vesicular storage and exocytosis of zinc ions.
- 176Maret, W.; Li, Y. Coordination dynamics of zinc in proteins. Chem. Rev. 2009, 109, 4682– 4707, DOI: 10.1021/cr800556uGoogle Scholar176Coordination dynamics of zinc in proteinsMaret, Wolfgang; Li, YuanChemical Reviews (Washington, DC, United States) (2009), 109 (10), 4682-4707CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)A review. In thousands of proteins, Zn participates in enzymic catalysis, structural organization, and/or regulation of function. The great no. and variety of Zn-contg. proteins has stimulated research on Zn2+ regulatory and chem. mechanisms that safeguard the distribution of Zn to proteins within the cell in a timely and spatially coordinated manner. The chem. properties of Zn in enzymes is largely attributed to its function as a strong Lewis acid. Generally, fast ligand exchange, stereochem. flexibility, and redox-inertness are addnl. characteristics for the selection of Zn2+ ions in the function of so many proteins. Here, the authors address the dynamics of Zn2+ coordination and the cellular distribution of Zn, namely, how proteins control Zn2+ (Zn2+ metalloregulation), how Zn2+ controls proteins (Zn2+ signaling), and how Zn2+ concns. are regulated and buffered intracellularly.
- 177Mattapalli, H.; Monteith, W. B.; Burns, C. S.; Danell, A. S. Zinc deposition during ESI-MS analysis of peptide-zinc-complexes. J. Am. Soc. Mass Spectrom. 2009, 20, 2199– 2205, DOI: 10.1016/j.jasms.2009.08.007Google Scholar177Zinc Deposition During ESI-MS Analysis of Peptide-Zinc ComplexesMattapalli, Haritha; Monteith, William B.; Burns, Colin S.; Danell, Allison S.Journal of the American Society for Mass Spectrometry (2009), 20 (12), 2199-2205CODEN: JAMSEF; ISSN:1044-0305. (Elsevier Inc.)Electrospray ionization (ESI) mass spectrometry (MS) has proven to be an extremely powerful technique for studying the stoichiometry and binding strength of peptide-metal complexes. We have found a significant new problem in the ESI-MS of zinc-peptide systems involving the deposition of zinc in the ESI emitter. This deposition of zinc during the expt. removes a significant amt. of zinc ions from the soln., impacting the resulting mass spectral intensities used to quantify the amt. of the zinc-bound species. Anal. of infused zinc-peptide samples with at. absorption spectrometry and with a custom-built nanoflow ESI source confirms the alteration of the analyte solns. with pos. or neg. or no potential applied to the emitter. Ultimately, the location of the zinc deposition was detd. to be the stainless steel emitter. The use of a custom-built nanoESI interface using glass emitters was found to mitigate the zinc deposition problem. The phenomenon of metal deposition warrants further investigation as it may not be limited to just zinc and may represent a significant obstacle in the ESI-MS anal. of all protein-metal systems.
- 178Kostyukevich, Y.; Kononikhin, A.; Popov, I.; Indeykina, M.; Kozin, S. A.; Makarov, A. A.; Nikolaev, E. Supermetallization of peptides and proteins during electrospray ionization. J. Mass Spectrom. 2015, 50, 1079– 1087, DOI: 10.1002/jms.3622Google Scholar178Supermetallization of peptides and proteins during electrospray ionizationKostyukevich, Yury; Kononikhin, Alexey; Popov, Igor; Indeykina, Maria; Kozin, Sergey A.; Makarov, Alexander A.; Nikolaev, EugeneJournal of Mass Spectrometry (2015), 50 (9), 1079-1087CODEN: JMSPFJ; ISSN:1076-5174. (John Wiley & Sons Ltd.)The formation of metal-peptide complexes during electrospray ionization (ESI) is a widely known phenomenon and is often considered to be undesirable. Such effect considerably limits the use of ESI mass spectrometry for the study of biol. relevant metal-peptide compds. that are present in the soln. and play crit. roles in many bioprocesses such as progression of neurodegenerative diseases. Under specific conditions such as high temp. of the desolvating capillary, an interesting effect, which can be called supermetallization, occurs. Using a model peptide Aβ amyloid domain 1-16, an increase in the temp. of the desolvating capillary results in multiple substitutions of hydrogen atoms by Zn atoms in this peptide. At high temps. (T ∼ 400°), up to 11 zinc atoms can be covalently bound to (1-16) Aβ. Supermetallization of (1-16) Aβ depends on the solvent compn. and pH. Supermetallization was also demonstrated for proteins, such as ubiquitin and cytochrome C. That proves that the supermetallization is a general phenomenon for peptides and proteins. For the structural study of supermetallized complexes, electron-capture dissocn. (ECD) fragmentation was applied. The effect of hydrogen rearranging during ECD was obsd. In addn., quantum chem. calcns. were used to est. the possible structures of different supermetallized complexes. These results allow a more deep understanding of the limitations of the use of ESI mass spectrometry for the study of biol. relevant metal-peptide complexes.
- 179Blindauer, C. A.; Leszczyszyn, O. I. Metallothioneins: unparalleled diversity in structures and functions for metal ion homeostasis and more. Nat. Prod. Rep. 2010, 27, 720– 741, DOI: 10.1039/b906685nGoogle Scholar179Metallothioneins: Unparalleled diversity in structures and functions for metal ion homeostasis and moreBlindauer, Claudia A.; Leszczyszyn, Oksana I.Natural Product Reports (2010), 27 (5), 720-741CODEN: NPRRDF; ISSN:0265-0568. (Royal Society of Chemistry)A review. Metallothioneins have been the subject of intense study for five decades, and have greatly inspired the development of bio-anal. methodologies including multi-dimensional and multi-nuclear NMR. With further advancements in mol. biol., protein science, and instrumental techniques, recent years have seen a renaissance of research into metallothioneins. The current report focuses on in vitro studies of so-called class II metallothioneins from a variety of phyla, highlighting the diversity of metallothioneins in terms of structure, biol. functions, and mol. functions such as metal ion specificity, thermodn. stabilities, and kinetic reactivity. We are still far from being able to predict any of these properties, and further efforts will be required to generate the knowledge that will enable a better understanding of what governs the biol. and chem. properties of these unusual and intriguing small proteins.
- 180Chan, J.; Huang, Z.; Merrifield, M. E.; Salgado, M. T.; Stillman, M. J. Studies of metal binding reactions in metallothioneins by spectroscopic, molecular biology, and molecular modeling techniques. Coord. Chem. Rev. 2002, 233, 319– 339, DOI: 10.1016/S0010-8545(02)00176-5Google Scholar180Studies of metal binding reactions in metallothioneins by spectroscopic, molecular biology, and molecular modeling techniquesChan, Jayna; Huang, Zuyun; Merrifield, Maureen E.; Salgado, Maria T.; Stillman, Martin J.Coordination Chemistry Reviews (2002), 233-234 (), 319-339CODEN: CCHRAM; ISSN:0010-8545. (Elsevier Science B.V.)A review with refs. Metallothioneins (MTs) are a class of metal-binding proteins characterized by a high cysteine content (up to 30% of the amino acid residues), low mol. wt., and lack of arom. amino acid residues. Remarkable metal-binding properties have been reported both in vivo and in vitro by a no. of different anal. techniques. Chem. and spectroscopic studies have shown that an unusually wide range of metals bind to MT. 113Cd and 1H-NMR techniques have been used successfully to det. the structures in a no. of different proteins. Together with X-ray diffraction results, analyses of these NMR data have established that in mammalian Cd7-MT and Zn7-MT, the metals are tetrahedrally coordinated in two isolated domains with stoichiometries of M4S11 and M3S9. Recently, Stillman, collaborators, and coworkers have characterized the Zn(II), Cd(II), Cu(I), Ag(I), Au(I) and Hg(II) contg. forms of MTs from mammalian, yeast, and recombinant human sources using a no. of different spectroscopic techniques. Optical spectroscopy, and in particular CD and luminescence, have provided rich details of a complicated metal binding chem., whether metals are added directly to the metal free, apo-MT, or to the Zn-contg. protein. Electrospray ionization mass spectrometry is a powerful technique for the characterization of proteins in general directly from soln. For MTs, both from mammalian and recombinant sources, use of this technique allows study of the details of metal binding to the protein as a function of metal loading and pH. Kinetic studies allow detn. of the metal binding mechanism when Cu(I) binds to both the metal-free and Zn(II) protein. Scanning tunneling microscopy is a surface anal. technique capable of producing nanoscale images with at. resoln. and is being applied to obtain images of MT on Au(111) surfaces. Mol. modeling, together with XANES, XAFS, and structural information from 2D-1H-NMR data allow the detn. of 3D structures of a range of MTs. Finally, mol. dynamics (MD) calcns. have been carried out to investigate the motion of the metal-contg. and metal free peptides, with special interest in the region of the metal binding site.
- 181Krężel, A.; Maret, W. The functions of metamorphic metallothioneins in zinc and copper metabolism. Int. J. Mol. Sci. 2017, 18, 1237, DOI: 10.3390/ijms18061237Google Scholar181The functions of metamorphic metallothioneins in zinc and copper metabolismKrezel, Artur; Maret, WolfgangInternational Journal of Molecular Sciences (2017), 18 (6), 1237/1-1237/20CODEN: IJMCFK; ISSN:1422-0067. (MDPI AG)A REVIEW. Recent discoveries in zinc biol. provide a new platform for discussing the primary physiol. functions of mammalian metallothioneins (MTs) and their exquisite zinc-dependent regulation. It is now understood that the control of cellular zinc homeostasis includes buffering of Zn2+ ions at picomolar concns., extensive subcellular re-distribution of Zn2+, the loading of exocytotic vesicles with zinc species, and the control of Zn2+ ion signalling. In parallel, characteristic features of human MTs became known: their graded affinities for Zn2+ and the redox activity of their thiolate coordination environments. Unlike the single species that structural models of mammalian MTs describe with a set of seven divalent or eight to twelve monovalent metal ions, MTs are metamorphic. In vivo, they exist as many species differing in redox state and load with different metal ions. The functions of mammalian MTs should no longer be considered elusive or enigmatic because it is now evident that the reactivity and coordination dynamics of MTs with Zn2+ and Cu+ match the biol. requirements for controlling-binding and delivering-these cellular metal ions, thus completing a 60-yr search for their functions. MT represents a unique biol. principle for buffering the most competitive essential metal ions Zn2+ and Cu+. How this knowledge translates to the function of other families of MTs awaits further insights into the specifics of how their properties relate to zinc and copper metab. in other organisms.
- 182Li, Y.; Maret, W. Human metallothionein metallomics. J. Anal. At. Spectrom. 2008, 23, 1055– 1062, DOI: 10.1039/b802220hGoogle Scholar182Human metallothionein metallomicsLi, Yuan; Maret, WolfgangJournal of Analytical Atomic Spectrometry (2008), 23 (8), 1055-1062CODEN: JASPE2; ISSN:0267-9477. (Royal Society of Chemistry)A review. Research on metallothionein (MT), unlike studies on any other metalloprotein, captures best the difficulties that investigators faced, and continue to face, when relating protein structure to protein function. This article discusses the structural complexity of the family of human MTs with the goal of providing a background for future investigations of their functions in health and disease by mol. and at. spectroscopies. The anal. (bio)chemist needs to overcome formidable challenges in terms of sample prepn. and speciation. MTs are expressed tissue- and isoprotein-specifically; their expression responds to a variety of physiol. and environmental stimuli; they are present as closely related products of at least ten different genes, several of which can be expressed simultaneously at considerably different levels in a given tissue; they have addnl. polymorphic forms, variable metal load, oxidn. states, and d.p.; they react with different metals and thiol-modifying agents; they are localized in the extracellular and intracellular space and re-distribute subcellularly. Results from the application of highly sensitive fluorimetric techniques suggest that the active form of the zinc/thiolate clusters in MT is not the one described by x-ray crystallog. or NMR spectroscopy and that the thiol chem. is the central aspect of MT's mechanism of action. MT is not satd. with zinc ions under normal physiol. conditions, and it exists to variable extent in the apoform (thionein) and in partially oxidized forms (thionin). The complexity of human MTs calls for metallomics and metalloproteomics approaches that integrate the knowledge from several scientific disciplines. Any interpretation of MT functions is based on the linkage between the metal ions and their sulfur ligands from the cysteines. Studies on the antioxidant or reactive species-scavenging properties of the cysteine sulfurs of MT need to take the effects on zinc metab. into account, while any studies with transition metal ions need to consider the effect of metal binding on the reactivity of the cysteine sulfurs.
- 183Maret, W.; Vallee, B. L. Cobalt as probe and label of proteins. Methods Enzymol. 1993, 226, 52– 71, DOI: 10.1016/0076-6879(93)26005-TGoogle Scholar183Cobalt as probe and label of proteinsMaret, Wolfgang; Vallee, Bert L.Methods in Enzymology (1993), 226 (Metallobiochemistry, Pt. C), 52-71CODEN: MENZAU; ISSN:0076-6879.A review with 68 refs. Crystallog. studies of 2 cobalt-substituted zinc enzymes that have now proved that the isomorphous replacements of the native zinc atoms provide a firm structural basis for this metal substitution. Whether cobalt could also serve as an isostructural probe in metalloenzymes other than zinc enzymes has not yet been ascertained. Cobalt derivs. of zinc enzymes have, for the first time, visualized dynamic events that occur in the coordination environment of zinc active sites during catalysis. The cobalt enzymes are almost as efficient catalysts as are the native zinc enzymes. Despite these characteristics, however, zinc and cobalt enzymes are not strictly isofunctional. They differ in substrate specificity and in some kinetic steps of the catalytic cycle.
- 184Freisinger, E.; Vašák, M. Cadmium in metallothioneins. Met. Ions Life Sci. 2013, 11, 339– 371, DOI: 10.1007/978-94-007-5179-8_11Google Scholar184Cadmium in metallothioneinsFreisinger, Eva; Vasak, MilanMetal Ions in Life Sciences (2013), 11 (Cadmium: From Toxicity to Essentiality), 339-371CODEN: MILSCT; ISSN:1559-0836. (Springer)Metallothioneins (MTs) are low-mol.-mass cysteine-rich proteins with the ability to bind mono- and divalent metal ions with the electron configuration d10 in form of metal-thiolate clusters. MTs are thought, among others, to play a role in the homeostasis of essential Zn(II) and Cu(I) ions. Besides these metal ions also Cd(II) can be bound to certain MTs in vivo, giving rise to the perception that another physiol. role of MTs is in the detoxification of heavy metal ions. Substitution of the spectroscopically silent Zn(II) ions in metalloproteins by Cd(II) proved to be an indispensable tool to probe the Zn(II) sites in vitro. In this review, methods applied in the studies of structural and chem. properties of Cd-MTs are presented. The first section focuses on the phys. basis of spectroscopic techniques such as electronic absorption, CD (CD), magnetic CD, X-ray absorption, and perturbed angular correlation of γ-rays spectroscopy, as well as mass spectrometry, and their applications to Cd-MTs from different organisms. The following is devoted to the discussion of metal binding affinities of Cd-MTs, cluster dynamics, the reactivity of bound Cd(II) ions with metal ion chelators and of thiolate ligands with alkylating and oxidizing agents. Finally, a brief summary of the known three-dimensional structures of Cd-MTs, detd. almost exclusively by multinuclear NMR techniques, is presented. Besides Cd-MTs, the described methods can also be applied to the study of metal binding sites in other metalloproteins.
- 185Stillman, M. J.; Cai, W.; Zelazowski, A. J. Cadmium binding to metallothioneins. Domain specificity in reactions of alpha and beta fragments, apometallothionein, and zinc metallothionein with Cd2+. J. Biol. Chem. 1987, 262, 4538– 4548, DOI: 10.1016/S0021-9258(18)61226-8Google Scholar185Cadmium binding to metallothioneins. Domain specificity in reactions of α and β fragments, apometallothionein, and zinc metallothionein with cadmium(2+)Stillman, Martin J.; Cai, Wuhua; Zelazowski, Andrzej J.Journal of Biological Chemistry (1987), 262 (10), 4538-48CODEN: JBCHA3; ISSN:0021-9258.The Cd-binding properties of rabbit liver Zn7-metallothionein (MT) 2 and apo-MT, rat liver apo-α MT and Zn4-α MT, and calf liver apo-β MT, were studied using CD and magnetic CD spectroscopies. Both sets of spectra recorded during the titrn. of Zn7-MT 2 with Cd exhibit a complicated pattern that is quite unexpected. Such behavior is not found at all in sets of spectra recorded during titrns. of the apo-species (apo-MT, apo-α MT, and apo-β MT), and is obsd. to a much lesser extent in the titrn. of Zn-α MT. Comparison between the band centers of the Cd-α MT and Cd-β MT indicates that the CD spectrum of Cd7-MT is dominated by intensity from transitions that originate on Cd-S chromophores in the α domain, with little direct contribution from the β domain. Anal. of the spectra recorded during titrns. of Zn7-MT 2 with Cd suggests: (i) that Cd replaces Zn in Zn7-MT isomorphously; (ii) that Cd binds in a nonspecific, distributed manner across both domains; (iii) that cluster formation in the α domain only occurs after 4 mol eq of Cd were added and is indicated by the presence of a cluster-sensitive, CD spectral feature; (iv) that the characteristic deriv. CD spectrum of native Cd4,Zn3-Met is only obtained from synthetic Cd4,Zn3-MT following a treatment cycle that allows the redistribution of Cd into the α domain; warming the synthetic native, Cd4,Zn3-MT, to 65° results in Cd being preferentially bound in the α domain; and (v) Zn7-MT will bind Cd quite normally at up to 65° but with greater specificity for the α domain compared with titrns. carried out at 25°. Apparently, the initial presence of Zn in both domains is an important factor in the lack of any domain specificity during Cd binding to Zn-MT which contrasts the domain specific manner obsd. for Cd binding to apo-MT.
- 186Vašák, M.; Kägi, J. H. R.; Holmquist, B.; Vallee, B. L. Spectral studies of cobalt(II)- and nickel(II)-metallothionein. Biochemistry 1981, 20, 6659– 6664, DOI: 10.1021/bi00526a021Google Scholar186Spectral studies of cobalt(II)- and nickel(II)-metallothioneinVasak, Milan; Kaegi, Jeremias H. R.; Holmquist, Barton; Vallee, Bert L.Biochemistry (1981), 20 (23), 6659-64CODEN: BICHAW; ISSN:0006-2960.The Zn and Cd of native rabbit metallothionein-1 were replaced stoichiometrically with either Co(II) or Ni(II). The electronic, MCD, and ESR spectra of Co(II)-metallothionein reflect distorted tetrahedral coordination of the Co atoms. Both the d-d and charge-transfer spectral regions closely resemble those of simple Co-tetrathiolate complexes, implying that their coordination chem. is analogous. Ni(II) complex ions and Ni(II)-metallothionein similarly exhibit analogous MCD bands in the d-d region. The CD bands assocd. with ligand-metal charge-transfer transitions in the non-d-d region of Co(II)- and Ni(II)-metallothionein afford addnl. evidence for the similarity in tetrahedral microsymmetry of the 2 metal derivs. The known ratio of 20 thiolate ligands to 7 metal ions, in conjunction with the spectral evidence for tetrathiolate coordination in metallothionein, represents good evidence that these metal thiolates are organized in clusters.
- 187Bertini, I.; Luchinat, C.; Messori, L.; Vašák, M. A two-dimensional NMR study of Co(II)7 rabbit liver metallothionein. Eur. J. Biochem. 1993, 211, 235– 240, DOI: 10.1111/j.1432-1033.1993.tb19891.xGoogle Scholar187A two-dimensional NMR study of cobalt(II)7 rabbit liver metallothioneinBertini, Ivano; Luchinat, Claudio; Messori, Luigi; Vasak, MilanEuropean Journal of Biochemistry (1993), 211 (1-2), 235-40CODEN: EJBCAI; ISSN:0014-2956.The 600-MHz 1H-NMR NOESY spectra on Co(II)7-reconstituted metallothionein (Co7MT), exhibiting hyperfine signals in the range 350 ppm to -50 ppm, with nuclear relaxation times of the order of a few milliseconds, have been measured and several interproton connectivities have been detected. This is the largest spectral window ever reported for a two-dimensional 1H-NMR spectrum in the case of a paramagnetic metalloprotein. No scalar connectivities could be detected. The hyperfine-shifted signals belong to the cysteine-ligand protons of the Co4S11 cluster of Co7MT. Together with results from one-dimensional NOE expts., the two-dimensional expts. permitted the pairwise assignment of the isotropically shifted signals of the CβH2 groups of the metal-coordinated cysteines. With the aid of computer-graphics inspection of the four-metal-cluster domain, based on the NMR soln. structure of Cd7MT, it is possible to propose sequence-specific assignments of a few hyperfine-shifted 1H-NMR signals. In particular, a tentative assignment is given for the six signals whose shifts exhibit an antiCurie temp. dependence. The assignment relies on the theor. model that qual. rationalizes the isotropic-shift pattern and its temp. dependence. Inferences on the soln. structure of the Co4S11 cluster are drawn.
- 188Pearce, L. L.; Gandley, R. E.; Han, W.; Wasserloos, K.; Stitt, M.; Kanai, A. J.; McLaughlin, M. K.; Pitt, B. R.; Levitan, E. S. Role of metallothionein in nitric oxide signaling as revealed by a green fluorescent fusion protein. Proc. Natl. Acad. Sci. U. S. A. 2000, 97, 477– 482, DOI: 10.1073/pnas.97.1.477Google Scholar188Role of metallothionein in nitric oxide signaling as revealed by a green fluorescent fusion proteinPearce, Linda L.; Gandley, Robin E.; Han, Weiping; Wasserloos, Karla; Stitt, Molly; Kanai, Anthony J.; McLaughlin, Margaret K.; Pitt, Bruce R.; Levitan, Edwin S.Proceedings of the National Academy of Sciences of the United States of America (2000), 97 (1), 477-482CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Although the function of metallothionein (MT), a 6- to 7-kDa cysteine-rich metal binding protein, remains unclear, it has been suggested from in vitro studies that MT is an important component of intracellular redox signaling, including being a target for nitric oxide (NO). To directly study the interaction between MT and NO in live cells, we generated a fusion protein consisting of MT sandwiched between two mutant green fluorescent proteins (GFPs). In vitro studies with this chimera (FRET-MT) demonstrate that fluorescent resonance energy transfer (FRET) can be used to follow conformational changes indicative of metal release from MT. Imaging expts. with live endothelial cells show that agents that increase cytoplasmic Ca2+ act via endogenously generated NO to rapidly and persistently release metal from MT. A role for this interaction in intact tissue is supported by the finding that the myogenic reflex of mesenteric arteries is absent in MT knockout mice (MT-/-) unless endogenous NO synthesis is blocked. These results are the first application of intramol. green fluorescent protein (GFP)-based FRET in a native protein and demonstrate the utility of FRET-MT as an intracellular surrogate indicator of NO prodn. In addn., an important role of metal thiolate clusters of MT in NO signaling in vascular tissue is revealed.
- 189Hong, S. H.; Maret, W. A fluorescence resonance energy transfer sensor for the beta-domain of metallothionein. Proc. Natl. Acad. Sci. U. S. A. 2003, 100, 2255– 2260, DOI: 10.1073/pnas.0438005100Google Scholar189A fluorescence resonance energy transfer sensor for the β-domain of metallothioneinHong, Sung-Hye; Maret, WolfgangProceedings of the National Academy of Sciences of the United States of America (2003), 100 (5), 2255-2260CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)We have designed a nanosensor to study the potential function of metallothionein (MT) in metal transfer and its interactions with redox partners and ligands by attaching two fluorescent probes to recombinant human MT. The specific labeling takes advantage of two different modification reactions. One is based on the fact that recombinant MT has a free N-terminal amino group when produced by the IMPACT T7 expression and purifn. system, the other on the observation that one human MT isoform (1b) contains an addnl. cysteine at position 32. It is located in the linker region of the mol., allowing the introduction of a probe between the two domains. An S32C mutation was introduced into hMT-2. Its thiol reactivity, metal binding capacity, and CD and UV spectra all demonstrate that the addnl. cysteine contains a free thiol(ate); it perturbs neither the overall structure of the protein nor the formation of the metal/thiolate clusters. MT contg. only cadmium was labeled stoichiometrically with Alexa 488 succinimidyl ester at the N terminus and with Alexa 546 maleimide at the free thiol group, followed by conversion to MT contg. only zinc. Energy transfer between Alexa 488 (donor) and Alexa 546 (acceptor) in double-labeled MT allows the monitoring of metal binding and conformational changes in the N-terminal β-domain of the protein.
- 190Otvos, J. D.; Armitage, I. M. Structure of the metal clusters in rabbit liver metallothionein. Proc. Natl. Acad. Sci. U. S. A. 1980, 77, 7094– 7098, DOI: 10.1073/pnas.77.12.7094Google Scholar190Structure of the metal clusters in rabbit liver metallothioneinOtvos, James D.; Armitage, Ian M.Proceedings of the National Academy of Sciences of the United States of America (1980), 77 (12), 7094-8CODEN: PNASA6; ISSN:0027-8424.Cd-113 NMR was used to det. the structures of the multiple Cd-binding sites in the 2 major isoproteins of rabbit liver metallothionein. Isotopically labeled metallothionein was sepd. from the livers of rabbits that had been subjected to repeated injections of 113CdCl2. The native protein isolated from these livers contains an appreciable amt. of Zn in addn. to Cd: 2-3 mol/mol protein out of a total metal content of 7 mol/mol protein. The 113Cd NMR spectrum of Cd, Zn-contg. metallothionein is quite complex, reflecting the fact that the native protein is a heterogeneous mixt. of species contg. different relative amts. of Zn and Cd. Replacement of the native Zn with 113Cd in vitro gave a protein whose 113Cd NMR spectrum was much simpler, contg. 8 distinct multiplets with chem. shifts ranging from 611 to 670 ppm. The multiplet structures were due to 113Cd-113Cd scalar coupling arising from 2-bond interactions between 113Cd ions linked to one another by bridging cysteine thiolate ligands. The sizes and structures of the metal clusters in the protein were detd. by the application of selective homonuclear 113Cd decoupling techniques. Rabbit liver metallothionein contains 2 sep. metal clusters, one contg. 4 Cd2+ ions and the other contg. 3. These 2 clusters, whose structures are the same in both isoproteins, were designated cluster A and cluster B, resp. Structures for the clusters are proposed that account for the 113Cd spin-coupling data and for the participation of all 20 of the cysteine residues in metal ligation, 11 in cluster A and 9 in cluster B. The appearance in the spectrum of 8 multiplets rather than the 7 that would be expected on the basis of the no. of metal-binding sites in the protein is an indication of some residual heterogeneity in the 113Cd-labeled metallothionein sample. The origin of this heterogeneity is suggested to be the presence of a protein species that lacks metal ions at its cluster B binding sites.
- 191Boulanger, Y.; Armitage, I. M.; Miklossy, K. A.; Winge, D. R. 113Cd NMR study of a metallothionein fragment. Evidence for a two-domain structure. J. Biol. Chem. 1982, 257, 13717– 13719, DOI: 10.1016/S0021-9258(18)33506-3Google Scholar191Cadmium-113 NMR study of a metallothionein fragment. Evidence for a two-domain structureBoulanger, Yvan; Armitage, Ian M.; Miklossy, Kathy Anne; Winge, Dennis R.Journal of Biological Chemistry (1982), 257 (22), 13717-19CODEN: JBCHA3; ISSN:0021-9258.A 32-residue polypeptide fragment, designated αI, of rat liver metallothionein obtained by subtilisin digestion was studied by 113Cd NMR. The amino acid compn. of the fragment corresponded to residues 30-61 of the metallothionein primary structure, and it contained 3.4 g atoms of Cd2+/mol of αI-fragment. Four 113Cd resonances were obsd., 3 of which had identical chem. shifts to those assigned to the 4-metal cluster in human liver metallothionein-2 under the same pH and buffer conditions. The 5-ppm chem. shift difference between the remaining resonance assigned to the 4-metal cluster in the intact protein can be explained to result from the removal of the N-terminal polypeptide fragment contg. the 3-metal cluster. These results provide unambiguous evidence for the 2-domain structure of metallothionein, contg. a sep. 3- and a 4-metal cluster.
- 192Zangger, K.; Shen, G.; Öz, G.; Otvos, J. D.; Armitage, I. M. Oxidative dimerization in metallothionein is a result of intermolecular disulphide bonds between cysteines in the alpha-domain. Biochem. J. 2001, 359, 353– 360, DOI: 10.1042/bj3590353Google Scholar192Oxidative dimerization in metallothionein is a result of intermolecular disulphide bonds between cysteines in the α-domainZangger, Klaus; Shen, Gong; Oz, Gulin; Otvos, James D.; Armitage, Ian M.Biochemical Journal (2001), 359 (2), 353-360CODEN: BIJOAK; ISSN:0264-6021. (Portland Press Ltd.)Upon storage under aerobic conditions metallothioneins (MTs) form a new species, which is characterized by a mol. mass approx. twice the size of monomeric MT and shifted 113/111Cd- and 1H-NMR resonances. The investigation of this oxidative dimerization process by NMR spectroscopy allowed us to structurally characterize this MT species that has been described to occur in vivo and might be synthesized under conditions of oxidative stress. The oxidative dimer was characterized by the formation of an intermol. cysteine disulfide bond involving the α-domain, and a detailed anal. of chem. shift changes and intermol. nuclear Overhauser effects points towards a disulfide bond involving Cys36. In contrast to the metal-bridged (non-oxidative) dimerization, the metal-cysteine cluster structures in both MT domains remain intact and no conformational exchange or metal-metal exchange was obsd. Also in contrast to the many recently reported oxidative processes which involve the β-domain cystcine groups and result in the increased dynamics of the bound metal ions in this N-terminal domain, we found no evidence for any increased dynamics in the α-domain metals following this oxidn. Therefore these findings provide addnl. corroboration that metal binding in the C-terminal α-domain is rather tight, even under conditions of a changing cellular oxidn. potential; compared with the more labile/dynamic nature of the metals in the N-terminal β-domain cluster under similar conditions.
- 193Gan, T.; Munoz, A.; Shaw, C. F., 3rd; Petering, D. H. Reaction of 111Cd7-metallothionein with EDTA. A reappraisal. J. Biol. Chem. 1995, 270, 5339– 5345, DOI: 10.1074/jbc.270.10.5339Google Scholar193Reaction of 111Cd7-metallothionein with EDTA. A reappraisalGan, Tong; Munoz, Amalia; Shaw, C. Frank, III; Petering, David H.Journal of Biological Chemistry (1995), 270 (10), 5339-45CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)The ligand substitution reaction of EDTA with Cd7-metallothionein (Cd7-MT) has been reinvestigated. NMR titrn. of the 111Cd-protein with EDTA showed that the ligand interacts preferentially and cooperatively with Cd2+ ions in the β-domain cluster. NMR and ultrafiltration kinetic anal. of this reaction using 5.6 mM Cd2+ as 111Cd7-MT and 56 mM EDTA indicated that cadmium-EDTA formed less rapidly than 111Cd peak intensity declined. Spectrophotometric and gel filtration studies of the reaction with 20 μM Cd2+ as Cd7-MT with various concns. of EDTA revealed biphasic kinetics with much larger rate consts. than obsd. in the NMR expts. The fraction of total ligand substitution occurring in each kinetic step varied with EDTA concn. The EDTA concn. dependence of both kinetic steps was consistent with the initial formation of protein·EDTA adducts, followed by their breakdown into products. Kinetic measurements were also made for the reactions of the isolated Cd4-α- and Cd3-β-domains with EDTA. The Cd4 domain reacted with EDTA with biphasic kinetics, in which one Cd2+ was removed rapidly with first-order kinetics, which were zero-order in EDTA. The other three reacted with kinetics like those for the slower step of the holoprotein. Cd3-β reacted with EDTA like the faster rate process assocd. with the Cd7-protein. The obsd. rate consts. for the reaction of Cd7-metallothionein with EDTA and the fraction of reaction in the faster rate process were sensitive to protein concn. These results are consistent with the hypothesis that the monomer-dimer equil. of the protein controls its kinetic reactivity with EDTA.
- 194Yu, X.; Wu, Z.; Fenselau, C. Covalent sequestration of melphalan by metallothionein and selective alkylation of cysteines. Biochemistry 1995, 34, 3377– 3385, DOI: 10.1021/bi00010a029Google Scholar194Covalent Sequestration of Melphalan by Metallothionein and Selective Alkylation of CysteinesYu, Xiaolan; Wu, Zhuchun; Fenselau, CatherineBiochemistry (1995), 34 (10), 3377-85CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)Rabbit liver metallothionein-2 is shown to form covalent bonds with the anticancer agent melphalan, in support of the hypothesis that covalent sequestration by metallothionein constitutes one mechanism for the cross-resistance acquired by cancer patients to therapeutic alkylating agents. Among 20 cysteines in the 2-domain protein, 89% of the first alkylation reaction occurs with 2 that co-chelate a zinc cation in the carboxy domain. Computer-supported docking studies indicate a favorable binding site for melphalan near these cysteine sulfhydryl groups. Although folded metallothionein-2 is resistant to trypsin cleavage, alkylation by 1 mol of melphalan allows cleavage by trypsin between the two globular domains.
- 195Zaia, J.; Jiang, L.; Han, M. S.; Tabb, J. R.; Wu, Z.; Fabris, D.; Fenselau, C. A binding site for chlorambucil on metallothionein. Biochemistry 1996, 35, 2830– 2835, DOI: 10.1021/bi952243nGoogle Scholar195A Binding Site for Chlorambucil on MetallothioneinZaia, Joseph; Jiang, Licong; Han, Mark S.; Tabb, Jeremiah R.; Wu, Zuchun; Fabris, Daniele; Fenselau, CatherineBiochemistry (1996), 35 (9), 2830-5CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)It is of interest to test the hypothesis that induced metallothionein (MT) acts in acquired drug resistance by covalent sequestration. In this study MT was incubated in vitro with chlorambucil (CHB) under conditions where only 1:1 covalent adducts were formed. The proteolytic products of these adducts were analyzed by HPLC and mass spectrometry to reveal two major sites of modification. These were the sulfur atoms of cysteines 33 and 48, which cochelate the same metal atom in native MT. The time course of the reaction was followed using online electrospray ionization with a double-focusing mass spectrometer. These expts. showed that drug-modified MT binds seven metal ions, as does the unmodified protein. Mol. docking expts. showed that the selectivity of drug binding is influenced by the presence of the aziridinium ion in the drug structure and complementary charge densities in the protein structure.
- 196Yu, X.; Wojciechowski, M.; Fenselau, C. Assessment of metals in reconstituted metallothioneins by electrospray mass spectrometry. Anal. Chem. 1993, 65, 1355– 1359, DOI: 10.1021/ac00058a010Google Scholar196Assessment of metals in reconstituted metallothioneins by electrospray mass spectrometryYu, Xiaolan; Wojciechowski, Marek; Fenselau, CatherineAnalytical Chemistry (1993), 65 (10), 1355-9CODEN: ANCHAM; ISSN:0003-2700.A method was developed that combines electrospray-ionization mass spectrometry with pH control to provide anal. of metals in native or reconstituted metallothioneins. These metalloproteins cooperatively bind 7 divalent metal ions, most commonly Zn2+ and Cd2+. Since the protein is denatured and metal ions are lost below pH 3, the pH of the electrospray soln. is crit. to successful results. The metal-free apoprotein was detected with its most abundant ions in a charge state of 6+, while the folded metallothionein-metal complexes were obsd. with lower charge states. The retention of 7 metals in the mol. ions detected is consistent with the hypothesis that metallothionein retains its conformation in the gas phase. This mass spectrometric technique can be used to det. rapidly and accurately how many and what cations are incorporated per mol. of protein. Information about molar distributions and ests. of relative abundances of various complexes in the sample can be acquired in a single measurement.
- 197Zaia, J.; Fabris, D.; Wei, D.; Karpel, R. L.; Fenselau, C. Monitoring metal ion flux in reactions of metallothionein and drug-modified metallothionein by electrospray mass spectrometry. Protein Sci. 1998, 7, 2398– 2404, DOI: 10.1002/pro.5560071117Google Scholar197Monitoring metal ion flux in reactions of metallothionein and drug-modified metallothionein by electrospray mass spectrometryZaia, Joseph; Fabris, Daniele; Wei, Dong; Karpel, Richard L.; Fenselau, CatherineProtein Science (1998), 7 (11), 2398-2404CODEN: PRCIEI; ISSN:0961-8368. (Cambridge University Press)The capabilities of electrospray ionization mass spectrometry are demonstrated for monitoring the flux of metal ions out of and into the metalloprotein rabbit liver metallothionein and, in one example, chlorambucil-alkylated metallothionein. Metal ion transfers may be followed as the reactions proceed in situ to provide kinetic information. More uniquely to this technique, metal ion stoichiometries may be detd. for reaction intermediates and products. Partners used in these studies include EDTA, carbonic anhydrase, a zinc-bound hexamer of insulin, and the core domain of bacteriophage T4 gene 32 protein, a binding protein for single-stranded DNA.
- 198Gehrig, P. M.; You, C.; Dallinger, R.; Gruber, C.; Brouwer, M.; Kägi, J. H.; Hunziker, P. E. Electrospray ionization mass spectrometry of zinc, cadmium, and copper metallothioneins: evidence for metal-binding cooperativity. Protein Sci. 2000, 9, 395– 402, DOI: 10.1110/ps.9.2.395Google Scholar198Electrospray ionization mass spectrometry of zinc, cadmium, and copper metallothioneins: evidence for metal-binding cooperativityGehrig, Peter M.; You, Chunhui; Dallinger, Reinhard; Gruber, Christine; Brouwer, Marius; Kagi, Jeremias H. R.; Hunziker, Peter E.Protein Science (2000), 9 (2), 395-402CODEN: PRCIEI; ISSN:0961-8368. (Cambridge University Press)Electrospray ionization (ESI) mass spectra of both well-characterized and novel metallothioneins (MTs) from various species were recorded to explore their metal-ion-binding modes and stoichiometries. The ESI mass spectra of the zinc- and cadmium-binding MTs showed a single main peak corresponding to metal-to-protein ratios of 4, 6, or 7. These findings combined with data obtained by other methods suggest that these MTs bind zinc or cadmium in a single predominant form and are consistent with the presence of three- and four-metal clusters. An unstable copper-specific MT isoform from Roman snails (Helix pomatia) could be isolated intact and was shown to preferentially bind 12 copper ions. To obtain addnl. information on the formation and relative stability of metal-thiolate clusters in MTs, a mass spectrometric titrn. study was conducted. One to seven molar equivalents of zinc or of cadmium were added to metal-free human MT-2 at neutral pH, and the resulting complexes were measured by ESI mass spectrometry. These expts. revealed that the formation of the four-metal cluster and of the thermodynamically less stable three-metal cluster is sequential and largely cooperative for both zinc and cadmium. Minor intermediate forms between metal-free MT, Me4MT, and fully reconstituted Me7MT were also obsd. The addn. of increasing amts. of cadmium to metal-free blue crab MT-I resulted in prominent peaks whose masses were consistent with apoMT, Cd3MT, and Cd6MT, reflecting the known structure of this MT with two Me3Cys9 centers. In a similar reconstitution expt. performed with Caenorhabditis elegans MT-II, a series of signals corresponding to apoMT and Cd3MT to Cd6MT species were obsd.
- 199Meloni, G.; Zovo, K.; Kazantseva, J.; Palumaa, P.; Vašák, M. Organization and assembly of metal-thiolate clusters in epithelium specific metallothionein-4. J. Biol. Chem. 2006, 281, 14588– 14595, DOI: 10.1074/jbc.M601724200Google Scholar199Organization and Assembly of Metal-Thiolate Clusters in Epithelium-specific Metallothionein-4Meloni, Gabriele; Zovo, Kairit; Kazantseva, Jekaterina; Palumaa, Peep; Vasak, MilanJournal of Biological Chemistry (2006), 281 (21), 14588-14595CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)Mammalian metallothionein-4 (MT-4) was found to be specifically expressed in stratified squamous epithelia where it plays an essential but poorly defined role in regulating zinc or copper metab. Here we report on the organization, stability, and the pathway of metal-thiolate cluster assembly in MT-4 reconstituted with Cd2+ and Co2+ ions. Both the 113Cd NMR studies of 113Cd7MT-4 and the spectroscopic characterization of Co7MT-4 showed that, similar to the classical MT-1 and MT-2 proteins, metal ions are organized in two independent Cd4Cys11 and Cd3Cys9 clusters with each metal ion tetrahedrally coordinated by terminal and bridging cysteine ligands. Moreover, we have demonstrated that the cluster formation in Cd7MT-4 is cooperative and sequential, with the Cd4Cys11 cluster being formed first, and that a distinct single-metal nucleation intermediate Cd1MT-4 is required in the cluster formation process. Conversely, the absorption and CD features of metal-thiolate clusters in Cd7MT-4 indicate that marked differences in the cluster geometry exist when compared with those in Cd7MT-1/2. The biol. implication of our studies as to the role of MT-4 in zinc metab. of stratified epithelia is discussed.
- 200Wan, X.; Freisinger, E. The plant metallothionein 2 from Cicer arietinum forms a single metal-thiolate cluster. Metallomics 2009, 1, 489– 500, DOI: 10.1039/b906428aGoogle Scholar200The plant metallothionein 2 from Cicer arietinum forms a single metal-thiolate clusterWan, Xiaoqiong; Freisinger, EvaMetallomics (2009), 1 (6), 489-500CODEN: METAJS; ISSN:1756-591X. (Royal Society of Chemistry)The plant metallothionein 2 from Cicer arietinum (chickpea; cicMT2) is a typical member of this subfamily and features two cysteine-rich regions contg. eight and six cysteine residues, resp., sepd. by a linker region 41 amino acids in length. This metallothionein thus differs significantly from the well-studied vertebrate forms. A synthetic gene encoding cicMT2 was designed, cloned into a suitable vector, and the protein was over-expressed in Escherichia coli. For the first time, an in-depth spectroscopic characterization of cicMT2 in the presence of divalent metal ions is performed showing a binding capacity for five ZnII, CdII, or CoII ions and the typical features of metal-thiolate clusters. Based on proteolytic digestion expts., the cluster arrangement formed by the divalent metal ions and the cysteine thiolate groups connects the amino-terminal with the carboxy-terminal cysteine-rich region. The cluster formation process, put into effect with the addn. of the fourth metal ion to the apo protein, was investigated using the characteristic shift of absorption bands obsd. in the UV/Vis spectra upon titrn. with CoII. The pH-dependent ZnII- and CdII-thiolate cluster stability is one of the highest obsd. for plant MTs so far, but lower than that usually found in vertebrate metallothioneins. The dependence of the pH stability on the ionic strength of the soln. is more pronounced for the CdII- than for the ZnII-form of the protein.
- 201Artells, E.; Palacios, Ò.; Capdevila, M.; Atrian, S. In vivo-folded metal-metallothionein 3 complexes reveal the Cu-thionein rather than Zn-thionein character of this brain-specific mammalian metallothionein. FEBS J. 2014, 281, 1659– 1678, DOI: 10.1111/febs.12731Google Scholar201In vivo-folded metal-metallothionein 3 complexes reveal the Cu-thionein rather than Zn-thionein character of this brain-specific mammalian metallothioneinArtells, Ester; Palacios, Oscar; Capdevila, Merce; Atrian, SilviaFEBS Journal (2014), 281 (6), 1659-1678CODEN: FJEOAC; ISSN:1742-464X. (Wiley-Blackwell)Metallothionein-3 (MT3) is one of the 4 mammalian metallothioneins (MT), and is constitutively synthesized in the brain. MT3 acts both intracellularly and extracellularly in this organ, performing functions related to neuronal growth and physiol. metal (Zn and Cu) handling. It appears to be involved in the prevention of neurodegenerative disorders caused by insol. Cu-peptide aggregates, as it triggers a Zn-Cu swap that may counteract the deleterious presence of Cu in neural tissues. The literature data on MT3 coordination come from studies either on apo-MT3 reconstitution or the reaction of Zn-MT3 with Cu2+, an ion that is hardly present inside cells. To ascertain the MT3 metal-binding features in a scenario closer to the reductive cell cytoplasm, a study of the recombinant Zn2+, Cd2+ and Cu+ complexes of MT3, βMT3, and αMT3, as well as the in vitro Zn2+-Cd2+ and Zn2+-Cu+ replacement processes, is presented here. The authors conclude that MT3 has a Cu-thionein character that is stronger than that of the MT1 and MT2 isoforms, which are also present in the mammalian brain, which is mainly contributed by its β domain. In contrast, the α domain retains a high capacity to bind Zn2+ ions, and, consequently, the entire MT3 peptide shows a peculiar dual ability to handle both metal ions. The nature of the formed Cu+-MT3 complexes oscillates from heterometallic Cu6Zn4-MT3 to homometallic Cu10-MT3 major species, in a narrow Cu concn. range. Therefore, the entire MT3 peptide shows a high capacity to bind Cu+, provided that this occurs in a nonoxidative milieux. This reflects a peculiar property of this MT isoform, which accurately senses different Cu contents in the environment in which it is synthesized.
- 202Breuker, K.; McLafferty, F. W. Stepwise evolution of protein native structure with electrospray into the gas phase, 10–12 to 102 s. Proc. Natl. Acad. Sci. U. S. A. 2008, 105, 18145– 18152, DOI: 10.1073/pnas.0807005105Google Scholar202Stepwise evolution of protein native structure with electrospray into the gas phase, 10-12 to 102 sBreuker, Kathrin; McLafferty, Fred W.Proceedings of the National Academy of Sciences of the United States of America (2008), 105 (47), 18145-18152CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)A review. Mass spectrometry (MS) has been revolutionized by electrospray ionization (ESI), which is sufficiently "gentle" to introduce nonvolatile biomols. such as proteins and nucleic acids (RNA or DNA) into the gas phase without breaking covalent bonds. Although in some cases noncovalent bonding can be maintained sufficiently for ESI/MS characterization of the soln. structure of large protein complexes and native enzyme/substrate binding, the new gaseous environment can ultimately cause dramatic structural alterations. The temporal (picoseconds to minutes) evolution of native protein structure during and after transfer into the gas phase, as proposed here based on a variety of studies, can involve side-chain collapse, unfolding, and refolding into new, non-native structures. Control of individual exptl. factors allows optimization for specific research objectives.
- 203Konermann, L.; Ahadi, E.; Rodriguez, A. D.; Vahidi, S. Unraveling the mechanism of electrospray ionization. Anal. Chem. 2013, 85, 2– 9, DOI: 10.1021/ac302789cGoogle Scholar203Unraveling the Mechanism of Electrospray IonizationKonermann, Lars; Ahadi, Elias; Rodriguez, Antony D.; Vahidi, SiavashAnalytical Chemistry (Washington, DC, United States) (2013), 85 (1), 2-9CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)A review. Electrospray ionization (ESI) generates intact gas phase ions from analytes in soln. for mass spectrometric studies. ESI can proceed via different mechanisms. Low mol. wt. analytes follow the ion evapn. model (IEM), whereas the charged residue model (CRM) applies to large globular species. A chain ejection model (CEM) was proposed for disordered polymers.
- 204Jayawardena, D. P.; Heinemann, I. U.; Stillman, M. J. Zinc binds non-cooperatively to human liver metallothionein 2a at physiological pH. Biochem. Biophys. Res. Commun. 2017, 493, 650– 653, DOI: 10.1016/j.bbrc.2017.08.137Google Scholar204Zinc binds non-cooperatively to human liver metallothionein 2a at physiological pHJayawardena, Devika P.; Heinemann, Ilka U.; Stillman, Martin J.Biochemical and Biophysical Research Communications (2017), 493 (1), 650-653CODEN: BBRCA9; ISSN:0006-291X. (Elsevier B.V.)Maintenance of the homeostasis of zinc is very important in regulating bodily functions. There are over 300 Zn-dependent enzymes identified where Zn(II) plays a structural or catalytic role. However, an excess of Zn(II) in a cell is toxic and free Zn(II) is tightly controlled. Metallothioneins (MTs) are small cysteine rich proteins that can bind up to seven Zn(II) and act as a Zn(II) reservoir. The MT2a isoform is predominantly found in the liver. This study focused on designing an MT2a construct of recombinant human MT2a to det. the Zn(II) binding profile of MT2a in vitro. We analyzed the pH dependence of Zn-MT2a speciation from electrospray ionization mass spectral data. At physiol. pH, Zn(II) is terminally bound to the cysteine thiols of MT2a, making bead-like structures (non-cooperative metal binding), while at low pH, Zn(II) formed Zn4S11-MT2a clusters involving bridged cysteinyl thiols to the Zn(II) (cooperative metal binding). The Zn(II) binding profile of MT2a was compared to Zn(II) binding profile of human kidney MT1a, which was reported in literature, and found that the Zn(II) binding profile of MT2a is similar to that of MT1a. The facility of forming bead-like structures at physiol. pH for Zn5-MT2a means that Zn7-MT2a can donate up to two Zn(II) to Zn-dependent enzymes.
- 205Scheller, J. S.; Irvine, G. W.; Stillman, M. Unraveling the mechanistic details of metal binding to mammalian metallothioneins from stoichiometric, kinetic, and binding affinity data. Dalton Trans. 2018, 47, 3599– 4002, DOI: 10.1039/C7DT03319BGoogle ScholarThere is no corresponding record for this reference.
- 206Korkola, N. C.; Scarrow, P. M.; Stillman, M. J. pH dependence of the non-cooperative binding of Bi3+ to human apo-metallothionein 1A: kinetics, speciation, and stoichiometry. Metallomics 2020, 12, 435– 448, DOI: 10.1039/C9MT00285EGoogle Scholar206pH dependence of the non-cooperative binding of Bi3+ to human apo-metallothionein 1A: kinetics, speciation, and stoichiometryKorkola, Natalie C.; Scarrow, Patti M.; Stillman, Martin J.Metallomics (2020), 12 (3), 435-448CODEN: METAJS; ISSN:1756-591X. (Royal Society of Chemistry)In this paper, we explored the binding of [Bi(cit)]- and [Bi(EDTA)]- to apo-MT 1a as the most basic of binding motifs. It was found that both Bi3+ salts bound in a non-cooperative stepwise manner to terminal cysteinal thiolates at pH 2.6, 5.0, and 7.4. We report that [Bi(EDTA)]- only binds stepwise up to Bi6MT, whereas [Bi(cit)]- forms up to Bi8MT, where the 7th and 8th Bi3+ appear to be adducts. Stepwise speciation anal. provided the 7 binding consts. that decreased systematically from K1 to K7 indicating a non-cooperative binding profile. Cysteine modifications with benzoquinone and iodoacetamide revealed that when apoMT is fully metalated with Bi3+ there are two free cysteines, meaning 18 cysteines are used in binding the 6 Bi3+. Kinetic studies showed that [Bi(EDTA)]- binds very slowly at pH 2.6 (k = 0.0290 x 106 M-1 s-1) and approx. 2000 times faster at pH 7.4 (k = 66.5 x 106 M-1 s-1). [Bi(cit)]- binding at pH 2.6 was faster than [Bi(EDTA)]- (k = 672 x 106 M-1 s-1) at either pH level. The data strongly support a non-clustered binding motif, emphasizing the non-traditional pathway reported previously for As3+.
- 207Peris-Díaz, M. D.; Guran, R.; Zitka, O.; Adam, V.; Krężel, A. Mass spectrometry-based structural analysis of cysteine-rich metal-binding sites in proteins with MetaOdysseus R software. J. Proteome Res. 2021, 20, 776– 785, DOI: 10.1021/acs.jproteome.0c00651Google Scholar207Mass Spectrometry-Based Structural Analysis of Cysteine-Rich Metal-Binding Sites in Proteins with MetaOdysseus R SoftwarePeris-Diaz, Manuel David; Guran, Roman; Zitka, Ondrej; Adam, Vojtech; Krezel, ArturJournal of Proteome Research (2021), 20 (1), 776-785CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)Identification of metal-binding sites in proteins and understanding metal-coupled protein folding mechanisms are aspects of high importance for the structure-to-function relationship. Mass spectrometry (MS) has brought a powerful adjunct perspective to structural biol., obtaining from metal-to-protein stoichiometry to quaternary structure information. Currently, the different exptl. and/or instrumental setups usually require the use of multiple data anal. software, and in some cases, they lack some of the main data anal. steps (MS processing, scoring, identification). Here, we present a comprehensive data anal. pipeline that addresses charge-state deconvolution, statistical scoring, and mass assignment for native MS, bottom-up, and native top-down with emphasis on metal-protein complexes. We have evaluated all of the approaches using assemblies of increasing complexity, including free and chem. labeled proteins, from low- to high-resoln. MS. In all cases, the results have been compared with common software and proved how MetaOdysseus outperformed them.
- 208Laganowsky, A.; Reading, E.; Allison, T. M.; Ulmschneider, M. B.; Degiacomi, M. T.; Baldwin, A. J.; Robinson, C. V. Membrane proteins bind lipids selectively to modulate their structure and function. Nature 2014, 510, 172– 175, DOI: 10.1038/nature13419Google Scholar208Membrane proteins bind lipids selectively to modulate their structure and functionLaganowsky, Arthur; Reading, Eamonn; Allison, Timothy M.; Ulmschneider, Martin B.; Degiacomi, Matteo T.; Baldwin, Andrew J.; Robinson, Carol V.Nature (London, United Kingdom) (2014), 510 (7503), 172-175CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)Previous studies have established that the folding, structure and function of membrane proteins are influenced by their lipid environments and that lipids can bind to specific sites, for example, in potassium channels. Fundamental questions remain however regarding the extent of membrane protein selectivity towards lipids. Here we report a mass spectrometry approach designed to det. the selectivity of lipid binding to membrane protein complexes. We investigate the mechanosensitive channel of large conductance (MscL) from Mycobacterium tuberculosis and aquaporin Z (AqpZ) and the ammonia channel (AmtB) from Escherichia coli, using ion mobility mass spectrometry (IM-MS), which reports gas-phase collision cross-sections. We demonstrate that folded conformations of membrane protein complexes can exist in the gas phase. By resolving lipid-bound states, we then rank bound lipids on the basis of their ability to resist gas phase unfolding and thereby stabilize membrane protein structure. Lipids bind non-selectively and with high avidity to MscL, all imparting comparable stability; however, the highest-ranking lipid is phosphatidylinositol phosphate (PI), in line with its proposed functional role in mechanosensation. AqpZ is also stabilized by many lipids, with cardiolipin (CDL) imparting the most significant resistance to unfolding. Subsequently, through functional assays we show that cardiolipin modulates AqpZ function. Similar expts. identify AmtB as being highly selective for phosphatidylglycerol (PG), prompting us to obtain an X-ray structure in this lipid membrane-like environment. The 2.3 Å resoln. structure, when compared with others obtained without lipid bound, reveals distinct conformational changes that re-position AmtB residues to interact with the lipid bilayer. Our results demonstrate that resistance to unfolding correlates with specific lipid-binding events, enabling a distinction to be made between lipids that merely bind from those that modulate membrane protein structure and/or function. We anticipate that these findings will be important not only for defining the selectivity of membrane proteins towards lipids, but also for understanding the role of lipids in modulating protein function or drug binding.
- 209Przygońska, K.; Poznański, J.; Mistarz, U. H.; Rand, K. D.; Dadlez, M. Side-chain moieties from the N-terminal region of Aβ are involved in an oligomer-stabilizing network of interactions. PLoS One 2018, 13, e0201761 DOI: 10.1371/journal.pone.0201761Google Scholar209Side-chain moieties from the N-terminal region of Aβ are Involved in an oligomer stabilizing network of interactionsPrzygonska, Kaja; Poznanski, Jarosoaw; Mistarz, Ulrik H.; Rand, Kasper D.; Dadlez, MichaoPLoS One (2018), 13 (8), e0201761/1-e0201761/25CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)Oligomeric forms of the Aβ peptide represent the most probable neurotoxic agent in Alzheimer's disease. The dynamic and heterogeneous character of these oligomers makes their structural characterization by classic methods difficult. Native mass spectrometry, when supported by addnl. gas phase techniques, like ion mobility sepn. and hydrogendeuterium exchange (IM-HDX-MS), enable anal. of different oligomers coexisting in the sample and may provide species-specific structural information for each oligomeric form populated in the gas phase. Here, we have combined these three techniques to obtain insight into the structural properties of oligomers of Aβ1-40 and two variants with scrambled sequences. Gas-phase HDX-MS revealed a sequence-specific engagement of the sidechains of residues located at the N-terminal part of the peptide in a network of oligomer-stabilizing interactions. Oligomer-specific interactions were no longer obsd. in the case of the fully scrambled sequence. Also, the ability to form alternative structures, obsd. for WT Aβ peptide, was lost upon scrambling. Our data underscore a role for the N-terminal residues in shaping the equil. of oligomeric forms. Although the peptide lacking the N-terminal 1-16 residues (p3 peptide) is thought to be benign, the role of the N-terminus has not been sufficiently characterized yet. We speculate that the interaction networks revealed here may be crucial for enabling structural transitions necessary to obtain mature parallel cross-β structures from smaller antiparallel oligomers. We provide a hypothetical mol. model of the trajectory that allows a gradual conversion from antiparallel to parallel oligomers without decompn. of oligomers. Oligomer-defining interactions involving the Aβ peptide N-terminus may be important in prodn. of the neurotoxic forms and thus should not be neglected.
- 210Guo, Y.; Ling, Y.; Thomson, B. A.; Siu, K. W. Combined ion-mobility and mass-spectrometry investigations of metallothionein complexes using a tandem mass spectrometer with a segmented second quadrupole. J. Am. Soc. Mass Spectrom. 2005, 16, 1787– 1794, DOI: 10.1016/j.jasms.2005.07.011Google Scholar210Combined Ion-Mobility and Mass-Spectrometry Investigations of Metallothionein Complexes Using a Tandem Mass Spectrometer with a Segmented Second QuadrupoleGuo, Yuzhu; Ling, Yun; Thomson, Bruce A.; Siu, K. W. MichaelJournal of the American Society for Mass Spectrometry (2005), 16 (11), 1787-1794CODEN: JAMSEF; ISSN:1044-0305. (Elsevier Inc.)Rabbit metallothionein (MT) 2A complexes with Cd(II), Zn(II), Ag(I), Cu(I), Hg(II), arsenite, monomethylarsonous acid (MMA), and dimethylarsinous acid (DMA) have been examd. using ion-mobility measurements and mass spectrometry in a triple-quadrupole mass spectrometer equipped with a segmented second quadrupole that doubled as an ion-mobility cell (2005). The metal ions confer conformational rigidity on the MT complexes, which counteracts Coulombic repulsion among protons added as a result of electrospray. Triply and quadruply protonated Cd7MT2A have smaller cross-sections than the Cd7MT2A structure deduced from published NMR data. For the 6+ ions, the As6MT2A complex has a cross-section of 790 Å2; the MMA10MT2A complex, 920 Å2; and the DMA20MT2A complex, 1220 Å2. This increase in cross-section of the As(III) species, from As3+ to MMA to DMA, is interpreted as a consequence of decreasing multiple coordination and increasing no. of Me groups.
- 211Dupuis, N. F.; Wu, C.; Shea, J. E.; Bowers, M. T. The amyloid formation mechanism in human IAPP: dimers have β-strand monomer-monomer interfaces. J. Am. Chem. Soc. 2011, 133, 7240– 7243, DOI: 10.1021/ja1081537Google Scholar211The Amyloid Formation Mechanism in Human IAPP: Dimers Have β-Strand Monomer-Monomer InterfacesDupuis, Nicholas F.; Wu, Chun; Shea, Joan-Emma; Bowers, Michael T.Journal of the American Chemical Society (2011), 133 (19), 7240-7243CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Early oligomerization of human IAPP (hIAPP) is responsible for β-cell death in the pancreas and is increasingly considered a primary pathol. process linked to Type II Diabetes (T2D). Yet, the assembly mechanism remains poorly understood, largely due to the inability of conventional techniques to probe distributions or detailed structures of early oligomeric species. Here, we describe the first exptl. data on the isolated and unmodified dimers of human (hIAPP) and nonamyloidogenic rat IAPP (rIAPP). The expts. reveal that the human IAPP dimers are more extended than those formed by rat IAPP and likely descend from extended monomers. Independent all-atom mol. dynamics simulations show that rIAPP forms compact helix and coil rich dimers, whereas hIAPP forms β-strand rich dimers that are generally more extended. Also, the simulations reveal that the monomer-monomer interfaces of the hIAPP dimers are dominated by β-strands and that β-strands can recruit coil or helix structured regions during the dimerization process. Our β-rich interface contrasts with an N-terminal helix-to-helix interface proposed in the literature but is consistent with existing exptl. data on the self-interaction pattern of hIAPP, mutation effects, and inhibition effects of the N-methylation in the mutation region.
- 212Pagel, K.; Natan, E.; Hall, Z.; Fersht, A. R.; Robinson, C. V. Intrinsically disordered p53 and its complexes populate compact conformations in the gas phase. Angew. Chem., Int. Ed. 2013, 52, 361– 365, DOI: 10.1002/anie.201203047Google Scholar212Intrinsically Disordered p53 and Its Complexes Populate Compact Conformations in the Gas PhasePagel, Kevin; Natan, Eviatar; Hall, Zoe; Fersht, Alan R.; Robinson, Carol V.Angewandte Chemie, International Edition (2013), 52 (1), 361-365CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)The use of ion mobility mass spectrometry (IM-MS) to study the topol. of proteins and their complexes is attracting considerable attention. Under the appropriate conditions results have shown that unique insight can be gained for intractable systems such as protein aggregates, viral capsids, and membrane complexes. Here we applied IM-MS to one of the most studied natively disordered tetrameric complexes, that of the tumor suppressor protein p53. This protein complex, discovered more than 30 years ago, is a major chemotherapeutic target, and has continued to thwart structural biologists largely because disordered regions comprise ca. 40 % of the protein. For our study we systematically introduced intrinsically disordered regions (flexible linker, N and C termini) to the two folded domains. This enabled us to probe the effects of disordered regions on the various constructs as well as full-length tetrameric p53 bound to cognate DNA.
- 213Jurneczko, E.; Cruickshank, F.; Porrini, M.; Clarke, D. J.; Campuzano, I. D. G.; Morris, M.; Nikolova, P. V.; Barran, P. E. Probing the conformational diversity of cancer-associated mutations in p53 with ion-mobility mass spectrometry. Angew. Chem., Int. Ed. 2013, 52, 4370– 4374, DOI: 10.1002/anie.201210015Google Scholar213Probing the Conformational Diversity of Cancer-Associated Mutations in p53 with Ion-Mobility Mass SpectrometryJurneczko, Ewa; Cruickshank, Faye; Porrini, Massimiliano; Clarke, David J.; Campuzano, Iain D. G.; Morris, Michael; Nikolova, Penka V.; Barran, Perdita E.Angewandte Chemie, International Edition (2013), 52 (16), 4370-4374CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)Suppressor p53 is the most mutated protein in human cancers. Herein ion mobility mass spectrometry studies are reported on the conformational diversity of wild type p53 and its cancer-assocd. mutants.
- 214Ruotolo, B. T.; Giles, K.; Campuzano, I.; Sandercock, A. M.; Bateman, R. H.; Robinson, C. V. Evidence for macromolecular protein rings in the absence of bulk water. Science 2005, 310, 1658– 1661, DOI: 10.1126/science.1120177Google Scholar214Evidence for Macromolecular Protein Rings in the Absence of Bulk WaterRuotolo, Brandon T.; Giles, Kevin; Campuzano, Iain; Sandercock, Alan M.; Bateman, Robert H.; Robinson, Carol V.Science (Washington, DC, United States) (2005), 310 (5754), 1658-1661CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)We have examd. the architecture of a protein complex in the absence of bulk water. By detg. collision cross sections of assemblies of the trp RNA binding protein, TRAP, we established that the 11-membered ring topol. of the complex can be maintained within a mass spectrometer. We also found that the binding of tryptophan enhances the stability of the ring structure and that addn. of a specific RNA mol. increases the size of the complex and prevents structural collapse. These results provide definitive evidence that protein quaternary structure can be maintained in the absence of bulk water and highlight the potential of ion mobility sepn. for defining shapes of heterogeneous macromol. assemblies.
- 215Dong, S.; Shirzadeh, M.; Fan, L.; Laganowsky, A.; Russell, D. H. Ag+ ion binding to human metallothionein-2A is cooperative and domain specific. Anal. Chem. 2020, 92, 8923– 8932, DOI: 10.1021/acs.analchem.0c00829Google Scholar215Ag+ Ion Binding to Human Metallothionein-2A Is Cooperative and Domain SpecificDong, Shiyu; Shirzadeh, Mehdi; Fan, Liqi; Laganowsky, Arthur; Russell, David H.Analytical Chemistry (Washington, DC, United States) (2020), 92 (13), 8923-8932CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)Metallothioneins (MTs) constitute a family of cysteine-rich proteins that play key biol. roles for a wide range of metal ions, but unlike many other metalloproteins, the structures of apo- and partially metalated MTs are not well understood. Here, we combine nano-electrospray ionization-mass spectrometry (ESI-MS) and nano-ESI-ion mobility (IM)-MS with collision-induced unfolding (CIU), chem. labeling using N-ethylmaleimide (NEM), and both bottom-up and top-down proteomics in an effort to better understand the metal binding sites of the partially metalated forms of human MT-2A, viz., Ag4-MT. The results for Ag4-MT are then compared to similar results obtained for Cd4-MT. The results show that Ag4-MT is a cooperative product, and data from top-down and bottom-up proteomics mass spectrometry anal. combined with NEM labeling revealed that all four Ag+ ions of Ag4-MT are bound to the β-domain. The binding sites are identified as Cys13, Cys15, Cys19, Cys21, Cys24, and Cys26. While both Ag+ and Cd2+ react with MT to yield cooperative products, i.e., Ag4-MT and Cd4-MT, these products are very different; Ag+ ions of Ag4-MT are located in the β-domain, whereas Cd2+ ions of Cd4-MT are located in the α-domain. Ag6-MT has been reported to be fully metalated in the β-domain, but our data suggest the two addnl. Ag+ ions are more weakly bound than are the other four. Higher order Agi-MT complexes (i = 7-17) are formed in solns. that contain excess Ag+ ions, and these are assumed to be bound to the α-domain or shared between the two domains. Interestingly, the excess Ag+ ions are displaced upon addn. of NEM to this soln. to yield predominantly Ag4NEM14-MT. Results from CIU suggest that Agi-MT complexes are structurally more ordered and that the energy required to unfold these complexes increases as the no. of coordinated Ag+ increases.
- 216Peris-Díaz, M. D.; Guran, R.; Domene, C.; de los Rios, V.; Zitka, O.; Adam, V.; Krężel, A. An integrated mass spectrometry and molecular dynamics simulations approach reveals the spatial organization impact of metal-binding sites on the stability of metal-depleted metallothionein-2 species. J. Am. Chem. Soc. 2021, DOI: 10.1021/jacs.1c05495Google ScholarThere is no corresponding record for this reference.
- 217Riordan, J. R.; Richards, V. Human foetal liver contains both zinc- and copper-rich forms of metallothionein. J. Biol. Chem. 1980, 255, 5380– 5383, DOI: 10.1016/S0021-9258(19)70797-2Google Scholar217Human fetal liver contains both zinc- and copper-rich forms of metallothioneinRiordan, John R.; Richards, VivienneJournal of Biological Chemistry (1980), 255 (11), 5380-3CODEN: JBCHA3; ISSN:0021-9258.Metallothionein was purified under anaerobic conditions from livers of human fetuses ranging from 19 wk gestational age to term. Homogeneous metallothionein obtained in the absence of reducing agents from the sol. fraction of the tissue which contained 24% and 85% of the total liver Cu and Zn, resp., had <1 g atom of Cu and ∼3.1 g atoms of Zn/mol. Extn. with 1% 2-mercaptoethanol of the insol. fraction of the tissue (contg. 76% and 15% of the liver Cu and Zn, resp.) yielded a metallothionein with ∼2.5 g atoms of Cu and 1.3 g atoms of Zn. When the whole tissue was extd. similarly by the procedure of L. Ryden and H. F. Deutsch (1978), nearly equal proportions of the 2 metals were obtained in the sol. fraction from which metallothionein, with ∼3.0 g atoms of Zn, and 1.5 g atoms of Cu, was purified. Thus, Zn- and Cu-rich forms of metallothionein are differently distributed between the sol. and insol. fractions of fetal liver. Nevertheless, the predominant metal in metallothionein of human fetal liver is Zn as is the case in the adult; significant amts. of Cu are also present, consistent with the elevated quantity of this metal in the fetal tissue relative to the adult. Cd was not detected. Zn is much more easily removed from the protein than is Cu.
- 218Klein, D.; Scholz, G. A.; Drasch, G. A.; Müller-Höcker, J.; Summer, K. H. Metallothionein, copper and zinc in fetal and neonatal human liver: changes during development. Toxicol. Lett. 1991, 56, 61– 67, DOI: 10.1016/0378-4274(91)90090-SGoogle Scholar218Metallothionein, copper and zinc in fetal and neonatal human liver: changes during developmentKlein, D.; Scholz, P.; Drasch, G. A.; Mueller-Hoecker, J.; Summer, Karl H.Toxicology Letters (1991), 56 (1-2), 61-7CODEN: TOLED5; ISSN:0378-4274.Total and cytosolic Zn and Cu, cytosolic metallothionein (MT) and the Cu-load of MT were investigated in fetal (22, 24 and 32 gestational weeks) and neonatal (2-15 mo) human liver. Whereas the fraction of cytosolic Zn remained const. at 66% of the total independent of the stage of development, the fraction of cytosolic Cu increased from 26% in preterm liver to about 100% within 12 mo postnatally. The MT content was higher in fetal than in neonatal liver. There was a linear correlation between cytosolic MT and Zn in both fetal and neonatal liver but not between MT and Cu. In contrast to fetal liver, the Cu-load of MT in neonatal liver seems to be detd. by the Zn/Cu ratio in the cytosol. The results suggest that MT is involved in the regulation of Cu and Zn metab. during fetal and neonatal development.
- 219Kägi, J. H. R.; Vašák, M.; Lerch, K.; Gilg, D. E. O.; Hunziker, P.; Bernhard, W. R.; Good, M. Structure of mammalian metallothionein. Environ. Health Persp. 1984, 54, 93– 103, DOI: 10.2307/3429795Google Scholar219Structure of mammalian metallothioneinKagi J H; Vasak M; Lerch K; Gilg D E; Hunziker P; Bernhard W R; Good MEnvironmental health perspectives (1984), 54 (), 93-103 ISSN:0091-6765.All mammalian metallothioneins characterized contain a single polypeptide chain of 61 amino acid residues, among them 20 cysteines providing the ligands for seven metal-binding sites. Native metallothioneins are usually heterogeneous in metal composition, with Zn, Cd, and Cu occurring in varying proportions. However, forms containing only a single metal species, i.e., Zn, Cd, Ni, Co, Hg, Pb, Bi, have now been prepared by in vitro reconstitution from the metal-free apoprotein. By spectroscopic analysis of such derivatives it was established that all cysteine residues participate in metal binding, that each metal ion is bound to four thiolate ligands, and that the symmetry of each complex is close to that of a tetrahedron. To satisfy the requirements of the overall Me7(Cys-)20 stoichiometry, the complexes must be combined to form metal-thiolate cluster structures. Experimental proof for the occurrence of such clusters comes from the demonstration of metal-metal interactions by spectroscopic and magnetic means. Thus, in Co(II)7-metallothionein, the Co(II)-specific ESR signals are effectively suppressed by antiferromagnetic coupling of juxtaposed paramagnetic metal ions. By monitoring changes in ESR signal size occurring on stepwise incorporation of Co(II) into the protein, it is possible to follow the building up of the clusters. This process is biphasic. Up to binding of four equivalents of Co(II), the ESR amplitude increases in proportion to the metal content, indicating generation of magnetically noninteracting high-spin complexes. However, upon addition of the remaining three equivalents of Co(II), these features are progressively suppressed, signaling the formation of clusters. The same mode of cluster formation has also been documented for Cd and Hg. The actual spatial organization of the clusters and the polypeptide chain remains to be established. An attractive possibility is the arrangement of the tetrahedral metal-thiolates in adamantane-like structures surrounded by properly folded segments of the chain providing the ligands. 1H-NMR data and infrared absorption measurements are consistent with a tightly folded structure rich in beta-type conformation.
- 220Alvarez, L.; Gonzalez-Iglesias, H.; Garcia, M.; Ghosh, S.; Sanz-Medel, A.; Coca-Prados, M. The stoichiometric transition from Zn6Cu1-metallothionein to Zn7-metallothionein underlies the up-regulation of metallothionein (MT) expression: Quantitative analysis of MT-metal load in eye cells. J. Biol. Chem. 2012, 287, 28456– 28469, DOI: 10.1074/jbc.M112.365015Google Scholar220The Stoichiometric Transition from Zn6Cu1-Metallothionein to Zn7-Metallothionein Underlies the Up-regulation of Metallothionein (MT) Expression. Quantitative Analysis of MT-Metal Load in Eye Cells.Alvarez, Lydia; Gonzalez-Iglesias, Hector; Garcia, Montserrat; Ghosh, Sikha; Sanz-Medel, Alfredo; Coca-Prados, MiguelJournal of Biological Chemistry (2012), 287 (34), 28456-28469CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)We examd. the profiling of gene expression of metallothioneins (MTs) in human tissues from cadaver eyes with microarray-based anal. All MT1 isoforms, with the exception of MT1B, were abundantly expressed in lens and corneal tissue. Along with MT1B, MT4 was not detected in any tissues. Antibodies to MT1/2 labeled the corneal epithelial and endothelial cells, whereas MT3 label the retinal ganglion cells. We studied the effects of zinc and cytokines on the gene expression of MT isoforms in a corneal epithelial cell line (HCEsv). Zinc exerted an up-regulation of the expression of MT isoforms, and this effect was further potentiated in the presence of IL1α or TNFα. Zinc also elicited a strong down-regulation of the expression of inflammatory cytokines, and this effect was blocked in the presence of TNFα or IL1α. The concn. of MTs, bound zinc, and the metal stoichiometry of MTs in cultured HCEsv were detd. by mass spectrometry. The total concn. of MTs was 0.24 ± 0.03 μm and, after 24 h of zinc exposure, increased to 0.96 ± 0.01 μm. The combination of zinc and IL1α further enhanced the level of MTs to 1.13 ± 0.03 μm. The av. metal stoichiometry of MTs was Zn6Cu1-MT, and after exposure to the different treatments, it changed to Zn7-MT. Actinomycin D blocked transcription, and cycloheximide attenuated synthesis of MTs in the presence or absence of zinc, suggesting transcriptional regulation. Overall the data provide mol. and anal. evidence on the interplay between zinc, MTs, and proinflammatory cytokines in HCEsv cells, with potential implications on cell-based inflammatory eye diseases.
- 221Jara-Biedma, R.; González-Dominguez, R.; García-Barrera, T.; Lopez-Barea, J.; Pueyo, C.; Gómez-Ariza, J. L. Evolution of metallothionein isoforms complexes in hepatic cells of Mus musculus along cadmium exposure. BioMetals 2013, 26, 639– 650, DOI: 10.1007/s10534-013-9636-0Google Scholar221Evolution of metallotionein isoforms complexes in hepatic cells of Mus musculus along cadmium exposureJara-Biedma, R.; Gonzalez-Dominguez, R.; Garcia-Barrera, T.; Lopez-Barea, J.; Pueyo, C.; Gomez-Ariza, J. L.BioMetals (2013), 26 (4), 639-650CODEN: BOMEEH; ISSN:0966-0844. (Springer)Characterization of Cd-binding proteins has great anal. interest due to the high toxicity of Cd to living organisms. Metallothioneins (MTs), as Cd(II)-binding proteins are of increasing interest, since they form very stable Cd chelates and are involved in many detoxification processes. In this work, inductively coupled plasma octopole reaction cell mass spectrometry and nanospray ionization time-of-flight mass spectrometry were used in parallel and combined with two-dimensional chromatog.: size exclusion followed by reversed-phase high performance liq. chromatog., to study metal complexes of MT isoforms produced in hepatic cytosols of Mus musculus during exposure expts. to Cd. Exposure expts. were carried out by s.c. injection of a growing dose of the toxic element ranging from 0.1 to 1.0 mg of Cd per kg of body wt. per day during 10 days. A control group and three exposure groups at days 2, 6 and 10 of exposure were studied, and different cadmium, copper and zinc complexes with MTs isoforms were isolated and characterized from the two most exposed groups. The results allow gaining insight into the mechanisms involved in metal detoxification by MTs, showing the changes in the stoichiometry of metal complexes-MTs along cadmium exposure.
- 222Suzuki, Y. Metal-binding properties of metallothionein in extracellular fluids and its role in cadmium-exposed rats. In Biological Roles of Metallothionein; Foulkes, E. C., Ed.; Elsevier: Amsterdam, The Netherlands, 1982; pp 27– 35.Google ScholarThere is no corresponding record for this reference.
- 223Tio, L.; Villarreal, L.; Atrian, S.; Capdevila, M. Functional differentiation in the mammalian metallothionein gene family: Metal-binding features of mouse MT-4 and comparison with its paralog MT-1. J. Biol. Chem. 2004, 279, 24403– 24413, DOI: 10.1074/jbc.M401346200Google Scholar223Functional Differentiation in the Mammalian Metallothionein Gene Family: Metal binding features of mouse MT4 and comparison with its paralog MT1Tio, Laura; Villarreal, Laura; Atrian, Silvia; Capdevila, MerceJournal of Biological Chemistry (2004), 279 (23), 24403-24413CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)This paper reports on the characterization of the metal binding abilities of mammalian MT4 and their comparison with those of the well known MT1. Heterologous Escherichia coli expression in cultures supplemented with zinc, cadmium, or copper was achieved for MT4 and for its sep. αMT4 and βMT4 domains as well as for MT1 and its αMT1 domain in cadmium-enriched medium. The in vivo conformed metal complexes and the in vitro substituted zinc/cadmium and zinc/copper MT4 aggregates were characterized. Biosynthesis of MT4 and βMT4 in Cd(II)-supplemented medium revealed that these peptides failed to form the same homometallic species as MT1, thus appearing less effective for cadmium coordination. Conversely, the entire MT4 and both of its domains showed better Cu(I) binding properties than MT1, affording Cu10-MT4, Cu5-αMT4 and Cu7-βMT4, stoichiometries that make the domain dependence toward Cu(I) clear. Overall results allow consideration of MT4 as a novel copper-thionein, made up of two copper-thionein domains, the first of this class reported in mammals, and by extension in vertebrates. Furthermore, the in silico protein sequence analyses corroborated the copper-thionein nature of the MT4 peptides. As a consequence, there is the suggestion of a possible physiol. role played by MT4 related with copper requirements in epithelial differentiating tissues, where MT4 is expressed.
- 224Włostowski, T. Postnatal changes in subcellular distribution of copper, zinc and metallothionein in the liver of bank vole (Clethrionomys glareolus): a possible involvement of metallothionein and copper in cell proliferation. Comp. Biochem. Physiol., C: Comp. Pharmacol. 1992, 103, 285– 290, DOI: 10.1016/0742-8413(92)90009-VGoogle Scholar224Postnatal changes in subcellular distribution of copper, zinc and metallothionein in the liver of bank vole (Clethrionomys glareolus): a possible involvement of metallothionein and copper in cell proliferationWlostowski, T.Comparative Biochemistry and Physiology, Part C: Pharmacology, Toxicology & Endocrinology (1992), 103C (2), 285-90CODEN: CBPCEE; ISSN:0742-8413.Dramatic interdependent changes in the intracellular concns. of Cu, Zn and metallothionein (MT) in the liver of bank voles during the first 30 days of their life were obsd. The post-mitochondrial Cu, Zn and MT (Zn-MT) abruptly decreased between 1 and 3 days following birth but the nuclear MT (Cu-MT) and Cu increased at the same time, suggesting that Cu displaced Zn already bound to MT in the cytoplasm and subsequently the complex Cu-MT was translocated to the nuclei. The nuclear Cu concn. reached the highest level (62-71% of the total tissue Cu) in the period from day 3 to 20 post-partum, just prior to and during a rapid growth of the liver. The data indicate that MT and Cu may be involved in the hepatocyte proliferation.
- 225Tottey, S.; Waldron, K. J.; Firbank, S. J.; Reale, B.; Bessant, C.; Sato, K.; Cheek, T. R.; Gray, J.; Banfield, M. J.; Dennison, C.; Robinson, N. J. Protein-folding location can regulate manganese-binding versus copper- or zinc-binding. Nature 2008, 455, 1138– 1145, DOI: 10.1038/nature07340Google Scholar225Protein-folding location can regulate manganese-binding versus copper- or zinc-bindingTottey, Steve; Waldron, Kevin J.; Firbank, Susan J.; Reale, Brian; Bessant, Conrad; Sato, Katsuko; Cheek, Timothy R.; Gray, Joe; Banfield, Mark J.; Dennison, Christopher; Robinson, Nigel J.Nature (London, United Kingdom) (2008), 455 (7216), 1138-1142CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)Metals are needed by at least 25% of all proteins. Although metallochaperones insert the correct metal into some proteins, they have not been found for the vast majority, and the view is that most metalloproteins acquire their metals directly from cellular pools. However, some metals form more stable complexes with proteins than do others. For instance, as described in the Irving-Williams series, Cu2+ and Zn2+ typically form more stable complexes than Mn2+. Thus, it is unclear what cellular mechanisms manage metal acquisition by most nascent proteins. To investigate this question, the authors identified the most abundant Cu2+-protein, CucA (Cu2+-cupin A), and the most abundant Mn2+-protein, MncA (Mn2+-cupin A), in the periplasm of the cyanobacterium, Synechocystis PCC 6803. Each of these newly identified proteins binds its resp. metal via identical ligands within a cupin fold. Consistent with the Irving-Williams series, MncA only binds Mn2+ after folding in solns. contg. at least a 104-fold molar excess of Mn2+ over Cu2+ or Zn2+. However, once MncA has bound Mn2+, the metal does not exchange with Cu2+. MncA and CucA had signal peptides for different export pathways into the periplasm, Tat and Sec, resp. Export by the Tat pathway allows MncA to fold in the cytoplasm, which contains only tightly bound Cu2+ or Zn2+, but micromolar Mn2+. In contrast, CucA folds in the periplasm to acquire Cu2+. These results reveal a mechanism whereby the compartment in which a protein folds overrides its binding preference to control its metal content. They explain why the cytoplasm must contain only tightly bound and buffered Cu2+ and Zn2+.
- 226Pountney, D. L.; Schauwecker, I.; Zarn, J.; Vašák, M. Formation of mammalian Cu8-metallothionein in vitro: Evidence for the existence of two Cu(I)4-thiolate clusters. Biochemistry 1994, 33, 9699– 9705, DOI: 10.1021/bi00198a040Google Scholar226Formation of Mammalian Cu8-Metallothionein in vitro: Evidence for the Existence of Two Cu(I)4-Thiolate ClustersPountney, Dean L.; Schauwecker, Ivo; Zarn, Juerg; Vasak, MilanBiochemistry (1994), 33 (32), 9699-705CODEN: BICHAW; ISSN:0006-2960.Cu accumulates in metallothionein (Cu-MT) in Cu overload diseases, such as Wilson's disease and Bedlington Terriers disease. The in vitro formation of the Cu12-MT form comprising 2 Cu(I)6(CysS)9,11 cores is well documented. However, lysosomal Cu-MT isolated from canine liver was previously shown to contain 8 Cu(I) ions in 2 proposed adamantane-like Cu4-thiolate clusters. The present studies were carried out in an effort to learn more about the Cu(I)-thiolate cluster species formed upon the sequential incorporation of Cu(I) ions into metal-free MT from rabbit liver. On the basis of changes in the electronic absorption, CD, MCD, and luminescence spectra, besides the formation of a mol. species with 12 Cu(I) equiv., evidence for the existence of a distinct MT complex with 8 Cu(I) equiv. (Cu8-MT) was obtained. Anal. of the metal-dependent absorption envelope of Cu(I)-MT between 240 and 360 nm permitted the discrimination between predominantly CysS-Cu(I) charge-transfer (LMCT) (240-260 nm) and cluster-localized Cu(I) (d-s) transitions (260-360 nm). Accordingly, the decrease in the ratio of intensities of LMCT to d-s bands from 2.6 to 2.4 on going from 8 to 12 Cu(I) equiv. was attributed to the formation of Cu-MT species with different cysteine ligand to metal stoichiometries. The results suggested that while in Cu12-MT all 20 thiolate ligands participate in metal binding, in the Cu8-MT species between 12 and 14 cysteines take part in Cu(I) coordination. The low-temp. luminescence spectrum (77 K) of Cu8-MT was characterized by low- and high-energy emission bands at 610 nm (τ = ∼130 μs) and 425 nm (τ = ∼50 μs), resp. In contrast, the corresponding spectrum of the 2 Cu6 clusters in Cu12-MT exhibited only the low-energy band at 610 nm (τ = ∼130 μs). The measured lifetimes (τ) were consistent with emissions from triplet excited states assigned to mixed d-s/LMCT in origin. Similar luminescence behavior was previously obsd. with crystallog. defined inorg. Cu(I)6 and Cu(I)4 cluster models where it was attributed to a shorter Cu···Cu sepn. in the latter case. Accordingly, the occurrence of the high-energy band in Cu8-MT provided evidence for the existence of Cu(I)4 clusters in this species. On the basis of these data, it was concluded that the Cu8-MT species contains 2 Cu(I)4(CysS)6-7 clusters and is thus analogous to that isolated from canine liver (Bedlington Terriers disease). It was suggested that the formation of distinct Cu8- and Cu12-MT species reflects the relative concns. of Cu and protein present in soln.
- 227Roschitzki, B.; Vašák, M. A distinct Cu4-thiolate cluster of human metallothionein-3 is located in the N-terminal domain. JBIC, J. Biol. Inorg. Chem. 2002, 7, 611– 616, DOI: 10.1007/s00775-002-0339-1Google Scholar227A distinct Cu4-thiolate cluster of human metallothionein-3 is located in the N-terminal domainRoschitzki, Bernd; Vasak, MilanJBIC, Journal of Biological Inorganic Chemistry (2002), 7 (6), 611-616CODEN: JJBCFA; ISSN:0949-8257. (Springer-Verlag)Metallothionein-3 (MT-3), also known as neuronal growth inhibitory factor, is a metalloprotein expressed almost exclusively in the brain. Isolated MT-3 contains four Cu(I) and three Zn(II) ions organized in homometallic metal-thiolate clusters located in two independent protein domains. In this work a Cu(I) binding to metal-free MT-3 has been studied, aiming at the better understanding of the domain specificity for this metal ion. The cluster formation was followed by electronic absorption, CD, and by luminescence spectroscopy at room temp. and 77 K. The stepwise incorporation of Cu(I) into recombinant human apo-MT-3 revealed the cooperative formation of two Cu4S9 clusters in succession, formed in both protein domains, i.e. Cu4- and Cu8-MT-3. Further binding of four Cu(I) caused an expansion of these Cu(I) cores, leading to fully metal-loaded Cu12-MT-3 contg. Cu6S9 and Cu6S11 clusters in the β- and α-domains of the protein, resp. The location of the preferentially formed Cu4 cluster in the protein was established by immunochem. Using domain-specific antibodies, in combination with limited tryptic digestion of a partially metal-occupied Cu4-MT-3, we could demonstrate that the Cu4S9 cluster is located in the N-terminal β-domain of the protein that contains a total of nine cysteine ligands. Electronic supplementary material to this paper, comprising Table S1 (amino acid sequences of peptides used in immunization) and Fig. S1 (luminescence spectra of Cu(I) titrated apo-MT-3).
- 228Hofbeck, T.; Monkowius, U.; Yersin, H. Highly efficient luminescence of Cu(I) compounds – TADF combined with short-lived phosphorescence. J. Am. Chem. Soc. 2015, 137, 399– 404, DOI: 10.1021/ja5109672Google Scholar228Highly Efficient Luminescence of Cu(I) Compounds: Thermally Activated Delayed Fluorescence Combined with Short-Lived PhosphorescenceHofbeck, Thomas; Monkowius, Uwe; Yersin, HartmutJournal of the American Chemical Society (2015), 137 (1), 399-404CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Luminescent materials showing thermally-activated delayed fluorescence (TADF) have gained high attractiveness as emitters in org. light emitting diodes (OLEDs) and other photonic applications. Even use of TADF can be further improved, introducing a novel concept. This is demonstrated by a new class of brightly luminescent low-cost Cu(I) compds., for which the emission stems from both the lowest excited triplet T1 and singlet S1 state. At T = 300 K, these materials exhibit quantum yields of more than ΦPL = 90% at short emission decay times. About 80% of the emission intensity stems from the singlet due to TADF, but importantly, an addnl. 20% is contributed by the lower lying triplet state according to effective spin-orbit coupling (SOC). SOC induces also a relatively large zero-field splitting of the triplet being unusual for Cu(I) complexes. Thus, the overall emission decay time is distinctly reduced. Combined use of both decay paths opens novel photonic applications, in particular, for OLEDs. Crystallog. data are given.
- 229Scheller, J. S.; Irvine, G. W.; Wong, D. L.; Hartwig, A.; Stillman, M. J. Stepwise copper(I) binding to metallothionein: A mixed cooperative and non-cooperative mechanism for all 20 copper ions. Metallomics 2017, 9, 447– 462, DOI: 10.1039/C7MT00041CGoogle Scholar229Stepwise copper(I) binding to metallothionein: a mixed cooperative and non-cooperative mechanism for all 20 copper ionsScheller, Judith S.; Irvine, Gordon W.; Wong, Daisy L.; Hartwig, Andrea; Stillman, Martin J.Metallomics (2017), 9 (5), 447-462CODEN: METAJS; ISSN:1756-591X. (Royal Society of Chemistry)Copper is a ubiquitous trace metal of vital importance in that it serves as a cofactor in many metalloenzymes. Excess copper becomes harmful if not sequestered appropriately in the cell. As a metal ion chaperone, metallothionein (MT) has been proposed as a key player in zinc and copper homeostasis within the cell. The underlying mechanisms by which MT sequesters and transfers copper ions, and subsequently achieves its proposed biol. function remain unknown. Using a combination of electrospray ionization mass spectrometry (ESI-MS), CD (CD), and emission spectroscopy, we report that the Cu(I) to human apo-MT1a binding mechanism is highly pH-dependent. The 20 relative Kf-values for the binding of 1 to 20 Cu(I) to the 20 cysteines of MT were obtained from computational simulation of the exptl. mass spectral results. These data identified the pH-dependent formation of three sequential but completely different Cu-SCYS clusters, as a function of Cu(I) loading. These data provide the first overall sequence for Cu(I) binding in terms of domain specificity and transient binding site structures. Under cooperative binding at pH 7.4, a series of four clusters form: Cu4SCYS-6, followed by Cu6SCYS-9 (β), then a second Cu4SCYS-6 (α), and finally Cu7SCYS-x (α) (x = up to 11). Upon further addn. of Cu(I), a mixt. of species is formed in a non-cooperative mechanism, satg. the 20 cysteines of MT1a. Using benzoquinone, a cysteine modifier, we were able to confirm that Cu6SCYS-9 formed solely in the N-terminal β-domain, as well as confirming the existence of the presumed Cu4SCYS-6 cluster in the α-domain. Based on the results of ESI-MS and computational simulation we were able to identify Cu:MT speciation that resulted in specific emission and CD spectral properties.
- 230Nielson, K. B.; Winge, D. R. Preferential binding of copper to the β-domain metallothionein. J. Biol. Chem. 1984, 259, 4941– 4946, DOI: 10.1016/S0021-9258(17)42937-1Google Scholar230Preferential binding of copper to the β domain of metallothioneinNielson, Kirk B.; Winge, Dennis R.Journal of Biological Chemistry (1984), 259 (8), 4941-6CODEN: JBCHA3; ISSN:0021-9258.Proteolytic studies of rat liver metallothionein reconstituted in vitro with Cu salts revealed that the 2 metal centers fill in an ordered fashion. The B cluster in the N-terminal β domain fills prior to Cu binding in cluster A. This is contrary to cluster formation induced by the binding of Cd2+ or Zn2+ in which cluster A is the center of initial binding. The formation of metal cluster B by Cu occurs in a cooperative fashion yielding a satd. cluster with ∼6 Cu+ bound. The B cluster is satd. with Cd or Zn after binding of only 3 metal ions. The preferential binding of Cd and Cu to the α and β domains, resp., and the tolerance toward proteolysis of these 2 different half-satd. mols. permit the isolation of each domain. The metal cluster in each isolated domain can be reversibly formed with predicted stoichiometries of Cd and Cu. The folding of the polypeptide therefore appears to create each cluster independently. The metal binding data suggest that Cu-metallothionein contains 11-12 Cu ions, 6 bound in the β domain and 5-6 in the α domain. In contrast, Cd-metallothionein contains 7 Cd ions, 3 bound to β and 4 to α.
- 231Young, T. R.; Xiao, Z. Principles and practice of determining metal-protein affinities. Biochem. J. 2021, 478, 1085– 1116, DOI: 10.1042/BCJ20200838Google Scholar231Principles and practice of determining metal-protein affinitiesYoung, Tessa R.; Xiao, ZhiguangBiochemical Journal (2021), 478 (5), 1085-1116CODEN: BIJOAK; ISSN:0264-6021. (Portland Press Ltd.)A review. Metal ions play many crit. roles in biol., as structural and catalytic cofactors, and as cell regulatory and signalling elements. The metal-protein affinity, expressed conveniently by the metal dissocn. const., KD, describes the thermodn. strength of a metal-protein interaction and is a key parameter that can be used, for example, to understand how proteins may acquire metals in a cell and to identify dynamic elements (e.g. cofactor binding, changing metal availabilities) which regulate protein metalation in vivo. Here, we outline the fundamental principles and practical considerations that are key to the reliable quantification of metal-protein affinities. We review a selection of spectroscopic probes which can be used to det. protein affinities for essential biol. transition metals (including Mn(II), Fe(II), Co(II), Ni(II), Cu(I), Cu(II) and Zn(II)) and, using selected examples, demonstrate how rational probe selection combined with prudent exptl. design can be applied to det. accurate KD values.
- 232Morgan, M. T.; Yang, B.; Harankhedkar, S.; Nabatilan, A.; Bourassa, D.; McCallum, A. M.; Sun, F.; Wu, R.; Forest, C. R.; Fahrni, C. J. Stabilization of aliphatic phosphines by auxiliary phosphine sulfides offers zeptomolar affinity and unprecedented selectivity for probing biological CuI. Angew. Chem., Int. Ed. 2018, 57, 9711– 9715, DOI: 10.1002/anie.201804072Google Scholar232Stabilization of Aliphatic Phosphines by Auxiliary Phosphine Sulfides Offers Zeptomolar Affinity and Unprecedented Selectivity for Probing Biological CuIMorgan, M. Thomas; Yang, Bo; Harankhedkar, Shefali; Nabatilan, Arielle; Bourassa, Daisy; McCallum, Adam M.; Sun, Fangxu; Wu, Ronghu; Forest, Craig R.; Fahrni, Christoph J.Angewandte Chemie, International Edition (2018), 57 (31), 9711-9715CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)Full elucidation of the functions and homeostatic pathways of biol. copper requires tools that can selectively recognize and manipulate this trace nutrient within living cells and tissues, where it exists primarily as CuI. Buffered at attomolar concns., intracellular CuI is, however, not readily accessible to commonly employed amine and thioether-based chelators. Herein, the authors reveal a chelator design strategy in which phosphine sulfides aid in CuI coordination while simultaneously stabilizing aliph. phosphine donors, producing a charge-neutral ligand with low-zeptomolar dissocn. const. and 1017-fold selectivity for CuI over ZnII, FeII, and MnII. As illustrated by reversing ATP7A trafficking in cells and blocking long-term potentiation of neurons in mouse hippocampal brain tissue, the ligand is capable of intercepting copper-dependent processes. The phosphine sulfide-stabilized phosphine (PSP) design approach, which confers resistance towards protonation, dioxygen, and disulfides, could be readily expanded towards ligands and probes with tailored properties for exploring CuI in a broad range of biol. systems.
- 233Bagchi, P.; Morgan, M. T.; Bacsa, J.; Fahrni, C. J. Robust affinity standards for Cu(I) biochemistry. J. Am. Chem. Soc. 2013, 135, 18549– 18559, DOI: 10.1021/ja408827dGoogle Scholar233Robust Affinity Standards for Cu(I) BiochemistryBagchi, Pritha; Morgan, M. Thomas; Bacsa, John; Fahrni, Christoph J.Journal of the American Chemical Society (2013), 135 (49), 18549-18559CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)The measurement of reliable Cu-(I) protein binding affinities requires competing ref. ligands with similar binding strengths; however, the literature on such ref. ligands is not only sparse but often conflicting. To address this deficiency, we have created and characterized a series of water-sol. monovalent copper ligands, MCL-1,/ MCL-2, and MCL-3, that form well-defined, air-stable, and colorless complexes with Cu-(I) in aq. soln. X-ray structural data, electrochem. measurements, and an extensive network of equil. titrns. showed that all three ligands form discrete Cu-(I) complexes with 1:1 stoichiometry and are capable of buffering Cu-(I) concns. between 10-10 and 10-17 M. As most Cu-(I) protein affinities have been obtained from competition expts. with bathocuproine disulfonate or 2,2'-bicinchoninic acid, we further calibrated their Cu-(I) stability consts. against the MCL series. To demonstrate the application of these reagents, we detd. the Cu-(I) binding affinity of CusF (log K = 14.3 ± 0.1), a periplasmic metalloprotein required for the detoxification of elevated copper levels in Escherichia coli. Altogether, this interconnected set of affinity stds. establishes a reliable foundation that will facilitate the precise detn. of Cu-(I) binding affinities of proteins and small-mol. ligands.
- 234Hemmerich, P.; Sigwart, C. Cu(CH3CN)2+, ein Mittel zum Studium homogener Reaktionen des einwertigen Kupfers in wässriger Lösung. Experientia 1963, 19, 488– 489, DOI: 10.1007/BF02150666Google Scholar234Cu(CH3CN)2+, a method for the study of the homogeneous reactions of univalent copper in aqueous solutionHemmerich, P.; Sigwart, C.Experientia (1963), 19 (9), 488-9CODEN: EXPEAM; ISSN:0014-4754.In the presence of excess MeCN, the formation consts. of Cu(I) complexes may be detd. by pH titration. Log β2 for Cu(CH3CN)2 is 4.35.
- 235Xiao, Z.; Loughlin, F.; George, G. N.; Howlett, G. J.; Wedd, A. G. C-terminal domain of the membrane copper transporter Ctr1 from Saccharomyces cerevisiae binds four Cu(I) ions as a cuprous-thiolate polynuclear cluster: sub-femtomolar Cu(I) affinity of three proteins involved in copper trafficking. J. Am. Chem. Soc. 2004, 126, 3081– 3090, DOI: 10.1021/ja0390350Google Scholar235C-Terminal Domain of the Membrane Copper Transporter Ctr1 from Saccharomyces cerevisiae Binds Four Cu(I) Ions as a Cuprous-Thiolate Polynuclear Cluster: Sub-femtomolar Cu(I) Affinity of Three Proteins Involved in Copper TraffickingXiao, Zhiguang; Loughlin, Fionna; George, Graham N.; Howlett, Geoffrey J.; Wedd, Anthony G.Journal of the American Chemical Society (2004), 126 (10), 3081-3090CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)The cytosolic C-terminal domain of the membrane copper transporter Ctr1 from the yeast Saccharomyces cerevisiae, Ctr1c, was expressed in E. coli as an oxygen-sensitive sol. protein with no significant secondary structure. Visible-UV spectroscopy demonstrated that Ctr1c bound four Cu(I) ions, structurally identified as a CuI4(μ-S-Cys)6 cluster by X-ray absorption spectroscopy. This was the only metalated form detected by electrospray ionization mass spectrometry. An av. dissocn. const. Ka = (K1K2K3K4)1/4 = 10-19 for binding of Cu(I) to Ctr1c was estd. via competition with the ligand bathocuproine disulfonate bcs (β2 = 1019.8). Equivalent expts. for the yeast chaperone Atx1 and an N-terminal domain of the yeast Golgi pump Ccc2, which both bind a single Cu(I) ion, provided similar KD values. The ests. of KD were supported by independent ests. of the equil. consts. Kex for exchange of Cu(I) between pairs of these three proteins. It is apparent that, in vitro, the three proteins buffer "free" Cu(I) concns. in a narrow range around 10-19 M. The results provide quant. support for the proposals that, in yeast, (a) "free" copper concns. are very low in the cytosol and (b) the Cu(I) trafficking gradient is shallow along the putative Ctrlc → Atx1 → Ccc2n metabolic pathway. In addn., both Ctr1c and its copper-responsive transcription factor Mac1 contain similar clusters which may be important in signaling copper status in yeast.
- 236Banci, L.; Bertini, I.; Ciofi-Baffoni, S.; Kozyreva, T.; Zovo, K.; Palumaa, P. Affinity gradients drive copper to cellular destinations. Nature 2010, 465, 645– 648, DOI: 10.1038/nature09018Google Scholar236Affinity gradients drive copper to cellular destinationsBanci, Lucia; Bertini, Ivano; Ciofi-Baffoni, Simone; Kozyreva, Tatiana; Zovo, Kairit; Palumaa, PeepNature (London, United Kingdom) (2010), 465 (7298), 645-648CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)Copper is an essential trace element for eukaryotes and most prokaryotes. However, intracellular free copper must be strictly limited because of its toxic side effects. Complex systems for copper trafficking evolved to satisfy cellular requirements while minimizing toxicity. The factors driving the copper transfer between protein partners along cellular copper routes are, however, not fully rationalized. Until now, inconsistent, scattered and incomparable data on the copper-binding affinities of copper proteins have been reported. Here we det., through a unified electrospray ionization mass spectrometry (ESI-MS)-based strategy, in an environment that mimics the cellular redox milieu, the apparent Cu(I)-binding affinities for a representative set of intracellular copper proteins involved in enzymic redox catalysis, in copper trafficking to and within various cellular compartments, and in copper storage. The resulting thermodn. data show that copper is drawn to the enzymes that require it by passing from one copper protein site to another, exploiting gradients of increasing copper-binding affinity. This result complements the finding that fast copper-transfer pathways require metal-mediated protein-protein interactions and therefore protein-protein specific recognition. Together with Cu,Zn-SOD1, metallothioneins have the highest affinity for copper(I), and may play special roles in the regulation of cellular copper distribution; however, for kinetic reasons they cannot demetallate copper enzymes. Our study provides the thermodn. basis for the kinetic processes that lead to the distribution of cellular copper.
- 237Krężel, A.; Leśniak, W.; Jeżowska-Bojczuk, M.; Młynarz, P.; Brasuń, J.; Kozłowski, H.; Bal, W. Coordination of heavy metals by dithiothreitol, a commonly used thiol group protectant. J. Inorg. Biochem. 2001, 84, 77– 88, DOI: 10.1016/S0162-0134(00)00212-9Google Scholar237Coordination of heavy metals by dithiothreitol, a commonly used thiol group protectantKrezel, A.; Lesniak, W.; Jezowska-Bojczuk, M.; Mlynarz, P.; Brasun, J.; Kozlowski, H.; Bal, W.Journal of Inorganic Biochemistry (2001), 84 (1-2), 77-88CODEN: JIBIDJ; ISSN:0162-0134. (Elsevier Science Inc.)D,l-Dithiothreitol (DTT), known also as Cleland reagent, is a thiol group protectant, used commonly in peptide and protein chem. Therefore, it is often added at high concns. in prepns. of proteins relevant to heavy metal biochem. The coordination of five of these metal ions, Zn(II), Cd(II), Pb(II), Ni(II) and Cu(I) to DTT was studied by potentiometric titrns., and UV-Vis and NMR spectroscopies. It was found that DTT forms specific and very stable polymeric and monomeric complexes with all of these metal ions, using both of its sulfur donors. The quant. description of these complexes in soln. and the solid state provides the basis for predictions of interference from DTT in studies of metal ion binding of thiol-contg. biomols.
- 238Smirnova, J.; Kabin, E.; Järving, I.; Bragina, O.; Tõugu, V.; Plitz, T.; Palumaa, P. Copper(I)-binding properties of de-coppering drugs for the treatment of Wilson disease. α-Lipoic acid as a potential anti-copper agent. Sci. Rep. 2018, 8, 1463, DOI: 10.1038/s41598-018-19873-2Google Scholar238Copper(I)-binding properties of de-coppering drugs for the treatment of Wilson disease. α-Lipoic acid as a potential anti-copper agentSmirnova Julia; Kabin Ekaterina; Jarving Ivar; Bragina Olga; Tougu Vello; Palumaa Peep; Plitz ThomasScientific reports (2018), 8 (1), 1463 ISSN:.Wilson disease is an autosomal recessive genetic disorder caused by loss-of-function mutations in the P-type copper ATPase, ATP7B, which leads to toxic accumulation of copper mainly in the liver and brain. Wilson disease is treatable, primarily by copper-chelation therapy, which promotes copper excretion. Although several de-coppering drugs are currently available, their Cu(I)-binding affinities have not been quantitatively characterized. Here we determined the Cu(I)-binding affinities of five major de-coppering drugs - D-penicillamine, trientine, 2,3-dimercapto-1-propanol, meso-2,3-dimercaptosuccinate and tetrathiomolybdate - by exploring their ability to extract Cu(I) ions from two Cu(I)-binding proteins, the copper chaperone for cytochrome c oxidase, Cox17, and metallothionein. We report that the Cu(I)-binding affinity of these drugs varies by four orders of magnitude and correlates positively with the number of sulfur atoms in the drug molecule and negatively with the number of atoms separating two SH groups. Based on the analysis of structure-activity relationship and determined Cu(I)-binding affinity, we hypothesize that the endogenous biologically active substance, α-lipoic acid, may be suitable for the treatment of Wilson disease. Our hypothesis is supported by cell culture experiments where α-lipoic acid protected hepatic cells from copper toxicity. These results provide a basis for elaboration of new generation drugs that may provide better therapeutic outcomes.
- 239Meloni, G.; Faller, P.; Vašák, M. Redox silencing of copper in metal-linked neurodegenerative disorders. J. Biol. Chem. 2007, 282, 16068– 16078, DOI: 10.1074/jbc.M701357200Google Scholar239Redox Silencing of Copper in Metal-linked Neurodegenerative Disorders: Reaction of Zn7metallothionein-3 with Cu2+ ionsMeloni, Gabriele; Faller, Peter; Vasak, MilanJournal of Biological Chemistry (2007), 282 (22), 16068-16078CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)Dysregulation of copper and zinc homeostasis in the brain plays a crit. role in Alzheimer disease (AD). Copper binding to amyloid-β peptide (Aβ) is linked with the neurotoxicity of Aβ and free radical damage. Metallothionein-3 (MT-3) is a small cysteine- and metal-rich protein expressed in the brain and found down-regulated in AD. This protein occurs intra- and extracellularly, and it plays an important role in the metab. of zinc and copper. In cell cultures Zn7MT-3, by an unknown mechanism, protects neurons from the toxicity of Aβ. We have, therefore, used a range of complementary spectroscopic and biochem. methods to characterize the interaction of Zn7MT-3 with free Cu2+ ions. We show that Zn7MT-3 scavenges free Cu2+ ions through their redn. to Cu+ and binding to the protein. In this reaction thiolate ligands are oxidized to disulfides concomitant with Zn2+ release. The binding of the first four Cu2+ is cooperative forming a Cu(I)4-thiolate cluster in the N-terminal domain of Cu4,Zn4MT-3 together with two disulfides bonds. The Cu4-thiolate cluster exhibits an unusual stability toward air oxygen. The results of UV-visible, CD, and Cu(I) phosphorescence at 77 K suggest the existence of metal-metal interactions in this cluster. We have demonstrated that Zn7MT-3 in the presence of ascorbate completely quenches the copper-catalyzed hydroxyl radical (OH•) prodn. Thus, zinc-thiolate clusters in Zn7MT-3 can efficiently silence the redox-active free Cu2+ ions. The biol. implication of our studies as to the protective role of Zn7MT-3 from the Cu2+ toxicity in AD and other neurodegenerative disorders is discussed.
- 240Ohta, T.; Tachiyama, T.; Yoshizawa, K.; Yamabe, T.; Uchida, T.; Kitagawa, T. Synthesis, structure, and H2O2-dependent catalytic functions of disulfide-bridged dicopper(I) and related thioether-copper(I) and thioether-copper(II) complexes. Inorg. Chem. 2000, 39, 4358– 4369, DOI: 10.1021/ic000018aGoogle Scholar240Synthesis, Structure, and H2O2-Dependent Catalytic Functions of Disulfide-Bridged Dicopper(I) and Related Thioether-Copper(I) and Thioether-Copper(II) ComplexesOhta, Takehiro; Tachiyama, Takashi; Yoshizawa, Kazunari; Yamabe, Tokio; Uchida, Takeshi; Kitagawa, TeizoInorganic Chemistry (2000), 39 (19), 4358-4369CODEN: INOCAJ; ISSN:0020-1669. (American Chemical Society)A disulfide-bridged dicopper(I) complex, [Cu2(Py2SSPy2)](ClO4)2 (1) (Py2SSPy2 = bis{2-[N,N-bis(2-pyridylethyl)amino]-1,1-dimethylethyl}disulfide), a thioether-Cu(I) complex, [Cu(iPrSPy2)](ClO4) (2) (iPrSPy2 = N-(2-isopropylthio-2-methyl)propyl-N,N-bis-2-(2-pyridyl)ethylamine), and a thioether-Cu(II) complex, [Cu(PheSPy2)(H2O)](ClO4)2 (3) (PheSPy2 = N-(2-methyl-2-phenethylthio)propyl-N,N-bis-2-(2-pyridyl)ethylamine), were newly synthesized by the reactions of Cu(ClO4)2·6H2O with a thiol ligand of Py2SH (N,N-bis[2-(2-pyridyl)ethyl]-1,1-dimethyl-2-mercaptoethylamine) and thioether ligands of iPrSPy2 and PheSPy2, resp. For complexes 1 and 2, x-ray analyses were performed. Complex 1 crystallizes in the triclinic space group P‾1, and complex 2 crystallizes in the orthorhombic space group Pbca with the following unit cell parameters: for 1, a 15.165(3) Å, b 22.185(4) Å, c 14.989(3) Å, α 105.76(1), β 90.82(2), γ 75.23(1)°, and Z = 2; for 2, a 17.78(2) Å, b 17.70(1) Å, c 15.75(1) Å, and Z = 8. Complex 1 is the 1st structurally characterized example obtained by the redox reaction Cu(II) + RSH → Cu(I) + RSSR and has two independent structures (1a, 1b) which mainly differ in S-S bond distances, Cu(I)···Cu(I) sepns., and C-S-S-C dihedral angles of the disulfide units. The S-S bond distances of 2.088(7) Å in 1a and 2.070(7) Å in 1b are indicative of significant activation of the S-S bonds by the dicopper centers. Fragment MO (FMO) analyses and MO overlap population (MOOP) analyses based on the extended Huckel method clarify the preferable formation of the disulfide S-S bond in 1 rather than the formation of a thiolate-Cu(II) complex within the Py2S- ligand framework. Catalytic functions of complexes 1-3 were studied with peroxides (H2O2 and tBuOOH) as oxidants. Complex 1 catalyzed the selective oxidn. of cyclohexane to cyclohexanol and mediated the cyclohexene epoxidn. in the presence of H2O2. A transient dark green intermediate obsd. in the reaction of 1 with H2O2 was characterized by UV-visible, EPR, and resonance Raman spectroscopies, identifying it as a Cu(II)-OOH species, 1(OOH). The resonance Raman features of the ν(O-O) bands at 822 and 836 cm-1, which are red shifted to 781 and 791 cm-1, resp., upon introduction of H218O2, are indicative of formation of two kinds of Cu-OOH species rather than the Fermi doublet and the significant weakening of the O-O bonds. These mechanistic studies demonstrate that by virtue of the electron-donating ability of the disulfide unit the Cu-OOH species can be actually activated for 1-electron oxidn., which is reported so far unfavorable for other vibrationally characterized Cu-OOH species.
- 241Belle, C.; Rammal, W.; Pierre, J. L. Sulfur ligation in copper enzymes and models. J. Inorg. Biochem. 2005, 99, 1929– 1936, DOI: 10.1016/j.jinorgbio.2005.06.013Google Scholar241Sulfur ligation in copper enzymes and modelsBelle, Catherine; Rammal, Wassim; Pierre, Jean-LouisJournal of Inorganic Biochemistry (2005), 99 (10), 1929-1936CODEN: JIBIDJ; ISSN:0162-0134. (Elsevier B.V.)A review. Biol. Cu-S entities display versatile and unusual coordination chem. The role of S ligation is briefly reviewed through examples from selected Cu-contg. enzymes and relevant biomimetic models. Copper thiolate complexes are of particular interest because of their key roles in a no. of ubiquitous metalloenzymes such as type I (blue Cu proteins) or in the binuclear CuA electron transfer site found in both cytochrome c oxidase (CcO) and nitrous oxide reductase (N2OR). The possible roles of the S(Met) ligand in monoxygenases are described in relation to recently proposed pathways. Some perspectives regarding the biol. relevance of disulfide copper ligation and possible radical Cu bonds in catalytic cycle are also discussed.
- 242Weser, U. Redox reactions of sulphur-containing amino-acid residues in proteins and metalloproteins, an XPS study. In Cation Ordering and Electron Transfer. Structure and Bonding; Springer, Berlin, Germany, 1985; Vol. 61, pp 145– 160.Google ScholarThere is no corresponding record for this reference.
- 243Sievers, C.; Deters, D.; Hartman, H. J.; Weser, U. Stable thiyl radicals in dried yeast Cu(I)6-thionein. J. Inorg. Biochem. 1996, 62, 199– 205, DOI: 10.1016/0162-0134(95)00153-0Google Scholar243Stable thiyl radicals in dried yeast Cu(I)6-thioneinSievers, Christian; Deters, Dirk; Hartmann, Hans-Juergen; Weser, UlrichJournal of Inorganic Biochemistry (1996), 62 (3), 199-205CODEN: JIBIDJ; ISSN:0162-0134. (Elsevier)It was of interest to obtain long-lived thiyl radicals embedded in org. matrixes. Solid thiol compds. including penicillamine, glutathione, and cysteine were UV irradiated under anaerobic conditions at 293 K for 60 min. The formed radicals were identified by ESR (EPR) (g = 2.0265 ± 0.0015) at 293 K as thiyl radicals. The blue-colored radical species were subjected to reflection spectrometry (λmax = 601 ± 3 nm). The color and the EPR signal remained unchanged for six months. At the same time, UV irradn. of lyophilized yeast Cu(I)6-thionein generated stable EPR detectable thiyl radicals at a g-value of 2.2026 ± 0.001. Unlike irradiated cysteine, a five times higher concn. of thiyl radicals was seen when the Cu(I)-thiolate protein was used. No EPR detectable thiyl radicals were measured in the Cu(I)-thiolates of penicillamine, glutathione, and thiophenole, indicating that the hexanuclear copper arrangement in Cu(I)-thionein is most suitable for both the formation and stabilization of this sulfur radical species.
- 244Roschitzki, B.; Vašák, M. Redox labile site in a Zn4 cluster of Cu4,Zn4-metallothionein-3. Biochemistry 2003, 42, 9822– 9828, DOI: 10.1021/bi034816zGoogle Scholar244Redox Labile Site in a Zn4 Cluster of Cu4,Zn4-Metallothionein-3Roschitzki, Bernd; Vasak, MilanBiochemistry (2003), 42 (32), 9822-9828CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)Human metallothionein-3 (MT-3) is a neuronal inhibitory factor mainly expressed in brain and downregulated in Alzheimer's disease. The neuroinhibitory activity has been established for native Cu4,Zn3-MT-3 and recombinant Zn7-MT-3. However, there is only limited knowledge about the structure and properties of the former metalloform. The authors have now generated native-like MT-3 through direct Cu(I) and Zn(II) incorporation into the recombinant apoprotein. Its characterization revealed monomeric Cu4,Zn4-MT-3 contg. metal-thiolate clusters located in two mutually interacting protein domains, a Cu4 cluster in the β-domain and a Zn4 cluster in the α-domain. Using the PC12 cell line, the nontoxic nature of the protein was demonstrated. The results of electronic absorption and Cu(I) luminescence at 77 K showed that the Cu4 cluster possesses an unprecedented stability in air. In contrast, the Zn4 cluster is air sensitive. Its oxidn. in the release of one Zn(II) and the formation of a Zn3 cluster, i.e., Cu4,-Zn3-MT-3. This process can be prevented or reversed under reducing conditions. The detd. apparent stability const. for the Zn4 cluster of 2.4 × 1011 M-1 is similar to that obtained for other zinc-contg. MTs. This suggests that a substantially increased nucleophilic reactivity of specific thiolate ligands is responsible for this effect. Thus, the Zn4 cluster in MT-3 may play a redox-dependent regulatory role.
- 245Bogumil, R.; Faller, P.; Binz, P. A.; Vašák, M.; Charnock, J. M.; Garner, C. D. Structural characterization of Cu(I) and Zn(II) sites in neuronal-growth-inhibitory factor by extended X-ray absorption fine structure (EXAFS). Eur. J. Biochem. 1998, 255, 172– 177, DOI: 10.1046/j.1432-1327.1998.2550172.xGoogle Scholar245Structural characterization of Cu(I) and Zn (II) sites in neuronal-growth-inhibitory factor by extended x-ray absorption fine structure (EXAFS)Bogumil, Ralf; Faller, Peter; Binz, Pierre-Alain; Vasak, Milan; Charnock, John M.; Garner, C. DavidEuropean Journal of Biochemistry (1998), 255 (1), 172-177CODEN: EJBCAI; ISSN:0014-2956. (Springer-Verlag)Neuronal-growth-inhibitory factor (GIF) is a metalloprotein specific to the central nervous system which has been linked to Alzheimer's disease. The high metal content, approx. seven metal atoms/protein mol., and 70% sequence identity to mammalian metallothioneins (MT), including a preserved array of 20 cysteinyl residues, place GIF in the family of MT. In contrast to MT, native GIF isolated from human or bovine brain contains an unusual metal compn., viz. four Cu(I) and three Zn(II) per polypeptide chain. Cu and/or Zn K-edge x-ray absorption spectra have been recorded for native Cu, Zn-GIF, Zn-substituted GIF, and these metals bound to the 32-residue N-terminal domain, Cu4-, Cu6- or Zn3- GIF-(1-32) at 77 K. The results are consistent with the metals being bound to the protein by cysteinyl residues in every case. The Cu-S distance is approx. 2.25 Å and the EXAFS is considered to be consistent with primarily trigonal coordination of the Cu(I); Cu···Cu backscattering is obsd. at approx. 2.67 Å, indicative of the formation of Cux(Scys)y clusters. Thus, the Cu(I) environment is similar to that obsd. in MT. This is also the case for Zn(II), with 4 S at approx. 2.34 Å. However, in contrast to Zn-MT for Zn-substituted GIF and Zn3-GIF-(1-32), Zn···Zn backscattering is obsd. at approx. 3.28 Å. The significance of these results are discussed with respect to the specific biol. activity of GIF.
- 246Meloni, G.; Sonois, V.; Delaine, T.; Guilloreau, L.; Gillet, A.; Teissié, J.; Faller, P.; Vašák, M. Metal swap between Zn7-metallothionein-3 and amyloid-β-Cu protects against amyloid-β toxicity. Nat. Chem. Biol. 2008, 4, 366– 372, DOI: 10.1038/nchembio.89Google Scholar246Metal swap between Zn7-metallothionein-3 and amyloid-β-Cu protects against amyloid-β toxicityMeloni, Gabriele; Sonois, Vanessa; Delaine, Tamara; Guilloreau, Luc; Gillet, Audrey; Teissie, Justin; Faller, Peter; Vasak, MilanNature Chemical Biology (2008), 4 (6), 366-372CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)Aberrant interactions of copper and zinc ions with the amyloid-β peptide (Aβ) potentiate Alzheimer's disease (AD) by participating in the aggregation process of Aβ and in the generation of reactive oxygen species (ROS). The ROS prodn. and the neurotoxicity of Aβ are assocd. with copper binding. Metallothionein-3 (Zn7MT-3), an intra- and extracellularly occurring metalloprotein, is highly expressed in the brain and downregulated in AD. This protein protects, by an unknown mechanism, cultured neurons from the toxicity of Aβ. Here, the authors show that a metal swap between Zn7MT-3 and sol. and aggregated Aβ1-40-Cu(II) abolishes the ROS prodn. and the related cellular toxicity. In this process, copper is reduced by the protein thiolates forming Cu(I)4Zn4MT-3, in which an air-stable Cu(I)4-thiolate cluster and two disulfide bonds are present. The discovered protective effect of Zn7MT-3 from the copper-mediated Aβ1-40 toxicity may lead to new therapeutic strategies for treating AD.
- 247Wezynfeld, N. E.; Stefaniak, E.; Stachucy, K.; Drozd, A.; Płonka, D.; Drew, S. C.; Krężel, A.; Bal, W. Resistance of Cu(Aβ4–16) to copper capture by metallothionein-3 supports a function for the Aβ4–42 peptide as a synaptic Cu(II) scavenger. Angew. Chem., Int. Ed. 2016, 55, 8235– 8238, DOI: 10.1002/anie.201511968Google Scholar247Resistance of Cu(Aβ4-16) to copper capture by metallothionein-3 supports a function for the Aβ4-42 peptide as a synaptic CuII scavengerWezynfeld, Nina E.; Stefaniak, Ewelina; Stachucy, Kinga; Drozd, Agnieszka; Plonka, Dawid; Drew, Simon C.; Krezel, Artur; Bal, WojciechAngewandte Chemie, International Edition (2016), 55 (29), 8235-8238CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)Aβ4-42 is a major species of amyloid-β (Aβ) peptide in the brains of both healthy individuals and those affected by Alzheimer's disease. It has recently been demonstrated to bind Cu2+ with an affinity ∼3000-fold higher than the commonly studied Aβ1-42 and Aβ1-40 peptides, which are implicated in the pathogenesis of Alzheimer's disease. Metallothionein-3, a protein considered to orchestrate Cu2+ and Zn2+ metab. in the brain and provide antioxidant protection, was shown to ext. Cu2+ from Aβ1-40 when acting in its native Zn7MT-3 form. This reaction is assumed to underlie the neuroprotective effect of Zn7MT-3 against Aβ toxicity. Here, the authors used truncated model peptides Aβ1-16 and Aβ4-16 to demonstrate that the high-affinity Cu2+ complex of Aβ4-16 is resistant to Zn7MT-3 reactivity. This indicated that the analogous complex of the full-length peptide, Cu(Aβ4-42), will not yield Cu2+ to MT-3 in the brain, thus supporting the concept of a physiol. role for Aβ4-42 as a Cu2+ scavenger in the synaptic cleft.
- 248Santoro, A.; Wezynfeld, N. E.; Stefaniak, E.; Pomorski, A.; Płonka, D.; Krężel, A.; Bal, W.; Faller, P. Cu transfer from amyloid-β4–16 to metallothionein-3: the role of the neurotransmitter glutamate and metallothionein-3 Zn(II)-load states. Chem. Commun. (Cambridge, U. K.) 2018, 54, 12634– 12637, DOI: 10.1039/C8CC06221HGoogle Scholar248Cu transfer from amyloid-β4-16 to metallothionein-3: the role of the neurotransmitter glutamate and metallothionein-3 Zn(II)-load statesSantoro, Alice; Wezynfeld, Nina Ewa; Stefaniak, Ewelina; Pomorski, Adam; Plonka, Dawid; Krezel, Artur; Bal, Wojciech; Faller, PeterChemical Communications (Cambridge, United Kingdom) (2018), 54 (89), 12634-12637CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)Copper transfer from Cu(II)amyloid-β4-16 to human Zn7-metallothionein-3 can be accelerated by glutamate and by lowering the Zn-load of metallothionein-3 with EDTA. Glutamate facilitates the Cu(II) release, and Zn4-6-metallothionein-3 react more rapidly. These mechanisms are additive, proving the intricate and interconnected network of zinc and copper trafficking between biomols.
- 249Santoro, A.; Calvo, J. S.; Peris-Díaz, M. D.; Krężel, A.; Meloni, G.; Faller, P. The glutathione/metallothionein system challenges the design of efficient O2-activating copper complexes. Angew. Chem., Int. Ed. 2020, 59, 7830– 7835, DOI: 10.1002/anie.201916316Google Scholar249The Glutathione/Metallothionein System Challenges the Design of Efficient O2-Activating Copper ComplexesSantoro, Alice; Calvo, Jenifer S.; Peris-Diaz, Manuel David; Krezel, Artur; Meloni, Gabriele; Faller, PeterAngewandte Chemie, International Edition (2020), 59 (20), 7830-7835CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)Copper complexes are of medicinal and biol. interest, including as anticancer drugs designed to cleave intracellular biomols. by O2 activation. To exhibit such activity, the copper complex must be redox active and resistant to dissocn. Metallothioneins (MTs) and glutathione (GSH) are abundant in the cytosol and nucleus. Because they are thiol-rich reducing mols. with high CuI affinity, they are potential competitors for a copper ion bound in a copper drug. Herein, we report the investigation of a panel of CuI/CuII complexes often used as drugs, with diverse coordination chemistries and redox potentials. We evaluated their catalytic activity in ascorbate oxidn. based on redox cycling between CuI and CuII, as well as their resistance to dissocn. or inactivation under cytosolically relevant concns. of GSH and MT. O2-activating CuI/CuII complexes for cytosolic/nuclear targets are generally not stable against the GSH/MT system, which creates a challenge for their future design.
- 250Palumaa, P.; Mackay, E. A.; Vašák, M. Nonoxidative cadmium-dependent dimerization of Cd7-metallothionein from rabbit liver. Biochemistry 1992, 31, 2181– 2186, DOI: 10.1021/bi00122a040Google Scholar250Nonoxidative cadmium-dependent dimerization of cadmium-metallothionein from rabbit liverPalumaa, Peep; Mackay, Elaine A.; Vasak, MilanBiochemistry (1992), 31 (7), 2181-6CODEN: BICHAW; ISSN:0006-2960.The effect of free Cd(II) ions on monomeric Cd7-metallothionein-2 (MT) from rabbit liver has been studied. Slow, concn.-dependent dimerization of this protein was obsd. by gel filtration chromatog. studies. The dimeric MT form, isolated by gel filtration, contains approx. two addnl. and more weakly bound Cd(II) ions per monomer. The incubation of MT dimers with complexing agents EDTA and 2-mercaptoethanol leads to the dissocn. of dimers to monomers. The results of CD and electronic absorption studies indicate that the slow dimerization process is preceded by an initial rapid Cd-induced rearrangement of the monomeric Cd7-MT structure. The 113Cd NMR spectrum of the MT dimer revealed only four 113Cd resonances at chem. shift positions similar to those obsd. for the Cd4 cluster of the well-characterized monomeric 113Cd7-MT. This result suggests that on dimer formation major structural changes occur in the original three-metal cluster domain of Cd7-MT.
- 251Palumaa, P.; Vašák, M. Binding of inorganic phosphate to the cadmium-induced dimeric form of metallothionein from rabbit liver. Eur. J. Biochem. 1992, 205, 1131– 1135, DOI: 10.1111/j.1432-1033.1992.tb16882.xGoogle Scholar251Binding of inorganic phosphate to the cadmium-induced dimeric form of metallothionein from rabbit liverPalumaa, Peep; Vasak, MilanEuropean Journal of Biochemistry (1992), 205 (3), 1131-5CODEN: EJBCAI; ISSN:0014-2956.Recently it was demonstrated that the exposure of monomeric Cd7-metallothionein (MT) to Cd(II) ions in potassium phosphate buffer results in the nonoxidative formation of MT dimers contg. ∼2 addnl. Cd(II) ions/monomer subunit. The present data shows that inorg. phosphate participates in the Cd-induced dimerization of MT. In the absence of phosphate, Cd-induced oligomerization of MT still takes place, but a substantially lower apparent yield of the dimeric form and an addnl. peak of MT tetramers were detected in gel-filtration expts. Arsenate exhibits a similar effect to that of phosphate, whereas a no. of other anions, i.e. F-, NO3-, SO42-, ClO4-, BO3-, SCN-, HCOO-, and CH3COO- had no effect on Cd-induced oligomerization of MT. Studies on the pH dependence of MT dimerization indicate that the dianionic form of phosphate is involved in this process. Equil.-dialysis expts. using potassium [32P]phosphate established binding of 2 mols. of phosphate to the dimeric MT form with a dissocn. const., Kd, of 23 ± 3 μM (20 mM Tris-HCl and 0.1 M KCl, pH 8.0 at 25°), whereas binding of phosphate was not obsd. with the monomeric Cd7-MT. The noncovalent nature of phosphate binding to the Cd-induced MT dimers has been demonstrated. This is the first evidence for the binding of a nonmetallic cellular component to MT.
- 252Schmid, R. F. Kinetische Untersuchungen des Metallaustauschs in Metallothionein. Diploma Thesis, University of Zürich: Switzerland, 1991.Google ScholarThere is no corresponding record for this reference.
- 253Zangger, K.; Öz, G.; Otvos, J. D.; Armitage, I. M. Three-dimensional solution structure of mouse Cd7-metallothionein-1 by homonuclear and heteronuclear NMR spectroscopy. Protein Sci. 1999, 8, 2630– 2638, DOI: 10.1110/ps.8.12.2630Google Scholar253Three-dimensional solution structure of mouse [Cd7]-metallothionein-1 by homonuclear and heteronuclear NMR spectroscopyZangger, Klaus; Oz, Gulin; Otvos, James D.; Armitage, Ian M.Protein Science (1999), 8 (12), 2630-2638CODEN: PRCIEI; ISSN:0961-8368. (Cambridge University Press)Sequential 1H-NMR assignments of mouse [Cd7]-metallothionein-1 (MT1) have been carried out by std. homonuclear NMR methods and the use of an accordion-heteronuclear multiple quantum correlation (HMQC) expt. for establishing the metal, 113Cd2+, to cysteine connectivities. The three-dimensional structure was then calcd. using the distance constraints from two-dimensional nuclear Overhauser effect (NOE) spectroscopy spectra and the Cys-Cd connectivities as input for a distance geometry-dynamical simulated annealing protocol in X-PLOR 3.851. Similar to the mammalian MT2 isoforms, the homologous primary structure of MT1 suggested two sep. domains, each contg. one metal cluster. Because there were no interdomain constraints, the structure calcn. for the N-terminal β- and the C-terminal α-domain were carried out sep. The structures are based on 409 NMR constraints, consisting of 381 NOEs and 28 cysteine-metal connectivities. The only elements of regular secondary structure found were two short stretches of 310 helixes along with some half-turns in the α-domain. Structural comparison with rat liver MT2 showed high similarity, with the β-domain structure in mouse MT1 showing evidence of increased flexibility compared to the same domain in MT2. The latter was reflected by the presence of fewer interresidue NOEs, no slowly exchanging backbone amide protons, and enhanced cadmium-cadmium exchange rates found in the β-domain of MT1.
- 254Vašák, M.; Bauer, R. Evidence for two types of binding sites in cadmium metallothionein determined by perturbed angular correlation of γ-rays. J. Am. Chem. Soc. 1982, 104, 3236– 3238, DOI: 10.1021/ja00375a059Google Scholar254Evidence for two types of binding sites in cadmium metallothionein determined by perturbed angular correlation of γ-raysVasak, Milan; Bauer, RogertJournal of the American Chemical Society (1982), 104 (11), 3236-8CODEN: JACSAT; ISSN:0002-7863.The metal-binding sites in rabbit metallothionein-1 were probed by perturbed angular correlation of γ-ray spectroscopy (PAC) with excited 114Cd. The presence of 2 distinct frequencies, ω1 ∼120 and ω2 ∼580 MHz, is consistent with the existence of 2 different coordination geometries for Cd binding in this protein. The 120 MHz frequency is comparable to the 65 MHz frequency displayed by excited 111Cd when substituted for Zn in the slightly distorted tetrahedral structural metal site of horse liver alc. dehydrogenase. Judged from the greater amplitude of the 120 MHz signal, it would appear that this symmetry type applies to ∼80% of all metal-binding sites in metallothionein. The 580 MHz frequency is close to the 880 MHz frequency calcd. for the square planar Cd (Cys-S)42- complex and, hence, it could arise from an extremely distorted tetrahedron. Less likely, it could originate also from an octahedral-type complex having 2 axial carboxylate ligands and yielding a frequency of 640 MHz. The 2 frequencies are obsd. both in partially and in fully complexed metallothionein and are, thus, independent of the degree of metal-binding site occupation. Hence, the clustering of the metals in metallothionein, as proposed from homonuclear 113Cd-decoupling expts. and from magnetic studies of the Co(II)-deriv., does not measurably alter the basic coordination feature of the metal environment.
- 255Vašák, M.; Meloni, G. Metallothionein structure and reactivity. In Metallothionein in Biochemistry and Pathology; Zatta, P., Ed.; World Scientific: Singapore, 2008; pp 3– 26.Google ScholarThere is no corresponding record for this reference.
- 256Jones, W. B.; Elgren, T. E.; Morelock, M. M.; Elder, R. C.; Wilcox, D. E. Technetium metallothionein: Spectroscopic and EXAFS study of 99TcO3+ binding to Zn7-metallothionein. Inorg. Chem. 1994, 33, 5571– 5578, DOI: 10.1021/ic00102a034Google Scholar256Technetium Metallothionein: Spectroscopic and EXAFS Study of 99TcO3+ Binding to Zn7-MetallothioneinJones, William B.; Elgren, Timothy E.; Morelock, Maurice M.; Elder, R. C.; Wilcox, Dean E.Inorganic Chemistry (1994), 33 (24), 5571-8CODEN: INOCAJ; ISSN:0020-1669.Vibrational, absorption, CD, and Tc K-edge extended x-ray absorption fine structure (EXAFS) measurements have been made on three samples of metallothionein (MT) contg. 1.6, 3.9, and 6.2 mol equiv of 99Tc, prepd. by the metal displacement (transchelation) reaction of TcO(glucoheptonate)2- with Zn7-MT. For all three samples the TcO3+ ion is bound to the protein in a square pyramidal coordination with equatorial ligands consisting of ∼2 cysteine thiolates and, surprisingly, ∼2 O/N donor ligands. As the Tc content increases, there is very little change in the Tc coordination, except for a decrease in the no. of cysteine ligands per TcO3+ for Tc6.2-MT. The unusual ligand set for this metal ion when it is bound to MT, the absence of bridging cysteine ligands, and the significant no. of cysteines not involved in binding TcO3+, indicate that transchelation does not involve a simple substitution of TcO3+ for Zn(II).
- 257Good, M.; Vašák, M. Iron(II)-substituted metallothionein: evidence for the existence of iron-thiolate clusters. Biochemistry 1986, 25, 8353– 8356, DOI: 10.1021/bi00374a003Google Scholar257Iron(II)-substituted metallothionein: evidence for the existence of iron-thiolate clustersGood, Meinrad; Vasak, MilanBiochemistry (1986), 25 (26), 8353-6CODEN: BICHAW; ISSN:0006-2960.Metallothioneins (MTs) are unique low-mol.-wt. (Mr 6000-7000) metal- and cysteine-rich proteins characterized by 2 tetrahedral tetrathiolate clusters contg. 3 and 4 metal ions. Naturally occurring proteins usually contain the diamagnetic metal ions Zn(II) and(or) Cd(II). These ions were substituted with paramagnetic Fe(II) and the resulting Fe-MT was characterized. Rabbit liver MT-1 in which all 7 metal binding sites were occupied by Fe(II) ions displays absorption features typical of tetrahedral tetrathiolate Fe(II) coordination. This is documented by the presence of a ligand field 5E→2T2 transition in the near-IR region centered at ∼1850 nm absorption coeff of Fe, (εFe, ≈100 M-1 cm-1) and a broad charge-transfer absorption in the UV region with a shoulder at 314 nm. A metal-thiolate cluster structure is inferred from the 7/20 ratio of metal ions to cysteine residues and from spectral studies in which successive increments of Fe(II) were incorporated into the metal-free protein. Thus, to ∼4 equiv, the charge-transfer absorption and magnetic CD (MCD) features of the complexes formed resemble closely those of reduced rubredoxin from Desulfovibro gigas, in which tetrahedral tetrathiolate Fe(II) coordination is documented. However, upon further addn. of Fe(II), the charge-transfer absorption bands undergo a progressive red-shift until the full metal occupancy of 7 Fe(II) ions/mol. is reached. The bathochromic shift which is also manifested in the MCD spectra can be ascribed to the transformation of some of the terminal thiolate ligands to bridging when the fall complement of Fe(II) is bound. The concomitant loss in amplitude of the MCD bands at >4 equiv is thought to arise from exchange coupling of vicinal Fe(II) via the thiolate bridges.
- 258Werth, M. T.; Johnson, M. K. Magnetic circular dichroism and electron paramagnetic resonance studies of iron(II)-metallothionein. Biochemistry 1989, 28, 3982– 3988, DOI: 10.1021/bi00435a053Google Scholar258Magnetic circular dichroism and electron paramagnetic resonance studies of iron(II)-metallothioneinWerth, Mark T.; Johnson, Michael K.Biochemistry (1989), 28 (9), 3982-8CODEN: BICHAW; ISSN:0006-2960.The electronic and magnetic properties of the Fe(II)-thiolate centers in Fe(II)-metallothionein were investigated by low-temp. MCD and EPR spectroscopies at various levels of Fe(II) incorporation. In agreement with previous results, rabbit liver metallothionein was found to bind a max. of 7 Fe(II) ions, with cluster formation occurring when >4 Fe(II) ions are bound at pH 8.5. The results indicate that all the Fe in fully loaded Fe(II)-metallothionein is accommodated in Fe(II)-thiolate clusters that have either S = 0 or S = 2 ground states as a result of antiferromagnetic coupling between high-spin Fe(II) ions. By analogy with the cluster compn. and mechanism of assembly that have been established for other divalent metal ions, the clusters with S = 0 and S = 2 ground states are attributed to tetranuclear and trinuclear centers, resp. EPR signals indicative of S = 2 species were obsd. for samples contg. monomeric tetrathiolate-Fe(II) centers and trinuclear Fe(II)-thiolate clusters. However, the nature of the zero field splitting of the S = 2 ground states that is indicated by the EPR signals is not consistent with that deduced from Moessbauer and MCD studies, suggesting heterogeneity in both types of center.
- 259Ding, X. Q.; Butzlaff, C.; Bill, E.; Pountney, D. L.; Henkel, G.; Winkler, H.; Vašák, M.; Trautwein, A. X. Mössbauer and magnetic susceptibility studies on iron(II) metallothionein from rabbit liver. Evidence for the existence of an unusual type of [M3(CysS)9]3- cluster. Eur. J. Biochem. 1994, 220, 827– 837, DOI: 10.1111/j.1432-1033.1994.tb18685.xGoogle Scholar259Moessbauer and magnetic susceptibility studies on iron(II) metallothionein from rabbit liver. Evidence for the existence of an unusual type of [M3(CysS)9]3- clusterDing, Xiao-Qi; Butzlaff, Christian; Bill, Eckhard; Pountney, Dean L.; Henkel, Gerald; Winkler, Heiner; Vasak, Milan; Trautwein, Alfred XaverEuropean Journal of Biochemistry (1994), 220 (3), 827-37CODEN: EJBCAI; ISSN:0014-2956.The magnetic properties of the Fe(II)-binding-sites in Fe(II)7-metallothionein (MT) have been studied using Moessbauer spectroscopy and magnetic-susceptibility measurements. In agreement with the authors' previous results, simulation of the Moessbauer spectra showed the presence of paramagnetic and diamagnetic subspectra in the ratio 3:4. By comparison with Moessbauer spectra of the inorg. adamantane-like (Et4N)2[Fe4(SEt)10] model compd., the diamagnetic component in Fe(II)7-MT has been assigned to a four-metal cluster in which there is antiferromagnetic coupling between the high-spin Fe(II) ions. It is suggested that the organization of this cluster is similar to that detd. in the three-dimensional structure of the protein, contg. diamagnetic Zn(II) and/or Cd(II) ions. From magnetic-susceptibility studies, an av. magnetic moment of approx. 8.5 μn was obtained for the three remaining bound Fe(II) ions, responsible for the paramagnetic component obsd. in the Moessbauer studies. This value is slightly lower than that for three completely uncoupled Fe(II) ions, suggesting the existence of a three-metal cluster within which there is weak exchange coupling between adjacent Fe(II) ions. The spin-Hamiltonian formalism including, besides zero-field and Zeeman interaction, also exchange interaction among the three Fe(II) ions in the three-metal cluster, H = -J12(S1·S2)-J23(S2·S3)-J13 (S1·S3), was applied to simulate both magnetic-Moessbauer and magnetic-susceptibility date. Reasonable fits were achieved only with values |J12| = |J23| = |J13| = |J| < 1 cm-1. Such a situation could not be reconciled with the chair-like geometry of the [M3(CysS)9]3- cluster detd. with paramagnetic metal ions, where significantly stronger coupling would be anticipated (|J| = 50-70 cm-1). However, modest exchange-coupling properties have been reported for a no. of crystallog. characterized trinuclear [Fe3(SR)3X6]3- clusters (X = Cl, Br; R = Phe, p-tolyl, 2,6-Me2C6H3) distinguished by the preferential formation of a planar Fe3(μ2-SR)3 ring. It is therefore more likely that a pseudo-planar geometry rather than a chair-like geometry is present in the Fe3 cluster of Fe(II)7-MT. This would represent the first example of structural differences on binding divalent metal ions to this protein.
- 260Orihuela, R.; Fernández, B.; Palacios, O.; Valero, E.; Atrian, S.; Watt, R. K.; Domínguez-Vera, J. M.; Capdevila, M. Ferritin and metallothionein: dangerous liaisons. Chem. Commun. (Cambridge, U. K.) 2011, 47, 12155– 12157, DOI: 10.1039/c1cc14819bGoogle Scholar260Ferritin and metallothionein: dangerous liaisonsOrihuela, Ruben; Fernandez, Belen; Palacios, Oscar; Valero, Elsa; Atrian, Silvia; Watt, Richard K.; Dominguez-Vera, Jose M.; Capdevila, MerceChemical Communications (Cambridge, United Kingdom) (2011), 47 (44), 12155-12157CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)Ferritin (Ft) interaction with the Zn-complexes of mammalian MT1, MT2 and MT3 metallothioneins (MT) leads to simultaneous FeII and ZnII release.
- 261Baird, S. K.; Kurz, T.; Brunk, U. T. Metallothionein protects against oxidative stress-induced lysosomal destabilization. Biochem. J. 2006, 394, 275– 283, DOI: 10.1042/BJ20051143Google Scholar261Metallothionein protects against oxidative stress-induced lysosomal destabilizationBaird, Sarah K.; Kurz, Tino; Brunk, Ulf T.Biochemical Journal (2006), 394 (1), 275-283CODEN: BIJOAK; ISSN:0264-6021. (Portland Press Ltd.)The introduction of apo-ferritin or the iron chelator DFO (desferrioxamine) conjugated to starch into the lysosomal compartment protects cells against oxidative stress, lysosomal rupture and ensuing apoptosis/necrosis by binding intralysosomal redox-active iron, thus preventing Fenton-type reactions and ensuing peroxidn. of lysosomal membranes. Because up-regulation of MTs (metallothioneins) also generates enhanced cellular resistance to oxidative stress, including X-irradn., and MTs were found to be capable of iron binding in an acidic and reducing lysosomal-like environment, we propose that these proteins might similarly stabilize lysosomes following autophagocytotic delivery to the lysosomal compartment. Here, we report that Zn-mediated MT up-regulation, assayed by Western blotting and immunocytochem., results in lysosomal stabilization and decreased apoptosis following oxidative stress, similar to the protection afforded by fluid-phase endocytosis of apo-ferritin or DFO. In contrast, the endocytotic uptake of an iron phosphate complex destabilized lysosomes against oxidative stress, but this was suppressed in cells with up-regulated MT. It is suggested that the resistance against oxidative stress, known to occur in MT-rich cells, may be a consequence of autophagic turnover of MT, resulting in reduced iron-catalyzed intralysosomal peroxidative reactions.
- 262Kennedy, M. C.; Gan, T.; Antholine, W. E.; Petering, D. H. Metallothionein reacts with Fe2+ and NO to form products with a g = 2.039 ESR signal. Biochem. Biophys. Res. Commun. 1993, 196, 632– 635, DOI: 10.1006/bbrc.1993.2296Google Scholar262Metallothionein reacts with iron(2+) and nitric oxide to form products with a g = 2.039 ESR signalKennedy, Mary Claire; Gan, Tong; Antholine, William E.; Petering, David H.Biochemical and Biophysical Research Communications (1993), 196 (2), 632-5CODEN: BBRCA9; ISSN:0006-291X.The interaction of metallothionein (MT), Fe2+, and nitric oxide has been examd. by ESR spectroscopy. When either apoMT 2 or Zn-MT 2 from rabbit liver was mixed with Fe2+ and NO, paramagnetic products with g values of 2.013 and 2.039 were found. These are characteristic of iron nitrosyl thiolates with stoichiometry, Fe(NO)2(SR)2. This product was not generated when beef heart mitochondrial aconitase was used as the source of iron.
- 263Sano, Y.; Onoda, A.; Sakurai, R.; Kitagishi, H.; Hayashi, T. Preparation and reactivity of a tetranuclear Fe(II) core in the metallothionein α-domain. J. Inorg. Biochem. 2011, 105, 702– 708, DOI: 10.1016/j.jinorgbio.2011.01.011Google Scholar263Preparation and reactivity of a tetranuclear Fe(II) core in the metallothionein α-domainSano, Yohei; Onoda, Akira; Sakurai, Rie; Kitagishi, Hiroaki; Hayashi, TakashiJournal of Inorganic Biochemistry (2011), 105 (5), 702-708CODEN: JIBIDJ; ISSN:0162-0134. (Elsevier)Metallothioneins (MTs) are small cysteine-rich proteins which exhibit high affinities for various metal ions and play roles in storage of essential metals and detoxification of toxic metals. Studies on the redox properties of MTs have been quite limited. Recently, the authors focused on the α-domain of MT (MTα) as a protein matrix and incorporated a tetranuclear metal cluster as a reductant. UV-visible, CD and MS data indicate the formation of the stable tetranuclear metal-cysteine cluster in the MTα matrix with FeII4-MTα and CoII4-MTα species existing in water. Furthermore, the FeII4-MTα species was found to promote the redn. of met-myoglobin and azobenzene derivs. under mild conditions. Particularly, the stoichiometric redn. of methyl red with FeII4-MTα (1:1) proceeds with a conversion of 98% over a period of 6 h at 25°. All four Fe(II) cores contribute to the redn. The authors describe the prepn. and reactivity of the tetranuclear iron cluster in the protein matrix in this paper.
- 264Irving, H.; Williams, R. J. Order of stability of metal complexes. Nature 1948, 162, 746– 747, DOI: 10.1038/162746a0Google Scholar264Order of stability of metal complexesIrving, H.; Williams, R. J. P.Nature (London, United Kingdom) (1948), 162 (), 746-7CODEN: NATUAS; ISSN:0028-0836.cf. Mellor and Maley, C.A. 41, 4398e; 42, 4809d. In the first transition series (Mn, Fe, Co, Ni, Cu, Zn) the stability of complexes increases steadily to a max. at Cu; ligands may be NH3, (CH2NH2)2, CH2(CH2NH2)2, or salicyl-aldehyde. Zn complexes are less stable than those of CuII (M-shell is completely filled); however, attachment of 6 groups through d2sp3 or dsp2 orbitals is possible from Ca to Cu(II). A similar trend is noted in the 2nd transition series with Pd(II) > Cd(II). In the 3rd transition series, the order is Pt(II) > Hg(II) and Pb(II). The stability of complexes increases with the electronegativity of the metal involved.
- 265Irving, H. M. N. H.; Williams, R. J. P. The stability of transition-metal complexes. J. Chem. Soc. 1953, 3192– 3210, DOI: 10.1039/jr9530003192Google Scholar265Stability of transition metal complexesIrving, H. M.; Williams, R. J. P.Journal of the Chemical Society (1953), (), 3192-3210CODEN: JCSOA9; ISSN:0368-1769.The published values of the stability consts. for the bivalent ions of Mn, Fe, Co, Ni, Cu, and Zn with ligands exhibiting NN, NO, and OO co.ovrddot.ordinating patterns are tabulated. In almost all cases the order of stability is Mn < Fe < Co < Ni < Cu > Zn irrespective of the nature of the ligand. This order is a consequence of the monotonic change in the 2nd ionization potentials and the reciprocal radii, the parameters serving as a guide to the magnitude of the covalent and electrostatic interactions, resp., of the ions involved. When other ions are inserted in the order of stability, the new order varies with the ligand because there is not generally the same relation for both factors throughout the series. Characteristic co.ovrddot.ordination no., stereochem. considerations, and entropy factors may, however, affect the series given here. Examples of the former two effects are the low value for k3 in the Cu++-(NH2CH2)2 system, the high values for the consts. for the Fe++-ο-phenanthroline system, and the low values for k2 in systems with (MeNHCH2)2. The entropy effect is such that the predominant factor, the entropy of hydration of M++, follows the order of stabilization and hence will not be a factor in exceptions.
- 266Williams, R. J. P.; Fraústo da Silva, J. J. R. The distribution of elements in cells. Coord. Chem. Rev. 2000, 200–202, 247– 348, DOI: 10.1016/S0010-8545(00)00324-6Google Scholar266The distribution of elements in cellsWilliams, R. J. P.; Frausto da Silva, J. J. R.Coordination Chemistry Reviews (2000), 200-202 (), 247-348CODEN: CCHRAM; ISSN:0010-8545. (Elsevier Science S.A.)A review, with 82 refs. The selective uptake of elements by proteins can be based on several routes: (1) the equil. binding of different elements by different protein ligands using charge, size, electron affinity, and stereochem. preferences; (2) kinetic insertion of an element into such a coordination site of a protein; (3) removal of the element to a special compartment by pumping followed by (1) or (2). A cellular system also limits the amt. of each type of metal-binding apoprotein by genetic regulation of its symbiosis with element uptake. Such a limitation generates much greater selectivity. Finally we consider how the obsd. selection of elements by proteins has changed in evolution through changes of availability of elements and their combinations in the environment1.
- 267Fraústo da Silva, J. J. R.; Williams, R. J. P. The natural selection of the chemical elements; Clarendon Press: Oxford, UK, 1997.Google ScholarThere is no corresponding record for this reference.
- 268Foster, A. W.; Osman, D.; Robinson, N. J. Metal preferences and metallation. J. Biol. Chem. 2014, 289, 28095– 28103, DOI: 10.1074/jbc.R114.588145Google Scholar268Metal preferences and metalationFoster, Andrew W.; Osman, Deenah; Robinson, Nigel J.Journal of Biological Chemistry (2014), 289 (41), 28095-28103CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)A review. The metal binding preferences of most metalloproteins do not match their metal requirements. Thus, metalation of an estd. 30% of metalloenzymes is aided by metal delivery systems, with ∼25% acquiring preassembled metal cofactors. The remaining ∼70% are presumed to compete for metals from buffered metal pools. Metalation is further aided by maintaining the relative concns. of these pools as an inverse function of the stabilities of the resp. metal complexes. For example, Mg2+-contg. enzymes always prefer to bind Zn2+, and these metals dominate the metalloenzymes without metal delivery systems. Therefore, the buffered concn. of Zn2+ is held at least a million-fold below Mg2+ inside most cells.
- 269Robinson, N. J.; Glasfeld, A. Metalation: Nature’s challenge in bioinorganic chemistry. JBIC, J. Biol. Inorg. Chem. 2020, 25, 543– 545, DOI: 10.1007/s00775-020-01790-3Google Scholar269Metalation: nature's challenge in bioinorganic chemistryRobinson, Nigel J.; Glasfeld, ArthurJBIC, Journal of Biological Inorganic Chemistry (2020), 25 (4), 543-545CODEN: JJBCFA; ISSN:0949-8257. (Springer)Abstr.: The assocn. of proteins with metals, metalation, is challenging because the tightest binding metals are rarely the correct ones. Inside cells, correct metalation is enabled by controlled bioavailability plus extra mechanisms for tricky combinations such as iron and manganese.
- 270Kepp, K. P. A quantitative scale of oxophilicity and thiophilicity. Inorg. Chem. 2016, 55, 9461– 9470, DOI: 10.1021/acs.inorgchem.6b01702Google Scholar270A Quantitative Scale of Oxophilicity and ThiophilicityKepp, Kasper P.Inorganic Chemistry (2016), 55 (18), 9461-9470CODEN: INOCAJ; ISSN:0020-1669. (American Chemical Society)Oxophilicity and thiophilicity are widely used concepts with no quant. definition. In this paper, a simple, generic scale is developed that solves issues with ref. states and system dependencies and captures empirically known tendencies toward oxygen. This enables a detailed anal. of the fundamental causes of oxophilicity. Notably, the notion that oxophilicity relates to Lewis acid hardness is invalid. Rather, oxophilicity correlates only modestly and inversely with abs. hardness and more strongly with electronegativity and effective nuclear charge. Since oxygen is highly electroneg., ionic bonding is stronger to metals of low electronegativity. Left-side d-block elements with low effective nuclear charges and electronegativities are thus highly oxophilic, and the f-block elements, not because of their hardness, which is normal, but as a result of the small ionization energies of their outermost valence electrons, can easily transfer electrons to fulfill the electron demands of oxygen. Consistent with empirical experience, the most oxophilic elements are found in the left part of the d block, the lanthanides, and the actinides. The d-block elements differ substantially in oxophilicity, quantifying their different uses in a wide range of chem. reactions; thus, the use of mixed oxo- and thiophilic (i.e., "mesophilic") surfaces and catalysts as a design principle can explain the success of many recent applications. The proposed scale may therefore help to rationalize and improve chem. reactions more effectively than current qual. considerations of oxophilicity.
- 271Martin, R. B. A stability ruler for metal ion complexes. J. Chem. Educ. 1987, 64, 402, DOI: 10.1021/ed064p402Google Scholar271A stability ruler for metal ion complexesMartin, R. BruceJournal of Chemical Education (1987), 64 (5), 402CODEN: JCEDA8; ISSN:0021-9584.A stability ruler is described that makes use of the Irving-Williams series, the regular increase in stability of the dipos. metal ions of the 1st transition row, from Mg2+ through Cu2+, against which metal ions with more variable stabilities such as Zn2+ may be compared. The stabilities of Pb2+, Cd2+, and Hg2+ are characterized using the stability ruler.
- 272Romans, A. Y.; Graichen, M. E.; Lochmüller, C. H.; Henkens, R. W. Kinetics and mechanism of dissociation of zinc ion from carbonic anhydrase. Bioinorg. Chem. 1978, 9, 217– 229, DOI: 10.1016/S0006-3061(78)80007-6Google Scholar272Kinetics and mechanism of dissociation of zinc ion from carbonic anhydraseRomans, Alice Y.; Graichen, Mary E.; Lochmueller, C. H.; Henkens, Robert W.Bioinorganic Chemistry (1978), 9 (3), 217-29CODEN: BICHBX; ISSN:0006-3061.The kinetics of dissocn. of Zn2+ from carbonic anhydrase was measured over a range of pH, temp., and acetate concn. The rate of dissocn. was extremely slow at neutral pH, but increased in almost direct proportion to the H+ concn. and was enhanced in the presence of 1,10-phenanthroline or acetate. The thermodn. stability of the Zn-apoenzyme complex was detd. over a range of pH from rate data on binding and dissocn. (stability consts. 109 to 1011 M-1, 25°. The great stability of the complex and slow exchange of the apoenzyme ligand was attributed, at least in part, to the rigidity of the multidentate protein ligand.
- 273Heinz, U.; Kiefer, M.; Tholey, A.; Adolph, H. W. On the competition for available zinc. J. Biol. Chem. 2005, 280, 3197– 3207, DOI: 10.1074/jbc.M409425200Google Scholar273On the Competition for Available ZincHeinz, Uwe; Kiefer, Martin; Tholey, Andreas; Adolph, Hans-WernerJournal of Biological Chemistry (2005), 280 (5), 3197-3207CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)Extended x-ray absorption fine structure (EXAFS) spectroscopy was combined with thermodn. and kinetic approaches to investigate zinc binding to a zinc finger (C2H2) and a tetrathiolate (C4) peptide. Both peptides represent structural zinc sites of proteins and rapidly bind a single zinc ion with picomolar dissocn. consts. In competition with EDTA the transfer of peptide-bound zinc ions proved to be 6 orders of magnitude faster than predicted for a dissocn.-assocn. mechanism thus requiring ligand exchange mechanisms via peptide-zinc-EDTA complexes. EXAFS spectra of C2H2 showed the expected Cys2His2-ligand geometry when fully loaded with zinc. For a 2-fold excess of peptide, however, the existence of zinc-bridged peptide-peptide complexes with dominating sulfur coordination could be clearly shown. Whereas zinc binding kinetics of C2H2 appeared as a simple second order process, the suggested mechanism for C4 comprises a zinc-bridged Zn-(C4)2 species as well as a Zn-C4 species with less than 4 metal-bound thiolates, which is supported by EXAFS results. A rapid equil. of bound and unbound states of individual ligands might explain the kinetic instability of zinc-peptide complexes, which enables fast ligand exchange during the encounter of occupied and unoccupied acceptor sites. Depending on relative concns. and stabilities, this results in a rapid transfer of zinc ions in the virtual absence of free zinc ions, as seen for the zinc transfer to EDTA, or in the formation of zinc-bridged complexes, as seen for both peptides with excess of peptides over available zinc.
- 274Colvin, R. A.; Holmes, W. R.; Fontaine, C. P.; Maret, W. Cytosolic zinc buffering and muffling: their role in intracellular zinc homeostasis. Metallomics 2010, 2, 306– 317, DOI: 10.1039/b926662cGoogle Scholar274Cytosolic zinc buffering and muffling: their role in intracellular zinc homeostasisColvin, Robert A.; Holmes, William R.; Fontaine, Charles P.; Maret, WolfgangMetallomics (2010), 2 (5), 306-317CODEN: METAJS; ISSN:1756-591X. (Royal Society of Chemistry)A review. Our knowledge of the mol. mechanisms of intracellular homeostatic control of zinc ions is now firmly grounded on exptl. findings gleaned from the study of zinc proteomes and metallomes, zinc transporters, and insights from the use of computational approaches. A cell's repertoire of zinc homeostatic mols. includes cytosolic zinc-binding proteins, transporters localized to cytoplasmic and organellar membranes, and sensors of cytoplasmic free zinc ions. Under steady state conditions, a primary function of cytosolic zinc-binding proteins is to buffer the relatively large zinc content found in most cells to a cytosolic zinc(II) ion concn. in the picomolar range. Under non-steady state conditions, zinc-binding proteins and transporters act in concert to modulate transient changes in cytosolic zinc ion concn. in a process that is called zinc muffling. For example, if a cell is challenged by an influx of zinc ions, muffling reactions will dampen the resulting rise in cytosolic zinc ion concn. and eventually restore the cytosolic zinc ion concn. to its original value by shuttling zinc ions into subcellular stores or by removing zinc ions from the cell. In addn., muffling reactions provide a potential means to control changes in cytosolic zinc ion concns. for purposes of cell signalling in what would otherwise be considered a buffered environment not conducive for signalling. Such intracellular zinc ion signals are known to derive from redox modifications of zinc-thiolate coordination environments, release from subcellular zinc stores, and zinc ion influx via channels. Recently, it has been discovered that metallothionein binds its seven zinc ions with different affinities. This property makes metallothionein particularly well positioned to participate in zinc buffering and muffling reactions. In addn., it is well established that metallothionein is a source of zinc ions under conditions of redox signalling. We suggest that the biol. functions of transient changes in cytosolic zinc ion concns. (presumptive zinc signals) complement those of calcium ions in both spatial and temporal dimensions.
- 275Foster, A. W.; Robinson, N. J. Promiscuity and preferencs of metallothioneins: the cell rules. BMC Biol. 2011, 9, 25, DOI: 10.1186/1741-7007-9-25Google Scholar275Promiscuity and preferences of metallothioneins: the cell rulesFoster, Andrew W.; Robinson, Nigel J.BMC Biology (2011), 9 (), 25CODEN: BBMIF7; ISSN:1741-7007. (BioMed Central Ltd.)A review. Metalloproteins are essential for many cellular functions, but it has not been clear how they distinguish between the different metals to bind the correct ones. A report in BMC Biol. finds that preferences of two metallothionein isoforms for two different cations are due to inherent properties of these usually less discriminating proteins. Here these observations are discussed in the context of the cellular mechanisms that regulate metal binding to proteins.
- 276Osman, D.; Martini, M. A.; Foster, A. W.; Chen, J.; Scott, A. J. P.; Morton, R. J.; Steed, J. W.; Lurie-Luke, E.; Huggins, T. G.; Lawrence, A. D.; Deery, E.; Warren, M. J.; Chivers, P. T.; Robinson, N. J. Bacterial sensors define intracellular free energies for correct enzyme metalation. Nat. Chem. Biol. 2019, 15, 241– 249, DOI: 10.1038/s41589-018-0211-4Google Scholar276Bacterial sensors define intracellular free energies for correct enzyme metalationOsman, Deenah; Martini, Maria Alessandra; Foster, Andrew W.; Chen, Junjun; Scott, Andrew J. P.; Morton, Richard J.; Steed, Jonathan W.; Lurie-Luke, Elena; Huggins, Thomas G.; Lawrence, Andrew D.; Deery, Evelyne; Warren, Martin J.; Chivers, Peter T.; Robinson, Nigel J.Nature Chemical Biology (2019), 15 (3), 241-249CODEN: NCBABT; ISSN:1552-4450. (Nature Research)There is a challenge for metalloenzymes to acquire their correct metals because some inorg. elements form more stable complexes with proteins than do others. These preferences can be overcome provided some metals are more available than others. However, while the total amt. of cellular metal can be readily measured, the available levels of each metal have been more difficult to define. Metal-sensing transcriptional regulators are tuned to the intracellular availabilities of their cognate ions. Here the authors detd. the std. free energy for metal complex formation to which each sensor, in a set of bacterial metal sensors, is attuned: the less competitive the metal, the less favorable the free energy and hence the greater availability to which the cognate allosteric mechanism is tuned. Comparing these free energies with values derived from the metal affinities of a metalloprotein reveals the mechanism of correct metalation exemplified here by a cobalt chelatase for vitamin B12.
- 277Nar, H.; Huber, R.; Messerschmidt, A.; Filippou, A. C.; Barth, M.; Jaquinod, M.; Kamp, M.; Canters, G. W. Characterization and crystal structure of zinc azurin, a by-product of heterologous expression in Escherichia coli of Pseudomonas aeruginosa copper azurin. Eur. J. Biochem. 1992, 205, 1123– 1129, DOI: 10.1111/j.1432-1033.1992.tb16881.xGoogle Scholar277Characterization and crystal structure of zinc azurin, a by-product of heterologous expression in Escherichia coli of Pseudomonas aeruginosa copper azurinNar, Herbert; Huber, Robert; Messerschmidt, Albrecht; Filippou, Alexander C.; Barth, Manfred; Jaquinod, Michel; Van de Kamp, Mart; Canters, Gerard W.European Journal of Biochemistry (1992), 205 (3), 1123-9CODEN: EJBCAI; ISSN:0014-2956.Azurin*, a byproduct of heterologous expression of the gene encoding the blue copper protein azurin from P. aeruginosa in E. coli, was characterized by chem. anal. and electrospray ionization mass spectrometry, and its structure detd. by x-ray crystallog. It was shown that azurin* is native azurin with its copper atom replaced by zinc in the metal binding site. Zinc is probably incorporated in the apo-protein after its expression and transport into the periplasm. Holo-azurin can be reconstituted from azurin* by prolonged exposure of the protein to high copper ion concns. or unfolding of the protein and refolding in the presence of copper ions. An x-ray crystallog. anal. of azurin* at 0.21-nm resoln. revealed that the overall structure of azurin is not perturbed by the metal exchange. However, the geometry of the coordination sphere changes from trigonal bipyramidal in the case of copper azurin to distorted tetrahedral for the zinc protein. The copper ligand Met121 is no longer coordinated to zinc which adopts a position close to the carbonyl oxygen atom from residue Gly45. The polypeptide structure surrounding the metal site undergoes moderate reorganization upon zinc binding. The largest displacement obsd. is for the carbonyl oxygen from residue Gly45, which is involved in copper and zinc binding. It moves by 0.03 nm towards the zinc, thereby reducing its distance to the metal from 0.29 nm in the copper protein to 0.23 nm in the deriv.
- 278Maret, W.; Shiemke, A. K.; Wheeler, W. D.; Loehr, T. M.; Sanders-Loehr, J. Resonance Raman spectroscopy of blue copper proteins: Ligand and coenzyme effects in Cu(II)-substituted liver alcohol dehydrogenase. J. Am. Chem. Soc. 1986, 108, 6351– 6359, DOI: 10.1021/ja00280a036Google Scholar278Resonance Raman spectroscopy of blue copper proteins: ligand and coenzyme effects in copper(II)-substituted liver alcohol dehydrogenaseMaret, Wolfgang; Shiemke, Andrew K.; Wheeler, William D.; Loehr, Thomas M.; Sanders-Loehr, JoannJournal of the American Chemical Society (1986), 108 (20), 6351-9CODEN: JACSAT; ISSN:0002-7863.Liver alc. dehydrogenase (LADH) with Cu(II) substituted for the native Zn(II) at the catalytic metal site exhibits a resonance Raman spectrum characteristic of type 1 Cu of blue Cu proteins when excited within its cysteinate → Cu(II) charge-transfer band (620 nm at 298 K, 570 nm at 125 K). The 2 most intense Raman peaks at 350 and 415 cm-1 and the 2 weaker peaks at 330 and 342 cm-1 are assigned to Cu-cysteinate vibrations of 1 or both cysteine ligands. The multiplicity of peaks indicates coupling of ν(Cu-S) with ligand modes. The peak at 254 cm-1 which is shifted 2 cm-1 to lower energy in D2O is assigned to a Cu-imidazole vibration of the histidine ligand. All of the resonance-enhanced modes display narrow excitation profiles are max. at ∼600 nm (at 15 and 90 K) and appear to coincide with 1 component of the 570-nm electronic absorption envelope. The peaks in the resonance Raman spectra of Ni(II)- and Fe(III)-substituted LADH are very close in frequency and intensity to those of the Cu(II) protein, supporting the view that the protein imposes a tetrahedral structure at the catalytic metal site with ≥1 short metal-S(cysteine) bond. Binding of ligands such as pyrazole, imidazole, β-mercaptoethanol, and cyanide to Cu(II)-LADH induces 3-20-cm-1 shifts in the 4 main Cu-cysteinate vibrations and causes a substantial decrease in the intensities of the 2 high-energy Cu-cysteinate peaks. Low-temp. electronic spectra of these binary complexes show absorption max. close to 500 nm which are significantly blue-shifted from the 570-nm max. in the low-temp. spectrum of Cu(II)-LADH. These spectral alterations can be interpreted in terms of a distortion toward a tetragonal geometry at the metal site. In contrast, the binary NADH complex and the ternary NAD/pyrazole complex both exhibit intensity increases in the 2 high-frequency Cu-cysteinate modes are well as a rich set of overtone and combination bands. These resonance Raman features are new signatures of tetrahedral coordination which correlate well with optical and ESR indicators of type 1 Cu sites. The Cu(II)-LADH/NADH complex further resembles the blue Cu proteins in that the Cu-cysteinate peaks are strongly affected by deuteration but differs in that there are D-induced alterations in intensities as well as frequencies. It is proposed that these D isotope effects are due to H bonding of the cysteinate ligand S atoms to suitable protein donors, as is typical of metal-S centers in other metalloproteins.
- 279Goch, W.; Bal, W. Stochastic or not? Method to predict and quantify the stochastic effects on the association equilibria in nanoscopic systems. J. Phys. Chem. A 2020, 124, 1421– 1428, DOI: 10.1021/acs.jpca.9b09441Google Scholar279Stochastic or Not? Method To Predict and Quantify the Stochastic Effects on the Association Reaction Equilibria in Nanoscopic SystemsGoch, Wojciech; Bal, WojciechJournal of Physical Chemistry A (2020), 124 (7), 1421-1428CODEN: JPCAFH; ISSN:1089-5639. (American Chemical Society)The stochastic nature of chem. reaction and impact of the stochasticity on their evolution is soundly documented. Both theor. predictions and emerging exptl. evidence indicate the influence of stochastic effects on the equil. state of assocn. reaction. In this work simple math. formulas are introduced to est. these effects. First, the dependence of the ratio of obsd. reactants (apparent assocn. const., equiv. of macroscopic assocn. const. in stochastic anal.) on the vol. and the no. of mols. of reagents is discussed and the limiting factors of this effect are shown. Next, the apparent assocn. const. is approximated for nanoscale systems by closed-form formulas derived for this purpose. Finally, an estn. for the macroscopic const. value from the apparent one is provided and validated on the published exptl. data. This work was inspired by chem. reactions occurring in biol. compartments, but the results can be used for all systems belonging to the stochastic regime of chem. reactions.
- 280Andreini, C.; Banci, L.; Bertini, I.; Rosato, A. Counting the zinc-proteins encoded in the human genome. J. Proteome Res. 2006, 5, 196– 201, DOI: 10.1021/pr050361jGoogle Scholar280Counting the zinc-proteins encoded in the human genomeAndreini, Claudia; Banci, Lucia; Bertini, Ivano; Rosato, AntonioJournal of Proteome Research (2006), 5 (1), 196-201CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)Metalloproteins are proteins capable of binding one or more metal ions, which may be required for their biol. function, or for regulation of their activities or for structural purposes. Genome sequencing projects have provided a huge no. of protein primary sequences, but, even though several different elaborate analyses and annotations have been enabled by a rich and ever-increasing portfolio of bioinformatic tools, metal-binding properties remain difficult to predict as well as to investigate exptl. Consequently, the present knowledge about metalloproteins is only partial. The present bioinformatic research proposes a strategy to answer the question of how many and which proteins encoded in the human genome may require zinc for their physiol. function. This is achieved by a combination of approaches, which include: (i) searching in the proteome for the zinc-binding patterns that, on their turn, are obtained from all available X-ray data; (ii) using libraries of metal-binding protein domains based on multiple sequence alignments of known metalloproteins obtained from the Pfam database; and (iii) mining the annotations of human gene sequences, which are based on any type of information available. It is found that 1684 proteins in the human proteome are independently identified by all three approaches as zinc-proteins, 746 are identified by two, and 777 are identified by only one method. By assuming that all proteins identified by at least two approaches are truly zinc-binding and inspecting the proteins identified by a single method, it can be proposed that ca. 2800 Human proteins are potentially zinc-binding in vivo, corresponding to 10% of the human proteome, with an uncertainty of 400 sequences. Available functional information suggests that the large majority of human zinc-binding proteins are involved in the regulation of gene expression. The most abundant class of zinc-binding proteins in humans is that of zinc-fingers, with Cys4 and Cys2His2 being the most common types of coordination environment.
- 281Andreini, C.; Banci, L.; Bertini, I.; Rosato, A. Zinc through the three domains of life. J. Proteome Res. 2006, 5, 3173– 3178, DOI: 10.1021/pr0603699Google Scholar281Zinc through the Three Domains of LifeAndreini, Claudia; Banci, Lucia; Bertini, Ivano; Rosato, AntonioJournal of Proteome Research (2006), 5 (11), 3173-3178CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)Zinc is one of the metal ions essential for life, as it is required for the proper functioning of a large no. of proteins. Despite its importance, the annotation of zinc-binding proteins in gene banks or protein domain databases still has significant room for improvement. In the present work, we compiled a list of known zinc-binding protein domains and of known zinc-binding sequence motifs (zinc-binding patterns), and then used them jointly to analyze the proteome of 57 different organisms to obtain an overview of zinc usage by archaeal, bacterial, and eukaryotic organisms. Zinc-binding proteins are an abundant fraction of these proteomes, ranging between 4% and 10%. The no. of zinc-binding proteins correlates linearly with the total no. of proteins encoded by the genome of an organism, but the proportionality const. of Eukaryota (8.8%) is significantly higher than that obsd. in Bacteria and Archaea (from 5% to 6%). Most of this enrichment is due to the larger portfolio of regulatory proteins in Eukaryota.
- 282Maret, W. New perspectives of zinc coordination environments in proteins. J. Inorg. Biochem. 2012, 111, 110– 116, DOI: 10.1016/j.jinorgbio.2011.11.018Google Scholar282New perspectives of zinc coordination environments in proteinsMaret, WolfgangJournal of Inorganic Biochemistry (2012), 111 (), 110-116CODEN: JIBIDJ; ISSN:0162-0134. (Elsevier)A review. Zn(II) is more widely used as a cofactor in proteins than any other transition metal ion. In addn. to catalytic and structural functions, zinc(II) ions play a role in information transfer and cellular control. They bind transiently when proteins regulate Zn(II) concns. and re-distribute Zn(II) and when proteins are regulated by Zn(II). Transient Zn-binding sites employ the same donors of amino acid side-chains as catalytic and structural sites, but differ in their coordination chem. that can modulate Zn(II) affinities over at least 10 orders of magnitude. Redox activity of Cys ligands, multiple binding modes of O, S, and N donors, and protein conformational changes induce coordination dynamics in Zn sites and Zn(II) ion mobility. Functional annotations of the marked variation of coordination environments in Zn proteomes need to consider how the primary coordination spheres interact with protein structure and dynamics, and the adaptation of coordination properties to the biol. context in extracellular, cellular, or subcellular locations.
- 283Kambe, T.; Taylor, K. M.; Fu, D. Zinc transporters and their functional integration into mammalian cells. J. Biol. Chem. 2021, 296, 100320, DOI: 10.1016/j.jbc.2021.100320Google Scholar283Zinc transporters and their functional integration in mammalian cellsKambe, Taiho; Taylor, Kathryn M.; Fu, DaxJournal of Biological Chemistry (2021), 296 (), 100320CODEN: JBCHA3; ISSN:1083-351X. (Elsevier Inc.)A review. Zinc is a ubiquitous biol. metal in all living organisms. The spatiotemporal zinc dynamics in cells provide crucial cellular signaling opportunities, but also challenges for intracellular zinc homeostasis with broad disease implications. Zinc transporters play a central role in regulating cellular zinc balance and subcellular zinc distributions. The discoveries of two complementary families of mammalian zinc transporters (ZnTs and ZIPs) in the mid-1990s spurred much speculation on their metal selectivity and cellular functions. After two decades of research, we have arrived at a biochem. description of zinc transport. However, in vitro functions are fundamentally different from those in living cells, where mammalian zinc transporters are directed to specific subcellular locations, engaged in dedicated macromol. machineries, and connected with diverse cellular processes. Hence, the mol. functions of individual zinc transporters are reshaped and deeply integrated in cells to promote the utilization of zinc chem. to perform enzymic reactions, tune cellular responsiveness to pathophysiol. signals, and safeguard cellular homeostasis. At present, the underlying mechanisms driving the functional integration of mammalian zinc transporters are largely unknown. This knowledge gap has motivated a shift of the research focus from in vitro studies of purified zinc transporters to in cell studies of mammalian zinc transporters in the context of their subcellular locations and protein interactions. In this review, we will outline how knowledge of zinc transporters has been accumulated from in-test-tube to in-cell studies, highlighting new insights and paradigm shifts in our understanding of the mol. and cellular basis of mammalian zinc transporter functions.
- 284Hirose, J.; Ohsaki, T.; Nishimoto, N.; Matuoka, S.; Hiromoto, T.; Yoshida, T.; Minoura, T.; Iwamoto, H.; Fukasawa, K. M. Characterization of the metal-binding site in aminopeptidase B. Biol. Pharm. Bull. 2006, 29, 2378– 2382, DOI: 10.1248/bpb.29.2378Google Scholar284Characterization of the metal-binding site in aminopeptidase BHirose, Junzo; Ohsaki, Takamichi; Nishimoto, Naoyo; Matuoka, Shouji; Hiromoto, Takashi; Yoshida, Takahide; Minoura, Takatosi; Iwamoto, Hiroyuki; Fukasawa, Kayoko M.Biological & Pharmaceutical Bulletin (2006), 29 (12), 2378-2382CODEN: BPBLEO; ISSN:0918-6158. (Pharmaceutical Society of Japan)A recombinant rat aminopeptidase-B (Ap-B) was expressed as a glutathione S-transferase (GST) fusion protein in Escherichia coli BL21 harboring a plasmid pGEX-Ap-B and was purified by glutathione-Sepharose 4B and Q-Sepharose columns. The metal-substituted derivs. of Ap-B, Co(II)- and Cu(II)-Ap-B contain almost 1 mol of cobalt(II) and copper(II) ions per enzyme mol., resp. The specific activity of Co(II)-Ap-B is very similar to that of recombinant Ap-B but that of Cu(II)-Ap-B is very low. The dissocn. consts. of the zinc ions of recombinant Ap-B and of the cobalt ions of Co(II)-Ap-B calcd. from the relationships between the free metal ions and the residual enzyme activities are 3.7(±1.0) × 10-13 and 4.7(±1.0) × 10-12 M, resp. The EPR parameters (g.perp., g‖ and A‖) of Cu(II)-Ap-B were 2.06, 2.27, and 156 × 10-4 cm-1. The A‖ value and the g‖ of Cu(II)-Ap-B are very similar to those of Cu(II)-thermolysin or Cu(II)-dipeptidyl peptidase III, in which the coordination geometry is a distorted tetrahedral.
- 285Kleemann, S. G.; Keung, W. M.; Riordan, J. F. Metal binding to angiotensin converting enzyme: implications for the metal binding site. J. Inorg. Biochem. 1986, 26, 93– 106, DOI: 10.1016/0162-0134(86)80002-2Google Scholar285Metal binding to angiotensin converting enzyme: implications for the metal binding siteKleemann, Stephan G.; Keung, Wing Ming; Riordan, James F.Journal of Inorganic Biochemistry (1986), 26 (2), 93-106CODEN: JIBIDJ; ISSN:0162-0134.Angiotensin-converting enzyme (ACE) interacts with the chelator 1,10-phenanthroline (OP) to form a OP-Zn-ACE ternary complex, which subsequently dissocs. to OP-Zn and apoenzyme. The assocn. and dissocn. rate consts. for the reaction OP + Zn-ACE ↹ OP-Zn-ACE have been detd. and compared with those of known OP-metal complexes. Such consts. were also used to calc. the rate const. for formation of the OP-Zn complex from OP-Zn-ACE. The rate of dissocn. of Zn from ACE has been measured in the presence of EDTA (which acts only as a metal scavenger) as a function of chelator concn., at different pH values, and with different buffers. The stability const. for the binding of Zn to apo-ACE, log Kc = 8.2, detd. by equil. dialysis using at. absorption spectroscopy to assess metal concn., is much smaller than that for Zn-carboxypeptidase A, Zn-thermolysin, or Zn-carbonic anhydrase. This weak binding is attributable to the Zn dissocn. rate const. of ACE, 7.5 × 10-3 sec-1 at pH 7.0, which is much greater than that of the other Zn metalloenzymes. These results lead to inferences regarding the metal binding site of ACE.
- 286Ippolito, J. A.; Baird, T. T., Jr.; McGee, S. A.; Christianson, D. W.; Fierke, C. A. Structure-assisted redesign of aprotein-zinc binding site with femtomolar affinity. Proc. Natl. Acad. Sci. U. S. A. 1995, 92, 5017– 5021, DOI: 10.1073/pnas.92.11.5017Google Scholar286Structure-assisted redesign of a protein-zinc-binding site with femtomolar affinityIppolito, Joseph A.; Baird, Teaster T., Jr.; McGee, Sharon A.; Christianson, David W.; Fierke, Carol A.Proceedings of the National Academy of Sciences of the United States of America (1995), 92 (11), 5017-21CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)The authors have inserted a fourth protein ligand into the zinc coordination polyhedron of carbonic anhydrase II (CAII) that increases metal affinity 200-fold (Kd = 20 fM). The three-dimensional structures of threonine-199 → aspartate (T199D) and threonine-199 → glutamate (T199E) CAIIs, detd. by x-ray crystallog. methods to resolns. of 2.35 Å and 2.2 Å, resp., reveal a tetrahedral metal-binding site consisting of H94, H96, H119, and the engineered carboxylate side chain, which displaces zinc-bound hydroxide. Although the stereochem. of neither engineered carboxylate-zinc interaction is comparable to that found in naturally occurring protein zinc-binding sites, protein-zinc affinity is enhanced in T199E CAII demonstrating that ligand-metal sepn. is a significant determinant of carboxylate-zinc affinity. In contrast, the three-dimensional structure of threonine-199 → histidine (T199H) CAII, detd. to 2.25-Å resoln., indicates that the engineered imidazole side chain rotates away from the metal and does not coordinate to zinc; this results in a weaker zinc-binding site. All three of these substitutions nearly obliterate CO2 hydrase activity, consistent with the role of zinc-bound hydroxide as catalytic nucleophile. The engineering of an addnl. protein ligand represents a general approach for increasing protein-metal affinity if the side chain can adopt a reasonable conformation and achieve inner-sphere zinc coordination. Moreover, this structure-assisted design approach may be effective in the development of high-sensitivity metal ion biosensors.
- 287McCall, K. A.; Fierke, C. A. Probing determinants of the metal ion selectivity in carbonic anhydrase using mutagenesis. Biochemistry 2004, 43, 3979– 3986, DOI: 10.1021/bi0498914Google Scholar287Probing Determinants of the Metal Ion Selectivity in Carbonic Anhydrase Using MutagenesisMcCall, Keith A.; Fierke, Carol A.Biochemistry (2004), 43 (13), 3979-3986CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)Few studies measuring thermodn. metal ion selectivity of metalloproteins have been performed, and the major determinants of metal ion selectivity in proteins are not yet well understood. Several features of metal ion binding sites and metal coordination have been hypothesized to alter the transition metal selectivity of chelators, including (1) the polarizability of the coordinating atom, (2) the relative sizes of the binding site and the metal ion, and (3) the metal ion binding site geometry. To test these hypotheses, we have measured the metal ion affinity and selectivity of a prototypical zinc enzyme, human carbonic anhydrase II (CAII), and a no. of active site variants where one of the coordinating ligands is substituted by another side chain capable of coordinating metal. CAII and almost all of the variants follow the inherent metal ion affinity trend suggested by the Irving-Williams series, demonstrating that this trend operates within proteins as well as within small mol. chelators and may be a dominant factor in metal ion selectivity in biol. Neither the polarizability of the liganding side chains nor the size of the metal ion binding site correlates strongly with metal ion specificity; instead, changes in metal ion specificity in the variants correlate with the preferred coordination no. and geometry of the metal ion. This correlation suggests that a primary feature driving deviations from the inherent ligand affinity trend is the positioning of active site groups such that a given metal ion can adopt a preferred coordination no./geometry.
- 288Coleman, J. E.; Vallee, B. L. Metallocarboxypeptidases: Stability constants and enzymatic characteristics. J. Biol. Chem. 1961, 236, 2244– 2249, DOI: 10.1016/S0021-9258(18)64065-7Google Scholar288Metallocarboxypeptidases: stability constants and enzymic characteristicsColeman, Joseph E.; Vallee, Bert L.Journal of Biological Chemistry (1961), 236 (), 2244-9CODEN: JBCHA3; ISSN:0021-9258.cf. CA 54, 13212c; 55, 4619d. Apocarboxypeptidase forms enzymically active complexes with a series of metal ions. The ranking order of stability consts. for the complexes of a series of metal ions with simple ligands is characteristic of its constituent donor atoms. Thus, when N and N or N and O function in this capacity, the ranking order for the stability consts. of metal complexes follows the sequence Hg(II) > Cu(II) ≥ Ni(II) > Co(II) ≥ Zn(II) ≥ Cd(II) > Fe(II) > Mn(II). The substitution of S for one of the donor atoms, to yield a S.sbd.N ligand markedly changes this sequence to Hg(II) » Cd(II) > Zn(II) > Ni(II) > Co(II) > Fe(II) > Mn. Cu is indeterminant because of the oxidn. of the mercapto group. The complexes of Cd and Zn are more distinctly stabilized over those of Co and Ni, the characteristic feature of the S-ligand series. The close correlation of both the order and magnitudes of the consts. imply that in carboxypeptidase a N.sbd.S site binds metal ions to yield enzymic activity. This interpretation is supported by titrations showing 2 metal-binding groups with pK values of 7.7 and 9.1, resp., compatible with published values for α-amino and SH groups. Mn, Co, Ni, and Zn carboxypeptidase hydrolyze both peptides and an ester substrate, hippuryl-DL,-β-phenyllactate. The ranking order of peptidase activities for these metal atoms varies as a function of the primary structure of the synthetic peptide substrate. Hg, Cd, and Pd carboxypeptidases exhibit marked esterase activity but do not hydrolyze the synthetic peptides tested. Thus, the metal atom known to function in substrate binding also plays a role in the detn. of enzymic specificity. The role of metal ions in the action of carboxypeptidase is apparent in yet another manner. Under standard conditions of assay, the relative order of the catalytic efficiencies of different metallocarboxypeptidases varies as a function of the primary structure of the synthetic peptide substrate. Thus the ranking order Co > Ni > Zn > Mn observed for carbobenzyloxyglycyl-L-phenylalanine is confirmed and is preserved over a wide range of substrate concns., ionic strengths, and other conditions of assay, but is inverted to Zn > Co > Ni > Mn for both carbobenzyloxyglycyl-L-tryptophan and benzoylglycyl-L-phenylalanine, and also the ester substrate, hippuryl-DL-β-phenylacetate. The spectral changes accompanying the formation of Co carboxypeptidase show the formation of a mercaptide linkage with apocarboxypeptidase. Co carboxypeptidase exhibits a distinctive red color with an absorption max. at 530 mμ and an extinction coeff. of 150. The shift in the absorption max. from 512 in the hydrated Co(II) to 530 mμ in Co carboxypeptidase, together with the increase in the extinction coeff. from 10 to 150, suggest binding to S. Similar binding of Ni, Mn, Hg, and Cd to a S atom is indicated by the relative order of the stability consts. for the resp. metallocarboxypeptidases which follow that expected for a S-contg. ligand. The S.sbd.N nature of the bidentate binding site is implied by the magnitude of the stability consts. and by the release of 2 H ions on combination of the apoenzyme with Zn ions. Equil. dialysis expts. in which the Zn atom of carboxypeptidase is exchanged for Hg or Cd demonstrate that the same site of the enzyme is involved in binding all of these, as shown previously for Co. The evidence suggests that the active center of native carboxypeptidase A includes 1 S, 1 N, and 1 Zn atom.
- 289Hirose, J.; Iwamoto, H.; Nagao, I.; Enmyo, K.; Sugao, H.; Kanemitu, N.; Ikeda, K.; Takeda, M.; Inoue, M.; Ikeda, T.; Matsuura, F.; Fukasawa, K. M.; Fukasawa, K. Characterization of the metal-substituted dipeptidyl peptidase III (rat liver). Biochemistry 2001, 40, 11860– 11865, DOI: 10.1021/bi0110903Google Scholar289Characterization of the Metal-Substituted Dipeptidyl Peptidase III (Rat Liver)Hirose, Junzo; Iwamoto, Hiroyuki; Nagao, Ikuko; Enmyo, Kanako; Sugao, Hidenori; Kanemitu, Nobuharu; Ikeda, Keiichi; Takeda, Mitsunori; Inoue, Masaki; Ikeda, Tomoyuki; Matsuura, Fumito; Fukasawa, Kayoko M.; Fukasawa, KatsuhikoBiochemistry (2001), 40 (39), 11860-11865CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)Dipeptidyl peptidase III (DPP III) (EC 3.4.14.4), which has a HELLGH-E (residues 450-455, 508) motif as the zinc binding site, is classified as a zinc metallopeptidase. The zinc dissocn. consts. of the wild type, Leu453-deleted, and E508D mutant of DPP III at pH 7.4 were 4.5 (±0.7) × 10-13, 5.8 (±0.7) × 10-12, and 3.2 (±0.9) × 10-10 M, resp. The recoveries of the enzyme activities by the addn. of various metal ions to apo-DPP III were also measured, and Co2+, Ni2+, and Cu2+ ions completely recovered the enzyme activities as did Zn2+. The dissocn. consts. of Co2+, Ni2+, and Cu2+ ions for apo-DPP III at pH 7.4 were 8.2 (±0.9) × 10-13, 2.7 (±0.3) × 10-12, and 1.1 (±0.1) × 10-14 M, resp. The shape of the absorption spectrum of Co2+-DPP III was very similar to that of Co2+-carboxypeptidase A or Co2+-thermolysin, in which the Co2+ is bound to two histidyl nitrogens, a water mol., and a glutamate residue. The absorption spectrum of Cu2+-DPP III is also very similar to that of Cu2+-thermolysin. The EPR spectrum and the EPR parameters of Cu2+-DPP III were very similar to those of Cu2+-thermolysin but slightly different from those of Cu2+-carboxypeptidase A. The five lines of the superfine structure in the perpendicular region of the EPR spectrum in Cu2+-DPP III suggest that nitrogen atoms should coordinate to the cupric ion in Cu2+-DPP III. All of these data suggest that the donor set and the coordination geometry of the metal ions in DPP III, which has the HExxxH motif as the metal binding site, are very similar to those of the metal ions in thermolysin, which has the HExxH motif.
- 290Sellin, S.; Mannervik, B. (1984) Metal dissociation constants for glyoxalase I reconstituted with Zn2+, Co2+, Mn2+, and Mg2+. J. Biol. Chem. 1984, 259, 11426– 11429, DOI: 10.1016/S0021-9258(18)90878-1Google Scholar290Metal dissociation constants for glyoxalase I reconstituted with zinc(2+), cobalt(2+), manganese(2+), and magnesium(2+)Sellin, Siv; Mannervik, BengtJournal of Biological Chemistry (1984), 259 (18), 11426-9CODEN: JBCHA3; ISSN:0021-9258.Metal dissocn. consts. for glyoxalase I from human erythrocytes were detd. by use of nitrilotriacetic acid as a metal buffer. The consts. for Zn2+, Co2+, Mn2+, and Mg2+ were 2.7 × 10-11, 3.0 × 10-10, 4.9 × 10-9, and 1.0 × 10-6M, resp., demonstrating that the natural cofactor, Zn2+, has the highest affinity for the apoprotein. The results were consistent with the proposal of N and O atoms as ligands to the metal in the active site of glyoxalase I. Both 1:1 and 1:2 complexes of the metal ions and nitrilotriacetic acid must be considered in the application of the metal buffer technique.
- 291Lasch, J. Kinetic properties of bovine lens leucine aminopeptidase. Ophthalmic Res. 1979, 11, 372– 376, DOI: 10.1159/000265036Google Scholar291Kinetic properties of bovine lens leucine aminopeptidaseLasch, JuergenOphthalmic Research (1979), 11 (5-6), 372-6CODEN: OPRSAQ; ISSN:0030-3747.Kinetic studies with bovine eye lens leucine aminopeptidase are reported. Particular attention is paid to the substrate specificity, the transferase activity, the correlation between binding and catalytic parameters, and the catalytic properties of the different metal ion hybrids of the enzyme.
- 292Simons, T. J. B. The affinity of human erythrocyte porphobilinogen synthase for Zn2+ and Pb2+. Eur. J. Biochem. 1995, 234, 178– 183, DOI: 10.1111/j.1432-1033.1995.178_c.xGoogle Scholar292The affinity of human erythrocyte porphobilinogen synthase for Zn2+ and Pb2+Simons, Timothy J. B.European Journal of Biochemistry (1995), 234 (1), 178-83CODEN: EJBCAI; ISSN:0014-2956. (Springer)Porphobilinogen synthase activity was measured in human erythrocyte lysates supplemented with metal-ion buffers to control free Zn2+ and Pb2+ concns. The enzyme was activated by Zn2+ with a Km of 1.6 pM and inhibited by Pb2+ with a Ki of 0.07 pM. Pb2+ and Zn2+ appear to compete for a single metal-binding site. The half-time for loss of Zn2+ from the active site, or replacement of Pb2+ by Zn2+, were in the 10-20 min range at 37°. Zn2+ did not affect the affinity for the substrate 5-aminolevulinate, but Pb2+ reduced it non-competitively. All the expts. were conducted with a blood sample of the common 1-1 phenotype.
- 293Day, E. S.; Wen, D.; Garber, E. A.; Hong, J.; Avedissian, L. S.; Rayhorn, P.; Shen, W.; Zeng, C.; Bailey, V. R.; Reilly, J. O.; Roden, J. A.; Moore, C. B.; Williams, K. P.; Galdes, A.; Whitty, A.; Baker, D. P. Zinc-dependent structural stability of human sonic hedgehog. Biochemistry 1999, 38, 14868– 14880, DOI: 10.1021/bi9910068Google Scholar293Zinc-Dependent Structural Stability of Human Sonic HedgehogDay, Eric S.; Wen, Dingyi; Garber, Ellen A.; Hong, Jin; Avedissian, Lena S.; Rayhorn, Paul; Shen, Weihong; Zeng, Chenhui; Bailey, Voilaine R.; Reilly, Jennifer O.; Roden, Julie A.; Moore, Claire B.; Williams, Kevin P.; Galdes, Alphonse; Whitty, Adrian; Baker, Darren P.Biochemistry (1999), 38 (45), 14868-14880CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)The role of the zinc site in the N-terminal fragment of human Sonic hedgehog (ShhN) was explored by comparing the biophys. and functional properties of wild-type ShhN with those of mutants in which the zinc-coordinating residues H140, D147, and H182, or E176 which interacts with the metal ion via a bridging water mol., were mutated to alanine. The wild-type and E176A mutant proteins retained 1 mol of zinc/mol of protein after extensive dialysis, whereas the H140A and D147A mutants retained only 0.03 and 0.05 mol of zinc/mol of protein, resp. Assay of the wild-type and mutant proteins in two activity assays indicated that the wild-type and E176A mutant proteins had similar activity, whereas the H140A and D147A mutants were significantly less active. These assays also indicated that the H140A and D147A mutants were susceptible to proteolysis. CD, fluorescence, and 1H NMR spectra of the H140A, D147A, and E176A mutants measured at 20 or 25° were very similar to those obsd. for wild-type ShhN. However, CD measurements at 37° showed evidence of some structural differences in the H140A and D147A mutants. Guanidine hydrochloride (GuHCl) denaturation studies revealed that the loss of zinc from the H140A and D147A mutants destabilized the folded proteins by ∼3.5 kcal/mol, comparable to the effect of removing zinc from wild-type ShhN by treatment with EDTA. Thermal melting curves of wild-type ShhN gave a single unfolding transition with a midpoint Tm of ∼59°, whereas both the H140A and D147A mutants displayed two distinct transitions with Tm values of 37-38 and 52-54°, similar to that obsd. for EDTA-treated wild-type ShhN. Addn. of zinc to the H140A and D147A mutants resulted in a partial restoration of stability against thermal and GuHCl denaturation. The ability of these mutants to bind zinc was confirmed using a fluorescence-based binding assay that indicated that they bound zinc with Kd values of ∼1.6 and ∼15 nM, resp., as compared to a value of ≤100 pM for wild-type ShhN. The properties of the E176A mutant were indistinguishable from those of wild-type ShhN in all biophys. and functional assays, indicating that this residue does not contribute significantly to stabilization of the zinc-binding site and that ShhN does not require hydrolase activity for in vitro biol. function.
- 294Masuoka, J.; Hegenauer, J.; Van Dyke, B. R.; Saltman, P. Intrinsic stoichiometric equilibrium constants for the binding of zinc(II) and copper(II) to the high affinity site of serum albumin. J. Biol. Chem. 1993, 268, 21533– 21537, DOI: 10.1016/S0021-9258(20)80574-2Google Scholar294Intrinsic stoichiometric equilibrium constants for the binding of zinc(II) and copper(II) to the high affinity site of serum albuminMasuoka, James; Hegenauer, Jack; Van Dyke, Bruce R.; Saltman, PaulJournal of Biological Chemistry (1993), 268 (29), 21533-7CODEN: JBCHA3; ISSN:0021-9258.Intrinsic stoichiometric equil. consts. were detd. for zinc(II) and copper(II) binding to bovine and human serum albumin. Data were obtained from equil. dialysis expts. Metals were presented to apoprotein as metal chelates in order to avoid metal hydrolysis and to minimize nonspecific metal-protein interactions. Scatchard anal. of the binding data indicated that the high affinity class for both zinc and copper was comprised of one site. Results of binding expts. done at several pH values suggested that while both histidyl and carboxyl groups appear to be involved in copper binding, histidyl residues alone were sufficient for zinc binding. These amino acid residues were used in combination to model several binding sites used in the formulation of equil. expressions from which stoichiometric consts. were calcd. The log10K for bovine serum albumin was calcd. to be 7.28 for Zn(II) and 11.12 for Cu(II). That for human serum albumin was detd. to be 7.53 and 11.18 for Zn(II) and Cu(II), resp. These consts. were used in equil. to simulate speciation of metal-albumin and metal-chelator and to illustrate relative binding affinities. This comparison of binding strengths was possible only through the calcn. of an intrinsic stoichiometric binding const.
- 295Krężel, A.; Maret, W. Thionein/metallothionein control Zn(II) availability and the activity of enzymes. JBIC, J. Biol. Inorg. Chem. 2008, 13, 401– 409, DOI: 10.1007/s00775-007-0330-yGoogle Scholar295Thionein/metallothionein control Zn(II) availability and the activity of enzymesKrezel, Artur; Maret, WolfgangJBIC, Journal of Biological Inorganic Chemistry (2008), 13 (3), 401-409CODEN: JJBCFA; ISSN:0949-8257. (Springer GmbH)Fundamental issues in zinc biol. are how proteins control the concns. of free Zn(II) ions and how tightly they interact with them. Since, basically, the Zn(II) stability consts. of only two cytosolic zinc enzymes, carbonic anhydrase and superoxide dismutase, have been reported, the affinity for Zn(II) of another zinc enzyme, sorbitol dehydrogenase (SDH), was detd. Its log K is 11.2±0.1, which is similar to the log K values of carbonic anhydrase and superoxide dismutase despite considerable differences in the coordination environments of Zn(II) in these enzymes. Protein tyrosine phosphatase 1B (PTP 1B), on the other hand, is not classified as a zinc enzyme but is strongly inhibited by Zn(II), with log K = 7.8±0.1. In order to test whether or not metallothionein (MT) can serve as a source for Zn(II) ions, it was used to control free Zn(II) ion concns. MT makes Zn(II) available for both PTP 1B and the apoform of SDH. However, whether or not Zn(II) ions are indeed available for interaction with these enzymes depends on the thionein (T) to MT ratio and the redox poise. At ratios [T/(MT + T) = 0.08-0.31] prevailing in tissues and cells, picomolar concns. of free Zn(II) are available from MT for reconstituting apoenzymes with Zn(II). Under conditions of decreased ratios, nanomolar concns. of free Zn(II) become available and affect enzymes that are not zinc metalloenzymes. The match between the Zn(II) buffering capacity of MT and the Zn(II) affinity of proteins suggests a function of MT in controlling cellular Zn(II) availability.
- 296Payne, J. C.; Rous, B. W.; Tenderholt, A. L.; Godwin, H. A. Spectroscopic determination of the binding affinity of zinc to the DNA-binding domains of nuclear hormone receptors. Biochemistry 2003, 42, 14214– 14224, DOI: 10.1021/bi035002lGoogle Scholar296Spectroscopic Determination of the Binding Affinity of Zinc to the DNA-Binding Domains of Nuclear Hormone ReceptorsPayne, John C.; Rous, Brian W.; Tenderholt, Adam L.; Godwin, Hilary ArnoldBiochemistry (2003), 42 (48), 14214-14224CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)Zinc binding to the two Cys4 sites present in the DNA-binding domain (DBD) of nuclear hormone receptor proteins is required for proper folding of the domain and for protein activity. By utilizing Co2+ as a spectroscopic probe, we have characterized the metal-binding properties of the two Cys4 structural zinc-binding sites found in the DBD of human estrogen receptor α (hERα-DBD) and rat glucocorticoid receptor (GR-DBD). The binding affinity of Co2+ to the two proteins was detd. relative to the binding affinity of Co2+ to the zinc finger consensus peptide, CP-1. Using the known dissocn. const. of Co2+ from CP-1, the dissocn. consts. of cobalt from hERα-DBD were calcd.: KCod1 = 2.2 (± 1.0) × 10-7 M and KCod2 = 6.1 (± 1.5) × 10-7 M. Similarly, the dissocn. consts. of Co2+ from GR-DBD were calcd.:KCod1 = 4.1 (± 0.6) × 10-7 M and KCod2 = 1.7 (± 0.3) × 10-7 M. Metal-binding studies conducted in which Zn2+ displaces Co2+ from the metal-binding sites of hERα-DBD and GR-DBD indicate that Zn2+ binds to each of the Cys4 metal-binding sites approx. 3 orders of magnitude more tightly than Co2+ does: the stoichiometric dissocn. consts. are KZnd1 = 1 (± 1) × 10-10 M and KZnd2 = 5 (± 1) × 10-10 M for hERα-DBD and KZnd1 = 2 (± 1) × 10-10 M and KZnd2 = 3 (± 1) × 10-10 M for GR-DBD. These affinities are comparable to those obsd. for most other naturally occurring structural zinc-binding sites. In contrast to the recent prediction by Low et. al. that zinc binding in these systems should be cooperative [Low, L. Y., Hernandez, H., Robinson, C. V., O'Brien, R., Grossmann, J. G., Ladbury, J. E., and Luisi, B. (2002) J. Mol. Biol. 319, 87-106], these data suggest that the zincs that bind to the two sites in the DBDs of hERα-DBD and GR-DBD do not interact.
- 297Dinkova-Kostova, A. T.; Holtzclaw, W. D.; Wakabayashi, N. Keap1, the sensor for electrophiles and oxidants that regulates the phase 2 response, is a zinc metalloprotein. Biochemistry 2005, 44, 6889– 6899, DOI: 10.1021/bi047434hGoogle Scholar297Keap1, the sensor for electrophiles and oxidants that regulates the phase 2 response, is a zinc metalloproteinDinkova-Kostova, Albena T.; Holtzclaw, W. David; Wakabayashi, NobunaoBiochemistry (2005), 44 (18), 6889-6899CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)Induction of the phase 2 response, a major cellular reaction to oxidative/electrophile stress depends on a protein triad: actin-tethered Keap1 that binds to Nrf2. Inducers react with Keap1 releasing Nrf2 for nuclear translocation and activation of the antioxidant response element (ARE), which regulates phase 2 genes. The primary sensors for inducers are certain uniquely reactive Cys SH groups of Keap1. Recombinant murine Keap1 contains 0.9 Zn atoms per monomer as detd. by inductively coupled plasma-optical emission spectrometry: its Zn content depends on the metal compn. of the overexpression medium. Simultaneous direct measurement of bound Zn using a pyridazoresorcinol chelator and protein SH groups using 4,4'-dipyridyl disulfide established that (1) Zn is bound to reactive Cys SH groups of Keap1 and is displaced stoichiometrically by inducers; (2) with these Cys residues mutated to Ala, the affinity for Zn was reduced by nearly 2 orders of magnitude; and (3) the assocn. const. of Keap1 for Zn2+ was 1.02 × 1011 M-1, consistent with a Zn-metalloprotein. Co substitution for Zn yielded an optical spectrum consistent with tetrahedral metal coordination. Coincident binding of inducers and release of Zn altered the conformation of Keap1, as shown by a profound decline of its Trp fluorescence and depression of fluorescence of a hydrophobicity probe. Therefore, regulation of the phase 2 response involves chem. modification of crit. Cys residues of Keap1, whose reactivity is modulated by Zn2+ binding. Thus, Keap1 is a Zn-thiol protein endowed with a delicate switch controlled by both metal-binding and thiol reactivity.
- 298Muller, H. N.; Skerra, A. Grafting of a high-affinity Zn(II)-binding site on the beta-barrel of retinol-binding protein results in enhanced folding stability and enables simplified purification. Biochemistry 1994, 33, 14126– 14135, DOI: 10.1021/bi00251a023Google Scholar298Grafting of a high-affinity Zn(II)-binding site on the beta-barrel of retinol-binding protein results in enhanced folding stability and enables simplified purificationMuller H N; Skerra ABiochemistry (1994), 33 (47), 14126-35 ISSN:0006-2960.In a rational protein design approach, the His3 Zn(II)-binding site from the active center of human carbonic anhydrase II was transplanted on the beta-barrel of mammalian serum retinol-binding protein (RBP) in a solvent-accessible location on the protein's outer surface. Several mutants of RBP were generated and produced in Escherichia coli, and their Zn(II)-binding properties were investigated in equilibrium dialysis experiments. One mutant, RBP/H3(A), with His residues introduced at the positions 46, 54, and 56 in the polypeptide sequence was shown to bind Zn(II) specifically with a stoichiometry of 1 and a corresponding dissociation constant equal to 36 +/- 10 nM. Binding of Zn(II) had no influence on the binding of retinoic acid, a natural ligand of RBP. In guanidinium chloride-induced unfolding experiments the mutant was found to be significantly stabilized in the presence of small concentrations of ZnSO4. This effect could be quantitatively explained using thermodynamic theory. Furthermore, it was demonstrated that the protein-bound Zn(II) is accessible to iminodiacetic acid as an additional chelating ligand without competition for the metal ion. Thus it was possible to use the grafted metal-binding site for the efficient purification of the engineered, bifunctional RBP via immobilized metal affinity chromatography from the bacterial protein extract.
- 299Guerrerio, A. L.; Berg, J. M. Metal ion affinities of the zinc finger domains of the metal responsive element-binding transcription factor-1 (MTF1). Biochemistry 2004, 43, 5437– 5444, DOI: 10.1021/bi0358418Google Scholar299Metal Ion Affinities of the Zinc Finger Domains of the Metal Responsive Element-Binding Transcription Factor-1 (MTF1)Guerrerio, Anthony L.; Berg, Jeremy M.Biochemistry (2004), 43 (18), 5437-5444CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)Metal response element (MRE) binding transcription factor-1 (MTF1) is a six Cys2His2 zinc finger-contg. transcription factor required for basal and zinc-induced transcription of metallothionein genes. The cobalt(II) and zinc(II) affinities of a protein fragment comprising the six zinc finger domains have been examd. to reveal apparent dissocn. consts. (for the six domains collectively) of 0.5±0.2 μM for cobalt(II) and 31±14 pM for zinc(II). Two approaches have been used to det. the metal ion affinities of the individual domains. First, the six domains have been examd. as single domain peptides revealing dissocn. consts. ranging from 0.3 to 1.7 μM for cobalt(II). The domains fall into two sets with peptides corresponding to domains 2, 3, and 4 showing relatively high affinity (Kd(Co(II)) 0.3-0.5 μM) and peptides corresponding to domains 1, 5, and 6 showing lower affinity (Kd(Co(II)) 1.6-1.7 μM). Second, we examd. the affinity of each domain in the context of the six zinc finger domain protein by individually mutating one metal-binding His residue to Cys to allow independent monitoring of the cobalt(II) occupancy of each site. The affinity of each domain was higher in this context than as a single domain peptide with affinities (cor. for the effect of the mutation) ranging from 0.02 to 0.5 μM. The increase in affinity for the individual domains ranged from factors of 1.1 to 20. The order of affinities (from higher to lowest) was obsd. to be 4>2 ≈ 5>6 ≈ 3≈1. These results reveal that none of the Cys2His2 zinc finger domains of MTF1 have dramatically low metal ion affinities, certainly none low enough to respond to changes in free zinc ion concns. in the micromolar range. Nonetheless, the metal ion affinities of some domains do differ by a factor of 25 with domains at both the amino- and carboxyl-termini showing lower intrinsic affinities for metal ions than the central domains.
- 300Miłoch, A.; Krężel, A. Metal binding properties of the zinc finger metallome – Insights into variations of stability. Metallomics 2014, 6, 2015– 2024, DOI: 10.1039/C4MT00149DGoogle Scholar300Metal binding properties of the zinc finger metallome - insights into variations in stabilityMiloch, Anna; Krezel, ArturMetallomics (2014), 6 (11), 2015-2024CODEN: METAJS; ISSN:1756-591X. (Royal Society of Chemistry)Zinc is one of the most widespread metal ions found in biol. systems. Of the expected 3000 zinc proteins in the human proteome, most contain zinc in structural sites. Among these structures, the most important are zinc fingers, which are well suited to facilitate interactions with DNA, RNA, proteins and lipid mols. Knowledge regarding their stability is a crit. issue in understanding the function of zinc fingers and their reactivity under fluxing cellular Zn(II) availability and different redox states. Zinc stability consts. that have been detd. using a variety of methods demonstrate wide diversity. Recent studies on the stability of consensus zinc fingers have demonstrated that the known metal-ion affinities for zinc fingers may have been underestimated by as much as three or more orders of magnitude. Here, using four natural ββα zinc fingers, we compare in detail several different methods that have been used for the detn. of zinc finger stability consts., such as common reverse-titrn., potentiometry, competition with metal chelators, and a new approach based on a three-step spectrophotometric titrn. We discuss why the stabilities of zinc fingers that are detd. spectrophotometrically are frequently underestimated due to the lack of effective equil. competition, which leads to large errors during the processing of the titrn. data. The literature stability consts. of many natural zinc fingers have been underestimated, and they are significantly lower when compared with the consensus peptides. Our data show that in the cell, some naturally occurring zinc fingers may potentially be unoccupied and are instead loaded transiently with Zn(II). Large variations in stability within the same class of zinc fingers have demonstrated that the thermodn. effects hidden in the sequence and structure are the key elements responsible for the differentiation of the stability of the zinc finger metallome.
- 301Sikorska, M.; Krężel, A.; Otlewski, J. Femtomolar Zn2+ affinity of LIM domain of PDLIM1 protein uncovers crucial contribution of protein-protein interactions to protein stability. J. Inorg. Biochem. 2012, 115, 28– 35, DOI: 10.1016/j.jinorgbio.2012.05.009Google Scholar301Femtomolar Zn2+ affinity of LIM domain of PDLIM1 protein uncovers crucial contribution of protein-protein interactions to protein stabilitySikorska, Malgorzata; Krezel, Artur; Otlewski, JacekJournal of Inorganic Biochemistry (2012), 115 (), 28-35CODEN: JIBIDJ; ISSN:0162-0134. (Elsevier)An individual LIM domain has approx. 55 amino acids with 8 highly conserved residues responsible for binding of two Zn2+ into two distinct zinc finger motifs. We examd. LIM domain stability of PDLIM1 protein (known also as Elfin protein), its C-terminally extended constructs as well as sep. zinc fingers, and several full domain mutants in terms of Zn2+ affinity and domain stability. Thermal denaturation, mass spectrometry, limited proteolysis, protein oxidn. and CD techniques were used to det. a set of thermodn. stability parameters. The results demonstrate unambiguously very high (femtomolar) affinity of both Zn2+ to the conserved LIM domain (Kdav = 2.5 × 10-14 M) and its addnl. elevation in the C-terminally extended domain construct (Kdav = 3.1 × 10-15 M). We demonstrate in the example of PDLIM1 using a set of LIM protein constructs and its zinc finger peptides that stability of the entire zinc-contg. domain is not only defined by the Zn2+ coordination environment but significantly depends on the set of protein-protein interactions with the C-terminus of the protein. We discuss structural similarities of LIM domains and suggest the prolongation of the conserved LIM sequence to its C-terminal helix that has a significant impact on domain stability. We also discuss the functionality of LIM domains in terms of different physiol. zinc and redox buffering capacity.
- 302Park, Y. B.; Hohl, M.; Padjasek, M.; Jeong, E.; Jin, K. S.; Krężel, A.; Petrini, J. H.; Cho, Y. Eukaryotic Rad50 functions as a rod-shaped dimer. Nat. Struct. Mol. Biol. 2017, 24, 248– 257, DOI: 10.1038/nsmb.3369Google Scholar302Eukaryotic Rad50 functions as a rod-shaped dimerPark, Young Bong; Hohl, Marcel; Padjasek, Michal; Jeong, Eunyoung; Jin, Kyeong Sik; Krezel, Artur; Petrini, John H. J.; Cho, YunjeNature Structural & Molecular Biology (2017), 24 (3), 248-257CODEN: NSMBCU; ISSN:1545-9993. (Nature Publishing Group)The Rad50 hook interface is crucial for assembly and various functions of the Mre11 complex. Previous analyses suggested that Rad50 mols. interact within (intracomplex) or between (intercomplex) dimeric complexes. In this study, we detd. the structure of the human Rad50 hook and coiled-coil domains. The data suggest that the predominant structure is the intracomplex, in which the two parallel coiled coils proximal to the hook form a rod shape, and that a novel interface within the coiled-coil domains of Rad50 stabilizes the interaction of Rad50 protomers in the dimeric assembly. In yeast, removal of the coiled-coil interface compromised Tel1 activation without affecting DNA repair, while simultaneous disruption of that interface and the hook phenocopied a null mutation. The results demonstrate that the hook and coiled-coil interfaces coordinately promote intracomplex assembly and define the intracomplex as the functional form of the Mre11 complex.
- 303Crow, J. P.; Sampson, J. B.; Zhuang, Y.; Thompson, J. A.; Beckman, J. S. Decreased zinc affinity of amyotrophic lateral sclerosis-associated superoxide dismutase mutants leads to enhanced catalysis of tyrosine nitration by peroxynitrite. J. Neurochem. 1997, 69, 1936– 1944, DOI: 10.1046/j.1471-4159.1997.69051936.xGoogle Scholar303Decreased zinc affinity of amyotrophic lateral sclerosis-associated superoxide dismutase mutants leads to enhanced catalysis of tyrosine nitration by peroxynitriteCrow, John P.; Sampson, Jacinda B.; Zhuang, Yingxin; Thompson, John A.; Beckman, Joseph S.Journal of Neurochemistry (1997), 69 (5), 1936-1944CODEN: JONRA9; ISSN:0022-3042. (Lippincott-Raven)Mutations to Cu/Zn superoxide dismutase (SOD) linked to familial amyotrophic lateral sclerosis (ALS) enhance an unknown toxic reaction that leads to the selective degeneration of motor neurons. However, the question of how >50 different missense mutations produce a common toxic phenotype remains perplexing. The authors found that the zinc affinity of four ALS-assocd. SOD mutants was decreased up to 30-fold compared to wild-type SOD but that both mutants and wild-type SOD retained copper with similar affinity. Neurofilament-L (NF-L), one of the most abundant proteins in motor neurons, bound multiple zinc atoms with sufficient affinity to potentially remove zinc from both wild-type and mutant SOD while having a lower affinity for copper. The loss of zinc from wild-type SOD approx. doubled its efficiency for catalyzing peroxynitrite-mediated tyrosine nitration, suggesting that one gained function by SOD in ALS may be an indirect consequence of zinc loss. Nitration of protein-bound tyrosines is a permanent modification that can adversely affect protein function. Thus, the toxicity of ALS-assocd. SOD mutants may be related to enhanced catalysis of protein nitration subsequent to zinc loss. By acting as a high-capacity zinc sink, NF-L could foster the formation of zinc-deficient SOD within motor neurons.
- 304Posewitz, M. C.; Wilcox, D. E. Properties of the Sp1 zinc finger 3 peptide: coordination chemistry, redox reactions, and metal binding competition with metallothionein. Chem. Res. Toxicol. 1995, 8, 1020– 1028, DOI: 10.1021/tx00050a005Google Scholar304Properties of the Sp1 Zinc Finger 3 Peptide: Coordination Chemistry, Redox Reactions, and Metal Binding Competition with MetallothioneinPosewitz, Matthew C.; Wilcox, Dean E.Chemical Research in Toxicology (1995), 8 (8), 1020-8CODEN: CRTOEC; ISSN:0893-228X. (American Chemical Society)Toxic and/or carcinogenic consequences may result from metal ion substitution for the Zn(II) in transcription factors contg. zinc fingers, and the small Cys-rich metal-binding protein metallothionein (MT) may play a role in this metal substitution. To begin to evaluate this hypothesis, with regard to the carcinogenic metal ion Ni(II), a peptide corresponding to the third finger of the transcription factor Sp1 (Sp1-3) has been synthesized and its metal binding and redox reactions have been studied. The peptide binds Zn(II), Co(II), and Ni(II), with spectroscopic data indicating a tetrahedral coordination for the latter two; metal ion affinities have been quantified (Kd = 6 × 10-10, 3 × 10-7, and 4 × 10-6, resp.) and less than those of an optimized zinc finger peptide (B. A. Krizek et al. (1993)) but greater than those of the second finger of transcription factor IIIA (J. M. Berg and D. L. Merkle (1989)). Reactions of the peptide and its metal-bound forms with dioxygen or hydrogen peroxide did not produce oxygen radical species; however, oxidn. of the two Sp1-3 cysteines was modulated by metal ions (Zn < Co = apo < Ni), suggesting a protective role for Zn(II) but an enhancing role for Ni(II). Metal binding competition between Sp1-3 and the α domain of human liver MT-2 (α-hMT2) indicates a similar affinity for Zn(II). However, α-hMT2 has a higher affinity for Ni(II), suggesting that MT may play a protective role by ensuring Zn(II), rather than Ni(II), coordination to zinc finger sequences of transcription factors.
- 305diTargiani, R. C.; Lee, S. J.; Wassink, S.; Michel, S. L. Functional characterization of iron-substituted tristetraprolin-2D (TTP-2D, NUP475–2D): RNA binding affinity and selectivity. Biochemistry 2006, 45, 13641– 13649, DOI: 10.1021/bi060747nGoogle Scholar305Functional characterization of iron-substituted tristetraprolin-2D (TTP-2D, NUP475-2D): RNA binding affinity and selectivitydiTargiani Robert C; Lee Seung Jae; Wassink Sarah; Michel Sarah L JBiochemistry (2006), 45 (45), 13641-9 ISSN:0006-2960.The protein tristetraprolin (TTP, also known as NUP475 and TIS11) is a nonclassical zinc finger protein that is involved in regulating the inflammatory response. Specifically, TTP binds to AU-rich sequence elements located at the 3'-untranslated region of cytokine mRNAs forming a complex that is degraded by the exosome. The nucleic acid binding region of TTP is comprised of two CysX(8)CysX(5)CysX(3)His domains that are activated in the presence of zinc. A two-domain construct of TTP (TTP-2D) has been cloned and overexpressed in E. coli. TTP-2D picks up visible red coloration from the expression media, unless it is expressed under iron-restricted conditions. The iron-binding properties of TTP-2D and the effect of iron substitution on RNA recognition have been investigated. Both Fe(II) and Fe(III) bind to TTP-2D and a full titration of Fe(III) with TTP-2D revealed that this metal ion binds with micromolar affinity. Upon reconstitution of TTP-2D with either Fe(II) or Fe(III), the protein recognizes a canonical RNA-binding sequence, UUUAUUUAUUU, with nanomolar affinity. Substitution of a single adenine or both adenines results in a decreased affinity of TTP-2D for the RNA molecule, demonstrating that both Fe(II)-TTP-2D and Fe(III)-TTP-2D selectively recognize a physiologically relevant RNA sequence. The relative affinities of Fe(II)-TTP-2D and Fe(III)-TTP-2D for the series of RNA sequences mirror those observed for Zn(II)-TTP-2D and suggest that iron is a viable substitute for zinc in this protein.
- 306Bal, W.; Schwerdtle, T.; Hartwig, A. Mechanism of nickel assault on the zinc finger of DNA repair protein XPA. Chem. Res. Toxicol. 2003, 16, 242– 248, DOI: 10.1021/tx025639qGoogle Scholar306Mechanism of Nickel Assault on the Zinc Finger of DNA Repair Protein XPABal, Wojciech; Schwerdtle, Tanja; Hartwig, AndreaChemical Research in Toxicology (2003), 16 (2), 242-248CODEN: CRTOEC; ISSN:0893-228X. (American Chemical Society)Xeroderma pigmentosum group A complementing protein (XPA) is a member of the protein complex of the nucleotide excision repair (NER) pathway of DNA repair, participating in the assembly of the incision complex. The 4S zinc finger domain of XPA is involved the interactions with other NER proteins. As demonstrated previously, the activity of XPA is compromised by several metal ions implicated in DNA repair inhibition, including Ni(II), Cd(II), and Co(II) (M. Asmuss et al., 2000). To study the possible mol. mechanisms of XPA inhibition, we investigated Zn(II) and Ni(II) interactions with the synthetic 37 peptide (XPAzf), representing the XPA zinc finger sequence AcDYVICEECGKEFMDSYLMNHFDLPTCDNCRDADDKHKam. The binding consts. were detd. using fluorescence and UV-vis spectroscopies, structural insights were provided by CD, and oxidative damage to XPAzf was studied with HPLC. The binding consts. for Zn(II) and Ni(II) are (8.5±1.5) × 108 (log value 8.93(7)) and (1.05±0.07) × 106 M-1 (6.02(3)), resp., in 10 mM phosphate buffer, pH 7.4, and (6±4) × 109 (9.8(2)) and (2.9±0.5) × 106 M-1 (6.46(8)) in 50 mM phosphate buffer, pH 7.4, yielding binding const. ratios Zn(II)/Ni(II) of 800±100 and 2300±500, resp. The Ni(II) ion forms a square planar complex with the sulfurs of XPAzf, opposed to the tetrahedral structure of the native Zn(II) complex. Consequently, the overall zinc finger structure is lost in the Ni(II)-substituted peptide. Zn(II)-satd. XPAzf is remarkably resistant to air oxidn. and is only slowly oxidized by 0.01 mM, 0.1 mM, and 1 mM H2O2 in a concn.-dependent fashion. However, the presence of just 10-fold molar excess of Ni(II) is sufficient to accelerate this process for all three H2O2 concns. tested. Overall, our results indicate that XPAzf can undergo Ni(II) assault in specific conditions.
- 307Kocyła, A.; Krężel, A. Zinc clasp-based reversible toolset for selective metal-mediated protein heterodimerization. Chem. Commun. (Cambridge, U. K.) 2018, 54, 13539– 13542, DOI: 10.1039/C8CC06301JGoogle Scholar307Zinc clasp-based reversible toolset for selective metal-mediated protein heterodimerizationKocyla, Anna; Krezel, ArturChemical Communications (Cambridge, United Kingdom) (2018), 54 (96), 13539-13542CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)Considering the complex biol. quandaries of the tightly woven networks of biol. macromols., we present an optimized zinc clasp-based toolset from the CD4 co-receptor and Lck protein tyrosine kinase complex for selective, tight and fully reversible protein heterodimerization (log K12 = 18.6). We demonstrated its utility on CD4-tagged proteins with capture from bacterial lysate and constructed mol. baits using a new small-mol. tether.
- 308Kocyła, A.; Adamczyk, J.; Krężel, A. Interdependence of free zinc changes and protein complex assembly - insights into zinc signal regulation. Metallomics 2018, 10, 120– 131, DOI: 10.1039/C7MT00301CGoogle Scholar308Interdependence of free zinc changes and protein complex assembly - insights into zinc signal regulationKocyla, Anna; Adamczyk, Justyna; Krezel, ArturMetallomics (2018), 10 (1), 120-131CODEN: METAJS; ISSN:1756-591X. (Royal Society of Chemistry)Cellular zinc (Zn(ii)) is bound with proteins that are part of the proteomes of all domains of life. It is mostly utilized as a catalytic or structural protein cofactor, which results in a vast no. of binding architectures. The Zn(ii) ion is also important for the formation of transient protein complexes with a Zn(ii)-dependent quaternary structure that is formed upon cellular zinc signals. The mechanisms by which proteins assoc. with and dissoc. from Zn(ii) and the connection with cellular Zn(ii) changes remain incompletely understood. In this study, we aimed to examine how zinc protein domains with various Zn(ii)-binding architectures are formed under free Zn(ii) concn. changes and how formation of the Zn(ii)-dependent assemblies is related to the protein concn. and reactivity. To accomplish these goals we chose four zinc domains with different Zn(ii)-to-protein binding stoichiometries: classical zinc finger (ZnP), LIM domain (Zn2P), zinc hook (ZnP2) and zinc clasp (ZnP1P2) folds. Our research demonstrated a lack of changes in the satn. level of intraprotein zinc binding sites, despite various peptide concns., while homo- and heterodimers indicated a concn.-dependent tendency. In other words, at a certain free Zn(ii) concn., the fraction of a formed dimeric complex increases or decreases with subunit concn. changes. Secondly, even small or local changes in free Zn(ii) may significantly affect protein satn. depending on its architecture, function and subcellular concn. In our paper, we indicate the importance of interdependence of free Zn(ii) availability and protein subunit concns. for cellular zinc signal regulation.
- 309Hogstrand, C.; Verbost, P. M.; Wendelaar Bonga, S. E. Inhibition of human erythrocyte Ca2+-ATPase by Zn2+. Toxicology 1999, 133, 139– 145, DOI: 10.1016/S0300-483X(99)00020-7Google Scholar309Inhibition of human erythrocyte Ca2+-ATPase by Zn2+Hogstrand, Christer; Verbost, Pieter M.; Wendelaar Bonga, Sjoerd E.Toxicology (1999), 133 (2,3), 139-145CODEN: TXCYAC; ISSN:0300-483X. (Elsevier Science Ireland Ltd.)Recent investigations suggest that Ca2+-ATPase from fish gills is very sensitive to Zn2+ (1996). The effect of free Zn2+ ion on the human erythrocyte plasma membrane Ca2+-ATPase was investigated to explore the possible extension of this finding to humans. Membrane vesicles were prepd. and the Ca2+-ATPase activity was measured as Ca2+-stimulated ATP hydrolysis and as ATP-dependent Ca2+ transport. The Zn2+ ion inhibited the erythrocyte Ca2+-ATPase by reducing Vmax and increasing the K0.5. While in the Ca2+ transport assay only the Vmax was affected at lower Zn2+ concns. (50-100 pM), redn. of Vmax was always accompanied by an affinity decrease in the ATP hydrolysis assay. The Ca2+-ATPase was found to be inhibited by Zn2+ at extremely low concns. The IC10 and IC50 for Zn2+, at a Ca2+ concn. of 1.0 μM, were estd. at 4 and 80 pM, resp. Although the Ca2+-ATPase might be more sensitive in vitro than in vivo conditions, the results suggest that physiol. concns. of Zn2+ may reduce the activity of the erythrocyte Ca2+-ATPase. Furthermore, disturbance of Ca homeostasis may be a mechanism causing Zn toxicity during exposure.
- 310Eron, S. J.; MacPherson, D. J.; Dagbay, K. B.; Hardy, J. A. Multiple mechanisms of zinc-mediated inhibition for the apoptotic caspases-3, −6, −7, and −8. ACS Chem. Biol. 2018, 13, 1279– 1290, DOI: 10.1021/acschembio.8b00064Google Scholar310Multiple Mechanisms of Zinc-Mediated Inhibition for the Apoptotic Caspases-3, -6, -7, and -8Eron, Scott J.; MacPherson, Derek J.; Dagbay, Kevin B.; Hardy, Jeanne A.ACS Chemical Biology (2018), 13 (5), 1279-1290CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)Zinc is emerging as a widely used and important biol. regulatory signal. Cellular zinc levels are tightly regulated by a complex array of zinc importer and exporters to control processes such as apoptotic cell death. While caspase inhibition by zinc has been reported previously, the reported inhibition consts. were too weak to suggest a crit. biol. role for zinc-mediated inhibition. In this work we have adopted a method of assessing available zinc. This allowed assessment of the accurate inhibition consts. for apoptotic caspases, caspase-3, -6, -7 and -8. Each of these caspases are inhibited by zinc at intracellular levels, however, with widely differing inhibition consts. and different zinc binding stoichiometries. Caspase -3, -6 and -8 appear to be constitutively inhibited by typical zinc levels and this inhibition must be lifted to allow activation. The inhibition const. for caspase-7 (76 nM) is much weaker than for the other apoptotic caspases (2.6-6.9 nM) suggesting that caspase-7 is not inactivated by normal zinc concns. but can be inhibited under conditions of zinc stress. Caspase-3, -7, and -8 were found to bind three, one, and two zincs resp. In each of these caspases, zinc was present in the active site, in contrast to caspase-6, which binds one zinc allosterically. The most notable new mechanism to emerge from this work is for zinc-mediated inhibition of caspase-8. Zinc binds caspase-8 directly at the active site and at a second site. Zinc binding inhibits formation of the caspase-8 dimer, the activated form of the enzyme. Together these findings suggest that zinc plays a crit. role in regulation of apoptosis by direct inactivation of caspases, in a manner that is unique for each caspase.
- 311Hao, Q.; Hong, S.-H.; Maret, W. Lipid-raft dependent endocytosis of metallothionein in HepG2 cells. J. Cell. Physiol. 2007, 210, 428– 435, DOI: 10.1002/jcp.20874Google Scholar311Lipid raft-dependent endocytosis of metallothionein in HepG2 cellsHao, Qiang; Hong, Sung-Hye; Maret, WolfgangJournal of Cellular Physiology (2007), 210 (2), 428-435CODEN: JCLLAX; ISSN:0021-9541. (Wiley-Liss, Inc.)Human hepatocellular carcinoma (HepG2) cells take up metallothionein (MT) by endocytosis. MT co-localizes with albumin but not with transferrin, indicating uptake via a non-classical pathway rather than via clathrin-mediated endocytosis. A lipid raft-dependent uptake is indicated by pravastatin inhibition of cholesterol synthesis and methyl-β-cyclodextrin inhibition of cholesterol translocation to the plasma membrane, reducing MT uptake by 29% and 69%, resp. Subcellular fractionation after MT uptake reveals significant amts. of MT in vesicular fractions including lysosomes but virtually no MT in the cytosol. Metals bound to MT are released into the cytosol, however. The findings define a pathway for cellular metal acquisition. Together with results from other studies demonstrating secretion of MT from different cells and the presence of MT in extracellular fluids, the results suggest a function of MT in intercellular communication.
- 312Knipp, M.; Charnock, J. M.; Garner, C. D.; Vašák, M. Structural and functional characterization of the Zn(II) site on dimethylarginase-1 (DDAH-1) from bovine brain. J. Biol. Chem. 2001, 276, 40449– 40456, DOI: 10.1074/jbc.M104056200Google Scholar312Structural and functional characterization of the Zn(II) site in dimethylargininase-1 (DDAH-1) from bovine brain: Zn(II) release activates DDAH-1Knipp, Markus; Charnock, John M.; Garner, C. David; Vasak, MilanJournal of Biological Chemistry (2001), 276 (44), 40449-40456CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)L-Nω,Nω-Dimethylarginine dimethylaminohydrolase-1 (DDAH-1) is a Zn(II)-contg. enzyme that, through hydrolysis of side-chain methylated L-arginines, regulates the activity of nitric-oxide synthase. Herein we report the structural and functional properties of the Zn(II)-binding site in DDAH-1 from bovine brain. Activity measurements of the native and metal-free enzyme have revealed that the endogenously bound Zn(II) inhibits the enzyme. Native DDAH-1 could be fully or partially activated using various concns. of phosphate, imidazole, histidine, and histamine, a process that is paralleled by the release of Zn(II). The slow activation of the enzyme by the bulky complexing agents EDTA and 1,10-phenanthroline suggests that the Zn(II)-binding site is partially buried in the protein structure. The apparent Zn(II)-dissocn. const. of 4.2 nM, detd. by 19F NMR using the chelator 5F-BAPTA (1,2-bis(2-amino-5-fluorophenoxy)ethane-N,N,N',N'-tetraacetic acid), lies in the range of intracellular free Zn(II) concns. These results suggest a regulatory role for the Zn(II)-binding site. The coordination environment of the Zn(II) in DDAH-1 has been examd. by Zn K-edge x-ray absorption spectroscopy. The extended x-ray absorption fine structure obsd. is consistent with Zn(II) being coordinated by 2 S and 2 N (or O) atoms. The biol. implications of these findings are discussed.
- 313Maret, W. Inhibitory zinc sites in enzymes. BioMetals 2013, 26, 197– 204, DOI: 10.1007/s10534-013-9613-7Google Scholar313Inhibitory zinc sites in enzymesMaret, WolfgangBioMetals (2013), 26 (2), 197-204CODEN: BOMEEH; ISSN:0966-0844. (Springer)A review. Several pathways increase the concns. of cellular free zinc(II) ions. Such fluctuations suggest that zinc(II) ions are signalling ions used for the regulation of proteins. One function is the inhibition of enzymes. It is quite common that enzymes bind zinc(II) ions with micro- or nanomolar affinities in their active sites that contain catalytic dyads or triads with a combination of glutamate (aspartate), histidine and cysteine residues, which are all typical zinc-binding ligands. However, for such binding to be physiol. significant, the binding consts. must be compatible with the cellular availability of zinc(II) ions. The affinity of inhibitory zinc(II) ions for receptor protein tyrosine phosphatase β is particularly high (K i = 21 pM, pH 7.4), indicating that some enzymes bind zinc almost as strongly as zinc metalloenzymes. The competitive pattern of zinc inhibition for this phosphatase implicates its active site cysteine and nearby residues in the coordination of zinc. Quant. biophys. data on both affinities of proteins for zinc and cellular zinc(II) ion concns. provide the basis for examg. the physiol. significance of inhibitory zinc-binding sites in proteins and the role of zinc(II) ions in cellular signalling. Regulatory functions of zinc(II) ions add a significant level of complexity to biol. control of metab. and signal transduction and embody a new paradigm for the role of transition metal ions in cell biol.
- 314Romero-Hernandez, A.; Simorowski, N.; Karakas, E.; Furukawa, H. Molecular Basis for subtype specificity and high-affinity zinc inhibition in the GluN1-GluN2A NMDA receptor amino-terminal domain. Neuron 2016, 92, 1324– 1336, DOI: 10.1016/j.neuron.2016.11.006Google Scholar314Molecular Basis for Subtype Specificity and High-Affinity Zinc Inhibition in the GluN1-GluN2A NMDA Receptor Amino-Terminal DomainRomero-Hernandez, Annabel; Simorowski, Noriko; Karakas, Erkan; Furukawa, HiroNeuron (2016), 92 (6), 1324-1336CODEN: NERNET; ISSN:0896-6273. (Cell Press)Zinc is vastly present in the mammalian brain and controls functions of various cell surface receptors to regulate neurotransmission. A distinctive characteristic of N-methyl-D-aspartate (NMDA) receptors contg. a GluN2A subunit is that their ion channel activity is allosterically inhibited by a nano-molar concn. of zinc that binds to an extracellular domain called an amino-terminal domain (ATD). Despite physiol. importance, the mol. mechanism underlying the high-affinity zinc inhibition has been incomplete because of the lack of a GluN2A ATD structure. Here we show the first crystal structures of the heterodimeric GluN1-GluN2A ATD, which provide the complete map of the high-affinity zinc-binding site and reveal distinctive features from the ATD of the GluN1-GluN2B subtype. Perturbation of hydrogen bond networks at the hinge of the GluN2A bi-lobe structure affects both zinc inhibition and open probability, supporting the general model in which the bi-lobe motion in ATD regulates the channel activity in NMDA receptors.
- 315Milstein, C. Inhibition of phosphoglucomutase by trace metals. Biochem. J. 1961, 79, 591– 596, DOI: 10.1042/bj0790591Google Scholar315Inhibition of phosphoglucomutase by trace metalsMilstein, C.Biochemical Journal (1961), 79 (), 591-6CODEN: BIJOAK; ISSN:0264-6021.A quant. study of the inhibition of phosphoglucomutase provides addnl. evidence that the necessity for a chelating agent is the result of the presence of minute traces of inhibitory metals. Zn is an inhibitor of phosphoglucomutase, competing with Mg ions, with dissocn. const. KzD 3.9 × 10-14M. Cu also competes with Mg ions, but the inhibition is not purely competitive. The inhibition by Cu++ ions can be explained by competition with Mg ions (KCu 2.3 × 10-17M), together with noncompetitive combination at a 2nd site (K'Cu 2.5 × 10-16M). Three to 4 SH groups are present in phosphoglucomutase. Although some inhibition is observed with high concns. of several SH reagents, phosphoglucomutase does not appear to be an enzyme requiring these groups for activity. The effects are discussed in the light of the amino acid sequence in the active center previously detd.
- 316Wilson, M.; Hogstrand, C.; Maret, W. Picomolar concentrations of free zinc(II) ions regulate receptor protein-tyrosine phosphatase β activity. J. Biol. Chem. 2012, 287, 9322– 9326, DOI: 10.1074/jbc.C111.320796Google Scholar316Picomolar concentrations of free zinc(II) ions regulate receptor protein-tyrosine phosphatase β activityWilson, Matthew; Hogstrand, Christer; Maret, WolfgangJournal of Biological Chemistry (2012), 287 (12), 9322-9326CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)As key enzymes in the regulation of biol. phosphorylations, protein tyrosine phosphatases are central to the control of cellular signaling and metab. Zn(II) ions are known to inhibit these enzymes, but the physiol. significance of this inhibition has remained elusive. Here, employing metal buffering for strict metal control and performing a kinetic anal., the authors demonstrate that Zn(II) ions are reversible inhibitors of the cytoplasmic catalytic domain of receptor protein tyrosine phosphatase β (also known as vascular endothelial protein tyrosine phosphatase). The Ki(Zn) value was 21 pM, 6 orders of magnitude lower than Zn(II) inhibition reported previously for this enzyme. It exceeded the affinity of the most potent synthetic small mol. inhibitors targeting these enzymes. The inhibition was in the range of cellular Zn(II) ion concns., suggesting that Zn(II) regulates this enzyme, which is involved in vascular physiol. and angiogenesis. Thus, for some enzymes that are not recognized as Zn-metalloenzymes, Zn(II) binding inhibits rather than activates as in classical Zn-enzymes. Activation then requires removal of the inhibitory Zn(II).
- 317Hardyman, J. E. J.; Tyson, J.; Jackson, K. A.; Aldridge, C.; Cockell, S. J.; Wakeling, L. A.; Valentine, R. A.; Ford, D. Zinc sensing by metal-responsive transcription factor 1 (MTF1) controls metallothionein and ZnT1 expression to buffer the sensitivity of the transcriptome response to zinc. Metallomics 2016, 8, 337– 343, DOI: 10.1039/C5MT00305AGoogle Scholar317Zinc sensing by metal-responsive transcription factor 1 (MTF1) controls metallothionein and ZnT1 expression to buffer the sensitivity of the transcriptome response to zincHardyman, J. E. J.; Tyson, J.; Jackson, K. A.; Aldridge, C.; Cockell, S. J.; Wakeling, L. A.; Valentine, R. A.; Ford, D.Metallomics (2016), 8 (3), 337-343CODEN: METAJS; ISSN:1756-591X. (Royal Society of Chemistry)Only a small no. of genes are known direct targets of the zinc-responsive transcription factor MTF1; therefore, the aim of this study was to gain a more complete understanding of the MTF-1 regulated zinc-responsive component of the transcriptome. A targeted siRNA was used to deplete MTF1 expression in the human intestinal cell line Caco-2. We predicted that the response to zinc of direct MTF1 target genes would be abrogated by MTF1 knockdown. Surprisingly, a greater no. of genes were regulated by zinc following MFT1 knockdown, and most genes that responded to zinc under both control and MTF1-depleted conditions had an augmented response in the latter condition. Exceptions were the zinc effluxer ZnT1 and a suite of metallothionein genes, suggesting that responses of other genes to zinc are usually buffered by increases in these proteins. We propose that MTF1 heads a hierarchy of zinc sensors, and through controlling the expression of a raft of metallothioneins and other key proteins involved in controlling intracellular zinc levels (e.g. ZnT1) alters zinc buffering capacity and total cellular zinc content. We tested and validated this model by overexpressing metallothionein and observing the predicted curtailment in response of the zinc-repressed SLC30A5 (ZnT5) promoter. The model provides the framework for an integrated understanding of cellular zinc homeostasis. Because MTs can bind metals other than zinc, this framework links with overall cellular metal homeostasis.
- 318Krężel, A.; Hao, Q.; Maret, W. The zinc/thiolate redox biochemistry of metallothionein and the control of zinc ion fluctuations in cell signalling. Arch. Biochem. Biophys. 2007, 463, 188– 200, DOI: 10.1016/j.abb.2007.02.017Google Scholar318The zinc/thiolate redox biochemistry of metallothionein and the control of zinc ion fluctuations in cell signalingKrezel, Artur; Hao, Qiang; Maret, WolfgangArchives of Biochemistry and Biophysics (2007), 463 (2), 188-200CODEN: ABBIA4; ISSN:0003-9861. (Elsevier)A review. Free Zn2+ ions are potent effectors of proteins. Their tightly controlled fluctuations ("Zn2+ signals") in the picomolar range of concns. modulate cellular signaling pathways. S (cysteine) donors generate redox-active coordination environments in proteins for the redox-inert Zn2+ ion and make it possible for redox signals to induce Zn2+ signals. The amplitudes of Zn2+ signals are detd. by the cellular Zn2+ buffering capacity, which itself is redox-sensitive. In part by interfering with Zn2+ and redox buffering, reactive species, drugs, toxins, and metal ions can elicit Zn2+ signals that initiate physiol. and pathobiochem. changes or lead to cellular injury when free Zn2+ ions are sustained at higher concns. These interactions establish redox-inert Zn2+ as an important factor in redox signaling. At the center of Zn2+/redox signaling are the Zn/thiolate clusters of metallothionein. They can transduce Zn2+ and redox signals and thereby attenuate or amplify these signals.
- 319Han, Y.; Sanford, L.; Simpson, D. M.; Dowell, R. D.; Palmer, A. E. Remodeling of Zn2+ homeostasis upon differentiation of mammary epithelial cells. Metallomics 2020, 12, 346– 362, DOI: 10.1039/C9MT00301KGoogle Scholar319Remodeling of Zn2+ homeostasis upon differentiation of mammary epithelial cellsHan, Yu; Sanford, Lynn; Simpson, David M.; Dowell, Robin D.; Palmer, Amy E.Metallomics (2020), 12 (3), 346-362CODEN: METAJS; ISSN:1756-591X. (Royal Society of Chemistry)Using mouse mammary epithelial cells as a model system, we investigated the remodeling of zinc homeostasis during differentiation induced by treatment with the lactogenic hormones cortisol and prolactin. RNA-Seq at different stages of differentiation revealed changes in global gene expression, including genes encoding zinc-dependent proteins and regulators of zinc homeostasis. Increases in mRNA levels of three zinc homeostasis genes, Slc39a14 (ZIP14) and metallothioneins (MTs) I and II were induced by cortisol but not by prolactin. The cortisol-induced increase was partially mediated by the nuclear glucocorticoid receptor signaling pathway. An increase in the cytosolic labile Zn2+ pool was also detected in lactating mammary cells, consistent with upregulation of MTs. We found that the zinc transporter ZIP14 was important for the expression of a major milk protein, whey acid protein (WAP), as knockdown of ZIP14 dramatically decreased WAP mRNA levels. In summary, our study demonstrated remodeling of zinc homeostasis upon differentiation of mammary epithelial cells resulting in changes in cytosolic Zn2+ and differential expression of zinc homeostasis genes, and these changes are important for establishing the lactation phenotype.
- 320Sanford, L.; Carpenter, M. C.; Palmer, A. E. Intracellular Zn2+ transients modulate global gene expression in dissociated rat hippocampal neurons. Sci. Rep. 2019, 9, 9411, DOI: 10.1038/s41598-019-45844-2Google Scholar320Intracellular Zn(2+) transients modulate global gene expression in dissociated rat hippocampal neuronsSanford Lynn; Carpenter Margaret C; Palmer Amy EScientific reports (2019), 9 (1), 9411 ISSN:.Zinc (Zn(2+)) is an integral component of many proteins and has been shown to act in a regulatory capacity in different mammalian systems, including as a neurotransmitter in neurons throughout the brain. While Zn(2+) plays an important role in modulating neuronal potentiation and synaptic plasticity, little is known about the signaling mechanisms of this regulation. In dissociated rat hippocampal neuron cultures, we used fluorescent Zn(2+) sensors to rigorously define resting Zn(2+) levels and stimulation-dependent intracellular Zn(2+) dynamics, and we performed RNA-Seq to characterize Zn(2+)-dependent transcriptional effects upon stimulation. We found that relatively small changes in cytosolic Zn(2+) during stimulation altered expression levels of 931 genes, and these Zn(2+) dynamics induced transcription of many genes implicated in neurite expansion and synaptic growth. Additionally, while we were unable to verify the presence of synaptic Zn(2+) in these cultures, we did detect the synaptic vesicle Zn(2+) transporter ZnT3 and found it to be substantially upregulated by cytosolic Zn(2+) increases. These results provide the first global sequencing-based examination of Zn(2+)-dependent changes in transcription and identify genes that may mediate Zn(2+)-dependent processes and functions.
- 321Zhang, C.; Maslar, D.; Minckley, T. F.; LeJeune, K. D.; Qin, Y. Spontaneous, synchronous zinc spikes oscillate with neural excitabiliity and calcium spikes in primary hippocampal neuron culture. J. Neurochem. 2021, 157, 1838– 1849, DOI: 10.1111/jnc.15334Google Scholar321Spontaneous, synchronous zinc spikes oscillate with neural excitability and calcium spikes in primary hippocampal neuron cultureZhang, Chen; Maslar, Drew; Minckley, Taylor F.; LeJeune, Kate D.; Qin, YanJournal of Neurochemistry (2021), 157 (6), 1838-1849CODEN: JONRA9; ISSN:0022-3042. (Wiley-Blackwell)Zinc has been suggested to act as an intracellular signaling mol. due to its regulatory effects on numerous protein targets including enzymes, transcription factors, ion channels, neurotrophic factors, and postsynaptic scaffolding proteins. However, intracellular zinc concn. is tightly maintained at steady levels under natural physiol. conditions. In this work, we made the novel observation that the developing neurons generated spontaneous and synchronous zinc spikes in primary hippocampal cultures using a fluorescent zinc sensor, FluoZin-3. Blocking of glutamate receptor-dependent calcium influx depleted the zinc spikes, suggesting that these zinc spikes were driven by the glutamate-mediated spontaneous neural excitability and calcium spikes that have been characterized in early developing neurons. Simultaneous imaging of calcium or pH together with zinc, we uncovered that a downward pH spike was evoked with each zinc spike and this transient cellular acidification occurred downstream of calcium spikes but upstream of zinc spikes. Our results suggest that spontaneous, synchronous zinc spikes were generated through calcium influx-induced cellular acidification, which liberates zinc from intracellular zinc binding ligands. Given that changes in zinc concn. can modulate activities of proteins essential for synapse maturation and neuronal differentiation, these zinc spikes might act as important signaling roles in neuronal development.
- 322Blockhuys, S.; Celauro, E.; Hildesjö, C.; Feizi, A.; Stål, O.; Fierro-Gonzalez, J. C.; Wittung-Stafshede, P. Defining the human copper proteome and analysis of its expression variation in cancers. Metallomics 2017, 9, 112– 123, DOI: 10.1039/C6MT00202AGoogle Scholar322Defining the human copper proteome and analysis of its expression variation in cancersBlockhuys, S.; Celauro, E.; Hildesjoe, C.; Feizi, A.; Staal, O.; Fierro-Gonzalez, J. C.; Wittung-Stafshede, P.Metallomics (2017), 9 (2), 112-123CODEN: METAJS; ISSN:1756-591X. (Royal Society of Chemistry)Copper (Cu) is essential for living organisms, and acts as a cofactor in many metabolic enzymes. To avoid the toxicity of free Cu, organisms have specific transport systems that 'chaperone' the metal to targets. Cancer progression is assocd. with increased cellular Cu concns., whereby proliferative immortality, angiogenesis and metastasis are cancer hallmarks with defined requirements for Cu. The aim of this study is to gather all known Cu-binding proteins and reveal their putative involvement in cancers using the available database resources of RNA transcript levels. Using the Uniprot.org database along with manual curation, we identified a total of 54 Cu-binding proteins (named the human Cu proteome). Next, we retrieved RNA expression levels in cancer vs. normal tissues from the TCGA database for the human Cu proteome in 18 cancer types, and noted an intricate pattern of up- and downregulation of the genes in different cancers. Hierarchical clustering in combination with bioinformatics and functional genomics analyses allowed for the prediction of cancer-related Cu-binding proteins; these were specifically inspected for the breast cancer data. Finally, for the Cu chaperone ATOX1, which is the only Cu-binding protein proposed to have transcription factor activities, we validated its predicted over-expression in patient breast cancer tissue at the protein level. This collection of Cu-binding proteins, with RNA expression patterns in different cancers, will serve as an excellent resource for mechanistic-mol. studies of Cu-dependent processes in cancer.
- 323Hartmann, H. J.; Weser, U. Copper-thionein from fetal bovine liver. Biochim. Biophys. Acta, Protein Struct. 1977, 491, 211– 222, DOI: 10.1016/0005-2795(77)90057-5Google Scholar323Copper-thionein from fetal bovine liverHartmann, Hans Juergen; Weser, UlrichBiochimica et Biophysica Acta, Protein Structure (1977), 491 (1), 211-22CODEN: BBPTBH; ISSN:0005-2795.An 8 Cu-2 Zn thionein, having a mol. wt. of 11,500, was isolated from bovine fetal liver. Sixteen percent of the total Cu present in the whole liver was recovered in this protein. This Cu-thionein was similar to the polymeric neonatal type mitochondrocuprein. A comparison of different Cu-thioneins contg. variable amts. of Cu was possible when εCu from 280 nm to longer wavelengths was detd. With respect to the UV properties, there were no detectable differences between Cu-thioneins prepd. either in vivo or in vitro and the fetal Cu-thionein. Furthermore, the positions of the Cotton effects were rather similar. X-ray photoelectron spectrometric studies revealed a Cu(2p3/2) binding energy value of 932.9 eV. Unlike the S(2p1/2,3/2) value near 162 eV using Cu-thioneins from chicken liver or yeast, the higher S(2p1/2,3/2) binding energy of 163.0 eV employing fetal Cu-thionein was attributed to partial oxidn. of the protein moiety and (or) a particular chem. environment. The 2nd S(2p1/2,3/2) peak was assigned to the Cu-catalyzed oxidn. of S via OH radicals to yield RSO3-. In the x-ray photoelectron spectrum of the apoprotein, 1 homogeneous S(2p1/2,3/2) band at 163.7 eV was attributable to RSSR.
- 324Luchinat, E.; Barbieri, L.; Banci, L. A molecular chaperone activity of CCS restores the maturation of SOD1 fALS mutants. Sci. Rep. 2017, 7, 17433, DOI: 10.1038/s41598-017-17815-yGoogle Scholar324A molecular chaperone activity of CCS restores the maturation of SOD1 fALS mutantsLuchinat Enrico; Barbieri Letizia; Banci Lucia; Luchinat Enrico; Barbieri Letizia; Banci LuciaScientific reports (2017), 7 (1), 17433 ISSN:.Superoxide dismutase 1 (SOD1) is an important metalloprotein for cellular oxidative stress defence, that is mutated in familiar variants of Amyotrophic Lateral Sclerosis (fALS). Some mutations destabilize the apo protein, leading to the formation of misfolded, toxic species. The Copper Chaperone for SOD1 (CCS) transiently interacts with SOD1 and promotes its correct maturation by transferring copper and catalyzing disulfide bond formation. By in vitro and in-cell NMR, we investigated the role of the SOD-like domain of CCS (CCS-D2). We showed that CCS-D2 forms a stable complex with zinc-bound SOD1 in human cells, that has a twofold stabilizing effect: it both prevents the accumulation of unstructured mutant SOD1 and promotes zinc binding. We further showed that CCS-D2 interacts with apo-SOD1 in vitro, suggesting that in cells CCS stabilizes mutant apo-SOD1 prior to zinc binding. Such molecular chaperone function of CCS-D2 is novel and its implications in SOD-linked fALS deserve further investigation.
- 325Skopp, A.; Boyd, S. D.; Ullrich, M. S.; Liu, L.; Winkler, D. D. Copper-zinc superoxide dismutase (Sod1) activation terminates interaction between its copper chaperone (Ccs) and the cytosolic metal-binding domain of the copper importer Ctr1. BioMetals 2019, 32, 695– 705, DOI: 10.1007/s10534-019-00206-3Google Scholar325Copper-zinc superoxide dismutase (Sod1) activation terminates interaction between its copper chaperone (Ccs) and the cytosolic metal-binding domain of the copper importer Ctr1Skopp, Amelie; Boyd, Stefanie D.; Ullrich, Morgan S.; Liu, Li; Winkler, Duane D.BioMetals (2019), 32 (4), 695-705CODEN: BOMEEH; ISSN:0966-0844. (Springer)Copper-zinc superoxide dismutase (Sod1) is a crit. antioxidant enzyme that rids the cell of reactive oxygen through the redox cycling of a catalytic copper ion provided by its copper chaperone (Ccs). Ccs must first acquire this copper ion, directly or indirectly, from the influx copper transporter, Ctr1. The three proteins of this transport pathway ensure careful trafficking of copper ions from cell entry to target delivery, but the intricacies remain undefined. Biochem. examn. of each step in the pathway detd. that the activation of the target (Sod1) regulates the Ccs·Ctr1 interaction. Ccs stably interacts with the cytosolic C-terminal tail of Ctr1 (Ctr1c) in a copper-dependent manner. This interaction becomes tripartite upon the addn. of an engineered immature form of Sod1 creating a stable Cu(I)-Ctr1c·Ccs·Sod1 heterotrimer in soln. This heterotrimer can also be made by the addn. of a preformed Sod1·Ccs heterodimer to Cu(I)-Ctr1c, suggestive of multiple routes to the same destination. Only complete Sod1 activation (i.e. active site copper delivery and intra-subunit disulfide bond formation) breaks the Sod1·Ccs·Ctr1c complex. The results provide a new and extended view of the Sod1 activation pathway(s) originating at cellular copper import.
- 326Maryon, E. B.; Molloy, S. A.; Kaplan, J. H. Cellular glutathione plays a key role in copper uptake mediated by human copper transporter 1. Am. J. Phyiol. Cell Physiol. 2013, 304, C768– 779, DOI: 10.1152/ajpcell.00417.2012Google ScholarThere is no corresponding record for this reference.
- 327Morgan, M. T.; Nguyen, L. A. H.; Hancock, H. L.; Fahrni, C. J. Glutathione limits aquacopper(I) to sub-femtomolar concentrations through cooperative assembly of a tetranuclear cluster. J. Biol. Chem. 2017, 292, 21558– 21567, DOI: 10.1074/jbc.M117.817452Google Scholar327Glutathione limits aquacopper(I) to sub-femtomolar concentrations through cooperative assembly of a tetranuclear clusterMorgan, M. Thomas; Nguyen, Lily Anh H.; Hancock, Haylie L.; Fahrni, Christoph J.Journal of Biological Chemistry (2017), 292 (52), 21558-21567CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)The tripeptide glutathione (GSH) is a crucial intracellular reductant and radical scavenger, but it may also coordinate the soft Cu(I) cation and thereby yield pro-oxidant species. The GSH-Cu(I) interaction is thus a key consideration for both redox and copper homeostasis in cells. However, even after nearly four decades of investigation, the nature and stability of the GSH-Cu(I) complexes formed under biol. relevant conditions remain controversial. Here, we revealed the unexpected predominance of a tetranuclear [Cu4(GS)6] cluster that is sufficiently stable to limit the effective free aquacopper(I) concn. to the sub-femtomolar regime. Combined spectrophotometric-potentiometric titrns. at biol. realistic GSH/Cu(I) ratios, enabled by our recently developed Cu(I) affinity stds. and corroborated by low-temp. phosphorescence studies, established cooperative assembly of [Cu4(GS)6] as the dominant species over a wide pH range, from 5.5 to 7.5. Our robust model for the glutathione-Cu(I) equil. system sets a firm upper limit on the thermodn. availability of intracellular copper that is 3 orders of magnitude lower than previously estd. Taking into account their ability to catalyze the prodn. of deleterious superoxide, the formation of Cu(I)-glutathione complexes might be avoided under normal physiol. conditions. The actual intracellular Cu(I) availability may thus be regulated a further 3 orders of magnitude below the GSH/Cu(I) affinity limit, consistent with the most recent affinity detns. of Cu(I) chaperones.
- 328Chung, C. Y.-S.; Posimo, J. M.; Lee, S.; Tsang, T.; Davis, J. M.; Brady, D. C.; Chang, C. J. Activity-based ratiometric FRET probe reveals oncogene-driven changes in labile copper pools induced by altered glutathione metabolism. Proc. Natl. Acad. Sci. U. S. A. 2019, 116, 18285– 18294, DOI: 10.1073/pnas.1904610116Google Scholar328Activity-based ratiometric FRET probe reveals oncogene-driven changes in labile copper pools induced by altered glutathione metabolismChung Clive Yik-Sham; Lee Sumin; Chang Christopher J; Posimo Jessica M; Brady Donita C; Tsang Tiffany; Davis Julianne M; Brady Donita C; Chang Christopher J; Chang Christopher JProceedings of the National Academy of Sciences of the United States of America (2019), 116 (37), 18285-18294 ISSN:.Copper is essential for life, and beyond its well-established ability to serve as a tightly bound, redox-active active site cofactor for enzyme function, emerging data suggest that cellular copper also exists in labile pools, defined as loosely bound to low-molecular-weight ligands, which can regulate diverse transition metal signaling processes spanning neural communication and olfaction, lipolysis, rest-activity cycles, and kinase pathways critical for oncogenic signaling. To help decipher this growing biology, we report a first-generation ratiometric fluorescence resonance energy transfer (FRET) copper probe, FCP-1, for activity-based sensing of labile Cu(I) pools in live cells. FCP-1 links fluorescein and rhodamine dyes through a Tris[(2-pyridyl)methyl]amine bridge. Bioinspired Cu(I)-induced oxidative cleavage decreases FRET between fluorescein donor and rhodamine acceptor. FCP-1 responds to Cu(I) with high metal selectivity and oxidation-state specificity and facilitates ratiometric measurements that minimize potential interferences arising from variations in sample thickness, dye concentration, and light intensity. FCP-1 enables imaging of dynamic changes in labile Cu(I) pools in live cells in response to copper supplementation/depletion, differential expression of the copper importer CTR1, and redox stress induced by manipulating intracellular glutathione levels and reduced/oxidized glutathione (GSH/GSSG) ratios. FCP-1 imaging reveals a labile Cu(I) deficiency induced by oncogene-driven cellular transformation that promotes fluctuations in glutathione metabolism, where lower GSH/GSSG ratios decrease labile Cu(I) availability without affecting total copper levels. By connecting copper dysregulation and glutathione stress in cancer, this work provides a valuable starting point to study broader cross-talk between metal and redox pathways in health and disease with activity-based probes.
- 329Rae, T. D.; Schmidt, P. J.; Pufahl, R. A.; Culotta, V. C.; O’Halloran, T. V. Undetectable free copper: The requirement of copper chaperone for superoxide dismutase. Science 1999, 284, 805– 808, DOI: 10.1126/science.284.5415.805Google Scholar329Undetectable intracellular free copper: the requirement of a copper chaperone for superoxide dismutaseRae, T. D.; Schmidt, P. J.; Pufahl, R. A.; Culotta, V. C.; O'Halloran, T. V.Science (Washington, D. C.) (1999), 284 (5415), 805-808CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)The copper chaperone for the superoxide dismutase (CCS) gene is necessary for expression of an active, copper-bound form of superoxide dismutase (SOD1) in vivo in spite of the high affinity of SOD1 for copper (dissocn. const. = 6 fM) and the high intracellular concns. of both SOD1 (10 μM in yeast) and copper (70 μM in yeast). In vitro studies demonstrated that purified Cu(I)-yCCS protein is sufficient for direct copper activation of apo-ySOD1 but is necessary only when the concn. of free copper ions ([Cu]free) is strictly limited. Moreover, the physiol. requirement for yCCS in vivo was readily bypassed by elevated copper concns. and abrogation of intracellular copper-scavenging systems such as the metallothioneins. This metallochaperone protein activates the target enzyme through direct insertion of the copper cofactor and apparently functions to protect the metal ion from binding to intracellular copper scavengers. These results indicate that intracellular [Cu]free is limited to less than one free copper ion per cell and suggest that a pool of free copper ions is not used in physiol. activation of metalloenzymes.
- 330Bethin, K. E.; Cimato, T. R.; Ettinger, M. J. Copper binding to mouse liver S-adenosylhomocysteine hydrolase and the effects of copper on its levels. J. Biol. Chem. 1995, 270, 20703– 20711, DOI: 10.1074/jbc.270.35.20703Google Scholar330Copper binding to mouse liver S-adenosylhomocysteine hydrolase and the effects of copper on its levelsBethin, Kathleen E.; Cimato, Thomas R.; Ettinger, Murray J.Journal of Biological Chemistry (1995), 270 (35), 20703-11CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)The dissocn. const. and stoichiometry of copper binding to mouse liver S-adenosylhomocysteine hydrolase (SAHH) was detd. as part of characterizing the possible roles of SAHH in copper metab. Copper (64Cu(II)) binding was measured by an ultrafiltration method in the presence of EDTA as a competing ligand. The KD was 3.9×10-16 M, and the stoichiometry was one g atom of copper per 48-kDa subunit. Western blots indicated that the liver contains ≈12 times more SAHH than the kidney, which in turn contains ≃5 times more SAHH than the brain. The high concn. and copper affinity of SAHH in the liver may contribute to the liver's ability to preferentially accumulate copper, and the low levels of SAHH in the brain may contribute to the sensitivity of the brain to copper deficiency. The effects of genetic defects of copper metab. and copper deficiency on SAHH were also detd. Normal SAHH levels were detected in brindled mouse liver, kidney, and brain. However, SAHH from brindled mouse liver eluted abnormally from Ph Superose columns implying an effect of the brindled mouse defect on SAHH protein structure. Hepatic cytosols from the toxic milk mouse contained ≈42% the amt. of SAHH detected in controls, and hepatic levels of SAHH were also decreased by 45% in copper-deficient mice. The binding properties of SAHH and the effects of abnormal states of copper metab. on its levels are consistent with significant roles for SAHH in normal and abnormal copper metab. SAHH may have roles in regulating tissue copper levels and the distribution of intracellular copper.
- 331Chen, P.; Onana, P.; Shaw, C. F., III; Petering, D. H. Characterization of calf liver Cu,Zn-metallothionein: naturally variable Cu and Zn stoichiometries. Biochem. J. 1996, 317, 389– 394, DOI: 10.1042/bj3170389Google Scholar331Characterization of calf liver Cu,Zn-metallothionein: naturally variable Cu and Zn stoichiometriesChen, Pu; Onana, Patrick; Shaw, C. Frank, III; Petering, David H.Biochemical Journal (1996), 317 (2), 389-394CODEN: BIJOAK; ISSN:0264-6021. (Portland Press)Cu,Zn-metallothioneins were purified from bovine calf liver in order to examine the stoichiometry of metal binding to the protein. Cu and Zn analyses were carried out by at. absorption spectrophotometry. Consistent quant. thiolate analyses were obtained spectrophotometrically with Ellman's reagent and amperometrically with phenylmercuric acetate. These were used to define protein concn. A complementary method to assess the sum of the thiol and Cu(I) content of metallothionein involved titrn. of the reducing equiv. of the protein with ferricyanide. The reaction stoichiometry was consistent with the oxidn. of all of the SH groups to disulfides and all of the bound Cu from the Cu(I) to the Cu(II) oxidn. state. According to these methods, the total no. of Zn plus Cu ions bound to metallothionein isolated from a no. of calf livers centered on approx. 7, 10-12, or 15 g-atoms of metal per mol of protein. The reactivity of ferricyanide and 4,7-phenylsulfonyl-2,9-dimethyl-1,10-phenanthroline (BCS) with Cu,Zn-metallothioneins of various metal ratios was assessed. Zn-metallothionein reacted almost entirely in 2 slow steps with ferricyanide. As the Cu content of the protein increased, the fraction of reaction occurring in the time of mixing increased in parallel. BCS was able to remove 70-80% of metallothionein-bound Cu as Cu(I). The rest was resistant to reaction.
- 332Chang, C. J. Searching for harmony in transition-metal signaling. Nat. Chem. Biol. 2015, 11, 744– 747, DOI: 10.1038/nchembio.1913Google Scholar332Searching for harmony in transition-metal signalingChang, Christopher J.Nature Chemical Biology (2015), 11 (10), 744-747CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)A review. The recent emergence of signaling roles for transition metals presages a broader contribution of these elements beyond their traditional functions as metabolic cofactors. New chem. approaches to identify the sources, targets and physiologies of transition-metal signaling can help expand understanding of the periodic table in a biol. context.
- 333Leary, S. C.; Ralle, M. Advances in visualization of copper in mammalian systems using X-ray fluorescence microscopy. Curr. Opin. Chem. Biol. 2020, 55, 19– 25, DOI: 10.1016/j.cbpa.2019.12.002Google Scholar333Advances in visualization of copper in mammalian systems using X-ray fluorescence microscopyLeary, Scot C.; Ralle, MartinaCurrent Opinion in Chemical Biology (2020), 55 (), 19-25CODEN: COCBF4; ISSN:1367-5931. (Elsevier B.V.)Synchrotron-based X-ray fluorescence microscopy (XFM) has become an important imaging technique to investigate elemental concns. and distributions in biol. specimens. Advances in technol. now permit imaging at resolns. rivaling that of electron microscopy, and researchers can now visualize elemental concns. in subcellular organelles when using appropriate correlative methods. XFM is an esp. valuable tool to det. the distribution of endogenous trace metals that are involved in neurodegenerative diseases. Here, we discuss the latest research on the unusual copper (Cu) storage vesicles that were originally identified in mouse brains and the involvement of Cu in Alzheimer's disease. Finally, we provide an outlook of how future improvements to XFM will drive current trace element research forward.
- 334Grubman, A.; White, A. R. Copper as a key regulator of cell signalling pathways. Expert Rev. Mol. Med. 2014, 16, e11, DOI: 10.1017/erm.2014.11Google Scholar334Copper as a key regulator of cell signalling pathwaysGrubman, Alexandra; White, Anthony R.Expert Reviews in Molecular Medicine (2014), 16 (), e11/1-e11/27CODEN: ERMMFS; ISSN:1462-3994. (Cambridge University Press)A review. Copper is an essential element in many biol. processes. The crit. functions assocd. with copper have resulted from evolutionary harnessing of its potent redox activity. This same property also places copper in a unique role as a key modulator of cell signal transduction pathways. These pathways are the complex sequence of mol. interactions that drive all cellular mechanisms and are often assocd. with the interplay of key enzymes including kinases and phosphatases but also including intracellular changes in pools of smaller mols. A growing body of evidence is beginning to delineate the how, when and where of copper-mediated control over cell signal transduction. This has been driven by research demonstrating crit. changes to copper homeostasis in many disorders including cancer and neurodegeneration and therapeutic potential through control of disease assocd. cell signalling changes by modulation of copper-protein interactions. This timely review brings together for the first time the diverse actions of copper as a key regulator of cell signalling pathways and discusses the potential strategies for controlling disease-assocd. signalling processes using copper modulators. It is hoped that this review will provide a valuable insight into copper as a key signal regulator and stimulate further research to promote our understanding of copper in disease and therapy.
- 335Kardos, J.; Heja, L.; Simon, A.; Jablonkai, I.; Kovacs, R.; Jemnitz, K. Copper signalling: causes and consequences. Cell Commun. Signaling 2018, 16, 71, DOI: 10.1186/s12964-018-0292-4Google Scholar335Copper signalling: causes and consequencesKardos, Julianna; Heja, Laszlo; Simon, Agnes; Jablonkai, Istvan; Kovacs, Richard; Jemnitz, KatalinCell Communication and Signaling (2018), 16 (), 71CODEN: CCSEC6; ISSN:1478-811X. (BioMed Central Ltd.)Copper-contg. enzymes perform fundamental functions by activating dioxygen (O2) and therefore allowing chem. energy-transfer for aerobic metab. The copper-dependence of O2 transport, metab. and prodn. of signalling mols. are supported by mol. systems that regulate and preserve tightly-bound static and weakly-bound dynamic cellular copper pools. Disruption of the reducing intracellular environment, characterized by glutathione shortage and ambient Cu(II) abundance drives oxidative stress and interferes with the bidirectional, copper-dependent communication between neurons and astrocytes, eventually leading to various brain disease forms. A deeper understanding of of the regulatory effects of copper on neuro-glia coupling via polyamine metab. may reveal novel copper signalling functions and new directions for therapeutic intervention in brain disorders assocd. with aberrant copper metab.
- 336Ackerman, C. M.; Chang, C. J. Copper signaling in the brain and beyond. J. Biol. Chem. 2018, 293, 4628– 4635, DOI: 10.1074/jbc.R117.000176Google Scholar336Copper signaling in the brain and beyondAckerman, Cheri M.; Chang, Christopher J.Journal of Biological Chemistry (2018), 293 (13), 4628-4635CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)A review. Transition metals have been recognized and studied primarily in the context of their essential roles as structural and metabolic cofactors for biomols. that compose living systems. More recently, an emerging paradigm of transition-metal signaling, where dynamic changes in transitional metal pools can modulate protein function, cell fate, and organism health and disease, has broadened our view of the potential contributions of these essential nutrients in biol. Using Cu as a canonical example of transition-metal signaling, we highlight key expts. where direct measurement and/or visualization of dynamic Cu pools, in combination with biochem., physiol., and behavioral studies, have deciphered sources, targets, and physiol. effects of Cu signals.
- 337Krishnamoorthy, L.; Cotruvo, J. A., Jr; Chan, J.; Kaluarachchi, H.; Muchenditsi, A.; Pendyala, V. S.; Jia, S.; Aron, A. T.; Ackerman, C. M.; Vander Wal, M. N.; Guan, T.; Smaga, L. P.; Farhi, S. L.; New, E. J.; Lutsenko, S.; Chang, C. J. Copper regulates cyclic AMP-dependent lipolysis. Nat. Chem. Biol. 2016, 12, 586– 592, DOI: 10.1038/nchembio.2098Google Scholar337Copper regulates cyclic-AMP-dependent lipolysisKrishnamoorthy, Lakshmi; Cotruvo, Joseph A., Jr.; Chan, Jefferson; Kaluarachchi, Harini; Muchenditsi, Abigael; Pendyala, Venkata S.; Jia, Shang; Aron, Allegra T.; Ackerman, Cheri M.; Vander Wal, Mark N.; Guan, Timothy; Smaga, Lukas P.; Farhi, Samouil L.; New, Elizabeth J.; Lutsenko, Svetlana; Chang, Christopher J.Nature Chemical Biology (2016), 12 (8), 586-592CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)Cell signaling relies extensively on dynamic pools of redox-inactive metal ions such as sodium, potassium, calcium and zinc, but their redox-active transition metal counterparts such as copper and iron have been studied primarily as static enzyme cofactors. Here we report that copper is an endogenous regulator of lipolysis, the breakdown of fat, which is an essential process in maintaining body wt. and energy stores. Using a mouse model of genetic copper misregulation, in combination with pharmacol. alterations in copper status and imaging studies in a 3T3-L1 white adipocyte model, we found that copper regulates lipolysis at the level of the second messenger, cAMP, by altering the activity of the cAMP-degrading phosphodiesterase PDE3B. Biochem. studies of the copper-PDE3B interaction establish copper-dependent inhibition of enzyme activity and identify a key conserved cysteine residue in a PDE3-specific loop that is essential for the obsd. copper-dependent lipolytic phenotype.
- 338Angeletti, B.; Waldron, K. J.; Freeman, K. B.; Bawagan, H.; Hussain, I.; Miller, C. C.; Lau, K. F.; Tennant, M. E.; Dennison, C.; Robinson, N. J.; Dingwall, C. BACE1 cytoplasmic domain interacts with the copper chaperone for superoxide dismutase-1 and binds copper. J. Biol. Chem. 2005, 280, 17930– 17937, DOI: 10.1074/jbc.M412034200Google Scholar338BACE1 Cytoplasmic Domain Interacts with the Copper Chaperone for Superoxide Dismutase-1 and Binds CopperAngeletti, Barbara; Waldron, Kevin J.; Freeman, Katie B.; Bawagan, Hinayana; Hussain, Ishrut; Miller, Christopher C. J.; Lau, Kwok-Fai; Tennant, Maria E.; Dennison, Christopher; Robinson, Nigel J.; Dingwall, ColinJournal of Biological Chemistry (2005), 280 (18), 17930-17937CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)The amyloidogenic pathway leading to the prodn. and deposition of Aβ peptides, major constituents of Alzheimer's disease senile plaques, is linked to neuronal metal homeostasis. The amyloid precursor protein binds copper and zinc in its extracellular domain, and the Aβ peptides also bind copper, zinc, and iron. The first step in the generation of Aβ is cleavage of amyloid precursor protein by the aspartic protease BACE1. Here we show that BACE1 interacts with CCS (the copper chaperone for superoxide dismutase-1 (SOD1)) through domain I and the proteins co-immunoppt. from rat brain exts. We have also been able to visualize the co-transport of membranous BACE1 and sol. CCS through axons. BACE1 expression reduces the activity of SOD1 in cells consistent with direct competition for available CCS as overexpression of CCS restores SOD1 activity. Finally, we demonstrate that the twenty-four residue C-terminal domain of BACE1 binds a single Cu(I) atom with high affinity through cysteine residues.
- 339Udom, A. O.; Brady, F. O. Reactivation in vitro of zinc-requiring apo-enzymes by rat liver zinc-thionein. Biochem. J. 1980, 187, 329– 335, DOI: 10.1042/bj1870329Google Scholar339Reactivation in vitro of zinc-requiring apo-enzymes by rat liver zinc-thioneinUdom, Albert O.; Brady, Frank O.Biochemical Journal (1980), 187 (2), 329-35CODEN: BIJOAK; ISSN:0264-6021.Liver Zn-thionein was as good as, or better than ZnSO4, Zn(OAc)2, or Zn(NO3)2 in donating Zn to apoenzymes of alc. dehydrogenase (I), aldolase, thermolysin, alk. phosphatase (II), and carbonic anhydrase (III). Apo-I could not be reactivated by Zn salts or Zn-thionein, whereas incubation of the other apoenzymes with near satg. amts. of Zn as ZnSO4, Zn(OAc)2, Zn(NO3)2, or Zn-thionein caused reactivation. Apo-aldolase was 100% reactivated by Zn-thionein in 30 min, whereas reactivation by ZnSO4 and Zn(OAc)2 was complete and instantaneous. Zn-thionein was better than Zn(NO3)2 in completely reactivating apo-thermolysin. Apo-II was 43 and 18% reactivated by Zn(OAc)2 and Zn-thionein, resp. Zn-thionein was better than ZnSO4, Zn(OAc)2, or Zn(NO3)2 in reactivating apo-III, giving max. reactivation of 54%. Zn was apparently being transferred from Zn-thionein to apo-III, as indicated by the effects of 2,6-pyridinedicarboxylic acid and 1,10-phenanthroline on the reactivation of apo-III. Thus, Zn-thionein can apparently function in Zn homeostasis as a Zn reservoir releasing Zn to Zn-requiring metalloenzymes according to need.
- 340Li, T. Y.; Kraker, A. J.; Shaw, C. F., 3rd; Petering, D. H. Ligand substitution reactions of metallothioneins with EDTA and apo-carbonic anhydrase. Proc. Natl. Acad. Sci. U. S. A. 1980, 77, 6334– 6338, DOI: 10.1073/pnas.77.11.6334Google Scholar340Ligand substitution reactions of metallothioneins with EDTA and apo-carbonic anhydraseLi, Ta-Yuen; Kraker, Alan J.; Shaw, C. Frank, III; Petering, David H.Proceedings of the National Academy of Sciences of the United States of America (1980), 77 (11), 6334-8CODEN: PNASA6; ISSN:0027-8424.The reactions of Zn-, Cd-, and Zn,Cd-thioneins with EDTA and apo-carbonic anhydrase were studied. The ligand substitution reaction of Zn with EDTA is multiphasic having both associative and dissociative components in the rate expression. The Cd sites are ∼2 orders of magnitude less reactive. In contrast, apo-carbonic anhydrase abstrs. Zn from Zn-thionein and Zn,Cd-thionein in 2nd-order processes that are 2-3 orders of magnitude more rapid than those involving EDTA and approach the rate for unligated Zn2+ with the apo-protein. In comparison with other Zn proteins, Zn-thionein contains unusually reactive metal sites, suggesting that this protein may be a physiol. Zn transfer protein, able to donate Zn to Zn-requiring apo macromols.
- 341Brady, F. O. The physiological function of metallothionein. Trends Biochem. Sci. 1982, 7, 143– 145, DOI: 10.1016/0968-0004(82)90206-7Google Scholar341The physiological function of metallothioneinBrady, Frank O.Trends in Biochemical Sciences (1982), 7 (4), 143-5CODEN: TBSCDB; ISSN:0968-0004.A review with 29 refs. on the physiol. function of metallothionein, the nature of its Zn and Cu binding, and the regulation of its gene.
- 342Hathout, Y.; Reynolds, K. J.; Szilagyi, Z.; Fenselau, C. Metallothionein dimers studied by nano-spray mass spectrometry. J. Inorg. Biochem. 2002, 88, 119– 122, DOI: 10.1016/S0162-0134(01)00375-0Google Scholar342Metallothionein dimers studied by nano-spray mass spectrometryHathout, Yetrib; Reynolds, Kristy J.; Szilagyi, Zoltan; Fenselau, CatherineJournal of Inorganic Biochemistry (2002), 88 (2), 119-122CODEN: JIBIDJ; ISSN:0162-0134. (Elsevier Science Inc.)Both transient and stable dimers of metallothionein have been characterized, based on earlier studies using NMR, CD and size-exclusion chromatog. Here addnl. characterization is provided by nanospray mass spectrometry. Rapid redistribution of metal ions between monomeric Cd7- and Zn7-metallothionein 2a is monitored by nanospray. An expt. in which theses two forms of the monomeric protein are sepd. by a dialysis membrane, which will pass metal ions but not proteins, confirms that a transient dimer must form for metal ions to be redistributed. On the other hand, size-exclusion chromatog. of reconstituted Zn7- or Cd7-metallothionein revealed the presence of monomeric and dimeric species. These dimers do not equilibrate readily to form monomers and they are shown to be covalent.
- 343Andersson, I.; Maret, W.; Zeppezauer, M.; Brown, R. D., III; Koenig, S. H. Metal ion substitution at the catalytic site of horse liver alcohol dehydrogenase: Results from solvent magnetic relaxation studies. 2. Binding of manganese(II) and competition with zinc(II) and cadmium(II) ions. Biochemistry 1981, 20, 3433– 3438, DOI: 10.1021/bi00515a020Google Scholar343Metal ion substitution at the catalytic site of horse-liver alcohol dehydrogenase: results from solvent magnetic relaxation studies. 2. Binding of manganese(2+) and competition with zinc(2+) and cadmium(2+) ionsAndersson, Inger; Maret, Wolfgang; Zeppezauer, Michael; Brown, Rodney D., III; Koenig, Seymour H.Biochemistry (1981), 20 (12), 3433-8CODEN: BICHAW; ISSN:0006-2960.The interaction of Mn2+ with native horse liver alc. dehydrogenase demetalized specifically at the catalytic sites has been investigated by studying the magnetic field dependence and time dependence of the magnetic spin-lattice relaxation rate of solvent water protons. No detectable binding of Mn2+ to the catalytic sites was found in times on the order of hs; however, it was found that these ions bound to the enzyme at 2 previously unreported types of sites: one characterized by a low dissocn. const. (0.01 mM at pH 7.7, 5°), low relaxivity, and a stoichiometry of 1/2 catalytic sites, and a 2nd, with a high dissocn. const. (1.5 mM at pH 7.7, 5°) and high relaxivity. The stoichiometry of the 2nd type of site could not be detd. because of the relatively weak binding of Mn2+ to these sites. Both Zn2+ and Cd2+ bound to the newly found tight-binding sites, displacing Mn2+ and thereby altering the relaxation rates of solvent protons. By monitoring the return to equil. of these altered rates, it was found that Zn2+ entered the catalytic sites from the new tight-binding sites with an on-rate of ∼0.1 M-1s-1. It was not clear whether binding to these new sites was an obligatory intermediate for reintroduction of Zn2+ into the catalytic sites, but a small excess of Zn2+ beyond 1/monomer caused the protein to ppt. Cd2+ by contrast, entered the catalytic sites at least 1 order of magnitude more rapidly than did Zn2+, a rate too rapid to observe by the techniques employed. However, once the catalytic sites were filled, Cd2+ displaced Mn2+ at the new sites as did Zn2+.
- 344Mason, A. Z.; Perico, N.; Moeller, R.; Thrippleton, K.; Potter, T.; Lloyd, D. Metal donation and apo-metalloenzyme activation by stable isotopically labelled metallothionein. Mar. Environ. Res. 2004, 58, 371– 375, DOI: 10.1016/j.marenvres.2004.03.082Google Scholar344Metal donation and apo-metalloenzyme activation by stable isotopically labeled metallothioneinMason, Andrew. Z.; Perico, Natalie; Moeller, Rhonda; Thrippleton, Kelly; Potter, Tiffany; Lloyd, DouglasMarine Environmental Research (2004), 58 (2-5), 371-375CODEN: MERSDW; ISSN:0141-1136. (Elsevier Science B.V.)Coupled HPLC-ICP-MS has been used to quant. study the effects of GSSG and GSH on the ability of metallothionein (MTII) to donate essential and non-essential metals to apo-carbonic anhydrase. Stable isotopically labeled 67Zn3Cd4 in gills and liver MTII was used to enable Zn donated from MTII to be differentiated from extraneous sources of Zn. Transfer of both 67Zn and Cd from MTII to apo-carbonic anhydrase was noted in the absence of either GSSG or GSH. GSSG increased the initial transfer of both Zn and Cd. Thereafter, a gradual increase in the 67Zn content at the expense of Cd was noted over 24-h indicating continued interaction and exchange between MTII and the enzyme commensurate with the relative preferences shown by the proteins for these two metals. Although GSH also increased transfer of 67Zn from MT it reduced the simultaneous transfer of Cd to the enzyme thereby conferring protection against Cd induced activation.
- 345Roesijadi, G. Metal transfer as a mechanism for metallothionein-mediated metal detoxification. Cell. Mol. Biol. 2000, 46, 393– 405Google Scholar345Metal transfer as a mechanism for metallothionein-mediated metal detoxificationRoesijadi, G.Cellular and Molecular Biology (Paris) (2000), 46 (2), 393-405CODEN: CMOBEF; ISSN:0145-5680. (C.M.B. Association)A review with 101 refs. High kinetic reactivity and high metal affinity of the metal-binding sites of metallothionein are characteristics that would facilitate involvement of the thionein-zinc metallothionein couple in metal transfer or exchange reactions. Studies demonstrating thionein-metallothionein-mediated activation or inhibition of various zinc metalloenzymes and transcription factors provide support for a potential role for metallothionein in metal transfer reactions with receptor mols. Although a role in basal zinc regulation is currently a topic of debate, less controversial is a role for metallothionein in the detoxification of metals such as cadmium. The toxicity of metals can, in part, be due to adventitious binding to charged sites of target proteins or the displacement of zinc bound to zinc metalloproteins. Zinc metallothionein has the capability of repairing such structures through abstraction of a toxic metal in the former case or through a reciprocal metal transfer reaction that involves abstraction of the toxic metal and donation of the essential metal zinc in the latter. This would confer on metallothionein an active role in the protective response to metal toxicity, rather than a passive one that is solely dependent on the high metal affinity for binding free metal ions. The efficacy of such a mechanism for metal detoxification has been demonstrated with enzymes, actin and zinc finger proteins. With zinc finger proteins, zinc metallothionein can restore both altered secondary structure and inhibited DNA-binding function to functional states through a zinc for cadmium exchange.
- 346Costello, L. C.; Liu, Y.; Franklin, R. B.; Kennedy, M. C. Zinc inhibition of mitochondrial aconitase and its importance in citrate metabolism of prostate epithelial cells. J. Biol. Chem. 1997, 272, 28875– 28881, DOI: 10.1074/jbc.272.46.28875Google Scholar346Zinc inhibition of mitochondrial aconitase and its importance in citrate metabolism of prostate epithelial cellsCostello L C; Liu Y; Franklin R B; Kennedy M CThe Journal of biological chemistry (1997), 272 (46), 28875-81 ISSN:0021-9258.Prostate epithelial cells possess a uniquely limiting mitochondrial (m-) aconitase activity that minimizes their ability to oxidize citrate. These cells also possess uniquely high cellular and mitochondrial zinc levels. Correlations among zinc, citrate, and m-aconitase in prostate indicated that zinc might be an inhibitor of prostate m-aconitase activity and citrate oxidation. The present studies reveal that zinc at near physiological levels inhibited m-aconitase activity of mitochondrial sonicate preparations obtained from rat ventral prostate epithelial cells. Corresponding studies conducted with mitochondrial sonicates of rat kidney cells revealed that zinc also inhibited the kidney m-aconitase activity. However the inhibitory effect of zinc was more sensitive with the prostate m-aconitase activity. Zinc inhibition fit the competitive inhibitor model. The inhibitory effect of zinc occurred only with citrate as substrate and was specific for the citrate --> cis-aconitate reaction. Other cations (Ca2+, Mn2+, Cd2+) did not result in the inhibitory effects obtained with zinc. The presence of endogenous zinc inhibited the m-aconitase activity of the prostate mitochondrial preparations. Kidney preparations that contain lower endogenous zinc levels exhibited no endogenous inhibition of m-aconitase activity. Studies with pig prostate and seminal vesicle mitochondrial preparations also revealed that zinc was a competitive inhibitor against citrate of m-aconitase activity. The effects of zinc on purified beef heart m-aconitase verified the competitive inhibitor action of zinc. In contrast, zinc had no inhibitory effect on purified cytosolic aconitase. These studies reveal for the first time that zinc is a specific inhibitor of m-aconitase of mammalian cells. In prostate epithelial cells, in situ mitochondrial zinc levels inhibit m-aconitase activity, which provides a mechanism by which citrate oxidation is limited.
- 347Feng, W.; Cai, J.; Pierce, W. M.; Franklin, R. B.; Maret, W.; Benz, F. M.; Kang, Y. J. Metallothionein transfers zinc to mitochondrial-aconitase through a direct interaction in mouse hearts. Biochem. Biophys. Res. Commun. 2005, 332, 853– 858, DOI: 10.1016/j.bbrc.2005.04.170Google Scholar347Metallothionein transfers zinc to mitochondrial aconitase through a direct interaction in mouse heartsFeng, Wenke; Cai, Jian; Pierce, William M.; Franklin, Renty B.; Maret, Wolfgang; Benz, Frederick W.; Kang, Y. JamesBiochemical and Biophysical Research Communications (2005), 332 (3), 853-858CODEN: BBRCA9; ISSN:0006-291X. (Elsevier)Previous studies have shown that in a cell-free system, metallothionein (MT) releases zinc when the environment becomes oxidized and the released zinc is transferred to a zinc-binding protein if such a protein is present. However, it is unknown whether and how zinc transfers from MT to other proteins in vivo. The present study was undertaken to test the hypothesis that if zinc transfer from MT to other proteins occurs in vivo, the transfer would proceed through a direct interaction between MT and a specific group of proteins. The heart ext. obtained from MT-null mice was incubated with 65Zn-MT or 65ZnCl2 and the proteins receiving 65Zn were sepd. by blue-native PAGE (BN-PAGE) or SDS-PAGE, and detected by autoradiog. A unique 65Zn-binding band was obsd. from the 65Zn-MT-incubated, but not the 65ZnCl2-incubated prepn. The anal. using matrix assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometry revealed that mitochondrial aconitase (m-aconitase) was among the proteins accepting Zn directly from Zn-MT. The m-aconitase, not the cytosolic aconitase (c-aconitase), was co-immunopptd. with MT. This study demonstrates that MT transfers zinc to m-aconitase through a direct interaction.
- 348Maret, W. Zinc biochemistry: from a single zinc enzyme to a key element of life. Adv. Nutr. 2013, 4, 82– 91, DOI: 10.3945/an.112.003038Google Scholar348Zinc biochemistry: from a single zinc enzyme to a key element of lifeMaret, WolfgangAdvances in Nutrition (2013), 4 (1), 82-91CODEN: ANDUAW; ISSN:2156-5376. (American Society for Nutrition)A review. The nutritional essentiality of zinc for the growth of living organisms had been recognized long before zinc biochem. began with the discovery of zinc in carbonic anhydrase in 1939. Painstaking anal. work then demonstrated the presence of zinc as a catalytic and structural cofactor in a few hundred enzymes. In the 1980s, the field again gained momentum with the new principle of "zinc finger" proteins, in which zinc has structural functions in domains that interact with other biomols. Advances in structural biol. and a rapid increase in the availability of gene/protein databases now made it possible to predict zinc-binding sites from metal-binding motifs detected in sequences. This procedure resulted in the definition of zinc proteomes and the remarkable est. that the human genome encodes ∼3000 zinc proteins. More recent developments focus on the regulatory functions of zinc(II) ions in intra- and intercellular information transfer and have tantalizing implications for yet addnl. functions of zinc in signal transduction and cellular control. At least three dozen proteins homeostatically control the vesicular storage and subcellular distribution of zinc and the concns. of zinc(II) ions. Novel principles emerge from quant. investigations on how strongly zinc interacts with proteins and how it is buffered to control the remarkably low cellular and subcellular concns. of free zinc (II) ions. It is fair to conclude that the impact of zinc for health and disease will be at least as far-reaching as that of iron.
- 349Maret, W.; Jacob, C.; Vallee, B. L.; Fischer, E. H. Inhibitory sites in enzymes: Zinc removal and reactivation by thionein. Proc. Natl. Acad. Sci. U. S. A. 1999, 96, 1936– 1940, DOI: 10.1073/pnas.96.5.1936Google Scholar349Inhibitory sites in enzymes: zinc removal and reactivation by thioneinMaret, Wolfgang; Jacob, Claus; Vallee, Bert L.; Fischer, Edmond H.Proceedings of the National Academy of Sciences of the United States of America (1999), 96 (5), 1936-1940CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Thionein (T) has not been isolated previously from biol. material. However, it is generated transiently in situ by removal of zinc from metallothionein under oxidoreductive conditions, particularly in the presence of selenium compds. T very rapidly activates a group of enzymes in which zinc is bound at an inhibitory site. The reaction is selective, as is apparent from the fact that T does not remove zinc from the catalytic sites of zinc metalloenzymes. T instantaneously reverses the zinc inhibition with a stoichiometry commensurate with its known capacity to bind seven zinc atoms in the form of clusters in metallothionein. The zinc inhibition is much more pronounced than was previously reported, with dissocn. consts. in the low nanomolar range. Thus, T is an effective, endogenous chelating agent, suggesting the existence of a hitherto unknown and unrecognized biol. regulatory system. T removes the metal from an inhibitory zinc-specific enzymic site with a resultant marked increase of activity. The potential significance of this system is supported by the demonstration of its operations in enzymes involved in glycolysis and signal transduction.
- 350Bellomo, E.; Birla Singh, K.; Massarotti, A.; Hogstrand, C.; Maret, W. The metal face of protein tyrosine phosphatase 1B. Coord. Chem. Rev. 2016, 327–328, 70– 83, DOI: 10.1016/j.ccr.2016.07.002Google Scholar350The metal face of protein tyrosine phosphatase 1BBellomo, Elisa; Birla Singh, Kshetrimayum; Massarotti, Alberto; Hogstrand, Christer; Maret, WolfgangCoordination Chemistry Reviews (2016), 327-328 (), 70-83CODEN: CCHRAM; ISSN:0010-8545. (Elsevier B.V.)A review. A new paradigm in metallobiochem. describes the activation of inactive metalloenzymes by metal ion removal. Protein tyrosine phosphatases (PTPs) do not seem to require a metal ion for enzymic activity. However, both metal cations and metal anions modulate their enzymic activity. One binding site is the phosphate binding site at the catalytic cysteine residue. Oxyanions with structural similarity to phosphate, such as vanadate, inhibit the enzyme with nanomolar to micromolar affinities. In addn., zinc ions (Zn2+) inhibit with picomolar to nanomolar affinities. We mapped the cation binding site close to the anion binding site and established a specific mechanism of inhibition occurring only in the closed conformation of the enzyme when the catalytic cysteine is phosphorylated and the catalytic aspartate moves into the active site. We discuss this dual inhibition by anions and cations here for PTP1B, the most thoroughly investigated protein tyrosine phosphatase. The significance of the inhibition in phosphorylation signaling is becoming apparent only from the functions of PTP1B in the biol. context of metal cations as cellular signaling ions. Zinc ion signals complement redox signals but provide a different type of control and longer lasting inhibition on a biol. time scale owing to the specificity and affinity of zinc ions for coordination environments. Inhibitor design for PTP1B and other PTPs is a major area of research activity and interest owing to their prominent roles in metabolic regulation in health and disease, in particular cancer and diabetes. Our results explain the apparent dichotomy of both cations (Zn2+) and oxyanions such as vanadate inhibiting PTP1B and having insulin-enhancing ("anti-diabetic") effects and suggest different approaches, namely targeting PTPs in the cell by affecting their physiol. modulators and considering a metallodrug approach that builds on the knowledge of the insulin-enhancing effects of both zinc and vanadium compds.
- 351Chong, C. R.; Auld, D. S. Inhibition of carboxypeptidase A by D-penicillamine: mechanism and implications for drug design. Biochemistry 2000, 39, 7580– 7588, DOI: 10.1021/bi000101+Google Scholar351Inhibition of carboxypeptidase A by D-penicillamine: mechanism and implications for drug designChong, Curtis R.; Auld, David S.Biochemistry (2000), 39 (25), 7580-7588CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)Zinc metalloprotease inhibitors are usually designed to inactivate the enzyme by forming a stable ternary complex with the enzyme and active-site zinc. D-Cysteine inhibits carboxypeptidase, ZnCPD, by forming such a complex, with a Ki of 2.3 μM. In contrast, the antiarthritis drug D-penicillamine, D-PEN, which differs from D-Cys only by the presence of two Me groups on the β-carbon, inhibits ZnCPD by promoting the release of the active-site zinc. We have given the name catalytic chelator to such inhibitors. Inhibition is a two-step process characterized by formation of a complex with the enzyme (Ki(initial) = 1.2 mM) followed by release of the active-site zinc at rates up to 420-fold faster than the spontaneous release. The initial rate of substrate hydrolysis at completion of the second step also depends on D-PEN concn., reflecting formation of a thermodn. equil. governed by the stability consts. of chelator and apocarboxypeptidase for zinc (Ki(final) = 0.25 mM). The interaction of D-PEN and D-Cys with the active-site metal has been examd. by replacing the active-site zinc by a chromophoric cobalt atom. Both inhibitors perturb the d-d transitions of CoCPD in the 500-600 nm region within milliseconds of mixing but only the CoCPD·D-Cys complex displays a strong S → Co(II) charge-transfer band at 340 nm indicative of a metal-sulfur bond. While the D-Cys complex is stable, the CoCPD·D-PEN complex breaks down to apoenzyme and Co(D-PEN)2 with a half-life of 0.5 s. D-PEN is the first drug found to inhibit a metalloprotease by increasing the dissocn. rate const. of the active-site metal. The ability of D-PEN to catalyze metal removal from carboxypeptidase A and other zinc proteases suggests a possible mechanism of action in arthritis and Wilson's disease and may also underlie complications assocd. with its clin. use.
- 352Chong, C. R.; Auld, D. S. Catalysis of zinc transfer by D-penicillamine to secondary chelators. J. Med. Chem. 2007, 50, 5524– 5527, DOI: 10.1021/jm070803yGoogle Scholar352Catalysis of Zinc Transfer by D-Penicillamine to Secondary ChelatorsChong, Curtis R.; Auld, David S.Journal of Medicinal Chemistry (2007), 50 (22), 5524-5527CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)The antiarthritis drug D-penicillamine (D-PEN) catalyzes zinc(II) transfer from carboxypeptidase A to chelators such as thionein and EDTA at a rate const. up to 400-fold faster than the uncatalyzed release. Once D-PEN releases zinc(II) from enzyme stronger chelators can tightly bind zinc(II) leading to complete and essentially irreversible inhibition. D-PEN is the first drug to inhibit a zinc protease by catalyzing metal removal, and the name "catalytic chelation" is proposed for this mechanism.
- 353Zeng, J.; Heuchel, R.; Schaffner, W.; Kägi, J. H. R. Thionein (apometallothionein) can modulate DNA binding and transcription activation by zinc finger containing Sp1. FEBS Lett. 1991, 279, 310– 312, DOI: 10.1016/0014-5793(91)80175-3Google Scholar353Thionein (apometallothionein) can modulate DNA binding and transcription activation by zinc finger containing factor Sp1Zeng, Jin; Heuchel, Rainer; Schaffner, Walter; Kaegi, Jeremias H. R.FEBS Letters (1991), 279 (2), 310-12CODEN: FEBLAL; ISSN:0014-5793.A no. of transcription factors contain so-called Zn finger domains for the interaction with their cognate DNA sequence. Removal of the Zn ions complexed in these Zn fingers abrogates DNA binding and transcription activation. The hypothesis that the activity of transcription factors could be regulated by physiol. chelators of Zn was tested. A prominent candidate for such a chelator is the cysteine-rich protein thionein (apometallothionein) that is inducible by heavy metal loads, and by other environmental stimuli. DNA binding and in vitro transcription assays show that thionein indeed can inactivate the Zn finger-contg. Sp1 in a reversible manner. By contrast, transcription factor Oct-1, which binds DNA via a homeo-domain, i.e. a helix-turn-helix motif not involving Zn ions, is refractory to thionein action. It is proposed that modulation of intracellular thionein concn. is used for the coordinated regulation of a large subset of genes whose transcription depends on Zn finger proteins.
- 354Zeng, J.; Vallee, B. L.; Kägi, J. H. R. Zinc transfer from transcription factor IIIA fingers to thionein clusters. Proc. Natl. Acad. Sci. U. S. A. 1991, 88, 9984– 9988, DOI: 10.1073/pnas.88.22.9984Google Scholar354Zinc transfer from transcription factor IIIA fingers to thionein clustersZeng, Jin; Vallee, Bert L.; Kaegi, Jeremias H. R.Proceedings of the National Academy of Sciences of the United States of America (1991), 88 (22), 9984-8CODEN: PNASA6; ISSN:0027-8424.The rapid induction of thionein (apometallothionein) by many endogenous stimuli such as steroid hormones, cytokines, and second messengers suggests that this cysteine-rich, metal-binding protein participates in an as yet undefined role in cellular regulatory processes. This study demonstrates with DNA and RNA binding assays and in vitro transcription measurements that thionein suppresses the binding of the Xenopus laevis zinc finger transcription factor IIIA (TFIIIA) to 5 S RNA and to the 5 S RNA gene and abrogates the capacity of synthesis of 5 S RNA. The effect is reversed by the addn. of zinc and is not obsd. in the TFIIIA-independent transcription of a tRNA gene by the same RNA polymerase. In view of the strong tendency of thionein to complex posttransition metals such as zinc, one effect of its enhanced synthesis in vivo could be to reduce the intracellular disposability of zinc and thus modulate the actions of zinc-dependent enzymes and proteins, most notably those of the zinc finger transcription factors.
- 355Cano-Gauci, D. F.; Sarkar, B. Reversible zinc exchange between metallothionein and the estrogen receptor zinc finger. FEBS Lett. 1996, 386, 1– 4, DOI: 10.1016/0014-5793(96)00356-0Google Scholar355Reversible zinc exchange between metallothionein and the estrogen receptor zinc fingerCano-Gauci D F; Sarkar BFEBS letters (1996), 386 (1), 1-4 ISSN:0014-5793.We report here the first demonstration that reversible metal exchange occurs between metallothionein (MT) and full-length estrogen receptor (ER). Specific binding of ER to estrogen response element is inhibited in the presence of 40 microM thionein and restored by 120 microM zinc. Moreover, ER in metal-depleted nuclear extracts exhibits reduced DNA binding which can be restored by 140 microM native MT. Hence, thionein inhibits DNA binding by abstracting zinc from functional ER while native MT is capable of restoring binding to metal-depleted extracts by donating metal to ER. This indicates MT may be an important physiological regulator of intracellular zinc and/or other metals.
- 356Petering, D. H.; Zhu, J.; Krezoski, S.; Meeusen, J.; Kiekenbush, C.; Krull, S.; Specher, T.; Dughish, M. Apo-metallothionein emerging as a major player in the cellular activities of metallothionein. Exp. Biol. Med. (London, U. K.) 2006, 231, 1528– 1534, DOI: 10.1177/153537020623100912Google Scholar356Apo-metallothionein emerging as a major player in the cellular activities of metallothioneinPetering, David H.; Zhu, Jianyu; Krezoski, Susan; Meeusen, Jeffrey; Kiekenbush, Christy; Krull, Sara; Specher, Todd; Dughish, MuniraExperimental Biology and Medicine (Maywood, NJ, United States) (2006), 231 (9), 1528-1534CODEN: EBMMBE; ISSN:1535-3702. (Society for Experimental Biology and Medicine)A review. Observations of apo-metallothionein (apo-MT) have been made under a variety of physiol. circumstances, including Zn deficiency in cell culture and in rodents, cellular induction of MT by dexamethasone with concurrent Zn deficiency, a variety of tumors under normal Zn conditions, MT induction by Zn and Bi citrate, induction of hepatic MT after tumor cell injection into nude mice, and overexpression of cardiac MT in MT transgenic mice. Expts. demonstrating both the lability of Zn and Cu bound to MT and the cellular stability of apo-MT are described to help rationalize the widespread presence of this metal-depleted species. Finally, comparative in vitro and cellular expts. have examd. the relative reactivity of Zn- and apo-MT with NO species, showing that apo-MT is much more reactive chem. and that in cells it may be a principal reactive species within the MT pool.
- 357Kluska, K.; Adamczyk, J.; Krężel, A. Metal binding properties of zinc fingers with a naturally altered metal binding site. Metallomics 2018, 10, 248– 263, DOI: 10.1039/C7MT00256DGoogle Scholar357Metal binding properties of zinc fingers with a naturally altered metal binding siteKluska, Katarzyna; Adamczyk, Justyna; Krezel, ArturMetallomics (2018), 10 (2), 248-263CODEN: METAJS; ISSN:1756-591X. (Royal Society of Chemistry)A review. Zinc fingers (ZFs) are among the most abundant motifs found in proteins, and are commonly known for their structural role. Classical ZFs (CCHH) are part of the transcription factors that participate in DNA binding. Although biochem. studies of classical ZFs have a long history, there is limited knowledge about the sequential and structural diversity of ZFs. We have found that classical ZFs, with metal binding sites consisting of amino acids other than conserved Cys or His residues, are frequently encoded in the human genome, and we refer to these peptides as ZFs with a naturally altered metal binding site. The biol. role of the altered ZFs remains undiscovered. In this study, we characterized nine natural XCHH, CXHH, CCXH and CCHX ZFs in terms of their Zn(II) and Co(II) binding properties, such as complex stoichiometry, spectroscopic properties and metal-to-peptide affinity. We revealed that XCHH and CXHH ZFs form ML complexes that are 4-5 orders of magnitude weaker in comparison to CCHH ZFs. Nevertheless, spectroscopic studies demonstrate that, depending on the altered position, they may adopt an open coordination geometry with one or two water mols. bound to a central metal ion, which has not been demonstrated in natural ZFs before. Stability data show that both CCXH and CCHX peptides have high Zn(II) affinity (with a Kd of 10-9 to 10-11 M), suggesting their potential biol. function. This study is a comprehensive overview of the relationship between the sequence, structure, and stability of ZFs.
- 358Apuy, J. L.; Chen, X.; Russell, D. H.; Baldwin, T. O.; Giedroc, D. P. Ratiometric pulsed alkylation/mass spectrometry of the cysteine pairs in individual zinc fingers of MRE-binding transcription factor-1 (MTF-1) as a probe of zinc chelate stability. Biochemistry 2001, 40, 15164– 15175, DOI: 10.1021/bi0112208Google Scholar358Ratiometric Pulsed Alkylation/Mass Spectrometry of the Cysteine Pairs in Individual Zinc Fingers of MRE-Binding Transcription Factor-1 (MTF-1) as a Probe of Zinc Chelate StabilityApuy, Julius L.; Chen, Xiaohua; Russell, David H.; Baldwin, Thomas O.; Giedroc, David P.Biochemistry (2001), 40 (50), 15164-15175CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)Metal-response element (MRE)-binding transcription factor-1 (MTF-1) is a zinc-regulated transcriptional activator of metallothionein (MT) genes in mammalian cells. The MRE-binding domain of MTF-1 (MTF-zf) has six canonical Cys2-His2 zinc finger domains that are distinguished on the basis of their apparent affinities for zinc and their specific roles in MRE-binding. In this paper, pulsed alkylation of the zinc-liganding cysteine thiolate pairs with the sulfhydryl-specific alkylating reagent d5-N-ethylmaleimide (d5-NEM) is used as a residue-specific probe of the relative stabilities of the individual zinc finger coordination complexes in Zn6 MTF-zf. A chase with excess H5-N-ethylmaleimide (H5-NEM) to fully derivatize MTF-zf concomitant with complete proteolysis, followed by MALDI-TOF mass spectrometry allows quantitation of the mole fraction of d5,d5-, d5,H5-, and H5,H5-NEM derivatized peptides corresponding to each individual zinc finger domain as a function of d5-NEM pulse time. This expt. establishes the hierarchy of cysteine thiolate reactivity in MTF-zf as F5 > F6 » F1 > F2 ≈ F3 ≈ F4. The apparent second-order rate of reaction of F1 thiolates is comparable to that detd. for the DNA binding domain of Sp1, Zn3 Sp1-zf, under identical soln. conditions. The reactivities of all Cys residues in MTF-zf are significantly reduced when bound to an MREd-contg. oligonucleotide. An identical expt. carried out with Zn5 MTF-zf26, an MTF-zf domain lacking the N-terminal F1 zinc finger, reveals that MTF-zf26 binds to the MREd very weakly, and is characterized by strongly increased reactivity of nonadjacent F4 thiolates. These findings are discussed in the context of existing models for metalloregulation by MTF-1.
- 359Potter, B. M.; Feng, L. S.; Parasuram, P.; Matskevich, V. A.; Wilson, J. A.; Andrews, G. K.; Laity, J. H. The six zinc fingers of metal-responsive element binding transcription factor-1 form stable and quasi-ordered structures with relatively small differences in zinc affinities. J. Biol. Chem. 2005, 280, 28529– 28540, DOI: 10.1074/jbc.M505217200Google Scholar359The Six Zinc Fingers of Metal-responsive Element Binding Transcription Factor-1 Form Stable and Quasi-ordered Structures with Relatively Small Differences in Zinc AffinitiesPotter, Belinda M.; Feng, Linda S.; Parasuram, Priya; Matskevich, Viktor A.; Wilson, Jed A.; Andrews, Glen K.; Laity, John H.Journal of Biological Chemistry (2005), 280 (31), 28529-28540CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)Six Cys2His2 zinc fingers (F1-6) comprise the DNA binding domain of metal-responsive element binding transcription factor-1 (MTF-1). F1-6 is necessary for basal and zinc-induced expression of metallothionein genes. Anal. of NMR structural and dynamic data for an F1-6 protein construct demonstrates that each zinc finger adopts a stable ββα fold in the presence of stoichiometric Zn(II), provided that all cysteine ligands are in a reduced state. Parallel studies of protein constructs spanning the four N-terminal core DNA binding fingers (F1-4) and two C-terminal low DNA affinity fingers (F5-6) reveal similar stable zinc finger structures. In both the F1-6 and F5-6 proteins, the finger 5 cysteines were found to readily oxidize at neutral pH. Detailed spectral d. and hydrodynamic anal. of 15N relaxation data revealed quasi-ordered anisotropic rotational diffusion properties of the six F1-6 zinc fingers that could influence MTF-1 DNA binding function. A more general effect on the rotational diffusion properties of Cys2His2 zinc fingers was also uncovered that is dependent upon the position of each finger within multifinger domains. Anal. of NMR 1H-15N-heteronuclear single quantum coherence spectral peak intensities measured as a function of added Zn(II) in conjunction with Zn(II) binding modeling studies indicated that the Zn(II) affinities of all MTF-1 zinc fingers are within ∼10-50-fold. These analyses further suggested that metal sensing by MTF-1 in eukaryotic cells involves multiple zinc fingers and occurs over a 100-fold or less range of accessible Zn(II) concn.
- 360Vallee, B. L. Historical review and perspectives. EXS 1979, 34, 19– 40, DOI: 10.1007/978-3-0348-6493-0_1Google ScholarThere is no corresponding record for this reference.
- 361Jiang, L.-J.; Maret, W.; Vallee, B. L. The ATP-metallothionein complex. Proc. Natl. Acad. Sci. U. S. A. 1998, 95, 9146– 9149, DOI: 10.1073/pnas.95.16.9146Google Scholar361The ATP-metallothionein complexJiang, Li-Juan; Maret, Wolfgang; Vallee, Bert L.Proceedings of the National Academy of Sciences of the United States of America (1998), 95 (16), 9146-9149CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)We have previously shown that glutathione (GSH) and glutathione disulfide interact with metallothionein (MT) and modulate its capacity to donate and transfer zinc. In this paper, we show that ATP also forms a 1:1 complex with MT (Kd = 176±33 μM, pH 7.4) that enhances the transfer of zinc to zinc-depleted sorbitol dehydrogenase, increases the rate of thiol-disulfide interchange with Ellman's reagent [5,5'-dithiobis (Z-nitrobenzoic acid)], and changes the apparent shape of the protein. GTP produces almost identical effects. The corresponding di- or monophosphates and pyrimidine nucleotides, however, neither bind as strongly as ATP nor enhance zinc transfer. Carbamoylation of MT lysines abolishes ATP binding, indicating that these highly conserved residues are part of the binding site. GSH decreases, whereas glutathione disulfide increases, ATP binding. The interaction of MT with two crit. cellular ligands, i.e., GSH and ATP, and ensuing effects on zinc transfer and reactivity suggest that MT is not merely a cellular zinc buffer but, rather, actively participates in zinc distribution. Apparently, when isolated, MT lacks two important effectors that affect its redox behavior and function. The magnitude of the binding const. and the cellular concn. of ATP indicate that in the cell MT could be essentially satd. with ATP at low concns. of GSH. Both the redox and energy states of the cell seem to control zinc distribution from MT, but their relative contributions require further studies.
- 362Zangger, K.; Öz, G.; Armitage, I. M. Re-evaluation of the binding of ATP to metallothioneins. J. Biol. Chem. 2000, 275, 7534– 7538, DOI: 10.1074/jbc.275.11.7534Google Scholar362Re-evaluation of the binding of ATP to metallothioneinZangger, Klaus; Oz, Gulin; Armitage, Ian M.Journal of Biological Chemistry (2000), 275 (11), 7534-7538CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)In a recent paper Jiang et al. reported that metallothionein interacts with ATP to form a 1:1 complex with a dissocn. const. of Kd = 176 ± 33 μM at pH 7.4. In an effort to characterize further this interaction using NMR spectroscopy, titrn. calorimetry, gel-filtration chromatog., affinity chromatog., and ultrafiltration, we were unable to find any evidence for the binding of ATP to metallothionein.
- 363Kangur, L.; Palumaa, P. The effects of physiologically important non-metallic ligands in the reactivity of metallothionein towards 5,5′-dithiobis(2-nitrobenzoic acid). Eur. J. Biochem. 2001, 268, 4979– 4984, DOI: 10.1046/j.0014-2956.2001.02430.xGoogle Scholar363The effects of physiologically important nonmetallic ligands in the reactivity of metallothionein towards 5,5'-dithiobis(2-nitrobenzoic acid). A new method for the determination of ligand interactions with metallothioneinKangur, Liina; Palumaa, PeepEuropean Journal of Biochemistry (2001), 268 (18), 4979-4984CODEN: EJBCAI; ISSN:0014-2956. (Blackwell Science Ltd.)The reaction of Cd5Zn2-metallothionein (MT) with 5,5'-dithiobis(2-nitrobenzoic acid) (Nbs2) has been studied at different reagent stoichiometries, pH and temp. conditions and in the presence of several ligands. At stoichiometries of Nbs2 to MT from 0.5 to 5, the reaction followed first order kinetics. The first order rate consts. obtained were independent from the concn. of Nbs2 but were linearly dependent on the concn. of MT. At higher Nbs2/MT stoichiometries, the reaction deviates from first order kinetics and the obsd. rate const. increases. The reactivity of MT towards Nbs2 has been probed at 4 μM concn. of both reagents where the reaction is monophasic and is characterized by a linear Arrhenius plot (Ea = 45.8±2.7 kJ·mol-1). It has been demonstrated that metal release at low pH or subtraction from MT by EDTA substantially increases the reactivity of MT towards Nbs2. At the same time, a no. of nonmetallic ligands moderately accelerate the reaction of MT with Nbs2 and hyperbolic dose-response curves were obtained. The data have been interpreted with the binding of ligands to MT and following MT. Ligand binding consts. were calcd. as follows: ATP, K = 0.31±0.06 mM; ADP, K = 0.26±0.07 mM. Several compds. such as AMP, S-methylglutathione, and phosphate had no effect on the reaction, but Zn2+ ions showed an inhibitory effect at micromolar concns.
- 364Kwok, F.; Churchich, J. E. Brain pyridoxal kinase. Purification, substrate specificities, and sensitized photodestruction of an essential histidine. J. Biol. Chem. 1979, 254, 6489– 6495, DOI: 10.1016/S0021-9258(18)50394-XGoogle Scholar364Brain pyridoxal kinase. Purification, substrate specificities, and sensitized photodestruction of an essential histidineKwok, Francis; Churchich, Jorge E.Journal of Biological Chemistry (1979), 254 (14), 6489-95CODEN: JBCHA3; ISSN:0021-9258.Pyridoxal kinase (I) was purified 2000-fold from pig brain. The enzyme prepn. migrates as a single protein and activity band on anal. gel electrophoresis. I, 60,000 mol. wt., catalyzes the phosphorylation of pyridoxal (Km = 2.5 × 10-5 M) and pyridoxine (Km = 1.7 × 10-5 M). Pyridoxamine is not a substrate of the purified kinase. Irradn. of I in the presence of riboflavin leads to irreversible loss of catalytic activity. Riboflavin binds to I with a KD = 5 μM as shown by fluorometric titrns. Singlet excited O, generated by energy transfer from the lowest triplet of riboflavin to O, acts as the oxidizing agent of approx. 1 histidine residue/mol of I. The amino acid residues tyrosine, tryptophan, and cysteine are not photooxidized by the sensitizer bound to the enzyme. Probably, histidine is involved in the binding of the substrate ATP to the catalytic site of I.
- 365Nakano, H.; McCormick, D. B. Stereospecificity of the metal.ATP complex in flavokinase from rat small intestine. J. Biol. Chem. 1991, 266, 22125– 22128, DOI: 10.1016/S0021-9258(18)54542-7Google Scholar365Stereospecificity of the metal·ATP complex in flavokinase from rat small intestineNakano, Hideko; McCormick, Donald B.Journal of Biological Chemistry (1991), 266 (33), 22125-8CODEN: JBCHA3; ISSN:0021-9258.Transfer of the γ-phosphoryl group of ATP to riboflavin is catalyzed by flavokinase, which prefers Zn(II), and is essential in the biosynthesis of the flavocoenzyme, FMN. To study the mechanism and steric disposition of ATP binding, adenosine 5'-O-(2-thiotriphosphate) (ATPβS) and adenosine 5'-O-(3-thiotriphosphate) (ATPγS) were tested in comparison to ATP with representative divalent metal ions. Activities with 0.1 mM Zn(II) for 1 mM ATP thio analogs compared to ATP with flavokinase were 60% for the S-isomer of ATPβS, 312% for the R-isomer of ATPβS, and 14% for ATPγS. With Mg(II), flavokinase showed stereospecificity for the R-isomer of ATPβS with a Vmax ratio for the stereoisomers of S/R = 0.125. With Cd(II), the enzyme showed preference for the S-isomer of ATPβS with a Vmax ratio, S/R = 1.261. The Vmax ratio progressively increases from Mg(II) to Cd(II) in the order of affinity for sulfur. The ratios, (Vmax/Km)Mg/(Vmax/Km)Cd, for the diastereomers of ATPβS were expectedly >1 for one diastereomer (R = 6.597) and <1 for the other (S = 0.292). Activities with ATPγS were much slower than with ATP or ATPβS. With Mg(II), the γ analog was a good substrate; however, with Cd(II), it was hardly active. Altogether these results indicate that flavokinase uses the Λ,β,γ-bidentate Zn·ATP as a substrate.
- 366Lerner, F.; Niere, M.; Ludwig, A.; Ziegler, M. Structural and functional characterization of human NAD kinase. Biochem. Biophys. Res. Commun. 2001, 288, 69– 74, DOI: 10.1006/bbrc.2001.5735Google Scholar366Structural and Functional Characterization of Human NAD KinaseLerner, Felicitas; Niere, Marc; Ludwig, Antje; Ziegler, MathiasBiochemical and Biophysical Research Communications (2001), 288 (1), 69-74CODEN: BBRCA9; ISSN:0006-291X. (Academic Press)NADP is essential for biosynthetic pathways, energy, and signal transduction. Its synthesis is catalyzed by NAD kinase. Very little is known about the structure, function, and regulation of this enzyme from multicellular organisms. We identified a human NAD kinase cDNA and the corresponding gene using available database information. A cDNA was amplified from a human fibroblast cDNA library and functionally overexpressed in Escherichia coli. The obtained cDNA, slightly different from that deposited in the database, encodes a protein of 49 kDa. The gene is expressed in most human tissues, but not in skeletal muscle. Human NAD kinase differs considerably from that of prokaryotes by subunit mol. mass (49 kDa vs. 30-35 kDa). The catalytically active homotetramer is highly selective for its substrates, NAD and ATP. It did not phosphorylate the nicotinic acid deriv. of NAD (NAAD) suggesting that the potent calcium-mobilizing pyridine nucleotide NAADP is synthesized by an alternative route. (c) 2001 Academic Press.
- 367McCormick, D. Micronutrient cofactor research with extensions to applications. Nutr. Res. Rev. 2002, 15, 245– 262, DOI: 10.1079/NRR200241Google Scholar367Micronutrient cofactor research with extensions to applicationsMcCormick, Donald B.Nutrition Research Reviews (2002), 15 (2), 245-262CODEN: NREREX; ISSN:0954-4224. (CABI Publishing)A review. Following identification of essential micronutrients, there has been a continuum of research aimed at revealing their absorption, transport, utilization as cofactors, and excretion and secretion. Among those cases that have received our attention are vitamin B6, riboflavin, biotin, lipoate, ascorbate, and certain metal ions. Circulatory transport and cellular uptake of the water-sol. vitamins exhibit relative specificity and facilitated mechanisms at physiol. concns. Isolation of enzymes and metabolites from micro-organisms and mammals has provided information on pathways involved in cofactor formation and metab. Kinases catalyzing phosphorylation of B6 and riboflavin have a preference for Zn2+ in stereospecific chelates with ATP. The synthetase for FAD prefers Mg2+. The FMN-dependent oxidase that converts the 5'-phosphates of pyridoxine and of pyridoxamine to pyridoxal phosphate is a connection between B6 and riboflavin and is a primary control point for conversion of B6 to its coenzyme. Sequencing and cloning of a side-chain oxidase for riboflavin was achieved. Details on binding and function have been delineated for some cofactor systems, esp. in several flavoproteins. There is both photochem. oxidn. and oxidative catabolism of B6 and riboflavin. Both biotin and lipoate undergo oxidn. of their acid side chains with redox cleavage of the rings. Applications from our findings include the development of affinity absorbents, enhanced drug delivery, delineation of residues in biopolymer modification, pathogen photoinactivation in blood components, and input into human dietary recommendations. Ongoing and future research in the cofactor arena can be expected to add to this panoply. At the mol. level, the way in which the same cofactor can participate in diverse catalytic reactions resides in interactions with surrounding enzyme strictures that must be detd. case by case. At the level of human intake, more knowledge is desirable for making micronutrient recommendations based on biochem. indicators, esp. for the span between infancy and adulthood.
- 368Li, M. H.; Kwok, F.; Chang, W. R.; Lau, C. K.; Zhang, J. P.; Lo, S. C.; Jiang, T.; Liang, D. C. Crystal structure of brain pyridoxal kinase, a novel member of the ribokinase superfamily. J. Biol. Chem. 2002, 277, 46385– 46390, DOI: 10.1074/jbc.M208600200Google Scholar368Crystal structure of brain pyridoxal kinase, a novel member of the ribokinase superfamilyLi, Ming-Hui; Kwok, Francis; Chang, Wen-Rui; Lau, Chi-Kong; Zhang, Ji-Ping; Lo, Samuel C. L.; Jiang, Tao; Liang, Dong-CaiJournal of Biological Chemistry (2002), 277 (48), 46385-46390CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)The 3-dimensional structures of sheep brain pyridoxal kinase (I) and its complex with ATP were elucidated in the dimeric form at 2.1 and 2.6 Å, resp. The results showed that I, as an enzyme obeying random sequential kinetics in catalysis, did not possess a lid shape structure common to all kinases in the ribokinase superfamily. This finding was shown to be in line with the condition that I binds substrates with variable sizes of chem. groups at position 4 of vitamin B6 and its derivs. In addn., I contained a 12-residue peptide loop in the active site for the prevention of premature hydrolysis of ATP. Conserved amino acid residues Asp-118 and Tyr-127 in the peptide loop could be moved to a position covering the nucleotide after its binding so that its chance to hydrolyze in the aq. environment of the active site was reduced. With respect to the evolutionary trend of kinases, the existence of this loop in I could be classified as an independent category in the ribokinase superfamily according to the structural feature found and mechanism followed in catalysis.
- 369Bauer, S.; Kemter, K.; Bacher, A.; Huber, R.; Fischer, M.; Steinbacher, S. Crystal structure of Schizosaccharomyces pombe riboflavin kinase reveals a novel ATP and riboflavin-binding fold. J. Mol. Biol. 2003, 326, 1463– 1473, DOI: 10.1016/S0022-2836(03)00059-7Google Scholar369Crystal Structure of Schizosaccharomyces pombe Riboflavin Kinase Reveals a Novel ATP and Riboflavin-Binding FoldBauer, Stefanie; Kemter, Kristina; Bacher, Adelbert; Huber, Robert; Fischer, Markus; Steinbacher, StefanJournal of Molecular Biology (2003), 326 (5), 1463-1473CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Science Ltd.)The essential redox cofactors riboflavin monophosphate (FMN) and FAD are synthesized from their precursor, riboflavin, in sequential reactions by the metal-dependent riboflavin kinase and FAD synthetase. Here, we describe the 1.6 Å crystal structure of the Schizosaccharomyces pombe riboflavin kinase. The enzyme represents a novel family of phosphoryl transferring enzymes. It is a monomer comprising a central β-barrel clasped on one side by two C-terminal helixes that display an L-like shape. The opposite side of the β-barrel serves as a platform for substrate binding as demonstrated by complexes with ADP and FMN. Formation of the ATP-binding site requires significant rearrangements in a short α-helix as compared to the substrate free form. The diphosphate moiety of ADP is covered by the glycine-rich flap I formed from parts of this α-helix. In contrast, no significant changes are obsd. upon binding of riboflavin. The ribityl side-chain might be covered by a rather flexible flap II. The unusual metal-binding site involves, in addn. to the ADP phosphates, only the strictly conserved Thr45. This may explain the preference for zinc obsd. in vitro.
- 370Churchich, J. E.; Scholz, G. F.; Kwok, F. Activation of pyridoxal kinase by metallothionein. Biochim. Biophys. Acta, Protein Struct. Mol. Enzymol. 1989, 996, 181– 186, DOI: 10.1016/0167-4838(89)90245-8Google Scholar370Activation of pyridoxal kinase by metallothioneinChurchich, Jorge E.; Scholz, Glen; Kwok, FrancisBiochimica et Biophysica Acta, Protein Structure and Molecular Enzymology (1989), 996 (3), 181-6CODEN: BBAEDZ; ISSN:0167-4838.Brain pyridoxal kinase, which uses ATP complexed to either Zn(II) or Co(II) as substrates, displays high catalytic activity in the presence of Zn-thionein and Co-thionein. Several steps intervene in the process of pyridoxal kinase activation, i.e., binding of Zn2+ to ATP and interaction between Zn-ATP and the enzyme. Equil. binding studies show that ATP mediates the release of Zn2+ from the metal-thiolate clusters of the thioneins, whereas spectroscopic measurements conducted on Co-thionein reveal that the absorption transitions corresponding to the metal-thiolate of the protein are perturbed by ATP. The binding Zn-ATP to the kinase proceeds with a ΔG = -6.3 kcal/mol as demonstrated by fluorometric titrns. Direct interaction between the kinase and derivatized-metallothionein could not be detected by emission anisotropy measurements, indicating that juxtaposition of the proteins does not influence the exchange of metal ions. Since the concn. of free Zn in several mammalian tissues is <1 nM, it is postulated that under in vivo conditions the concn. of metallothionein regulates the catalytic activity of pyridoxal kinase.
- 371Sigel, H.; Griesser, R. Nucleoside 5′triphosphates: self-association, acid-base, and metal ion-binding properties in solution. Chem. Soc. Rev. 2005, 34, 875– 900, DOI: 10.1039/b505986kGoogle Scholar371Nucleoside 5'-triphosphates: self-association, acid-base, and metal ion-binding properties in solutionSigel, Helmut; Griesser, RolfChemical Society Reviews (2005), 34 (10), 875-900CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)A review. ATP (ATP4-) and related nucleoside 5'-triphosphates (NTP4-) serve as substrates in the form of metal ion complexes in enzymic reactions taking part thus in central metabolic processes. With this in mind, the coordination chem. of NTPs is critically reviewed and the conditions are defined for studies aiming to describe the properties of monomeric complexes because at higher concns. (>1 mM) self-stacking may take place. The metal ion (M2+) complexes of purine-NTPs are more stable than those of pyrimidine-NTPs; this stability enhancement is attributed, in accord with NMR studies, to macrochelate formation of the phosphate-coordinated M2+ with N7 of the purine residue and the formation degrees of the resulting isomeric complexes are listed. Furthermore, the formation of mixed-ligand complexes (including also those with buffer mols.), the effect of a reduced solvent polarity on complex stability and structure (giving rise to selectivity), the use of nucleotide analogs as antiviral agents, and the effect of metal ions on group transfer reactions are summarized.
- 372Si, M.; Lang, J. The roles of metallothioneins in carcinogenesis. J. Hematol. Oncol. 2018, 11, 107, DOI: 10.1186/s13045-018-0645-xGoogle Scholar372The roles of metallothioneins in carcinogenesisSi, Manfei; Lang, JingheJournal of Hematology & Oncology (2018), 11 (), 107/1-107/20CODEN: JHOOAO; ISSN:1756-8722. (BioMed Central Ltd.)A review. Metallothioneins (MTs) are small cysteine-rich proteins that play important roles in metal homeostasis and protection against heavy metal toxicity, DNA damage, and oxidative stress. In humans, MTs have four main isoforms (MT1, MT2, MT3, and MT4) that are encoded by genes located on chromosome 16q13. MT1 comprises eight known functional (sub)isoforms (MT1A, MT1B, MT1E, MT1F, MT1G, MT1H, MT1M, and MT1X). Emerging evidence shows that MTs play a pivotal role in tumor formation, progression, and drug resistance. However, the expression of MTs is not universal in all human tumors and may depend on the type and differentiation status of tumors, as well as other environmental stimuli or gene mutations. More importantly, the differential expression of particular MT isoforms can be utilized for tumor diagnosis and therapy. This review summarizes the recent knowledge on the functions and mechanisms of MTs in carcinogenesis and describes the differential expression and regulation of MT isoforms in various malignant tumors. The roles of MTs in tumor growth, differentiation, angiogenesis, metastasis, microenvironment remodeling, immune escape, and drug resistance are also discussed. Finally, this review highlights the potential of MTs as biomarkers for cancer diagnosis and prognosis and introduces some current applications of targeting MT isoforms in cancer therapy. The knowledge on the MTs may provide new insights for treating cancer and bring hope for the elimination of cancer.
- 373Vasconcelos, M. H.; Tam, S. C.; Hesketh, J. E.; Reid, M.; Beattie, J. H. Metal and tissue-dependent relationship between metallothionein mRNA and protein. Toxicol. Appl. Pharmacol. 2002, 182, 91– 97, DOI: 10.1006/taap.2002.9428Google Scholar373Metal- and Tissue-Dependent Relationship between Metallothionein mRNA and ProteinVasconcelos, M. Helena; Tam, Shuk-Ching; Hesketh, John E.; Reid, Martin; Beattie, John H.Toxicology and Applied Pharmacology (2002), 182 (2), 91-97CODEN: TXAPA9; ISSN:0041-008X. (Elsevier Science)Metallothionein (MT) expression is transcriptionally regulated but recent evidence suggests that translation of MT mRNA may be regulated under some circumstances (1995). A systematic investigation of MT mRNA, protein, and metal levels in liver and kidney of cadmium- or copper-treated rats was made to further understand the relationship between mRNA and protein in particular. Adult rats were injected once with either Cd (8.9 μmol/kg) or Cu2+ (8.7 μmol/kg) as the chloride salts, and the liver and kidney concns. of MT-1 and MT-2 mRNA, total sol. MT protein, and tissue Cd, Cu, and Zn were monitored over 48-72 h. The metal compn. in the sol. MT protein fraction was also analyzed by online size-exclusion chromatog.-ICP/MS. Discrepancies between mRNA and protein levels were found in both tissues, but particularly in kidney. Cd treatment significantly increased renal MT-1 and MT-2 mRNA levels but protein was unaffected. In contrast, Cu actually decreased renal MT-1 and MT-2 mRNA but significantly increased MT protein. Cd induced considerably more MT-1 than MT-2 mRNA in liver, but induction of both isoforms was similar in kidney and in liver of Cu-treated rats. Changes in tissue metal levels tended to reflect MT protein levels and Cd appeared to bind to existing MT in the kidney. The results support the contention that MT protein levels often bear no clear relationship with mRNA levels and emphasizes the importance of measuring both in studies of MT expression.
- 374Raudenska, M.; Gumulec, J.; Podlaha, O.; Sztalmachova, M.; Babula, P.; Eckschlager, T.; Adam, V.; Kizek, R.; Masarik, M. Metallothionein polymorphisms in pathological processes. Metallomics 2014, 6, 55– 68, DOI: 10.1039/C3MT00132FGoogle Scholar374Metallothionein polymorphisms in pathological processesRaudenska, Martina; Gumulec, Jaromir; Podlaha, Ondrej; Sztalmachova, Marketa; Babula, Petr; Eckschlager, Tomas; Adam, Vojtech; Kizek, Rene; Masarik, MichalMetallomics (2014), 6 (1), 55-68CODEN: METAJS; ISSN:1756-591X. (Royal Society of Chemistry)A review. Metallothioneins (MTs) are a class of metal-binding proteins characterized by a high cysteine content and low mol. wt. MTs play an important role in metal metab. and protect cells against the toxic effects of radiation, alkylating agents and oxygen free radicals. The evidence that individual genetic characteristics of MTs play an important role in physiol. and pathol. processes assocd. with antioxidant defense and detoxification inspired targeted studies of genetic polymorphisms in a clin. context. In recent years, common MT polymorphisms were identified and assocd. with, particularly, western lifestyle diseases such as cancer, complications of atherosclerosis, and type 2 diabetes mellitus along with related complications. This review summarizes all evidence regarding MT polymorphisms of major human MTs (MT1, MT2, MT3 and MT4), their relation to pathol. processes, and outlines specific applications of MTs as a set of genetic markers for certain pathologies.
- 375West, A. K.; Stallings, R.; Hildebrand, C. E.; Chiu, R.; Karin, M.; Richards, R. I. Human metallothionein genes: structure of the functional locus at 16q13. Genomics 1990, 8, 513– 518, DOI: 10.1016/0888-7543(90)90038-VGoogle Scholar375Human metallothionein genes: structure of the functional locus at 16q13West, A. K.; Stallings, R.; Hildebrand, C. E.; Chiu, R.; Karin, M.; Richards, R. I.Genomics (1990), 8 (3), 513-18CODEN: GNMCEP; ISSN:0888-7543.The functional human metallothionein (MT) genes are located on chromosome 16q13. The functional human MT locus was mapped by isolation and restriction digest mapping of cloned DNA. The mapped region contains all sequences on chromosome 16 that hybridize to metallothionein gene probes and comprises 14 tightly linked MT genes, 6 of which have not been previously described. This anal. defines the genetic limits of metallothionein functional diversity in the human genome.
- 376Stennard, F. A.; Holloway, A. F.; Hamilton, J.; West, A. K. Characterisation of six additional human metallothionein genes. Biochim. Biophys. Acta, Gene Struct. Expression 1994, 1218, 357– 365, DOI: 10.1016/0167-4781(94)90189-9Google Scholar376Characterization of six additional human metallothionein genesStennard, Fiona A.; Holloway, Adele F.; Hamilton, Jenny; West, Adrian K.Biochimica et Biophysica Acta, Gene Structure and Expression (1994), 1218 (3), 357-65CODEN: BBGSD5; ISSN:0167-4781. (Elsevier B.V.)Human metallothionein (MT) genes are clustered in a locus on chromosome 16, and this report presents the characterization of the remaining six uninvestigated members of the family. Nucleotide sequencing in whole or part suggested that four of these genes, MT1I, MT1J, MT1K and MT1L do not encode expressed MT proteins, based on the presence of structural faults or atypical amino acid assignments. On the other hand, the structures of MT1H and MT1X are consistent with these genes being functional and encoding unique type 1 isoforms. The promoters of both genes conferred activity to CAT expression constructs when transfected into HeLa cells, and showed differential responses to inducers of MT synthesis. Endogenous MT1H and MT1X genes were expressed at the mRNA level in HeLa cells following cadmium treatment. This work brings the no. of functional class 1 and 2 MT genes in the human to eight, and confirms that each encodes structurally unique proteins.
- 377Rao, P. S.; Jaggi, M.; Smith, D. J.; Hemstreet, G. P.; Balaji, K. C. Metallothionein 2A interacts with the kinase domain of PKCmu in prostate cancer. Biochem. Biophys. Res. Commun. 2003, 310, 1032– 1038, DOI: 10.1016/j.bbrc.2003.09.118Google Scholar377Metallothionein 2A interacts with the kinase domain of PKCμ in prostate cancerRao, Prema S.; Jaggi, Meena; Smith, David J.; Hemstreet, George P.; Balaji, K. C.Biochemical and Biophysical Research Communications (2003), 310 (3), 1032-1038CODEN: BBRCA9; ISSN:0006-291X. (Elsevier Science)Prostate cancer (PC) patients die from progression to androgen independence (AI) and chemoresistance (CR). Protein kinase Cμ (PKCμ) a novel member of the PKC family of signal transduction proteins is downregulated in AI PC. Studying PKCμ interactors in the yeast two-hybrid system identified metallothionein 2A (MT 2A) as an interactor of PKCμ kinase domain (KD) in PC, which was quantified by β-gal assay, confirmed in PC cells by immunopptn., and PKCμ-MT 2A co-localization in vivo by immunofluorescence studies. PKCμ domain interaction studies revealed that MT 2A interacted strongly with KD, relatively weakly with C1, and failed to interact with C2, PH or kinase mutant domains. Peptide library and in silico anal. strongly suggest that MT 2A is a novel substrate of PKCμ and our data indicate that the PKCμ-MT 2A interaction depends on PKCμ kinase activity. Because metallothioneins are assocd. with cell proliferation and CR, the PKCμ-MT 2A interaction may contribute to CR and/or AI in PC.
- 378Aras, M. A.; Hara, H.; Hartnett, K. A.; Kandler, K.; Aizenman, E. Protein kinase C regulation of neuronal zinc signaling mediates survival during preconditioning. J. Neurochem. 2009, 110, 106– 117, DOI: 10.1111/j.1471-4159.2009.06106.xGoogle Scholar378Protein kinase C regulation of neuronal zinc signaling mediates survival during preconditioningAras, Mandar A.; Hara, Hirokazu; Hartnett, Karen A.; Kandler, Karl; Aizenman, EliasJournal of Neurochemistry (2009), 110 (1), 106-117CODEN: JONRA9; ISSN:0022-3042. (Wiley-Blackwell)Sublethal activation of cell death processes initiate pro-survival signaling cascades. As intracellular Zn2+ liberation mediates neuronal death pathways, we tested whether a sublethal increase in free Zn2+ could also trigger neuroprotection. Neuronal free Zn2+ transiently increased following preconditioning, and was both necessary and sufficient for conferring excitotoxic tolerance. Lethal exposure to NMDA led to a delayed increase in Zn2+ that contributed significantly to excitotoxicity in non-preconditioned neurons, but not in tolerant neurons, unless preconditioning-induced free Zn2+ was chelated. Thus, preconditioning may trigger the expression of Zn2+-regulating processes, which, in turn, prevent subsequent Zn2+-mediated toxicity. Indeed, preconditioning increased Zn2+-regulated gene expression in neurons. Examn. of the mol. signaling mechanism leading to this early Zn2+ signal revealed a crit. role for protein kinase C (PKC) activity, suggesting that PKC may act directly on the intracellular source of Zn2+. We identified a conserved PKC phosphorylation site at serine-32 (S32) of metallothionein (MT) that was important in modulating Zn2+-regulated gene expression and conferring excitotoxic tolerance. Importantly, we obsd. increased PKC-induced serine phosphorylation in immunopurified MT1, but not in mutant MT1(S32A). These results indicate that neuronal Zn2+ serves as an important, highly regulated signaling component responsible for the initiation of a neuroprotective pathway.
- 379Merlos-Rodrigo, M. A.; Jimenez Jimemez, A. M.; Haddad, Y.; Bodoor, K.; Adam, P.; Krizkova, S.; Heger, Z.; Adam, V. Metallothionein isoforms as double agents – Their roles in carcinogenesis, cancer progression and chemoresistance. Drug Resist. Updates 2020, 52, 100691, DOI: 10.1016/j.drup.2020.100691Google Scholar379Metallothionein isoforms as double agents - Their roles in carcinogenesis, cancer progression and chemoresistanceMerlos Rodrigo Miguel Angel; Adam Vojtech; Jimenez Jimemez Ana Maria; Haddad Yazan; Krizkova Sona; Heger Zbynek; Bodoor Khaldon; Adam PavlinaDrug resistance updates : reviews and commentaries in antimicrobial and anticancer chemotherapy (2020), 52 (), 100691 ISSN:.Metallothioneins (MTs) are small cysteine-rich intracellular proteins with four major isoforms identified in mammals, designated MT-1 through MT-4. The best known biological functions of MTs are their ability to bind and sequester metal ions as well as their active role in redox homeostasis. Despite these protective roles, numerous studies have demonstrated that changes in MT expression could be associated with the process of carcinogenesis and participation in cell differentiation, proliferation, migration, and angiogenesis. Hence, MTs have the role of double agents, i.e., working with and against cancer. In view of their rich biochemical properties, it is not surprising that MTs participate in the emergence of chemoresistance in tumor cells. Many studies have demonstrated that MT overexpression is involved in the acquisition of resistance to anticancer drugs including cisplatin, anthracyclines, tyrosine kinase inhibitors and mitomycin. The evidence is gradually increasing for a cellular switch in MT functions, showing that they indeed have two faces: protector and saboteur. Initially, MTs display anti-oncogenic and protective roles; however, once the oncogenic process was launched, MTs are utilized by cancer cells for progression, survival, and contribution to chemoresistance. The duality of MTs can serve as a potential prognostic/diagnostic biomarker and can therefore pave the way towards the development of new cancer treatment strategies. Herein, we review and discuss MTs as tumor disease markers and describe their role in chemoresistance to distinct anticancer drugs.
- 380Maret, W. Zinc in pancreatic islet biology, insulin sensitivity, and diabetes. Prev. Nutr. Food Sci. 2017, 22, 1– 8, DOI: 10.3746/pnf.2017.22.1.1Google Scholar380Zinc in pancreatic islet biology, insulin sensitivity, and diabetesMaret, WolfgangPreventive Nutrition and Food Science (2017), 22 (1), 1-8CODEN: PNFSBW; ISSN:2287-1098. (Korean Society of Food Science and Nutrition)About 20 chem. elements are nutritionally essential for humans with defined mol. functions. Several essential and nonessential biometals are either functional nutrients with antidiabetic actions or can be diabetogenic. A key question remains whether changes in the metab. of biometals and biominerals are a consequence of diabetes or are involved in its etiol. Exploration of the roles of zinc (Zn) in this regard is most revealing because 80 years of scientific discoveries link zinc and diabetes. In pancreatic β- and α-cells, zinc has specific functions in the biochem. of insulin and glucagon. When zinc ions are secreted during vesicular exocytosis, they have autocrine, paracrine, and endocrine roles. The membrane protein ZnT8 transports zinc ions into the insulin and glucagon granules. ZnT8 has a risk allele that predisposes the majority of humans to developing diabetes. In target tissues, increased availability of zinc enhances the insulin response by inhibiting protein tyrosine phosphatase 1B, which controls the phosphorylation state of the insulin receptor and hence downstream signalling. Inherited diseases of zinc metab., environmental exposures that interfere with the control of cellular zinc homeostasis, and nutritional or conditioned zinc deficiency influence the pathobiochem. of diabetes. Accepting the view that zinc is one of the many factors in multiple gene-environment interactions that cause the functional demise of β-cells generates an immense potential for treating and perhaps preventing diabetes. Personalized nutrition, bioactive food, and pharmaceuticals targeting the control of cellular zinc in precision medicine are among the possible interventions.
- 381Li, X.; Cai, L.; Feng, W. Diabetes and metallothionein. Mini-Rev. Med. Chem. 2007, 7, 761– 768, DOI: 10.2174/138955707781024490Google Scholar381Diabetes and metallothioneinLi, Xiaokun; Cai, Lu; Feng, WenkeMini-Reviews in Medicinal Chemistry (2007), 7 (7), 761-768CODEN: MMCIAE; ISSN:1389-5575. (Bentham Science Publishers Ltd.)A review. Diabetes is a widespread disease, and its development and toxic effects on various organs were attributed to increased oxidative stress. Metallothionein (MT) is a group of intracellular metal-binding and cysteine-rich proteins, being highly inducible in many tissues. Although it mainly acts as a regulator of metal homeostasis such as zinc and copper in tissues, MT was found to be a potent antioxidant and adaptive (or stress) protein to protect cells and tissues from oxidative stress. Studies showed that zinc-induced or genetically enhanced MT synthesis in the pancreas prevented the development of spontaneous or chem.-induced diabetes. Genetically or pharmacol. enhanced MT expression in various organs including heart and kidney provided significant protection from diabetes-induced organ dysfunction such as cardiomyopathy and nephropathy. These studies suggest that MT as an adaptive protein can prevent both diabetes development and diabetic complications. This mini-review will thus briefly describe MT's biochem. features and then summarize the data on the protective effect of MT against diabetes and diabetic complications. In addn., the coordinative role of MT with zinc in the prevention of diabetes and its complications will also be discussed.
- 382Bensellam, M.; Laybutt, D. R.; Jonas, H.-C. Emerging roles of metallothioneins in beta cell pathophysiology: Beyond and above metal homeostasis and antioxidant response. Biology 2021, 10, 176, DOI: 10.3390/biology10030176Google Scholar382Emerging roles of metallothioneins in beta cell pathophysiology: beyond and above metal homeostasis and antioxidant responseBensellam, Mohammed; Laybutt, D. Ross; Jonas, Jean-ChristopheBiology (Basel, Switzerland) (2021), 10 (3), 176CODEN: BBSIBX; ISSN:2079-7737. (MDPI AG)Metallothioneins (MTs) are low mol. wt., cysteine-rich, metal-binding proteins whose precise biol. roles have not been fully characterized. Existing evidence implicated MTs in heavy metal detoxification, metal ion homeostasis and antioxidant defense. MTs were thus categorized as protective effectors that contribute to cellular homeostasis and survival. This view has, however, been challenged by emerging evidence in different medical fields revealing novel pathophysiol. roles of MTs, including inflammatory bowel disease, neurodegenerative disorders, carcinogenesis and diabetes. In the present focused review, we discuss the evidence for the role of MTs in pancreatic beta-cell biol. and insulin secretion. We highlight the pattern of specific isoforms of MT gene expression in rodents and human beta-cells. We then discuss the mechanisms involved in the regulation of MTs in islets under physiol. and pathol. conditions, particularly type 2 diabetes, and analyze the evidence revealing adaptive and neg. roles of MTs in beta-cells and the potential mechanisms involved. Finally, we underscore the unsettled questions in the field and propose some future research directions. Keywords: metallothionein; pancreatic beta-cel.
- 383Koh, J.-Y.; Lee, S.-J. Metallothionein-3 as a multifunctional player in the control of cellular processes and diseases. Mol. Brain 2020, 13, 116, DOI: 10.1186/s13041-020-00654-wGoogle Scholar383Metallothionein-3 as a multifunctional player in the control of cellular processes and diseasesKoh, Jae-Young; Lee, Sook-JeongMolecular Brain (2020), 13 (1), 116CODEN: MBORAO; ISSN:1756-6606. (BioMed Central Ltd.)A review. Transition metals, such as iron, copper, and zinc, play a very important role in life as the regulators of various physiochem. reactions in cells. Abnormal distribution and concn. of these metals in the body are closely assocd. with various diseases including ischemic seizure, Alzheimer's disease, diabetes, and cancer. Iron and copper are known to be mainly involved in in vivo redox reaction. Zinc controls a variety of intracellular metab. via binding to lots of proteins in cells and altering their structure and function. Metallothionein-3 (MT3) is a representative zinc binding protein predominant in the brain. Although the role of MT3 in other organs still needs to be elucidated, many reports have suggested crit. roles for the protein in the control of a variety of cellular homeostasis. Here, we review various biol. functions of MT3, focusing on different cellular mols. and diseases involving MT3 in the body.
- 384Fung, S. J.; Chung, R. S.; West, A. The role of metallothionein and astrocyte-neuron interactions in injury to the CNS. In Metallothioneins in Biochemistry and Pathology; Zatta, P., Ed.; World Scientific Publishing Co.: Singapore, 2008; pp 117– 138.Google ScholarThere is no corresponding record for this reference.
- 385Wang, B.; Wood, I. S.; Trayhurn, P. PCR arrays identify metallthionein-3 as a highly hypoxia-inducible gene in human adipocytes. Biochem. Biophys. Res. Commun. 2008, 368, 88– 93, DOI: 10.1016/j.bbrc.2008.01.036Google Scholar385PCR arrays identify metallothionein-3 as a highly hypoxia-inducible gene in human adipocytesWang, Bohan; Stuart Wood, I.; Trayhurn, PaulBiochemical and Biophysical Research Communications (2008), 368 (1), 88-93CODEN: BBRCA9; ISSN:0006-291X. (Elsevier)Hypoxia-signalling pathway PCR arrays were used to examine the integrated response of human adipocytes to low O2 tension. Incubation of adipocytes in 1% O2 for 24 h resulted in no change in the expression of 63 of the 84 genes on the arrays, a redn. in expression of 9 genes (including uncoupling protein 2) and increased expression of 12 genes. Substantial increases (>10-fold) in leptin, angiopoietin-like protein 4, VEGF and GLUT-1 mRNA levels were obsd. The expression of one gene, metallothionein-3 (MT-3), was dramatically (>600-fold) and rapidly (by 60 min) increased by hypoxia. MT-3 gene expression was also substantially induced by hypoxia mimetics (CoCl2, desferrioxamine, dimethyloxalylglycine), indicating transcriptional regulation through HIF-1. Hypoxia addnl. induced MT-3 expression in preadipocytes, and MT-3 mRNA was detected in human (obese) s.c. and omental adipose tissue. MT-3 is a highly hypoxia-inducible gene in human adipocytes; the protein may protect adipocytes from hypoxic damage.
- 386Bousleiman, J.; Pinsky, A.; Ki, S.; Su, A.; Morozova, I.; Kalachikov, S.; Wiqas, A.; Silver, R.; Sever, M.; Austin, R. N. Function of metallothionein-3 in neuronal cells: Do metal ions alter expression levels of MT3?. Int. J. Mol. Sci. 2017, 18, 1133, DOI: 10.3390/ijms18061133Google Scholar386Function of metallothionein-3 in neuronal cells: do metal ions alter expression levels of MT3?Bousleiman, Jamie; Pinsky, Alexa; Ki, Sohee; Su, Angela; Morozova, Irina; Kalachikov, Sergey; Wiqas, Amen; Silver, Rae; Sever, Mary; Austin, Rachel NarehoodInternational Journal of Molecular Sciences (2017), 18 (6), 1133/1-1133/17CODEN: IJMCFK; ISSN:1422-0067. (MDPI AG)A study of factors proposed to affect metallothionein-3 (MT3) function was carried out to elucidate the opaque role MT3 plays in human metalloneurochem. Gene expression of Mt2 and Mt3 was examd. in tissues extd. from the dentate gyrus of mouse brains and in human neuronal cell cultures. The whole-genome gene expression anal. identified significant variations in the mRNA levels of genes assocd. with zinc homeostasis, including Mt2 and Mt3. Mt3 was found to be the most differentially expressed gene in the identified groups, pointing to the existence of a factor, not yet identified, that differentially controls Mt3 expression. To examine the expression of the human metallothioneins in neurons, mRNA levels of MT3 and MT2 were compared in BE(2)C and SH-SY5Y cell cultures treated with lead, zinc, cobalt, and lithium. MT2 was highly upregulated by Zn2+ in both cell cultures, while MT3 was not affected, and no other metal had an effect on either MT2 or MT3.
- 387Subramanian Vignesh, K.; Landero Figueroa, J. A.; Porollo, A.; Divanovic, S.; Caruso, J. A.; Deepe, G. S., Jr. IL-4 induces metallothionein3- and SLC30A4-dependent increase in intracellular Zn2+ that promotes pathogen persistence in macrophages. Cell Rep. 2016, 16, 3232– 3246, DOI: 10.1016/j.celrep.2016.08.057Google Scholar387IL-4 Induces Metallothionein 3- and SLC30A4-Dependent Increase in Intracellular Zn(2+) that Promotes Pathogen Persistence in MacrophagesSubramanian Vignesh Kavitha; Landero Figueroa Julio A; Caruso Joseph A; Porollo Aleksey; Divanovic Senad; Deepe George S JrCell reports (2016), 16 (12), 3232-3246 ISSN:.Alternative activation of macrophages promotes wound healing but weakens antimicrobial defenses against intracellular pathogens. The mechanisms that suppress macrophage function to create a favorable environment for pathogen growth remain elusive. We show that interleukin (IL)-4 triggers a metallothionein 3 (MT3)- and Zn exporter SLC30A4-dependent increase in the labile Zn(2+) stores in macrophages and that intracellular pathogens can exploit this increase in Zn to survive. IL-4 regulates this pathway by shuttling extracellular Zn into macrophages and by activating cathepsins that act on MT3 to release bound Zn. We show that IL-4 can modulate Zn homeostasis in both human monocytes and mice. In vivo, MT3 can repress macrophage function in an M2-polarizing environment to promote pathogen persistence. Thus, MT3 and SLC30A4 dictate the size of the labile Zn(2+) pool and promote the survival of a prototypical intracellular pathogen in M2 macrophages.
- 388Laukens, D.; Waeytens, A.; De Bleser, P.; Cuvelier, C.; De Vos, M. Human metallothionein expression under normal and pathological conditions: Mechanism of gene regulation based on in silico promoter analysis. Crit. Rev. Eukaryotic Gene Expression 2009, 19, 301– 317, DOI: 10.1615/CritRevEukarGeneExpr.v19.i4.40Google Scholar388Human metallothionein expression under normal and pathological conditions: mechanisms of gene regulation based on in silico promoter analysisLaukens, Debby; Waeytens, Anouk; De Bleser, Pieter; Cuvelier, Claude; De Vos, MartineCritical Reviews in Eukaryotic Gene Expression (2009), 19 (4), 301-317CODEN: CRGEEJ; ISSN:1045-4403. (Begell House, Inc.)A review. Metallothioneins (MTs) are ubiquitous metal-binding proteins that have been highly conserved throughout evolution. Although their physiol. function is not completely understood, they are involved in diverse processes including metal homeostasis and detoxification, the oxidative stress response, inflammation, and cell proliferation. The human MT gene family consists of at least 18 isoforms, contg. pseudogenes as well as genes encoding functional proteins. Most of the MT isoforms can be induced by a wide variety of substances, such as metals, cytokines, and hormones. Different cell types express discrete MT isoforms, which reflects the specifically adapted functions of MTs and a divergence in their regulation. The aberrant expression of MTs has been described in a no. of diseases, including Crohn's disease, cancer, Alzheimer's disease, amyotrophic lateral sclerosis, Menkes disease, and Wilson's disease. Therefore, a thorough understanding of MT gene regulation is imperative. To date, the transcriptional regulation of MTs has primarily been studied in mice. While only four murine MT isoforms exist, the homol. between murine and human MTs allows for the evaluation of the regulatory regions in their resp. promoters. Here, we review the aberrant expression of MTs in human diseases and the mechanisms that regulate MT1 expression based on an in silico evaluation of transcription factor binding sites.
- 389Laity, J. H.; Andrews, G. K. Understanding the mechanisms of zinc-sensing by metal-response element binding transcription factor-1 (MTF-1). Arch. Biochem. Biophys. 2007, 463, 201– 210, DOI: 10.1016/j.abb.2007.03.019Google Scholar389Understanding the mechanisms of zinc-sensing by metal-response element binding transcription factor-1 (MTF-1)Laity, John H.; Andrews, Glen K.Archives of Biochemistry and Biophysics (2007), 463 (2), 201-210CODEN: ABBIA4; ISSN:0003-9861. (Elsevier)A review. The regulation of Zn2+ has been obsd. in a wide range of organisms. Since this metal is an essential nutrient, but also toxic in excess, Zn2+ homeostasis is crucial for normal cellular functioning. Metal-responsive-element-binding transcription factor-1 (MTF-1) is a key regulator of Zn2+ in higher eukaryotes ranging from insects to mammals. MTF-1 controls the expression of metallothioneins (MTs) and a no. of other genes directly involved in the intracellular sequestration and transport of Zn2+. Although the diverse functions of MTF-1 extend well beyond Zn2+ homeostasis to include stress-responses to heavy metal toxicity, oxidative stress, and selected chem. agents, in this review the authors focus on recent advances in understanding the mechanisms whereby MTF-1 regulates MT gene expression to protect the cell from fluctuations in environmental Zn2+. Particular emphasis is devoted to recent studies involving the Cys2His2 zinc finger DNA-binding domain of MTF-1, which is an important contributor to the zinc-sensing and metal-dependent transcriptional activation functions of this protein.
- 390Günther, V.; Lindert, U.; Schaffner, W. The taste of heavy metals: gene regulation by MTF-1. Biochim. Biophys. Acta, Mol. Cell Res. 2012, 1823, 1416– 1425, DOI: 10.1016/j.bbamcr.2012.01.005Google ScholarThere is no corresponding record for this reference.
- 391Zhang, B.; Georgiev, O.; Hagmann, M.; Günes, C.; Cramer, M.; Faller, P.; Vašák, M.; Schaffner, W. Activity of metal-responsive transcription factor 1 by toxic heavy metals and H2O2 in vitro is modulated by metallothionein. Mol. Cell. Biol. 2003, 23, 8471– 8485, DOI: 10.1128/MCB.23.23.8471-8485.2003Google Scholar391Activity of metal-responsive transcription factor 1 by toxic heavy metals and H2O2 in vitro is modulated by metallothioneinZhang, Bo; Georgiev, Oleg; Hagmann, Michael; Guenes, Cagatay; Cramer, Mirjam; Faller, Peter; Vasak, Milan; Schaffner, WalterMolecular and Cellular Biology (2003), 23 (23), 8471-8485CODEN: MCEBD4; ISSN:0270-7306. (American Society for Microbiology)Metallothioneins are small, cysteine-rich proteins that avidly bind heavy metals such as zinc, copper, and cadmium to reduce their concn. to a physiol. or nontoxic level. Metallothionein gene transcription is induced by several stimuli, notably heavy metal load and oxidative stress. Transcriptional induction of metallothionein genes is mediated by the metal-responsive transcription factor 1 (MTF-1), an essential zinc finger protein that binds to specific DNA motifs termed metal-response elements. In cell-free DNA binding reactions with nuclear exts., MTF-1 requires elevated zinc concns. for efficient DNA binding but paradoxically is inactivated by other in vivo inducers such as cadmium, copper, and hydrogen peroxide. Here the authors have developed a cell-free, MTF-1-dependent transcription system which accurately reproduces the activation of metallothionein gene promoters not only by zinc but also by these other inducers. Whereas transcriptional induction by zinc can be achieved by elevated zinc concn. alone, induction by cadmium, copper, or H2O2 addnl. requires the presence of zinc-satd. metallothionein. This is explained by the preferential binding of cadmium or copper to metallothionein or its oxidn. by H2O2; the concomitant release of zinc in turn leads to the activation of transcription factor MTF-1. Conversely, thionein, the metal-free form of metallothionein, inhibits activation of MTF-1. The release of zinc from cellular components, including metallothioneins, and the sequestration of zinc by newly produced apometallothionein might be a basic mechanism to regulate MTF-1 activity upon cellular stress.
- 392Lichten, L. A.; Ryu, M.-S.; Guo, L.; Embury, J.; Cousins, R. J. MTF-1-mediated repression of the zinc transporter Zip10 is alleviated by zinc restriction. PLoS One 2011, 6, e21526 DOI: 10.1371/journal.pone.0021526Google Scholar392MTF-1-mediated repression of the zinc transporter Zip10 is alleviated by zinc restrictionLichten, Louis A.; Ryu, Moon-Suhn; Guo, Liang; Embury, Jennifer; Cousins, Robert J.PLoS One (2011), 6 (6), e21526CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)The regulation of cellular zinc uptake is a key process in the overall mechanism governing mammalian zinc homeostasis and how zinc participates in cellular functions. We analyzed the zinc transporters of the Zip family in both the brain and liver of zinc-deficient animals and found a large, significant increase in Zip10 expression. Addnl., Zip10 expression decreased in response to zinc repletion. Moreover, isolated mouse hepatocytes, AML12 hepatocytes, and Neuro 2A cells also respond differentially to zinc availability in vitro. Measurement of Zip10 hnRNA and actinomycin D inhibition studies indicate that Zip10 was transcriptionally regulated by zinc deficiency. Through luciferase promoter constructs and ChIP anal., binding of MTF-1 to a metal response element located 17 bp downstream of the transcription start site was shown to be necessary for zinc-induced repression of Zip10. Furthermore, zinc-activated MTF-1 causes down-regulation of Zip10 transcription by phys. blocking Pol II movement through the gene. Lastly, ZIP10 is localized to the plasma membrane of hepatocytes and neuro 2A cells. Collectively, these results reveal a novel repressive role for MTF-1 in the regulation of the Zip10 zinc transporter expression by pausing Pol II transcription. ZIP10 may have roles in control of zinc homeostasis in specific sites particularly those of the brain and liver. Within that context ZIP10 may act as an important survival mechanism during periods of zinc inadequacy.
- 393Suhy, D. A.; Simon, K. D.; Linzer, D. I.; O’Halloran, T. V. Metallothionein is part of a zinc-scavenging mechanism for cell survival under conditions of extreme zinc deprivation. J. Biol. Chem. 1999, 274, 9183– 9192, DOI: 10.1074/jbc.274.14.9183Google Scholar393Metallothionein is part of a zinc-scavenging mechanism for cell survival under conditions of extreme zinc deprivationSuhy, David A.; Simon, Kathryn D.; Linzer, Daniel I. H.; O'Halloran, Thomas V.Journal of Biological Chemistry (1999), 274 (14), 9183-9192CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)Metallothionein (MT) is a small cysteine-rich protein thought to play a crit. role in cellular detoxification of inorg. species by sequestering metal ions that are present in elevated concns. We demonstrate here that metallothionein can play an important role at the other end of the homeostatic spectrum by scavenging an essential metal in a mouse fibroblast cell line that has been cultured under conditions of extreme zinc deprivation (LZA-LTK-). These cells unexpectedly produce constitutively high levels of metallothionein mRNA; however, the MT protein accumulates only when high concns. of zinc are provided in the media. Until this MT pool is satd., no measurable zinc remains in the external media. In this case, zinc deprivation leads to amplification of the MT gene locus in the LZA-LTK- cell line. Furthermore, the intracellular zinc levels in the fully adapted cells remain at the normal level of 0.4 fmol zinc/cell, even when extracellular zinc concn. is decreased by 2 orders of magnitude relative to normal media.
- 394Karin, M.; Andersen, R. D.; Slater, E.; Smith, K.; Herschman, H. R. Metallothionein mRNA induction in HeLa cells in response to zinc or dexamethasone is a primary induction response. Nature 1980, 286, 295– 297, DOI: 10.1038/286295a0Google Scholar394Metallothionein mRNA induction in HeLa cells in response to zinc or dexamethasone is a primary induction responseKarin, Michael; Andersen, Robert D.; Slater, Emily; Smith, Karen; Herschman, Harvey R.Nature (London, United Kingdom) (1980), 286 (5770), 295-7CODEN: NATUAS; ISSN:0028-0836.Induction of translatable metallothionein (MT) mRNA in HeLa cells by either Zn2+ or dexamethasone [50-02-2] was independent of concomitant protein synthesis but not RNA synthesis. Elevation of MT mRNA by 10-7M dexamethasone or 3.7 × 10-5M Zn2+, 5- or 6-fold, resp., was prevented by actinomycin D (1 μg/mL), which also lowered MT mRNA levels relative to control cultures by 50-60%. Variable cycloheximide enhancement of MT synthesis (40-100%) was obsd. in Zn-treated and control cultures but not in dexamethasone-induced cultures. In contrast to dexamethasone induction of MT mRNA, dexamethasone stimulation of Zn2+ transport was blocked by cycloheximide. MT induction by both dexamethasone and Zn2+ was, therefore, a primary induction response.
- 395Murphy, B. J.; Laderoute, K. R.; Chin, R. J.; Sutherland, R. M. Metallothionein IIA is up-regulated by hypoxia in human A431 squamous carcinoma cells. Cancer Res. 1994, 54, 5808– 5810Google Scholar395Metallothionein IIA is up-regulated by hypoxia in human A431 squamous carcinoma cellsMurphy, Brian J.; Laderoute, Keith R.; Chin, Roxanne J.; Sutherland, Robert M.Cancer Research (1994), 54 (22), 5808-10CODEN: CNREA8; ISSN:0008-5472. (American Association for Cancer Research)The expression of metallothionein IIA (MT-IIA) was investigated in A431 human squamous carcinoma cells exposed to hypoxia (pO2 ≤ 0.01% of atm. pO2) and subsequent reoxygenation. Northern anal. showed that MT-IIA mRNA levels were significantly increased during 14 h of hypoxia and during reoxygenation. Western blotting confirmed that total MT protein levels were also increased in response to these stresses. Evidence of the transcriptional control of MT-IIA expression in hypoxic and in reoxygenated A431 cells was found using a 0.2-kilobase sequence of the proximal 5'-regulatory region of the MT-IIA gene in a chloramphenicol acetyltransferase reporter gene construct. Thus the proximal promoter of the human MT-IIA gene appears to contain a hypoxic response elements(s). These observations indicate that MT-IIA may have an important role in the stress responses of cells in solid tumors.
- 396Murphy, B. J.; Kimura, T.; Sato, B. G.; Shi, Y.; Andrews, G. K. Metallothionein induction by hypoxia involves cooperative interactions between metal-responsive transcription factor-1 and hypoxia-inducible transcription factor-1α. Mol. Cancer Res. 2008, 6, 483– 490, DOI: 10.1158/1541-7786.MCR-07-0341Google Scholar396Metallothionein Induction by Hypoxia Involves Cooperative Interactions between Metal-Responsive Transcription Factor-1 and Hypoxia-Inducible Transcription Factor-1αMurphy, Brian J.; Kimura, Tomoki; Sato, Barbara G.; Shi, Yihui; Andrews, Glen K.Molecular Cancer Research (2008), 6 (3), 483-490CODEN: MCROC5; ISSN:1541-7786. (American Association for Cancer Research)Mammalian metallothionein (MT) genes are transcriptionally activated by the essential metal zinc as well as by environmental stresses, including toxic metal overload and redox fluctuations. In addn. to playing a key role in zinc homeostasis, MT proteins can protect against metal- and oxidant-induced cellular damage, and may participate in other fundamental physiol. and pathol. processes such as cell survival, proliferation, and neoplasia. Previously, our group reported a requirement for metal-responsive transcription factor-1 (MTF-1) in hypoxia-induced transcription of mouse MT-I and human MT-IIA genes. Here, we provide evidence that the protumorigenic hypoxia-inducible transcription factor-1α (HIF-1α) is essential for induction of MT-1 by hypoxia, but not zinc. Chromatin immunopptn. assays revealed that MTF-1 and HIF-1α are both recruited to the mouse MT-I promoter in response to hypoxia, but not zinc. In the absence of HIF-1α, MTF-1 is recruited to the MT-I promoter but fails to activate MT-I gene expression in response to hypoxia. Thus, HIF-1α seems to function as a coactivator of MT-I gene transcription by interacting with MTF-1 during hypoxia. Coimmunopptn. studies suggest interaction between MTF-1 and HIF-1α, either directly or as mediated by other factors. It is proposed that assocn. of these important transcription factors in a multi-protein complex represents a common strategy to control unique sets of hypoxia-inducible genes in both normal and diseased tissue.
- 397Kojima, I.; Tanaka, T.; Inagi, R.; Nishi, H.; Aburatani, H.; Kato, H.; Miyata, T.; Fujita, T.; Nangaku, M. Metallothionein is upregulated by hypoxia and stabilizes hypoxia-inducible factor in the kidney. Kidney Int. 2009, 75, 268– 277, DOI: 10.1038/ki.2008.488Google Scholar397Metallothionein is upregulated by hypoxia and stabilizes hypoxia-inducible factor in the kidneyKojima, Ichiro; Tanaka, Tetsuhiro; Inagi, Reiko; Nishi, Hiroshi; Aburatani, Hiroyuki; Kato, Hideki; Miyata, Toshio; Fujita, Toshiro; Nangaku, MasaomiKidney International (2009), 75 (3), 268-277CODEN: KDYIA5; ISSN:0085-2538. (Nature Publishing Group)Recent studies underscore that chronic hypoxia in the tubulointerstitium is a final common pathway to progression to end-stage renal failure regardless of etiol. We used microarray anal. of rat kidneys made hypoxic by unilateral renal artery stenosis to measure transcriptomic events and clarify pathophysiol. mechanisms of renal injury induced by chronic hypoxia. Many genes were upregulated in the kidney by chronic hypoxia, but we focused on metallothionein due to its antioxidative properties. Using tubular epithelial cells transfected with a reporter construct of luciferase, driven by the hypoxia-responsive elements (HRE), we found that addn. of metallothionein to the culture media increased luciferase activity. This was assocd. with upregulation of the target genes of hypoxia-inducible factor (HIF), such as vascular endothelial growth factor and glucose transporter-1. Stimulation of the HIF-HRE pathway by metallothionein was confirmed by metallothionein overexpression. Hypoxia and exogenous metallothionein increased HIF-1α protein without changes in its mRNA levels, suggesting protein stabilization. Upregulation of the HIF-HRE system by metallothionein was assocd. with phosphorylation of ERK but not Akt. MEK inhibition and rapamycin decreased metallothionein-induced HIF activity. Our study shows that upregulation of metallothionein expression by hypoxia activates the HIF-HRE system through the ERK/mTOR pathway and may be a novel defense against hypoxia. Kidney International (2009) 75, 268-277. doi:10.1038/ki.2008.488.
- 398Sato, S.; Shirakawa, H.; Tomita, S.; Tohkin, M.; Gonzalez, F. J.; Komai, M. The aryl hydrocarbon receptor and glucocorticoid receptor interact to activate human metallothionein 2A. Toxicol. Appl. Pharmacol. 2013, 273, 90– 99, DOI: 10.1016/j.taap.2013.08.017Google Scholar398The aryl hydrocarbon receptor and glucocorticoid receptor interact to activate human metallothionein 2ASato, Shoko; Shirakawa, Hitoshi; Tomita, Shuhei; Tohkin, Masahiro; Gonzalez, Frank J.; Komai, MichioToxicology and Applied Pharmacology (2013), 273 (1), 90-99CODEN: TXAPA9; ISSN:0041-008X. (Elsevier Inc.)Although the aryl hydrocarbon receptor (AHR) and glucocorticoid receptor (GR) play essential roles in mammalian development, stress responses, and other physiol. events, crosstalk between these receptors has been the subject of much debate. Metallothioneins are classic glucocorticoid-inducible genes that were reported to increase upon treatment with AHR agonists in rodent tissues and cultured human cells. In this study, the mechanism of human metallothionein 2A (MT2A) gene transcription activation by AHR was investigated. Cotreatment with 3-methylcholanthrene and dexamethasone, agonists of AHR and GR resp., synergistically increased MT2A mRNA levels in HepG2 cells. MT2A induction was suppressed by RNA interference against AHR or GR. Coimmunopptn. expts. revealed a phys. interaction between AHR and GR proteins. Moreover, chromatin immunopptn. assays indicated that AHR was recruited to the glucocorticoid response element in the MT2A promoter. Thus, we provide a novel mechanism whereby AHR modulates expression of human MT2A via the glucocorticoid response element and protein-protein interactions with GR.
- 399Rasinger, J. D.; Carroll, T. S.; Lundebye, A. K.; Hogstrand, C. Cross-omics gene and protein expression profiling in juvenile female mice highlights disruption of calcium and zinc signalling in the brain following dietary exposure to CB-153, BDE-47, HBCD or TCDD. Toxicology 2014, 321, 1– 12, DOI: 10.1016/j.tox.2014.03.006Google Scholar399Cross-omics gene and protein expression profiling in juvenile female mice highlights disruption of calcium and zinc signalling in the brain following dietary exposure to CB-153, BDE-47, HBCD or TCDDRasinger, J. D.; Carroll, T. S.; Lundebye, A. K.; Hogstrand, C.Toxicology (2014), 321 (), 1-12CODEN: TXCYAC; ISSN:0300-483X. (Elsevier Ltd.)The present study assessed if eating a diet of fish, spiked with persistent org. pollutants (POPs), affects gene and protein expression in the maturing mouse brain. Juvenile female Balb/c mice (22 days of age) were exposed for 28 days to fish-based diets spiked with the dioxin 2,3,7,8-tetrachlorodibenzodioxin (TCDD) or the non dioxin-like (NDL) chems. hexabromocyclodocecane (HBCD), 2,2'4,4'-tetrabromodiphenylether (BDE-47) or 2,2'4,4',5,5'-hexachlorobiphenyl (CB-153) at doses approximating their resp. lowest obsd. adverse effect levels (LOAEL). It was found that all POPs elicited changes in neural gene and protein expression profiles. Bioinformatic anal. of gene expression data highlighted the importance of the aryl hydrocarbon receptor (AHR) in dioxin toxicity and revealed that zinc regulation in the brain is targeted by TCDD through the AHR. Calcium homeostasis was affected by both TCDD and the NDL chems. In contrast to the transcriptomic anal., the proteomics data did not allow for a clear distinction between DL and NDL responses in the juvenile brain but indicated that proteins assocd. with excitotoxicity were affected in all exposure groups. Integrated interpretation of data led to the conclusion that the dietary contaminants investigated in the present study breach the blood brain barrier (BBB) and accumulate in the juvenile brain where they may induce excitotoxic insults by dysregulation of the otherwise tightly controlled homeostasis of calcium and zinc. Overall, the findings of the present study highlight the need for further assessment of the risks assocd. with early life exposure to foodborne POPs.
- 400Dalton, T.; Palmiter, R. D.; Andrews, G. K. Transcriptional induction of the mouse metallothionein-I gene in hydrogen peroxide-treated Hepa cells involves a composite major late transcription factor/antioxidant response element and metal response promoter elements. Nucleic Acids Res. 1994, 22, 5016– 5023, DOI: 10.1093/nar/22.23.5016Google Scholar400Transcriptional induction of the mouse metallothionein-I gene in hydrogen peroxide-treated Hepa cells involves a composite major late transcription factor/antioxidant response element and metal response promoter elementsDalton, Tim; Palmiter, Richard D.; Andrews, Glen K.Nucleic Acids Research (1994), 22 (23), 5016-23CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)Synthesis of metallothionein-I (MT-I) and heme oxygenase mRNAs is rapidly and transiently induced by H2O2 in mouse hepatomna cells (Hepa) and this effect is blocked by catalase. Menadione, which generates free radicals, also induces these mRNAs. Deletion mutagenesis revealed that a region between -42 and -153 in the mouse MT-I promoter was essential for induction of a CAT reporter gene. A multimer of a 16 bp sequence (-101 to -86) that includes an antioxidant response element and overlapping adenovirus major late transcription factor binding site elevated basal expression and allowed induction by H2O2 when inserted upstream of a minimal promoter. However, deletion of this region (-100 to -89) from the intact MT-I promoter (-153) did not completely eliminate response. Multiple copies of a metal response element also permitted response to H2O2. These results suggest that induction of MT-I gene transcription by H2O2 is mediated by at least two different elements within the proximal MT-I gene promoter and suggest a previously undescribed function of the MRE. Induction of MT gene transcription by ROS and the subsequent scavenging of ROS by the MT peptide is reminiscent of the metal regulatory loop and is consistent with the hypothesized protective functions of MT.
- 401Abdel-Mageed, A. B.; Agrawal, K. C. Activation of nuclear factor kappaB: potential role in metallothioinein-mediated mitogenic response. Cancer Res. 1998, 58, 2335– 2338Google Scholar401Activation of nuclear factor κB: potential role in metallothionein-mediated mitogenic responseAbdel-Mageed, Asim B.; Agrawal, Krishna C.Cancer Research (1998), 58 (11), 2335-2338CODEN: CNREA8; ISSN:0008-5472. (American Association for Cancer Research)The antiapoptotic response and enhanced cellular proliferation obsd. in neoplastic cells on overexpression of metallothionein (MT) have been well documented. We have investigated the mechanisms assocd. with this phenomenon by using MT inducers that increased MT transcripts and stimulated growth in MCF-7 cells. A MT antisense phosphorothioate oligonucleotide inhibited growth induction by >50%, suggesting a potential role of MT in mediating the mitogenic effects of these agents. Mobility shift assays using oligonucleotides encompassing the consensus nuclear factor κB (NFκB) binding site and anti-MT antibody revealed activation and a specific interaction of NFκB with MT. Cotransfection expts. using expression and reporter constructs demonstrated that MT caused transactivation of NFκB. Gel shift assays using purified proteins showed a specific interaction between MT and the p50 subunit of NFκB. These data indicate that MT may be involved in the interaction of NFκB with the DNA-binding domain and further suggest a potential role for NFκB in mediating the antiapoptotic effects of MT.
- 402Cramer, M.; Nagy, I.; Murphy, B. J.; Gassmann, M.; Hottiger, M. O.; Georgiev, O.; Schaffner, W. NF-kappaB contributes to transcription of placenta growth factor and interacts with metal-response transcription factor-1 in hypoxic animal cells. Biol. Chem. 2005, 386, 865– 872, DOI: 10.1515/BC.2005.101Google Scholar402NF-κB contributes to transcription of placenta growth factor and interacts with metal responsive transcription factor-1 in hypoxic human cellsCramer, Mirjam; Nagy, Ivana; Murphy, Brian J.; Gassmann, Max; Hottiger, Michael O.; Georgiev, Oleg; Schaffner, WalterBiological Chemistry (2005), 386 (9), 865-872CODEN: BICHF3; ISSN:1431-6730. (Walter de Gruyter GmbH & Co. KG)Placenta growth factor (PIGF) is a member of the vascular endothelial growth factor family of cytokines that control vascular and lymphatic endothelium development. It has been implicated in promoting angiogenesis in pathol. conditions via signaling to vascular endothelial growth factor receptor-1. PIGF expression is induced by hypoxia and proinflammatory stimuli. Metal responsive transcription factor 1 (MTF-1) was shown to take part in the hypoxic induction of PIGF in Ras-transformed mouse embryonic fibroblasts. Here we report that PIGF expression is also controlled by NF-κB. We identified several putative binding sites for NF-κB in the PIGF promoter/enhancer region by sequence analyses, and show binding and transcriptional activity of NF-κB p65 at these sites. Expression of NF-κB p65 from a plasmid vector in HEK293 cells caused a substantial increase of PIGF transcript levels. Furthermore, we found that hypoxic conditions induce nuclear translocation and interaction of MTF-1 and NF-κB p65 proteins, suggesting a role for this complex in hypoxia-induced transcription of PIGF.
- 403Maret, W. Zinc and Human Disease. In Interrelations between essential metal ions and human diseases; Metal Ions in Life Sciences; Sigel, A., Sigel, H., Sigel, R. K. O., Eds.; Springer Science + Business Media B.V.: Dordrecht, The Netherlands, 2013; Vol. 13, pp 389– 414.Google ScholarThere is no corresponding record for this reference.
- 404Butcher, H. L.; Kennette, W. A.; Collins, O.; Zalups, R. K.; Koropatnick, J. Metallothionein mediates the level and activity of nuclear factor kappa B in murine fibroblasts. J. Pharmacol. Exp. Ther. 2004, 310, 589– 598, DOI: 10.1124/jpet.104.066126Google Scholar404Metallothionein mediates the level and activity of nuclear factor κB in murine fibroblastsButcher, Heather L.; Kennette, Wendy A.; Collins, Olga; Zalups, Rudolfs K.; Koropatnick, JamesJournal of Pharmacology and Experimental Therapeutics (2004), 310 (2), 589-598CODEN: JPETAB; ISSN:0022-3565. (American Society for Pharmacology and Experimental Therapeutics)The zinc-binding protein metallothionein (MT) is assocd. with resistance to apoptosis. We examd. whether MT regulates the zinc-dependent antiapoptotic transcription factor nuclear factor κB (NF-κB), which is up-regulated under many conditions that lead to elevated MT expression. NF-κB protein levels and NF-κB-dependent reporter gene activity were examd. in clonal MT(+) (MT-WT) and MT(-) (MT-KO) fibroblastic cell lines. The amt. of cellular NF-κB p65 protein in MT-KO was less than 20% of the amt. in MT-WT cells, in accord with increased sensitivity of MT-KO cells to apoptosis. NF-κB p65 mRNA levels, and NF-κB p50 subunit and IκBα protein levels, were unchanged. NF-κB activity assessed by expression of a transfected NF-κB reporter construct was less than half that obsd. in MT-KO cells. Decreased nuclear localization of NF-κB p65 in MT-KO clones was not responsible for differences in activity. In fact, MT-KO cells had higher nuclear levels of NF-κB p65 than did MT-WT cells, despite a lower cellular NF-κB level and function, suggesting that metallothionein mediated the specific activity of NF-κB. Reconstitution of MT by stable incorporation of an MT-1 expression vector in MT-KO cells resulted in increased NF-κB p65 (but not IκBα or NF-κB p50), increased NF-κB-dependent reporter activity, and increased resistance to apoptosis. These data support the hypothesis that metallothionein pos. regulates the cellular level and activity of NF-κB.
- 405Blanden, A. R.; Yu, X.; Blayney, A. J.; Demas, C.; Ha, J.-H.; Liu, Y.; Withers, T.; Carpizo, D. R.; Loh, S. N. Zinc shapes the folding landscape of p53 and established a pathway for reactivating structurally diverse cancer mutants. eLife 2020, 9, e61487 DOI: 10.7554/eLife.61487Google Scholar405Zinc shapes the folding landscape of p53 and establishes a pathway for reactivating structurally diverse cancer mutantsBlanden, Adam R.; Yu, Xin; Blayney, Alan J.; Demas, Christopher; Ha, Jeung-Hoi; Liu, Yue; Withers, Tracy; Carpizo, Darren R.; Loh, Stewart N.eLife (2020), 9 (), e61487CODEN: ELIFA8; ISSN:2050-084X. (eLife Sciences Publications Ltd.)Missense mutations in the p53 DNA-binding domain (DBD) contribute to half of new cancer cases annually. Here we present a thermodn. model that quantifies and links the major pathways by which mutations inactivate p53. We find that DBD possesses two unusual properties-one of the highest zinc affinities of any eukaryotic protein and extreme instability in the absence of zinc-which are predicted to poise p53 on the cusp of folding/unfolding in the cell, with a major determinant being available zinc concn. We analyze the 20 most common tumorigenic p53 mutations and find that 80% impair zinc affinity, thermodn. stability, or both. Biophys., cellbased, and murine xenograft expts. demonstrate that a synthetic zinc metallochaperone rescues not only mutations that decrease zinc affinity, but also mutations that destabilize DBD without impairing zinc binding. The results suggest that zinc metallochaperones have the capability to treat 120,500 patients annually in the U.S.
- 406Meplan, C.; Richard, M. J.; Hainaut, P. Metalloregulation of the tumor suppressor protein p53: zinc mediates the renaturation of p53 after exposure to metal chelators in vitro and in intact cells. Oncogene 2000, 19, 5227– 5236, DOI: 10.1038/sj.onc.1203907Google Scholar406Metalloregulation of the tumor suppressor protein p53: zinc mediates the renaturation of p53 after exposure to metal chelators in vitro and in intact cellsMeplan, Catherine; Richard, Marie-Jeanne; Hainaut, PierreOncogene (2000), 19 (46), 5227-5236CODEN: ONCNES; ISSN:0950-9232. (Nature Publishing Group)The tumor suppressor p53 is a transcription factor which binds DNA through a structurally complex domain stabilized by a zinc atom. Zinc chelation disrupts the architecture of this domain, inducing the protein to adopt an immunol. phenotype identical to that of many mutant forms of p53. In this report, we used 65Zn to show that incorporation of zinc within the protein was required for folding in the "wild-type" conformation capable of specific DNA-binding. Using a cellular assay, we show that addn. of extracellular zinc at concns. within the physiol. range (5 μM) was required for renaturation and reactivation of wild-type p53. Among other divalent metals tested (Cd2+, Cu2+, Co2+, Fe2+ and Ni2+), only Co2+ at 125 μM had a similar effect. Recombinant metallothionein (MT), a metal chelator protein, was found to modulate p53 conformation in vitro. In cultured cells, overexpression of MT by transfection could modulate p53 transcriptional activity. Taken together, these results suggest that zinc binding plays a regulatory role in the control of p53 folding and DNA-binding activity.
- 407Ostrakhovitch, E. A.; Olsson, P.-E.; Jiang, S.; Cherian, M. G. Interaction of metallothionein with tumor suppressor p53 protein. FEBS Lett. 2006, 580, 1235– 1238, DOI: 10.1016/j.febslet.2006.01.036Google Scholar407Interaction of metallothionein with tumor suppressor p53 proteinOstrakhovitch, Elena A.; Olsson, Per-Erik; Jiang, Sean; Cherian, M. GeorgeFEBS Letters (2006), 580 (5), 1235-1238CODEN: FEBLAL; ISSN:0014-5793. (Elsevier B.V.)Previous reports have shown that metallothionein (MT) may modulate p53 protein activity through Zn2+ exchange. However, little is known about a direct interaction between MT and p53 in cells. Here, the results demonstrated that an interaction between MT and p53 could occur in vitro. The complex between MT and p53 was obsd. in breast cancer epithelial cells with both wild-type (wt) and inactive type of p53. Furthermore, it was shown that wt-p53 was preferentially assocd. with apo-MT. The data suggested that co-expression of MT and p53 and their complex formation in tumor cells may be involved in regulation of apoptosis in these cells.
- 408Lee, B. M.; Buck-Koehntop, B. A.; Martinez-Yamout, M. A.; Dyson, H. J.; Wright, P. E. Embryonic neural inducing factor churchill is not a DNA-binding zinc finger protein: solution structure reveals a solvent-exposed beta-sheet and zinc binuclear cluster. J. Mol. Biol. 2007, 371, 1274– 1289, DOI: 10.1016/j.jmb.2007.06.021Google Scholar408Embryonic Neural Inducing Factor Churchill Is not a DNA-binding Zinc Finger Protein: Solution Structure Reveals a Solvent-exposed β-Sheet and Zinc Binuclear ClusterLee, Brian M.; Buck-Koehntop, Bethany A.; Martinez-Yamout, Maria A.; Dyson, H. Jane; Wright, Peter E.Journal of Molecular Biology (2007), 371 (5), 1274-1289CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Ltd.)Churchill is a zinc-contg. protein that is involved in neural induction during embryogenesis. At the time of its discovery, it was thought on the basis of sequence alignment to contain two zinc fingers of the C4 type. Further, binding of an N-terminal GST-Churchill fusion protein to a particular DNA sequence was demonstrated by immunopptn. selection assay, suggesting that Churchill may function as a transcriptional regulator by sequence-specific DNA binding. We show by NMR soln. structure detn. that, far from contg. canonical C4 zinc fingers, the protein contains three bound zinc ions in novel coordination sites, including an unusual binuclear zinc cluster. The secondary structure of Churchill is also unusual, consisting of a highly solvent-exposed single-layer β-sheet. Hydrogen-deuterium exchange and backbone relaxation measurements reveal that Churchill is unusually dynamic on a no. of time scales, with the exception of regions surrounding the zinc coordinating sites, which serve to stabilize the otherwise unstructured N terminus and the single-layer β-sheet. No binding of Churchill to the previously identified DNA sequence could be detected, and extensive searches using DNA sequence selection techniques could find no other DNA sequence that was bound by Churchill. Since the N-terminal amino acids of Churchill form part of the zinc-binding motif, the addn. of a fusion protein at the N terminus causes loss of zinc and unfolding of Churchill. This observation most likely explains the published DNA-binding results, which would arise due to non-specific interaction of the unfolded protein in the immunopptn. selection assay. Since Churchill does not appear to bind DNA, we suggest that it may function in embryogenesis as a protein-interaction factor.
- 409Bellon, S. F.; Rodgers, K. K.; Schatz, D. G.; Coleman, J. E.; Steitz, T. A. Crystal structure of the RAG1 dimerization domain reveals multiple zinc-binding motifs including a novel zinc binuclear cluster. Nat. Struct. Biol. 1997, 4, 586– 591, DOI: 10.1038/nsb0797-586Google Scholar409Crystal structure of the RAG1 dimerization domain reveals multiple zinc-binding motifs including a novel zinc binuclear clusterBellon, Steven F.; Rodgers, Karla K.; Schatz, David G.; Coleman, Joseph E.; Steitz, Thomas A.Nature Structural Biology (1997), 4 (7), 586-591CODEN: NSBIEW; ISSN:1072-8368. (Nature America)The crystal structure of the dimerization domain of the V(D)J recombination-activating protein, RAG1, was solved using zinc anomalous scattering. The structure reveals an unusual combination of multi-class zinc-binding motifs, including a zinc RING finger and a C2H2 zinc finger, that together form a single structural domain. The domain also contains a unique zinc binuclear cluster in place of normally mononuclear zinc site in the RING finger. Together, four zinc ions help organize the entire domain, including the two helixes that form the dimer interface.
- 410Wu, H.; Min, J.; Lunin, V. V.; Antoshenko, T.; Dombrovski, L.; Zeng, H.; Allali-Hassani, A.; Campagna-Slater, V.; Vedadi, M.; Arrowsmith, C. H.; Plotnikov, A. N.; Schapira, M. Structural biology of human H3K9 methyltransferases. PLoS One 2010, 5, e8570 DOI: 10.1371/journal.pone.0008570Google Scholar410Structural biology of human H3K9 methyltransferasesWu Hong; Min Jinrong; Lunin Vladimir V; Antoshenko Tatiana; Dombrovski Ludmila; Zeng Hong; Allali-Hassani Abdellah; Campagna-Slater Valerie; Vedadi Masoud; Arrowsmith Cheryl H; Plotnikov Alexander N; Schapira MatthieuPloS one (2010), 5 (1), e8570 ISSN:.SET domain methyltransferases deposit methyl marks on specific histone tail lysine residues and play a major role in epigenetic regulation of gene transcription. We solved the structures of the catalytic domains of GLP, G9a, Suv39H2 and PRDM2, four of the eight known human H3K9 methyltransferases in their apo conformation or in complex with the methyl donating cofactor, and peptide substrates. We analyzed the structural determinants for methylation state specificity, and designed a G9a mutant able to tri-methylate H3K9. We show that the I-SET domain acts as a rigid docking platform, while induced-fit of the Post-SET domain is necessary to achieve a catalytically competent conformation. We also propose a model where long-range electrostatics bring enzyme and histone substrate together, while the presence of an arginine upstream of the target lysine is critical for binding and specificity. ENHANCED VERSION: This article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the web plugin are available in Text S1.
- 411Takahashi, S. Positive and negative regulators of the metallothionein gene. Mol. Med. Rep. 2015, 12, 795– 799, DOI: 10.3892/mmr.2015.3459Google Scholar411Positive and negative regulators of the metallothionein gene (review)Takahashi, ShinichiroMolecular Medicine Reports (2015), 12 (1, Pt. B), 795-799CODEN: MMROA5; ISSN:1791-2997. (Spandidos Publications Ltd.)A review. Metallothioneins (MTs) are metal-binding proteins involved in diverse processes, including metal homeostasis and detoxification, the oxidative stress response and cell proliferation. Aberrant expression and silencing of these genes are important in a no. of diseases. Several pos. regulators of MT genes, including metal-responsive element-binding transcription factor (MTF)-1 and upstream stimulatory factor (USF)-1, have been identified and mechanisms of induction have been well described. However, the neg. regulators of MT genes remain to be elucidated. Previous studies from the group of the present review have revealed that the hematopoietic master transcription factor, PU.1, directly represses the expression levels of MT genes through its epigenetic activities, and upregulation of MT results in the potent inhibition of myeloid differentiation. The present review focuses on PU.1 and several other neg. regulators of this gene, including PZ120, DNA methyltransferase 3a with Mbd3 and Brg1 complex, CCAAT enhancer binding protein α and Ku protein, and describes the suppression of the MT genes through these transcription factors.
- 412Tang, C.-M.; Westling, J.; Seto, E. trans repression of the human metallothionein IIA gene promoter by PZ120, a novel 120-kilodalton zinc finger protein.. Mol. Cell. Biol. 1999, 19, 680– 689, DOI: 10.1128/MCB.19.1.680Google Scholar412Trans Repression of the human metallothionein IIA gene promoter by PZ120, a novel 120-kilodalton zinc finger proteinTang, Chih-Min; Westling, Jennifer; Seto, EdwardMolecular and Cellular Biology (1999), 19 (1), 680-689CODEN: MCEBD4; ISSN:0270-7306. (American Society for Microbiology)Metallothioneins are small, highly conserved, cysteine-rich proteins that bind a variety of metal ions. They are found in virtually all eukaryotic organisms and are regulated primarily at the transcriptional level. In humans, the predominant metallothionein gene is hMTIIA, which accounts for 50% of all metallothioneins expressed in cultured human cells. The hMTIIA promoter is quite complex. In addn. to cis-acting DNA sequences that serve as binding sites for trans-acting factors such as Sp1, AP1, AP2, AP4, and the glucocorticoid receptor, the hMTIIA promoter contains eight consensus metal response element sequences. We report here the cloning of a novel zinc finger protein with a mol. mass of 120 kDa (PZ120) that interacts specifically with the hMTIIA transcription initiation site. The PZ120 protein is ubiquitously expressed in most tissues and possesses a conserved poxvirus and zinc finger (POZ) motif previously found in several zinc finger transcription factors. Intriguingly, we found that a region of PZ120 outside of the zinc finger domain can bind specifically to the hMTIIA DNA. Using transient-transfection anal., we found that PZ120 repressed transcription of the hMTIIA promoter. These results suggest that the hMTIIA gene is regulated by an addnl. neg. regulator that has not been previously described.
- 413Sadhu, C.; Gedamu, L. Metal-specific posttranscriptional control of human metallothionein genes. Mol. Cell. Biol. 1989, 9, 5738– 5741, DOI: 10.1128/mcb.9.12.5738-5741.1989Google Scholar413Metal-specific posttranscriptional control of human metallothionein genesSadhu, Chanchal; Gedamu, LashitewMolecular and Cellular Biology (1989), 9 (12), 5738-41CODEN: MCEBD4; ISSN:0270-7306.During the initial 4 h of treatment, copper and zinc similarly activated the rates of transcription and mRNA accumulation from the 2 human metallothionein (MT) genes, viz., MTI-G and MTII-A, in the hepatoblastoma cell line HepG2. The levels of copper-induced MT mRNAs remained at a plateau for up to 15 h. In contrast, the levels of zinc-induced MT mRNAs gradually declined after about 4 h, despite substantial transcription. The decrease in the zinc-induced MT mRNA half-life is probably due to a posttranscriptional event(s).
- 414Vasconcelos, M. H.; Tam, S. C.; Beattie, J. H.; Hesketh, J. E. Evidence of differences in the post-transcritional regulation of rat metallothionein isoforms. Biochem. J. 1996, 315, 665– 671, DOI: 10.1042/bj3150665Google Scholar414Evidence for differences in the post-transcriptional regulation of rat metallothionein isoformsVasconcelos, M. Helena; Tam, Shuk-Ching; Beattie, John H.; Hesketh, John E.Biochemical Journal (1996), 315 (2), 665-71CODEN: BIJOAK; ISSN:0264-6021. (Portland Press)The expression of metallothionein (MT)-1 and -2 mRNAs in rat liver following administration of Cd or Cu was investigated using specific oligonucleotides. The specificity was confirmed using a competitive prehybridization assay. Cd injection caused a biphasic induction of both isoform mRNAs, whereas Cu induced a sustained, monophasic response. Anal. of polyribosomal RNA showed that, after both Cd and Cu treatments, the recruitment of MT-1 mRNA into polyribosomes paralleled the increase in transcription, but the increase of polyribosomal MT-2 mRNA was less than that of total MT-2 mRNA. This indicates that not all the MT-2 mRNA induced was translated, suggesting that there is translational control of MT-2 mRNA expression, but not of MT-1 mRNA. This hypothesis was supported by the observation that, after Cu treatment, the induction of MT-1 protein was induced to the same extent as MT-1 mRNA, whereas the total MT protein (MT-1 + MT-2) was increased far less (7-fold) than MT-2 mRNA (30-fold).
- 415Ehmke, V.; Winkler, E.; Banner, D. W.; Haap, W.; Schweizer, W. B.; Rottmann, M.; Kaiser, M.; Freymond, C.; Schirmeister, T.; Diederich, F. Optimization of triazine nitriles as rhodesain inhibitors: structure-activity relationships, bioisosteric imidazopyridine nitriles, and X-ray crystal structure analysis with human cathepsin L. ChemMedChem 2013, 8, 967– 975, DOI: 10.1002/cmdc.201300112Google Scholar415Optimization of Triazine Nitriles as Rhodesain Inhibitors: Structure-Activity Relationships, Bioisosteric Imidazopyridine Nitriles, and X-ray Crystal Structure Analysis with Human Cathepsin LEhmke, Veronika; Winkler, Edwin; Banner, David W.; Haap, Wolfgang; Schweizer, W. Bernd; Rottmann, Matthias; Kaiser, Marcel; Freymond, Celine; Schirmeister, Tanja; Diederich, FrancoisChemMedChem (2013), 8 (6), 967-975CODEN: CHEMGX; ISSN:1860-7179. (Wiley-VCH Verlag GmbH & Co. KGaA)The cysteine protease rhodesain of Trypanosoma brucei parasites causing African sleeping sickness has emerged as a target for the development of new drug candidates. Based on a triazine nitrile moiety as electrophilic headgroup, optimization studies on the substituents for the S1, S2, and S3 pockets of the enzyme were performed using structure-based design and resulted in inhibitors with inhibition consts. in the single-digit nanomolar range. Comprehensive structure-activity relationships clarified the binding preferences of the individual pockets of the active site. The S1 pocket tolerates various substituents with a preference for flexible and basic side chains. Variation of the S2 substituent led to high-affinity ligands with inhibition consts. down to 2 nM for compds. bearing cyclohexyl substituents. Systematic investigations on the S3 pocket revealed its potential to achieve high activities with arom. vectors that undergo stacking interactions with the planar peptide backbone forming part of the pocket. X-ray crystal structure anal. with the structurally related enzyme human cathepsin L confirmed the binding mode of the triazine ligand series as proposed by mol. modeling. Sub-micromolar inhibition of the proliferation of cultured parasites was achieved for ligands decorated with the best substituents identified through the optimization cycles. In cell-based assays, the introduction of a basic side chain on the inhibitors resulted in a 35-fold increase in antitrypanosomal activity. Finally, bioisosteric imidazopyridine nitriles were studied to prevent off-target effects with unselective nucleophiles by decreasing the inherent electrophilicity of the triazine nitrile headgroup. Using this ligand, the stabilization by intramol. hydrogen bonding of the thioimidate intermediate, formed upon attack of the catalytic cysteine residue, compensates for the lower reactivity of the headgroup. The imidazopyridine nitrile ligand showed excellent stability toward the thiol nucleophile glutathione in a quant. in vitro assay and fourfold lower cytotoxicity than the parent triazine nitrile.
- 416Ye, B.; Maret, W.; Vallee, B. L. Zinc metallothionein imported into liver mitochondria modulates respiration. Proc. Natl. Acad. Sci. U. S. A. 2001, 98, 2317– 2322, DOI: 10.1073/pnas.041619198Google Scholar416Zinc metallothionein imported into liver mitochondria modulates respirationYe, Bin; Maret, Wolfgang; Vallee, Bert L.Proceedings of the National Academy of Sciences of the United States of America (2001), 98 (5), 2317-2322CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Metallothionein (MT) localizes in the intermembrane space of liver mitochondria as well as in the cytosol and nucleus. Incubation of intact liver mitochondria with physiol., micromolar concns. of MT leads to the import of MT into the mitochondria where it inhibits respiration. This activity is caused by the N-terminal β-domain of MT; in this system, the isolated C-terminal α-domain is inactive. Free zinc inhibits respiration at concns. commensurate with the zinc content of either MT or the isolated β-domain, indicating that MT inhibition involves zinc delivery to mitochondria. Respiratory inhibition of uncoupled mitochondria identifies the electron transfer chain as the primary site of inhibition. The apo-form of MT, thionein, is an endogenous chelating agent and activates zinc-inhibited respiration with a 1:1 stoichiometry ([zinc binding sites]/[zinc]). Carbamoylation of the lysines of MT significantly attenuates the inhibitory effect, suggesting that these residues are crit. for the passage of MT through the outer mitochondrial membrane. Such an import pathway has been proposed for other proteins that also lack a mitochondrial targeting sequence, e.g., apo-cytochrome c, and possibly Cox17, a mitochondrial copper chaperone that is the only protein known so far to exhibit significant primary sequence homol. to MT. The presence and respiratory inhibition of MT in liver, but not heart, mitochondria suggest a hitherto unknown biol. modulating activity of MT in cellular respiration and energy metab. in a tissue-specific manner.
- 417Banerjee, D.; Onosaka, S.; George Cherian, M. Immunohistochemical localization and metallothionein in cell nucleus and cytoplasm of rat liver and kidney. Toxicology 1982, 24, 95– 105, DOI: 10.1016/0300-483X(82)90048-8Google Scholar417Immunohistochemical localization of metallothionein in cell nucleus and cytoplasm of rat liver and kidneyBanerjee, Diponkar; Onosaka, Satomi; Cherian, M. GeorgeToxicology (1982), 24 (2), 95-105CODEN: TXCYAC; ISSN:0300-483X.The intracellular distribution of metallothionein (Mt) in hepatic and renal cells from control and CdCl2-injected rats was investigated by immunohistochem. methods. Antiserum to purified rat liver Mt was prepd. in rabbits after partial polymn. of the protein. The unlabeled peroxidase-antiperoxidase method using the specific rabbit anti-rat liver Mt provided a sensitive technique to localize Mt in tissue sections. In control rats, Mt or thionein (metal-free protein) was mainly localized in the cytoplasm of hepatocytes, renal collecting duct epithelium, and distal convoluted tubular epithelium. In rats injected i.p. with CdCl2 (0.6 mg/kg) for 2 wk, Mt was present mainly in the nuclei which were largely neg. in control rats. Repeated injection with CdCl2 for 4-8 wk resulted in the appearance of Mt in the nucleus and cytoplasm. Intraluminal staining was also noted in proximal convoluted tubules along with marked vacuolation in the cytoplasm at 6 and 8 wk. High intensity staining was obsd. in proximal convoluted tubules and collecting duct epithelium of the kidneys in CdCl2-injected rats. The bile duct epithelium in liver samples, renal glomerular mesangial cells, glomerular visceral epithelial cells, and vascular smooth muscle cells showed weak to moderately intense staining. No staining was seen in vascular endothelial cells, fibroblasts, or leukocytes. The staining for Mt in this technique was abolished when the antibody was absorbed with rat liver Mt in vitro or by substitution of the antiserum with normal rabbit serum, demonstrating the specificity of the staining reaction for Mt. The results showed the presence of small amts. of Mt predominantly in the cytoplasm of control rat hepatocytes and renal tubular epithelium and its appearance in the nucleus and cytoplasm after its synthesis induced by CdCl2 injections.
- 418Tsujikawa, K.; Imai, T.; Kakutani, M.; Kayamori, Y.; Mimura, T.; Otaki, N.; Kimura, M.; Fukuyama, R.; Shimizu, N. Localization of metallothionein in nuclei of growing primary cultured adult rat hepatyocytes. FEBS Lett. 1991, 283, 239– 242, DOI: 10.1016/0014-5793(91)80597-VGoogle Scholar418Localization of metallothionein in nuclei of growing primary cultured adult rat hepatocytesTsujikawa, Kazutake; Imai, Takumi; Kakutani, Makoto; Kayamori, Yuzo; Mimura, Tsutomu; Otaki, Noriko; Kimura, Masami; Fukuyama, Ryuichi; Shimizu, NobuyoshiFEBS Letters (1991), 283 (2), 239-42CODEN: FEBLAL; ISSN:0014-5793.In primary cultured adult rat hepatocytes stimulated by EGF and insulin, dramatic changes in the subcellular distribution of metallothionein were clarified by indirect immunofluorescence using antisera specific for this protein. Metallothionein was detected only in the cytoplasm of cultured hepatocytes in the G0 and G1 phases, but was concd. in the cell nuclei in the early S phase. The strongest staining pattern in the nuclei was obsd. 12 h after stimulation. Subsequently, the intensity of metallothionein staining in the nuclei decreased. These results suggest that primary cultured hepatocytes are suitable for examg. the relation between subcellular localization of metallothionein and cell growth.
- 419Cherian, M. G.; Howell, S. B.; Imura, N.; Klaassen, C. D.; Koropatnick, J.; Lazo, J. S.; Waalkes, M. P. Role of metallothionein in carcinogenesis. Toxicol. Appl. Pharmacol. 1994, 126, 1– 5, DOI: 10.1006/taap.1994.1083Google Scholar419Role of metallothionein in carcinogenesisCherian, M. George; Howell, Stephen B.; Imura, Nobusama; Klaassen, Curtis D.; Koropatnick, James; Lazo, John S.; Waalkes, Michael P.Toxicology and Applied Pharmacology (1994), 126 (1), 1-5CODEN: TXAPA9; ISSN:0041-008X.A review, with 36 refs. Metallothionein (MT) is a low-mol.-wt. protein (6800 Da) and one-third of its amino acids are cysteine residues. The 20 cysteines coordinate 7 metal atoms (zinc, copper, and/or cadmium). This protein is extremely inducible by metals as well as a no. of org. compds. MT is thought to be an important intracellular storage site for zinc and possibly other essential trace elements. In addn., tolerance to cadmium toxicity is often due to the induction of MT, which sequesters cadmium and lowers its concn. at crit. intracellular sites. Recently it has been proposed that MT might play important roles in several aspects of the carcinogenic process. In this context a symposium was held recently on this topic at the 1993 Annual Society of Toxicol. Meeting. At this symposium Dr. Cherian discussed the expression of MT in various human tumors and its use as a potential marker of tumor differentiation or cell proliferation. Dr Imura provided data illustrating that induction of MT can be used as an adjunct in cancer chemotherapy, in preventing toxicity caused by γ-irradn. or cisplatin (CDDP) and other chemotherapeutics. Induction of MT has been suggested to be an important mechanism of resistance of tumor cells to chemotherapeutic agents, such as CDDP. This is controversial, and various views on this topic were presented by Drs. Howell, Lazo, and Koropatnick. Dr. Waalkes then discussed the role of MT in the carcinogenic and anticarcinogenic effects of metals.
- 420Nagel, W.; Vallee, B. L. Cell cycle regulation of metallothionein in human colonic cancer cells. Proc. Natl. Acad. Sci. U. S. A. 1995, 92, 579– 583, DOI: 10.1073/pnas.92.2.579Google Scholar420Cell cycle regulation of metallothionein in human colonic cancer cellsNagel, Wolfgang W.; Vallee, Bert L.Proceedings of the National Academy of Sciences of the United States of America (1995), 92 (2), 579-83CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Elevated levels of metallothionein (MT) found in rapidly growing tissues such as neonatal liver and various types of human tumors have suggested a role for MT in cell proliferation. To further explore this possibility we investigated the concn. of MT in human colonic cancer (HT-29) cells at different stages of proliferation by means of immunocytochem. and competitive binding. MT is increased in subconfluent proliferating cells relative to growth-inhibited confluent cells, much as it is in growing tissues. Cycling cells synchronized with compactin, an inhibitor of 3-hydroxy-3-methylglutaryl-CoA reductase, revealed an oscillation of cytoplasmic MT that reached a max. in successive late G1 phases and at the G1/S transition. Individual phases of the cell cycle were assessed by [3H]thymidine incorporation and by immunofluorescence employing an antibody that detects a nuclear antigen assocd. with proliferation. An ELISA was used to quantify the relative amts. of MT in homogenate supernatants of HT-29 cells. A 2- to 3-fold increase in MT in actively proliferating cells and the regulation of the protein during the mitotic cell cycle point to a physiol. role for MT in cellular proliferation and suggest that it may also serve as a proliferation marker.
- 421Apostolova, M. D.; Ivanova, I. A.; Cherian, M. G. Signal transduction pathways, and nuclear translocation of zinc and metallothionein during differentiation of myoblast. Biochem. Cell Biol. 2000, 78, 27– 37, DOI: 10.1139/o99-070Google Scholar421Signal transduction pathways, and nuclear translocation of zinc and metallothionein during differentiation of myoblastsApostolova, Margarita D.; Ivanova, Iordanka A.; Cherian, M. GeorgeBiochemistry and Cell Biology (2000), 78 (1), 27-37CODEN: BCBIEQ; ISSN:0829-8211. (National Research Council of Canada)The changes in subcellular localization of metallothionein during differentiation were studied in two myoblast cell lines, L6 and H9C2. Addn. of insulin like growth factor-I or lowering fetal bovine serum to 1% can induce differentiation of myoblasts to myotubes. Metallothionein and zinc were localized mainly in the cytoplasm in myoblasts but were translocated into the nucleus of newly formed myotubes during early differentiation. In fully differentiated myotubes, metallothionein content was decreased with a cytoplasmic localization. Addn. of an inhibitor of mitogen-activated protein kinase, PD 98059, did not affect differentiation but blocked nuclear translocation of metallothionein. LY 294092, an inhibitor of PI3 kinase, and rapamycin, an inhibitor of p70S6 serine/threonine kinase, abolished insulin-like growth factor-I induced differentiation of myoblasts, retained metallothionein in the cytoplasm, and decreased metallothionein content. These results demonstrate that the cytoplasmic-nuclear translocation of metallothionein occurs during the early stage of differentiation of myoblasts to myotubes and can be blocked by inhibition of certain signal transduction pathways. The transient nuclear localization of metallothionein and zinc may be related to a high requirement for zinc for metabolic activities during the early stage of differentiation.
- 422Levadoux, M.; Mahon, C.; Beattie, J. H.; Wallace, H. M.; Hesketh, J. E. Nuclear import of metallothionein requires its mRNA to be associated with the perinuclear cytoskeleton. J. Biol. Chem. 1999, 274, 34961– 34966, DOI: 10.1074/jbc.274.49.34961Google Scholar422Nuclear import of metallothionein requires its mRNA to be associated with the perinuclear cytoskeletonLevadoux, Marilyne; Mahon, Connor; Beattie, John H.; Wallace, Heather M.; Hesketh, John E.Journal of Biological Chemistry (1999), 274 (49), 34961-34966CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)The influence of mRNA localization on metallothionein-1 protein distribution was studied by immunocytochem. We used Chinese hamster ovary cells that had been transfected with either a native metallothionein-1 gene construct or metallothionein-1 5'-untranslated region and coding sequences linked to the 3'-untranslated region from glutathione peroxidase. The change in the 3'-untranslated region caused the delocalization of the mRNA with a loss of the perinuclear localization and assocn. with the cytoskeleton. Clones were selected which expressed similar levels of metallothionein-1 protein, as assessed by RIA. The results showed that loss of metallothionein-1 mRNA localization was assocd. with a loss of metallothionein-1 protein localization, most notably with a lack of metallothionein-1 protein in the nucleus of synchronized cells which were beginning to synthesize DNA. This indicates that the assocn. of metallothionein-1 mRNA with the cytoskeleton around the nucleus is essential for efficient shuttling of the protein into the nucleus during the G1 to S phase transition. This is the first demonstration of a physiol. role for perinuclear mRNA localization and we propose that such localization may be important for a wide range of nuclear proteins, including those that shuttle between nucleus and cytoplasm in a cell cycle dependent manner.
- 423Nagano, T.; Itoh, N.; Ebisutani, C.; Takatani, T.; Miyoshi, T.; Nakanishi, T.; Tanaka, K. The transport mechanism of metallothionein is different from that of classical NLS-bearing protein. J. Cell. Physiol. 2000, 185, 440– 446, DOI: 10.1002/1097-4652(200012)185:3<440::AID-JCP15>3.0.CO;2-NGoogle Scholar423The transport mechanism of metallothionein is different from that of classical NLS-bearing proteinNagano, Takayuki; Itoh, Norio; Ebisutani, Chikara; Takatani, Tomoka; Miyoshi, Tomoya; Nakanishi, Tsuyoshi; Tanaka, KeiichiJournal of Cellular Physiology (2000), 185 (3), 440-446CODEN: JCLLAX; ISSN:0021-9541. (Wiley-Liss, Inc.)A nuclear localization signal (NLS) has been detected in several nuclear proteins. Classical NLS-mediated nuclear pore targeting is performed by using the cytosolic factors, importin α and importin β, whereas nuclear translocation requires the small GTPase, Ran. In the present study, we demonstrated that nuclear localization of metallothionein (MT) differs from that of classical NLS-mediated substrates. In digitonin-permeabilized BALB/c3T3 cells, biotinylated MT was localized in the nucleus in the presence of ATP and erythrocyte cytosol in the same manner as for SV40 large T NLS-conjugated allophycocyanin (APC-NLS). Under ATP-free conditions, nuclear rim-binding was obsd. in both transport substrates. Rim-binding of labeled MT was competitively inhibited by the addn. of an excess amt. of unlabeled MT. Different elution profiles were obsd. for the localization-promotion activities of MT in the cytosol compared to those of APC-NLS. Furthermore, nuclear localization of MT was detd. to be a wheat germ agglutinin-insensitive, GTPγS-sensitive, and anti-Ran antibody-sensitive process. Green fluorescent protein-metallothionein (GFP-MT) fusion protein was also localized in the nucleus in the stable transformant of CHL-IU cells. These results strongly suggest that the targeting by MT of the nuclear pore is mediated by cytosolic factor(s) other than importins and that MT requires Ran for its nuclear localization.
- 424Woo, E. S.; Dellapiazza, D.; Wang, A. S.; Lazo, J. S. Energy-dependent nuclear binding dictates metallothionein localization. J. Cell. Physiol. 2000, 182, 69– 76, DOI: 10.1002/(SICI)1097-4652(200001)182:1<69::AID-JCP8>3.0.CO;2-9Google Scholar424Energy-dependent nuclear binding dictates metallothionein localizationWoo, Elizabeth S.; Dellapiazza, Dana; Wang, Angela S.; Lazo, John S.Journal of Cellular Physiology (2000), 182 (1), 69-76CODEN: JCLLAX; ISSN:0021-9541. (Wiley-Liss, Inc.)Metallothioneins (MTs) are low-mol.-wt., stress-activated proteins that protect cells against heavy metals, oxidants, and some electrophilic drugs. Both nuclear and cytoplasmic MT phenotypes have been obsd. in cells even though MTs (6 kDa) are well below the size exclusion limit for diffusion through the nuclear envelope. To study the factors controlling MT subcellular partitioning, the authors covalently linked MTII to a fluorescent label and examd. its subcellular distribution in response both to pharmacol. and phys. perturbations. Fluorescent MTII localized to the nucleus of digitonin-permeabilized human SCC25 carcinoma cells, consistent with its endogenous distribution in these cells. Nuclear sequestration of the fluorescent MTII was inhibited by a 100-fold molar excess of unlabeled MTII and by wheat germ agglutinin, indicating a saturable binding mechanism and the involvement of one or more glycoproteins, resp. Depletion of ATP (ATP) inhibited MTII nuclear localization, implying energy-dependent nuclear translocation or retention of MT. Neither chilling nor the absence of cytosolic exts. inhibited nuclear sequestration of MTII, supporting diffusion-based entry mechanism. In situ biochem. extns. of the nuclear MTII revealed at least two distinct binding activities. Collectively, these data indicate that MTII diffuses into the nucleus of SCC25 cells, where it is selectively and actively retained by nuclear binding factors, imparting its localization phenotype.
- 425Takahashi, Y.; Ogra, Y.; Suzuki, K. T. Nuclear trafficking of metallothionein requires oxidation of a cytosolic partner. J. Cell. Physiol. 2005, 202, 563– 569, DOI: 10.1002/jcp.20158Google Scholar425Nuclear trafficking of metallothionein requires oxidation of a cytosolic partnerTakahashi Yukihisa; Ogra Yasumitsu; Suzuki Kazuo TJournal of cellular physiology (2005), 202 (2), 563-9 ISSN:0021-9541.The present study revealed the mechanism underlying the nuclear trafficking of metallothionein (MT). Nuclear localization of MT in digitonin-permeabilized BALB 3T3 cells was enhanced in the presence of a cytosolic factor added as a rat red blood cell lysate by oxidation with H2O2 in a dose-dependent manner, but inhibited with excess glutathione. A cytosolic partner was assumed to bind MT and retain it in the cytoplasm, and its oxidation can mobilize MT to the nuclei on cellular oxidation. Pre-treatment of nuclei with H2O2 did not enhance the localization, and MT that had been localized in the nuclei was washed out, indicating that MT is in the nuclei as a result of a higher rate of uptake by the nuclei than the rate of diffusion from the nuclei. Nuclear localization of lysozyme and nuclear localization signal (NLS)-bearing allophycocyanin were not enhanced by the oxidation in the presence of cytosolic factor, suggesting that the nuclear traffic occurring on oxidation is specific to MT. Moreover, when cells were arrested the cell cycle at the S phase, MT was localized in the nuclei in response to coincidental generation of a feeble reactive oxygen species (ROS). These observations suggest that MT comes localized in the nuclei on the sensing of intracellular oxidation, whereby a cytosolic partner specific to MT comes oxidized as a cargo system, MT being localized as a result of enhanced uptake in the nuclei and re-localized in the cytoplasm diffusely. Nuclear MT was proposed to protect the nuclei from the oxidation occurring with progression of the cell cycle.
- 426Lee, S. J.; Park, M. H.; Kim, H. J.; Koh, J. Y. Metallothionein-3 regulates lysosomal function in cultured astrocytes under both normal and oxidative conditions. Glia 2010, 58, 1186– 1196, DOI: 10.1002/glia.20998Google Scholar426Metallothionein-3 regulates lysosomal function in cultured astrocytes under both normal and oxidative conditionsLee Sook-Jeong; Park Mi-Ha; Kim Hyun-Jae; Koh Jae-YoungGlia (2010), 58 (10), 1186-96 ISSN:.Cellular zinc plays a key role in lysosomal change and cell death in neurons and astrocytes under oxidative stress. Here, using astrocytes lacking metallothionein-3 (MT3), a potential source of labile zinc in the brain, we studied the role of MT3 in oxidative stress responses. H(2)O(2) induced a large increase in labile zinc in wild-type (WT) astrocytes, but stimulated only a modest rise in MT3-null astrocytes. In addition, H(2)O(2)-induced lysosomal membrane permeabilization (LMP) and cell death were comparably attenuated in MT3-null astrocytes. Expression and glycosylation of Lamp1 (lysosome-associated membrane protein 1) and Lamp2 were increased in MT3-null astrocytes, and the activities of several lysosomal enzymes were significantly reduced, indicating an effect of MT3 on lysosomal components. Consistent with lysosomal dysfunction in MT3-null cells, the level of LC3-II (microtubule-associated protein 1 light chain 3), a marker of early autophagy, was increased by oxidative stress in WT astrocytes, but not in MT3-null cells. Similar changes in Lamp1, LC3, and cathepsin-D were induced by the lysosomal inhibitors bafilomycin A1, chloroquine, and monensin, indicating that lysosomal dysfunction may lie upstream of changes observed in MT3-null astrocytes. Consistent with this idea, lysosomal accumulation of cholesterol and lipofuscin were augmented in MT3-null astrocytes. Similar to the results seen in MT3-null cells, MT3 knockdown by siRNA inhibited oxidative stress-induced increases in zinc and LMP. These results indicate that MT3 may play a key role in normal lysosomal function in cultured astrocytes.
- 427Nordberg, G. F.; Garvey, J. S.; Chang, C. C. Metallothionein in plasma and urine of cadmium workers. Environ. Res. 1982, 28, 179– 182, DOI: 10.1016/0013-9351(82)90167-0Google Scholar427Metallothionein in plasma and urine of cadmium workersNordberg, Gunnar F.; Garvey, Justine S.; Chang, Chin C.Environmental Research (1982), 28 (1), 179-82CODEN: ENVRAL; ISSN:0013-9351.Metallothionein (MT) and β-2-microglobulin (BM) concns. were detd. in samples of plasma and urine from workers in a Swedish factory manufg. Cd batteries. Concns. of metallothionein in plasma was 2-11 ng/g and in urine from 2-155 ng/g. In workers with urinary BM concns. >500 μg/L, the median value of MT in urine was statistically significantly higher than that in workers with urinary BM concns. <500 μg/L. In individual cases, however, a considerable increase in urinary BM may occur without any increase in urinary MT. The possible significance of increased MT concns. in plasma, as seen in some of the workers in the present study, the relation to transport of Cd-MT to the renal cortex is pointed out.
- 428Nordberg, M.; Nordberg, G. F. Metallothioneins: Historical developments and overview. Met. Ions Life Sci. 2009, 5, 1– 29, DOI: 10.1039/9781847559531-00001Google Scholar428Metallothioneins: historical development and overviewNordberg, Monica; Nordberg, Gunnar F.Metal Ions in Life Sciences (2009), 5 (Metallothioneins and Related Chelators), 1-29CODEN: MILSCT; ISSN:1559-0836. (Royal Society of Chemistry)A review. The history on research of metallothionein is reviewed. Various methods for isolation, characterization, and quantification are evaluated. The role of metallothionein in metal metab. and toxicity is explained. Gender differences and polymorphism as well as possible relationships with diseases are discussed. The review is based on data from the literature and on own original exptl. and epidemiol. data. Aspects on future research within the metallothionein field are indicated.
- 429Sabolić, I.; Breljak, D.; Skarica, M.; Herak-Kramberger, C. M. Role of metallothionein in cadmium traffic and toxicity in kidneys and other mammalian organs. BioMetals 2010, 23, 897– 926, DOI: 10.1007/s10534-010-9351-zGoogle Scholar429Role of metallothionein in cadmium traffic and toxicity in kidneys and other mammalian organsSabolic, Ivan; Breljak, Davorka; Skarica, Mario; Herak-Kramberger, Carol M.BioMetals (2010), 23 (5), 897-926CODEN: BOMEEH; ISSN:0966-0844. (Springer)A review. Metallothioneins are cysteine-rich, small metal-binding proteins present in various mammalian tissues. Of the four common metallothioneins, MT-1 and MT-2 (MTs) are expressed in most tissues, MT-3 is predominantly present in brain, whereas MT-4 is restricted to the squamous epithelia. The expression of MT-1 and MT-2 in some organs exhibits sex, age, and strain differences, and inducibility with a variety of stimuli. In adult mammals, MTs have been localized largely in the cell cytoplasm, but also in lysosomes, mitochondria and nuclei. The major physiol. functions of MTs include homeostasis of essential metals Zn and Cu, protection against cytotoxicity of Cd and other toxic metals, and scavenging free radicals generated in oxidative stress. The role of MTs in Cd-induced acute and chronic toxicity, particularly in liver and kidneys, is reviewed in more details. In acute toxicity, liver is the primary target, whereas in chronic toxicity, kidneys are major targets of Cd. The intracellular MTs bind Cd ions and form CdMT. In chronic intoxication, Cd stimulates de novo synthesis of MTs; it is assumed that toxicity in the cells starts when loading with Cd ions exceeds the buffering capacity of intracellular MTs. CdMT, released from the Cd-injured organs, or when applied parenterally for exptl. purposes, reaches the kidneys via circulation, where it is filtered, endocytosed in the proximal tubule cells, and degraded in lysosomes. Liberated Cd can immediately affect the cell structures and functions. The resulting proteinuria and CdMT in the urine can be used as biomarkers of tubular injury.
- 430Bremner, I.; Mehra, R. K. Assay of extracellular metallothionein. Methods Enzymol. 1991, 205, 60– 70, DOI: 10.1016/0076-6879(91)05086-BGoogle Scholar430Assay of extracellular metallothioneinBremner, Ian; Mehra, Rajesh K.Methods in Enzymology (1991), 205 (Metallobiochem. Pt. B), 60-70CODEN: MENZAU; ISSN:0076-6879.Purifn. of antigen, prepn. of antibodies, detection of antibodies, immunoassay procedure, and assay of metallothionein in extracellular fluids are discussed.
- 431Akintola, D. F.; Sampson, B.; Fleck, A. Development of an enzyme-linked immunosorbent assay for human metallothionein-1 in plasma and urine. J. Lab. Clin. Med. 1995, 126, 119– 127Google Scholar431Development of an enzyme-linked immunosorbent assay for human metallothionein-1 in plasma and urineAkintola, D. F.; Sampson, B.; Fleck, A.Journal of Laboratory and Clinical Medicine (1995), 126 (2), 119-27CODEN: JLCMAK; ISSN:0022-2143. (Mosby-Year Book)The development of a sensitive ELISA for human metallothionein-1 (MT-1) is reported. MT was purified from postmortem human liver and used to raise high-titer antibodies in rabbits. The assay was specific for human MT-1, and there was no significant cross-reaction with human MT-2. The detection limit (sensitivity) of the assay was 5 ng/mL, and the added MT-1 could be fully recovered from plasma and urine. The normal ref. range for MT-1 was 32 ng/mL in plasma and 10 ng MT-1 per μmol of creatinine in random samples of urine. No significant differences were found between the values for males and females. The concn. of MT-1 was greatly increased between 24 and 48 h after surgery, indicating that the protein behaves like an acute phase reactant in human subjects.
- 432Maret, W. Metallothionein and the acute phase response. J. Lab. Clin. Med. 1995, 126, 106– 107Google Scholar432Metallothionein and the acute phase responseMaret WThe Journal of laboratory and clinical medicine (1995), 126 (2), 106-7 ISSN:0022-2143.There is no expanded citation for this reference.
- 433Knipp, M.; Meloni, G.; Roschitzki, B.; Vašák, M. Zn7-metallothionein-3 and the synaptic vesicle cycle: interaction of metallothionein-3 with the small GTPase Rab3A. Biochemistry 2005, 44, 3159– 3165, DOI: 10.1021/bi047636dGoogle Scholar433Zn7Metallothionein-3 and the Synaptic Vesicle Cycle: Interaction of Metallothionein-3 with the Small GTPase Rab3AKnipp, Markus; Meloni, Gabriele; Roschitzki, Bernd; Vasak, MilanBiochemistry (2005), 44 (9), 3159-3165CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)In the central nervous system, a large amt. of chelatable Zn2+ is sequestered in presynaptic vesicles of certain glutamatergic nerve terminals. The exo-endocytic cycle of synaptic vesicles is strictly linked to the small GTPase Rab3A. Metallothionein-3 (Zn7MT-3) has been proposed to be involved in the intracellular trafficking of Zn2+ in zinc-contg. neurons, but its role in this process is not understood. By using affinity pptn. and surface plasmon resonance anal., we show that Zn7MT-3 binds reversibly to Rab3A·GDP (KD = 2.6 μM), but not to Rab3A·GTP. The binding of Zn7MT-3 to Rab3A·GDP is specific as no binding was obsd. with the metal-free form of MT-3. Mutational studies of Rab3A mapped the interaction site to the effector binding site of the protein. This location is further supported by the kinetics of GDP exchange, which was found to be unaffected by binding of Zn7MT-3 to Rab3A·GDP. The interaction of Zn7MT-3 with Rab3A indicates that Zn7MT-3 is not merely a cellular Zn2+ buffer, but actively participates in synaptic vesicle trafficking upstream of vesicle fusion.
- 434Chung, R. S.; Penkowa, M.; Dittmann, J.; King, C. E.; Bartlett, C.; Asmussen, J. W.; Hidalgo, J.; Carrasco, J.; Leung, Y. K.; Walker, A. K.; Fung, S. J.; Dunlop, S. A.; Fitzgerald, M.; Beazley, L. D.; Chuah, M. I.; Vickers, J. C.; West, A. K. Redefining the role of metallothionein within the injured brain: Extracellular metallothioneins play and important role in the astrocyte-neuron response to injury. J. Biol. Chem. 2008, 283, 15349– 15358, DOI: 10.1074/jbc.M708446200Google Scholar434Redefining the Role of Metallothionein within the Injured Brain: extracellular metallothioneins play an important role in the astrocyte-neuron response to injuryChung, Roger S.; Penkowa, Milena; Dittmann, Justin; King, Carolyn E.; Bartlett, Carole; Asmussen, Johanne W.; Hidalgo, Juan; Carrasco, Javier; Leung, Yee Kee J.; Walker, Adam K.; Fung, Samantha J.; Dunlop, Sarah A.; Fitzgerald, Melinda; Beazley, Lyn D.; Chuah, Meng I.; Vickers, James C.; West, Adrian K.Journal of Biological Chemistry (2008), 283 (22), 15349-15358CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)A no. of intracellular proteins that are protective after brain injury are classically thought to exert their effect within the expressing cell. The astrocytic metallothioneins (MT) are one example and are thought to act via intracellular free radical scavenging and heavy metal regulation, and in particular zinc. Indeed, we have previously established that astrocytic MTs are required for successful brain healing. Here we provide evidence for a fundamentally different mode of action relying upon intercellular transfer from astrocytes to neurons, which in turn leads to uptake-dependent axonal regeneration. First, we show that MT can be detected within the extracellular fluid of the injured brain, and that cultured astrocytes are capable of actively secreting MT in a regulatable manner. Second, we identify a receptor, megalin, that mediates MT transport into neurons. Third, we directly demonstrate for the first time the transfer of MT from astrocytes to neurons over a specific time course in vitro. Finally, we show that MT is rapidly internalized via the cell bodies of retinal ganglion cells in vivo and is a powerful promoter of axonal regeneration through the inhibitory environment of the completely severed mature optic nerve. Our work suggests that the protective functions of MT in the central nervous system should be widened from a purely astrocytic focus to include extracellular and intra-neuronal roles. This unsuspected action of MT represents a novel paradigm of astrocyte-neuronal interaction after injury and may have implications for the development of MT-based therapeutic agents.
- 435El Refaey, H.; Ebadi, M.; Kuszynski, C. A.; Sweeney, J.; Hamada, F. M.; Hamed, A. Identification of metallothionein receptors in human astrocytes. Neurosci. Lett. 1997, 231, 131– 134, DOI: 10.1016/S0304-3940(97)00548-XGoogle Scholar435Identification of metallothionein receptors in human astrocytesEl Refaey H; Ebadi M; Kuszynski C A; Sweeney J; Hamada F M; Hamed ANeuroscience letters (1997), 231 (3), 131-4 ISSN:0304-3940.Metallothionein (MT) isoforms are low molecular weight (6000-7000 Da) zinc binding proteins containing 60-68 amino acid residues, 25-30% cysteine, no aromatic amino acids, and binding between 5-7 g zinc/mol of protein. Since the synthesis of MT is induced by endotoxin, cytokines, and glucocorticoids, MT is now considered to be an acute phase protein protecting against oxygen radicals and oxidative damages caused by inflammation, tissue injury, and stress to the central nervous system. By postulating that a specific mechanism must exist to foster the induction of MTs I and II by numerous and diversified factors, we searched for and identified for the first time, MT receptors on U373MG cell membrane preparations, by using fluoresceinated MT I isoform probe; and by employing cysteine, glutathione, and four MT isoforms to determine high affinity and specific binding. MT receptors revealed a Kd value of 0.84 nM and a Bmax of 99.82 fmol/mg protein. Moreover, MT receptors were found in greater density on the surface of aggregated astrocytes. We postulate that conditions or agents generating reactive oxygen species may influence the expression of MT receptors.
- 436Wolff, N. A.; Abouhamed, M.; Verroust, P. J.; Thévenod, F. Megalin-dependent internalization of cadmium-metallothionein and cytotoxicity in cultured renal proximal tubule cells. J. Pharmacol. Exp. Ther. 2006, 318, 782– 787, DOI: 10.1124/jpet.106.102574Google Scholar436Megalin-dependent internalization of cadmium-metallothionein and cytotoxicity in cultured renal proximal tubule cellsWolff, Natascha A.; Abouhamed, Marouan; Verroust, Pierre J.; Thevenod, FrankJournal of Pharmacology and Experimental Therapeutics (2006), 318 (2), 782-791CODEN: JPETAB; ISSN:0022-3565. (American Society for Pharmacology and Experimental Therapeutics)Chronic cadmium (Cd2+) exposure results in renal proximal tubular cell damage. Delivery of Cd2+ to the kidney occurs mainly as complexes with metallothionein-1 (mol. mass ∼ 7 kDa), freely filtered at the glomerulus. For Cd2+ to gain access to the proximal tubule cells, these complexes are thought to be internalized via receptors for small protein ligands, such as megalin and cubilin, followed by release of Cd2+ from metallothionein-1 in endosomal/lysosomal compartments. To investigate the role of megalin in renal cadmium-metallothionein-1 resorption, megalin expression and dependence of cadmium-metallothionein-1 internalization and cytotoxicity on megalin were studied in a renal proximal tubular cell model (WKPT-0293 Cl.2 cells). Expression of megalin was detected by reverse transcriptase-polymerase chain reaction and visualized by immunofluorescence both at the cell surface (live staining) and intracellularly (permeabilized cells). Internalization of Alexa Fluor 488-coupled metallothionein-1 was concn.-dependent, satg. at approx. 15 μM. At 14.3 μM, metallothionein-1 uptake could be significantly attenuated by 30.9±6.6% (n = 4) by 1 μM of the receptor-assocd. protein (RAP) used as a competitive inhibitor of cadmium-metallothionein-1 binding to megalin and cubilin. Consistently, cytotoxicity of a 24-h treatment with 7.14 μM cadmium-metallothionein-1 was significantly reduced by 41.0±7.6%, 61.6±3.4%, and 26.2±1.8% (n = 4-5 each) by the presence of 1 μM RAP, 400 μg/mL anti-megalin antibody, or 5 μM of the cubilin-specific ligand, apo-transferrin, resp. Cubilin expression in proximal tubule cells was also confirmed at the mRNA and protein level. The data indicate that renal proximal tubular cadmium-metallothionein-1 uptake and cell death are mediated at least in part by megalin.
- 437Thévenod, F.; Fels, J.; Lee, W. K.; Zarbock, R. Channels, transporters and receptors for cadmium and cadmium complexes in eukaryotic cells: myths and facts. BioMetals 2019, 32, 469– 489, DOI: 10.1007/s10534-019-00176-6Google Scholar437Channels, transporters and receptors for cadmium and cadmium complexes in eukaryotic cells: myths and factsThevenod, Frank; Fels, Johannes; Lee, Wing-Kee; Zarbock, RalfBioMetals (2019), 32 (3), 469-489CODEN: BOMEEH; ISSN:0966-0844. (Springer)A review. Cadmium (Cd2+) is a toxic and non-essential divalent metal ion in eukaryotic cells. Cells can only be targeted by Cd2+ if it hijacks physiol. high-affinity entry pathways, which transport essential divalent metal ions in a process termed "ionic and mol. mimicry". Hence, "free" Cd2+ ions and Cd2+ complexed with small org. mols. are transported across cellular membranes via ion channels, carriers and ATP hydrolyzing pumps, whereas receptor-mediated endocytosis (RME) internalizes Cd2+-protein complexes. Only Cd2+ transport pathways validated by stringent methodol., namely electrophysiol., 109Cd2+ tracer studies, inductively coupled plasma mass spectrometry, at. absorption spectroscopy, Cd2+-sensitive fluorescent dyes, or specific ligand binding and internalization assays for RME are reviewed whereas indirect correlative studies are excluded. At toxicol. relevant concns. in the submicromolar range, Cd2+ permeates voltage-dependent Ca2+ channels ("T-type" CaV3.1, CatSper), transient receptor potential (TRP) channels (TRPA1, TRPV5/6, TRPML1), solute carriers (SLCs) (DMT1/SLC11A2, ZIP8/SLC39A8, ZIP14/SLC39A14), amino acid/cystine transporters (SLC7A9/SLC3A1, SLC7A9/SLC7A13), and Cd2+-protein complexes are endocytosed by the lipocalin-2/NGAL receptor SLC22A17. Cd2+ transport via the mitochondrial Ca2+ uniporter, ATPases ABCC1/2/5 and transferrin receptor 1 is likely but requires further evidence. Cd2+ flux occurs through the influx carrier OCT2/SLC22A2, efflux MATE proteins SLC47A1/A2, the efflux ATPase ABCB1, and RME of Cd2+-metallothionein by the receptor megalin (low d. lipoprotein receptor-related protein 2, LRP2):cubilin albeit at high concns. thus questioning their relevance in Cd2+ loading. Which Cd2+-protein complexes are internalized by megalin:cubilin in vivo still needs to be detd. A stringent conservative and reductionist approach is mandatory to verify relevance of transport pathways for Cd2+ toxicity and to overcome dissemination of unsubstantiated conjectures.
- 438Moltedo, O.; Verde, C.; Capasso, A.; Parisi, E.; Remondelli, P.; Bonatti, S.; Alvarez-Hernandez, X.; Glass, J.; Alvino, C. G.; Leone, A. Zinc transport and metallothionein secretion in the intestinal human cell line Caco-2. J. Biol. Chem. 2000, 275, 31819– 31825, DOI: 10.1074/jbc.M002907200Google Scholar438Zinc transport and metallothionein secretion in the intestinal human cell line Caco-2Moltedo, Ornella; Verde, Cinzia; Capasso, Antonio; Parisi, Elio; Remondelli, Paolo; Bonatti, Stefano; Alvarez-Hernandez, Xavier; Glass, Jonathan; Alvino, Claudio G.; Leone, ArturoJournal of Biological Chemistry (2000), 275 (41), 31819-31825CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)Caco-2, a human cell line, displays several biochem. and morphol. characteristics of differentiated enterocytes. Among these is the ability to transport zinc from the apical to the basal compartment. This process was enhanced following exposure by the apical compartment to increasing concns. of the metal. High pressure liq. chromatog. fractionation of the media obtained from cells labeled with radioactive zinc showed that metallothioneins (MTs), small metal-binding, cysteine-rich proteins, were present in the apical and basal media of controls as well as in cells grown in the presence of high concns. of zinc. Following exposure to the metal, the levels of Zn-MTs in the apical medium increased, while in the basal compartment the greatest part of zinc appeared in a free form with minor changes in the levels of basal MTs. Metabolic labeling expts. with radioactive cysteine confirmed the apical secretion of MTs. A stable transfectant clone of Caco-2 cells (CL11) was selected for its ability to express constitutively high levels of the mouse metallothionein I protein. This cell line showed an enhanced transport of the metal following exposure to high concns. of zinc and a constitutive secretion of the mouse metallothionein I protein in the apical compartment. Together, these findings strongly support the hypothesis of a functional role between the biosynthesis and secretion of MTs and the transport of zinc in intestinal cells.
- 439Trayhurn, P.; Beattie, J. H. Physiological role of adipose tissue: white adipose tissue as an endocrine and secretory organ. Proc. Nutr. Soc. 2001, 60, 329– 339, DOI: 10.1079/PNS200194Google Scholar439Physiological role of adipose tissue: white adipose tissue as an endocrine and secretory organTrayhurn, Paul; Beattie, John H.Proceedings of the Nutrition Society (2001), 60 (3), 329-339CODEN: PNUSA4; ISSN:0029-6651. (CABI Publishing)A review with refs. The traditional role attributed to white adipose tissue is energy storage, fatty acids being released when fuel is required. The metabolic role of white fat is, however, complex. For example, the tissue is needed for normal glucose homeostasis and a role in inflammatory processes has been proposed. A radical change in perspective followed the discovery of leptin; this crit. hormone in energy balance is produced principally by white fat, giving the tissue an endocrine function. Leptin is one of a no. of proteins secreted from white adipocytes, which include angiotensinogen, adipsin, acylation-stimulating protein, adiponectin, retinol-binding protein, tumor necrosis factor α, interleukin 6, plasminogen activator inhibitor-1 and tissue factor. Some of these proteins are inflammatory cytokines, some play a role in lipid metab., while others are involved in vascular hemostasis or the complement system. The effects of specific proteins may be autocrine or paracrine, or the site of action may be distant from adipose tissue. The most recently described adipocyte secretory proteins are fasting-induced adipose factor, a fibrinogenangiopoietin-related protein, metallothionein and resistin. Resistin is an adipose tissue-specific factor which is reported to induce insulin resistance, linking diabetes to obesity. Metallothionein is a metal-binding and stress-response protein which may have an antioxidant role. The key challenges in establishing the secretory functions of white fat are to identify the complement of secreted proteins, to establish the role of each secreted protein, and to assess the pathophysiol. consequences of changes in adipocyte protein prodn. with alterations in adiposity (obesity, fasting, cachexia). There is already considerable evidence of links between increased prodn. of some adipocyte factors and the metabolic and cardiovascular complications of obesity. In essence, white adipose tissue is a major secretory and endocrine organ involved in a range of functions beyond simple fat storage.
- 440Lynes, M. A.; Zaffuto, K.; Unfricht, D. W.; Marusov, G.; Samson, J. S.; Yin, X. The physiological roles of extracellular metallothionein. Exp. Biol. Med. 2006, 231, 1548– 1554, DOI: 10.1177/153537020623100915Google Scholar440The physiological roles of extracellular metallothioneinLynes, Michael A.; Zaffuto, Kristin; Unfricht, Darryn W.; Marusov, Gregory; Samson, Jacqueline S.; Yin, XiuyunExperimental Biology and Medicine (Maywood, NJ, United States) (2006), 231 (9), 1548-1554CODEN: EBMMBE; ISSN:1535-3702. (Society for Experimental Biology and Medicine)A review. Metallothionein (MT) is a low-mol.-wt. protein with a no. of roles to play in cellular homeostasis. MT is synthesized as a consequence of a variety of cellular stressors, and has been found in both intracellular compartments and in extracellular spaces. The intracellular pool of this cysteine-rich protein can act as a reservoir of essential heavy metals, as a scavenger of reactive O and N species, as an antagonist of toxic metals and org. mols., and as a regulator of transcription factor activity. The presence of MT outside of cells due to the influence of stressors suggests that this protein may make important contributions as a "danger signal" that influences the management of responses to cellular damage. While conventional wisdom has held that extracellular MT is the result of cell death or leakage from stressed cells, there are numerous examples of selective release of proteins by nontraditional mechanisms, including stress response proteins. This suggests that MT may similarly be selectively released, and that the pool of extracellular MT represents an important regulator of various cellular functions. For example, extracellular MT has effects both on the severity of autoimmune disease, and on the development of adaptive immune functions. Extracellular MT may operate as a chemotactic factor that governs the trafficking of inflammatory cells that move to resolve damaged tissues, as a counter to extracellular oxidant-mediated damage, and as a signal that influences the functional behavior of wounded cells. A thorough understanding of the mechanisms of MT release from cells, the conditions under which MT is released to the extracellular environment, and the ways in which MT interacts with sensitive cells may both illuminate the understanding of an important control mechanism that operates in stressful conditions, and should indicate new opportunities for therapeutic management via the manipulation of this pool of extracellular MT.
- 441Liu, H.; Liang, Z.; Wang, F.; Zhou, C.; Zheng, X.; Hu, T.; He, X.; Wu, X.; Lan, P. Exosomes from mesenchymal stromal cells reduce murine colonic inflammation via a macrophage-dependent mechanism. JCI Insight 2019, 4, 131273, DOI: 10.1172/jci.insight.131273Google ScholarThere is no corresponding record for this reference.
- 442Atrian, S.; Capdevila, M. Metallothionein–protein interactions. Biomol. Concepts 2013, 4, 143– 160, DOI: 10.1515/bmc-2012-0049Google Scholar442Metallothionein-protein interactionsAtrian, Silvia; Capdevila, MerceBiomolecular Concepts (2013), 4 (2), 143-160CODEN: BCIOB8; ISSN:1868-5021. (Walter de Gruyter GmbH)A review. Metallothioneins (MTs) are a family of universal, small proteins, sharing a high cysteine content and an optimal capacity for metal ion coordination. They take part in a plethora of metal ion-related events (from detoxification to homeostasis, storage, and delivery), in a wide range of stress responses, and in different pathol. processes (tumorigenesis, neurodegeneration, and inflammation). The information on both intracellular and extracellular interactions of MTs with other proteins is here comprehensively reviewed. In mammalian kidney, MT1/MT2 interact with megalin and related receptors, and with the transporter transthyretin. Most of the mammalian MT partners identified concern interactions with central nervous system (mainly brain) proteins, both through phys. contact or metal exchange reactions. Phys. interactions mainly involve neuronal secretion multimers. Regarding metal swap events, brain MT3 appears to control the metal ion load in peptides whose aggregation leads to neurodegenerative disorders, such as Aβ peptide, α-synuclein, and prion proteins (Alzheimer's and Parkinson's diseases, and spongiform encephalopathies, resp.). Interaction with ferritin and bovine serum albumin are also documented. The intercourse of MTs with zinc-dependent enzymes and transcription factors is capable to activate/deactivate them, thus conferring MTs the role of metabolic and gene expression regulators. As some of these proteins are involved in cell cycle and proliferation control (p53, nuclear factor κB, and PKCμ), they are considered in the context of oncogenesis and tumor progression. Only one non-mammalian MT interaction, involving Drosophila MtnA and MtnB major isoforms and peroxiredoxins, has been reported. The prospective use for biomedical applications of the MT-interaction information is finally discussed.
- 443El Ghazi, I.; Martin, B. L.; Armitage, I. M. New proteins found interacting with brain metallothionein-3 are linked to secretion. Int. J. Alzheimer's Dis. 2011, 2011, 208634, DOI: 10.4061/2011/208634Google ScholarThere is no corresponding record for this reference.
- 444Binz, P.-A.; Kägi, J. H. R. Metallothionein: Molecular Evolution and Classification. In Metallothionein IV; Klaassen, C. D., Ed.; Birkhäuser: Basel, Switzerland, 1999; pp 7– 14.Google ScholarThere is no corresponding record for this reference.
- 445Blindauer, C. Metallothioneins. In Binding, Transport, and Storage of Metal Ions in Biological Cells; Maret, W., Wedd, A. G., Eds.; RSC Cambridge, 2014; pp 606– 665.Google ScholarThere is no corresponding record for this reference.
- 446Serén, N.; Glaberman, S.; Carretero, M. A.; Chiari, Y. Molecular evolution and functional divergence of the metallothionein gene family in vertebrates. J. Mol. Evol. 2014, 78, 217– 233, DOI: 10.1007/s00239-014-9612-5Google Scholar446Molecular evolution and functional divergence of the metallothionein gene family in VertebratesSeren, Nina; Glaberman, Scott; Carretero, Miguel A.; Chiari, YleniaJournal of Molecular Evolution (2014), 78 (3-4), 217-233CODEN: JMEVAU; ISSN:0022-2844. (Springer)The metallothionein (MT) gene superfamily consists of metal-binding proteins involved in various metal detoxification and storage mechanisms. The evolution of this gene family in vertebrates has mostly been studied in mammals using sparse taxon or gene sampling. Genomic databases and available data on MT protein function and expression allow a better understanding of the evolution and functional divergence of the different MT types. We recovered 77 MT coding sequences from 20 representative vertebrates with annotated complete genomes. We found multiple MT genes, also in reptiles, which were thought to have only one MT type. Phylogenetic and synteny analyses indicate the existence of a eutherian MT1 and MT2, a tetrapod MT3, an amniote MT4, and fish MT. The optimal gene-tree/species-tree reconciliation analyses identified the best root in the fish clade. Functional analyses reveal variation in hydropathic index among protein domains, likely correlated with their distinct flexibility and metal affinity. Analyses of functional divergence identified amino acid sites correlated with functional divergence among MT types. Uncovering the no. of genes and sites possibly correlated with functional divergence will help to design cost-effective MT functional and gene expression studies. This will permit further understanding of the distinct roles and specificity of these proteins and to properly target specific MT for different types of functional studies. Therefore, this work presents a crit. background on the mol. evolution and functional divergence of vertebrate MTs to carry out further detailed studies on the relationship between heavy metal metab. and tolerances among vertebrates.
- 447Thomas, P. D. The Gene Ontology and the Meaning of Biological Function. Methods Mol. Biol. 2017, 1446, 15– 24, DOI: 10.1007/978-1-4939-3743-1_2Google Scholar447The Gene Ontology and the Meaning of Biological FunctionThomas, Paul D.Methods in Molecular Biology (New York, NY, United States) (2017), 1446 (Gene Ontology Handbook), 15-24CODEN: MMBIED; ISSN:1940-6029. (Springer)The Gene Ontol. (GO) provides a framework and set of concepts for describing the functions of gene products from all organisms. It is specifically designed for supporting the computational representation of biol. systems. A GO annotation is an assocn. between a specific gene product and a GO concept, together making a statement pertinent to the function of that gene. However, the meaning of the term "function" is not as straightforward as it might seem, and has been discussed at length in both philosophical and biol. circles. Here, I first review these discussions. I then present an explicit formulation of the biol. model that underlies the GO and annotations, and discuss how this model relates to the broader debates on the meaning of biol. function.
- 448Abdin, A. Y.; Jacob, C.; Kästner, L. The enigmatic metallothioneins. A case of upward-looking research. Int. J. Mol. Sci. 2021, 22, 5984, DOI: 10.3390/ijms22115984Google Scholar448The enigmatic metallothioneins: a case of upward-looking researchAbdin, Ahmad Yaman; Jacob, Claus; Kaestner, LenaInternational Journal of Molecular Sciences (2021), 22 (11), 5984CODEN: IJMCFK; ISSN:1422-0067. (MDPI AG)In the mid-1950s, Bert Lester Vallee and his colleague Marvin Margoshes discovered a mol. referred to today as metallothionein (MT). Meanwhile, MTs have been shown to be common in many biol. organisms. Despite their prevalence, however, it remains unclear to date what exactly MTs do and how they contribute to the biol. function of an organism or organ. We investigate why biochem. research has not yet been able to pinpoint the function(s) of MTs. We shall systematically examine both the discovery of and recent research on Dr. Vallee's beloved family of MT proteins utilizing tools from philosophy of science. Our anal. highlights that Vallee's initial work exhibited features prototypical of a developing research tradition: it was upward-looking, exploratory, and utilized mere interactions. Since the 1960s, MT research has increasingly become intervention- and hypothesis-based while it remained largely upward-looking in character. While there is no reason to think that upward-looking research cannot successfully yield structure-function mappings, it has not yet been successful in the case of MTs. Thus, we suggest it might be time to change track and consider other research strategies looking into the evolution of MTs. Recent studies in mollusks render research in this direction worthy of pursuit.
- 449Klaassen, C. D.; Liu, J.; Diwan, B. W. Metallothionein protection of cadmium toxicity. Toxicol. Appl. Pharmacol. 2009, 238, 215– 220, DOI: 10.1016/j.taap.2009.03.026Google Scholar449Metallothionein protection of cadmium toxicityKlaassen, Curtis D.; Liu, Jie; Diwan, Bhalchandra A.Toxicology and Applied Pharmacology (2009), 238 (3), 215-220CODEN: TXAPA9; ISSN:0041-008X. (Elsevier B.V.)A review. The discovery of the cadmium (Cd)-binding protein from horse kidney in 1957 marked the birth of research on this low-mol. wt., cysteine-rich protein called metallothionein (MT) in Cd toxicol. MT plays minimal roles in the gastrointestinal absorption of Cd, but MT plays important roles in Cd retention in tissues and dramatically decreases biliary excretion of Cd. Cd-bound to MT is responsible for Cd accumulation in tissues and the long biol. half-life of Cd in the body. Induction of MT protects against acute Cd-induced lethality, as well as acute toxicity to the liver and lung. Intracellular MT also plays important roles in ameliorating Cd toxicity following prolonged exposures, particularly chronic Cd-induced nephrotoxicity, osteotoxicity, and toxicity to the lung, liver, and immune system. There is an assocn. between human and rodent Cd exposure and prostate cancers, esp. in the portions where MT is poorly expressed. MT expression in Cd-induced tumors varies depending on the type and the stage of tumor development. For instance, high levels of MT are detected in Cd-induced sarcomas at the injection site, whereas the sarcoma metastases are devoid of MT. The use of MT-transgenic and MT-null mice has greatly helped define the role of MT in Cd toxicol., with the MT-null mice being hypersensitive and MT-transgenic mice resistant to Cd toxicity. Thus, MT is crit. for protecting human health from Cd toxicity. There are large individual variations in MT expression, which might in turn predispose some people to Cd toxicity.
- 450Calvo, J.; Jung, H.; Meloni, G. Copper metallothioneins. IUBMB Life 2017, 69, 236– 245, DOI: 10.1002/iub.1618Google Scholar450Copper metallothioneinsCalvo, Jenifer; Jung, Hunmin; Meloni, GabrieleIUBMB Life (2017), 69 (4), 236-245CODEN: IULIF8; ISSN:1521-6543. (John Wiley & Sons, Inc.)A review. Metallothioneins (MTs) are a class of low mol. wt. and cysteine-rich metal binding proteins present in all the branches of the tree of life. MTs efficiently bind with high affinity several essential and toxic divalent and monovalent transition metals by forming characteristic polynuclear metal-thiolate clusters within their structure. MTs fulfil multiple biol. functions related to their metal binding properties, with essential roles in both Zn(II) and Cu(I) homeostasis as well as metal detoxification. Depending on the organism considered, the primary sequence, and the specific physiol. and metabolic status, Cu(I)-bound MT isoforms have been isolated, and their chem. and biol. characterized. Besides the recognized role in the biochem. of divalent metals, it is becoming evident that unique biol. functions in selectively controlling copper levels, its reactivity as well as copper-mediated biochem. processes have evolved in some members of the MT superfamily. Selected examples are reviewed to highlight the peculiar chem. properties and biol. functions of copper MTs.
- 451Andrews, G. Regulation of metallothionein gene expression by oxidative stress and metal ions. Biochem. Pharmacol. 2000, 59, 95– 104, DOI: 10.1016/S0006-2952(99)00301-9Google Scholar451Regulation of metallothionein gene expression by oxidative stress and metal ionsAndrews G KBiochemical pharmacology (2000), 59 (1), 95-104 ISSN:0006-2952.The metallothioneins (MT) are small, cysteine-rich heavy metal-binding proteins which participate in an array of protective stress responses. Although a single essential function of MT has not been demonstrated, MT of higher eukaryotes evolved as a mechanism to regulate zinc levels and distribution within cells and organisms. These proteins can also protect against some toxic metals and oxidative stress-inducing agents. In mice, among the four known MT genes, the MT-I and -II genes are most widely expressed. Transcription of these genes is rapidly and dramatically up-regulated in response to zinc and cadmium, as well as in response to agents which cause oxidative stress and/or inflammation. The six zinc-finger metal-responsive transcription factor MTF-1 plays a central role in transcriptional activation of the MT-I gene in response to metals and oxidative stress. Mutation of the MTF-1 gene abolishes these responses, and MTF-1 is induced to bind to the metal response elements in proximal MT promoter in cells treated with zinc or during oxidative stress. The exact molecular mechanisms of action of MTF-1 are not fully understood. Our studies suggest that the DNA-binding activity of MTF-1 in vivo and in vitro is reversibly activated by zinc interactions with the zinc-finger domain. This reflects heterogeneity in the structure and function of the six zinc fingers. We hypothesize that MTF-1 functions as a sensor of free zinc pools in the cell. Changes in free zinc may occur in response to chemically diverse inducers. MTF-1 also exerts effects on MT-I gene transcription which are independent of a large increase in MTF-1 DNA-binding activity. For example, cadmium, which has little effect on the DNA-binding activity of MTF-1 in vivo or in vitro, is a more potent inducer of MT gene expression than is zinc. The basic helix-loop-helix-leucine zipper protein, USF (upstream stimulatory factor family), also plays a role in regulating transcription of the mouse MT-I gene in response to cadmium or H2O2. Expression of dominant negative USF-1 or deletion of its binding site from the proximal promoter attenuates induction of the mouse MT-I gene. USF apparently functions in this context by interacting with as yet unidentified proteins which bind to an antioxidant response element which overlaps the USF-binding site (USF/ARE). Interestingly, this composite element does not participate in the induction of MT-I gene transcription by zinc or redox-cycling quinones. Thus, regulation of the mouse MT-I gene by metals and oxidative stress involves multiple signaling pathways which depend on the species of metal ion and the nature of the oxidative stress.
- 452Vallee, B. L. Introduction to metallothionein. Methods Enzymol. 1991, 205, 3– 7, DOI: 10.1016/0076-6879(91)05077-9Google Scholar452Introduction to metallothioneinVallee, Bert L.Methods in Enzymology (1991), 205 (Metallobiochem. Pt. B), 3-7CODEN: MENZAU; ISSN:0076-6879.A brief description of metallothionein is given, including structural features such as zinc coordination. Possible functional roles of the protein are also discussed.
- 453Palmiter, R. D. The elusive function of metallothioneins. Proc. Natl. Acad. Sci. U. S. A. 1998, 95, 8428– 8430, DOI: 10.1073/pnas.95.15.8428Google Scholar453The elusive function of metallothioneinsPalmiter, Richard D.Proceedings of the National Academy of Sciences of the United States of America (1998), 95 (15), 8428-8430CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)A review with 55 refs. Biochem. and genetics are both required to elucidate the function of macromols. There is no question that metallothioneins (MTs) have unique biochem. properties, but genetic expts. have not substantiated the importance of MTs under physiol. conditions. Even after thousands of studies describing the structure, biochem. characteristics, tissue distribution, induction, and consequences of genetic disruption and deliberate overexpression, the evolutionary forces that led to the initial appearance, gene duplications, and nearly ubiquitous expression of MTs remain enigmatic.
- 454Kelly, E. J.; Palmiter, R. D. A murine model of Menkes disease reveals a physiological function of metallothionein. Nat. Genet. 1996, 13, 219– 222, DOI: 10.1038/ng0696-219Google Scholar454A murine model of Menkes disease reveals a physiological function of metallothioneinKelly, Edward J.; Palmiter, Richard D.Nature Genetics (1996), 13 (2), 219-222CODEN: NGENEC; ISSN:1061-4036. (Nature Publishing Co.)The authors investigated the role of metallothionine in copper homeostasis by using mice bearing inactivated alleles of both Mt1 and Mt2 genes (Mt-/-) which were crossed with Mo-brJ mice. Their results suggest that metallothionine is essential to protect against copper toxicity in the embryonic placenta. They also predict that metallothionine would continue to be required to protect intestinal cells after birth.
- 455Gudekar, N.; Shanbhag, V.; Wang, Y.; Ralle, M.; Weisman, G. A.; Petris, M. J. Metallothioneins regulate ATP7A trafficking and control cell viability during copper deficiency and excess. Sci. Rep. 2020, 10, 7856, DOI: 10.1038/s41598-020-64521-3Google Scholar455Metallothioneins regulate ATP7A trafficking and control cell viability during copper deficiency and excessGudekar, Nikita; Shanbhag, Vinit; Wang, Yanfang; Ralle, Martina; Weisman, Gary A.; Petris, Michael J.Scientific Reports (2020), 10 (1), 7856CODEN: SRCEC3; ISSN:2045-2322. (Nature Research)Abstr.: Copper (Cu) is an essential, yet potentially toxic nutrient, as illustrated by inherited diseases of copper deficiency and excess. Elevated expression of the ATP7A Cu exporter is known to confer copper tolerance, however, the contribution of metal-binding metallothioneins is less clear. In this study, we investigated the relative contributions of ATP7A and the metallothioneins MT-I and MT-II to cell viability under conditions of Cu excess or deficiency. Although the loss of ATP7A increased sensitivity to low Cu concns., the absence of MTs did not significantly affect Cu tolerance. However, the absence of all three proteins caused a synthetic lethal phenotype due to extreme Cu sensitivity, indicating that MTs are crit. for Cu tolerance only in the absence of ATP7A. A lack of MTs resulted in the trafficking of ATP7A from the trans-Golgi complex in a Cu-dependent manner, suggesting that MTs regulate the delivery of Cu to ATP7A. Under Cu deficiency conditions, the absence of MTs and / or ATP7A enhanced cell proliferation compared to wild type cells, suggesting that these proteins compete with essential Cu-dependent pathways when Cu is scarce. These studies reveal new roles for ATP7A and metallothioneins under both Cu deficiency and excess.
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Abstract
Figure 1
Figure 1. Amino acid sequence of horse MT1b with the Cys-containing motifs indicated, including CC (blue), CXC (red), CXXXC (green), SC (gray), and CK, KC, CR (underlined).
Figure 2
Figure 2. Sequences of human MT1–4 with conservation of Cys residues. There is one exception, though: human MT1b has an extra cysteine, yielding a total of 21 Cys. Only some of the Lys (K) residues are conserved, one CKC motif in the N-terminal part and three (KCA, CKG, and KCS) in the C-terminal part─KCS is not conserved in MT1m and MT1b, though. The KK motif in the middle of the protein is conserved with the exception of MT4, where it is RK. Blue codes on the right denote UniProt entries. Alignment was performed using MAFFT software (20) and visualized using Jalview. (21) Yellow and black bars stand for conservation and consensus, respectively.
Figure 3
Figure 3. Phylogram (left) and cladogram (right) of human metallothioneins (based on protein structures, translation of pseudogenes is not included). MT2 is part of the MT1 branch, while MT3 and MT4 have a separate root. The tree was generated in Clustal Omega using the neighbor-joining clustering method and visualized by iTOL. (23,24) The bar indicates the number of changes per residue. The number 0.01 corresponds in length to a 1% difference in sequences.
Figure 4
Figure 4. Alignment of MT sequences from representative species of vertebrates. In this selection, 23 amino acids are strictly conserved, but among them only 16 Cys are strictly conserved. Besides Coelacanth and African chameleon MTs, all other MTs contain 20 Cys residues. Yellow and black bars below the alignment show the patterns of conservation and consensus. Blue codes on the right denote UniProt entries. Alignment was performed using MAFFT software (20) and visualized using Jalview. (21) Yellow and black bars stand for conservation and consensus, respectively.
Figure 5
Figure 5. NMR assignments of Cd(II)/thiolate coordination environments in the MT clusters. Cluster A (or α) is in the C-terminal α-domain, and cluster B (or β) is in the N-terminal β-domain. Black and red numbers indicate Cys residues in the rabbit MT2 sequence and the Cd(II) ions assigned from 113Cd NMR (inset). (35,39)
Figure 6
Figure 6. NMR structures of mammalian α-domains (PDB: 1MRB, 1MRT, 1MHU) and β-domains (PDB: 2MRB, 2MRT, 2MHU) of MT2. (A) Comparison of the structures of β (left) and α (right) domains of rabbit (blue ribbon), rat (beige ribbon), and human (pink ribbon) Cd7MT2. (B) Sequences of both domains with criss-cross Cd(II) (M) binding sites. (44−46)
Figure 7
Figure 7. Structure of the entire rat MT2 molecule solved by X-ray crystallography. (A) Structure of Cd5Zn2MT2 with the indication of Cd(II) (beige color) and Zn(II) (gray color) ions in both domains. (B) Connectivities of the metal ions with the Cys residues in the β-cluster. (47)
Figure 8
Figure 8. NMR structure of the α-domain of human MT3 in the complex with Cd(II). (A) Comparison of the MT2 (blue) and MT3 (beige) structures of the individual domains (PDB: 1MHU vs 2FJ4). (B) Comparison of the MT3 structures of the individual domain (beige) and the one in the entire protein (pink) (PDB: 2F5H). (50)
Figure 9
Figure 9. NMR structures of Cu(I)-containing mouse MT1 domains. (A) 20-structure family of ZnyCu4βMT1; (B) 20 best structures of ZnxCu3αMT1; (C) superposition of the mean Cd3βMT1 structure (red) and the structure family of ZnyCu4βMT1 (blue); (D) superposition of the mean Cd4αMT1 structure (red) and the structure family of ZnxCu3αMT1 (blue). (A, B) Gray, blue, and yellow colors represent the polypeptide backbone, cysteinyl side chain, and sulfur atoms, respectively. Figures were adopted with permission from ref (51), copyright 2007 by John Wiley and Sons.
Figure 10
Figure 11
Figure 11. Function of the MT/T couple as a homeostatic Zn(II) system. An increase in the amount of available Zn(II) induces the synthesis of T through the action of Zn(II) on Zn(II)-dependent transcription factors and leads to the formation of MT and a sequestration of Zn(II) (left). When available Zn(II) is low and needed for the synthesis of zinc proteins, Zn(II) dissociates from MT and T is formed (right). For clarity, the effects of oxidants and reductants are omitted. The redox effects are illustrated in Figure 13.
Figure 12
Figure 12. Differential modification of MT with ABD-F uncovers the presence of significant amounts of metal-depleted protein. (A) Schematic reaction of ABD-F with the sulfhydryl group resulting in a fluorescent adduct. (B) Amounts of MT and T (TR+TO) found in various rat tissues. (88) (C) Changes of MT and T (TR+TO) content in HT-29 cells pretreated with ZnSO4. (88) (D) Percentage of MT, T, TR, and TO in rat liver stored on ice for up to 30 h as determined with a modified differential ABD-F assay. (89) Gray, red, blue, and green bars correspond to MT, T (TR+TO), TR, and TO species. MT, T, TR, and TO correspond to fully metal-loaded, fully depleted, reduced and depleted, and oxidized MT, respectively.
Figure 13
Figure 13. MT and T redox cycles. In this expanded dual cycle, TR and TO represent the fully reduced (thionein) and oxidized (thionin) metal-free protein. Zn7MT, Zn7–xMTR, and Zn7–xMTO refer to fully loaded, partially Zn(II)-depleted reduced, and partially Zn(II)-depleted oxidized protein, respectively. It is presumed that thionein/thionin can serve its own role as a redox couple in vivo under conditions of restricted metal ion availability.
Figure 14
Figure 14. Affinity series of metal ions binding to metallothioneins. The order of affinities is based on experimental data and estimations from exchange experiments and model studies. (28,135−142) The series also defines the tendency of free metal ion concentrations buffered by MTs. Its implication for the metalation of proteins is discussed in section 4.1.
Figure 15
Figure 16
Figure 16. Illustration of how the metal-to-protein affinity shifts when the pH increases or decreases. The case corresponds to a model metal site composed of Cys residues with a −log Kd value = 11.5. Inflection points of the binding isotherms numerically correspond to −log Kd values under the conditions used.
Figure 17
Figure 17. Influence of the composition of the coordination environment in zinc sites on the metal affinity and the pH dependence of conditional dissociation constants. With HySS software, the relationships were simulated based on the assumed protonation and stability constants in such a way that all formed complexes reach −log Kd = 12 at pH 7.4. (162) The pKa values of Cys thiols and His imidazoles have been limited to the 8.6–8.9 and 6.1–6.4 ranges, respectively, based on previously determined data. (135,160,161)
Figure 18
Figure 18. Experimental and simulated UV-monitored pH titrations of Zn7MT2 and Cd7MT2. (A) Experimental pH titration of 1 μM Zn7MT2 from pH 3 to 7. (B) The simulations of pH-dependent saturation of the protein with Zn(II) (see main text for details). The dashed line corresponds to a case where all seven Zn(II) are bound with the same affinity (−log Kd = 11.7). The solid line indicates a case where four sites are bound with high affinity and the remaining three with gradually lower affinities. (73) (C) Experimental pH titration of 1 μM Cd7MT2 from pH 3 to 7.
Figure 19
Figure 19. Speciation of zinc metallothionein (Zn7MT, MT) according to three thermodynamic models in the literature. (left) Model with all seven Zn(II) ions bound with the same affinity (Kd1–7 = 10–11.8 M). (141) (middle) Stepwise model with Kd values ranging from 10–11.8 to 10–7.8 M for human MT2. (73) (right) Stepwise model with Kd values close to each other in the range from 10–12.5 to 10–11.4 M for a modified human MT1a. (172) The upper panels show the relationship between ratios of the apo-form (T) over total protein and free Zn(II) concentrations (−log[Zn(II)]). (lower) Speciation of the apo-form, partially and fully Zn(II)-saturated species. The heuristic value of the models is discussed in the main text.
Figure 20
Figure 20. Order of the stepwise Zn(II) dissociation for the unfolding pathway for (A) Zn7MT2 and (B) Cd7MT2 obtained by constant-speed steered molecular dynamics simulations that involve Zn7MT2 and Cd7MT2 with the C-termini fixed. (216) The bars show the binding/unbinding of the metal ion for each metal-loaded species. Representative conformations of the protein when six, five, and four Zn(II) ions (C) or Cd(II) ions (D) are bound. Zn(II) and Cd(II) are represented by gray spheres, and the sulfur atoms are shown in yellow. (57) The figure is adopted from ref (216) with permission of the American Chemical Society.
Figure 21
Figure 21. Free metal ion concentrations and buffering of the most competitive essential metal ions Zn(II) and Cu(I) and nonessential and toxic Cd(II). Estimated steady-state concentrations for Zn(II) and Cu(I) are picomolar and zeptomolar, respectively. Cd(II) interacts with the metabolism of both. Very low concentrations require stochastics when the volume limits the definition of concentrations. (279) Physiological and pathophysiological redox changes and other triggering events can increase the concentrations (arrows). Zn(II) fluctuations are used in cellular signaling at concentrations lower than those in calcium signaling. Recent work suggests a dynamic copper pool that is involved in copper signaling as further discussed in section 4.5.
Figure 22
Figure 22. Two modes of action in which MT buffers Cu(I) either independent of Zn(II) or dependent on Zn(II).
Figure 23
Figure 23. Pathways for the formation of mixed CuyZn7–xMT in reduced and oxidized forms.
Figure 24
Figure 24. Interaction of pyridoxal kinase with ZnATP (PDB code: 1LHR). The crystal structure of pyridoxal kinase (PK) shows complexation with ZnATP. (368) There is not enough free Zn(II) available to form the ZnATP needed to activate PK directly. However, the interaction of Zn(II)-MT with ATP could provide the ZnATP to activate PK. (370)
Figure 25
Figure 25. Combined gene and protein regulation of the MT system. (left) Many signal transduction pathways converge at the MT promoters to induce the expression of at least 11 human MT genes. Various steps in these pathways depend on Zn(II). A Zn(II)-sensing transcription factor (MTF-1) controls the metal-dependent transcription at metal regulatory elements. Among the other inducers are interferons, glucocorticoids, and redox signals acting on the respective regulatory elements (ISRE, GRE, HRE, XRE, and ARE). DNA methylation can silence the effects of these cis-acting factors. (right) Newly synthesized thioneins bind metal ions to form metallothioneins. The availability of metal ions from MT proteins is linked to redox changes and redox signaling. Protein modifications, interactions with low molecular ligands and other proteins, and protein degradation afford additional layers of regulation as discussed in the main text. With regard to the induction of gene expression, it is a generalized scheme, as not all the respective regulator elements are present in a given MT gene.
Figure 26
Figure 26. Structures of mammalian proteins containing Znx(Cys/His)y clusters. (top left and right) Human embryonic neural inducing factor churchill (2JOX) (408) and dimerization domain (1RMD) (409) of mouse V(D)J recombination-activating protein RAG1, respectively. In both structures unique Zn2HisCys6 clusters with one bridging thiolate sulfur are present. (bottom) Structure of human euchromatic histone methyltransferase 2 containing a Zn3Cys6 cluster. (410)
References
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- 1Drinker, K. R.; Collier, E. S. The significance of zinc in the living organism. J. Industr. Hygiene 1926, 8, 257– 269There is no corresponding record for this reference.
- 2Margoshes, M.; Vallee, B. L. A Cadmium protein from equine kidney cortex. J. Am. Chem. Soc. 1957, 79, 4813– 4814, DOI: 10.1021/ja01574a0642A cadmium protein from equine kidney cortexMargoshes, Marvin; Vallee, Bert L.Journal of the American Chemical Society (1957), 79 (), 4813-14CODEN: JACSAT; ISSN:0002-7863.cf. C.A. 50, 5817c. Fractionation of horse-kidney cortex with EtOH and (NH4)2SO4 gave a product contg. 20-5 mg./g. dry wt. of Cd in Cl3CCO2H-precipitable material in succesive fractionations. Ultracentrifugation in a synthetic-boundary cell showed the final products of 4 successive fractionations to be unidisperse with a sedimentation (uncor. for viscosity of diffusion) varying from 0.94 to 1.22 × 10-13. Paper electrophoresis at pH 8.5 of the product of 1 fractionation showed 3 components moving toward the cathode; the slowest comprised about 70% of the material. The Cd content rose 30-fold throughout the fractionation from the 1st ext. to the product. Cd was not removable by dialysis at pH 7, but was removed by treatment with hot Cl3CCO2H. With the exception of Zn, other metals initially present or introduced during fractionation were removed and occurred in low concn. in the final material. The possibility of isomorphism with Zn is discussed. The product contains 14% N, and reacts positively to ninhydrin and biuret. Hydrolysis and paper chromatography showed serine, glycine, aspartic and glutamic acids among other nonidentifiable amino acids. The last fraction contained about 1% hexosamine.
- 3Kägi, J. H. R.; Vallee, B. L. Metallothionein: a cadmium- and zinc-containing protein from equine renal cortex. J. Biol. Chem. 1960, 235, 3460– 3465, DOI: 10.1016/S0021-9258(18)64490-43Metallothionein: a cadmium- and zinc-containing protein from equine renal cortexKagi, Jeremias H. R.; Vallee, Bert L.; Carlson, Janet M.Journal of Biological Chemistry (1960), 235 (), 3460-5CODEN: JBCHA3; ISSN:0021-9258.cf. CA 52, 7393c. Metallothionein, a protein of small mol. wt. isolated from equine renal cortex, contains 2.9% Cd, 0.6% Zn, and 4% S. The protein, homogeneous on ultracentrifugation but contg. minor impurities on electrophoresis, does not absorb radiation at or near 280 mμ; this finding indicates a very low aromatic amino acid content. A large no. of cysteine residues accounts for the high S content. The stoichiometry between titratable SH groups and metal atoms, the displacement of the metals by SH-specific agents, and the selective removal of Zn and Cd by H ions suggest isomorphous binding of the 2 metals to the protein through mercaptide linkages. Although the biol. function of metallothionein is not known thus far, the specific assocn. of Cd with this macromol. suggests a definite biol. role.
- 4Kägi, J. H. R.; Vallee, B. L.; Carlson, J. M. Metallothionein: a cadmium- and zinc-containing protein from equine renal cortex. II. Physicochemical properties. J. Biol. Chem. 1961, 236, 2435– 2442, DOI: 10.1016/S0021-9258(18)64017-74Metallothionein: a cadmium and zinc-containing protein from equine renal cortex. II. Physicochemical propertiesKagi, Jeremias H. R.; Vallee, Bert L.Journal of Biological Chemistry (1961), 236 (), 2435-42CODEN: JBCHA3; ISSN:0021-9258.cf. Federation Proc. 19, 340(1960); CA 55, 7595e. Lyophilized metallothionein, prepd. from horse-kidney cortex by EtOH and salt fractionation (loc. cit.), was purified further by DEAE cellulose chromatography. The specific absorption of metallothionein at 250 mμ is a convenient monitor for its isolation. The purest prepn. of metallothionein contained 14.9% N and 8.5% S with 26 titratable mercapto groups accounting for more than 95% of the total S content. The mol. wt. of metallothionein is 10,000 ± 260, an av. of the values obtained from velocity sedimentation and diffusion detns. and the approach to equil. centrifugation. The small mol. wt. accounts for losses on dialysis through membranes of large pore size. The partial sp. vol., 0.648 ml./g., is unusually low, in part because of the high metal and S contents. Approx. 1 of 3 or 4 amino acids in this small protein mol. is a cysteine residue; proline, serine, and lysine residues come next in that order of abundance. Tryptophan and tyrosine are absent. The diffusion const., D20,w, is 12.42 ± 0.07 × 10-7, and the frictional ratio, f:f0, is 1.28. The most highly purified prepn., homogeneous by ultracentrifugation and electrophoresis, contains 5.9% Cd, 2.2% Zn, 0.2% Fe, and 0.1% Cu. Metallothionein is formed through the interaction of 1 atom of Cd or Zn with 3 sulfhydryl groups. The characteristic charge-transfer band, with max. absorption at 250 mμ, is due to the Cd mercaptide; it is absent in thionein, the metal-free protein. A similar band at 215 mμ is apparently due to the Zn mercaptide. The conformation of thionein, its component cysteine residues, and the resultant steric organization of the free sulfhydryl groups, disallow a large no. of possible structures of this protein and are thought to det. both the remarkable avidity for Cd and the preponderance of Cd over Zn.
- 5Kägi, J. H. R.; Himmelhoch, S. R.; Whanger, P. D.; Bethune, J. L.; Vallee, B. L. Equine hepatic and renal metallothioneins. J. Biol. Chem. 1974, 249, 3537– 3542, DOI: 10.1016/S0021-9258(19)42605-75Equine hepatic and renal metallothioneins. Purification, molecular weight, amino acid composition, and metal contentKagi J H; Himmelhoch S R; Whanger P D; Bethune J L; Vallee B LThe Journal of biological chemistry (1974), 249 (11), 3537-42 ISSN:0021-9258.There is no expanded citation for this reference.
- 6Bühler, R. H. O.; Kägi, J. H. R. Human hepatic metallothioneins. FEBS Lett. 1974, 39, 229– 234, DOI: 10.1016/0014-5793(74)80057-86Human hepatic metallothioneinsBuehler, Rolf H. O.; Kaegi, Jeremias H. R.FEBS Letters (1974), 39 (2), 229-34CODEN: FEBLAL; ISSN:0014-5793.Two Zn-contg. metallothioneins were isolated from human liver. Each had a min. mol. wt. of ∼6600, and based on this mol. wt. one contained 6 Zn atoms and the other 7. This was proportional to the cysteine and titratable SH groups of the proteins. The amino acid compns. were detd. No other metal were bound to these proteins.
- 7Porter, H. The particulate half-cysteine-rich copper protein of newborn liver. Relationship to metallothionein and subcellular localization in non-mitochondrial particles possibly representing heavy lysosomes. Biochem. Biophys. Res. Commun. 1974, 56, 661– 668, DOI: 10.1016/0006-291X(74)90656-17Particulate half-cystine-rich copper protein of newborn liver. Relation to metallothionein and subcellular localization in nonmitochondrial particles possibly representing heavy lysosomesPorter, HuntingtonBiochemical and Biophysical Research Communications (1974), 56 (3), 661-8CODEN: BBRCA9; ISSN:0006-291X.The particulate half-cystine-rich Cu protein of newborn liver was partially purified by centrifugation of the heavy mitochondrial fraction through glycogen-sucrose or sucrose d. gradients. The resulting sediments contained ∼ 4% Cu, ∼ 20% half-cystine, and a 2-3-fold increase in β-glucuronidase specific activity. The Cu protein is not a true mitochondrial constituent and the data are consistent with its localization in a distinct population of heavy lysosomes. The amino acid composition of the polypeptide isolated from the crude insoluble Cu protein is strikingly similar to that of metallothionein, suggesting that the neonatal protein represents a Cu-rich form of metallothionein.
- 8Rydén, L.; Deutsch, H. F. Preparation and properties of the major copper─binding component in human fetal liver. J. Biol. Chem. 1978, 253, 519– 524, DOI: 10.1016/S0021-9258(17)38240-68Preparation and properties of the major copper-binding component in human fetal liver. Its identification as metallothioneinRyden, Lars; Deutsch, Harold F.Journal of Biological Chemistry (1978), 253 (2), 519-24CODEN: JBCHA3; ISSN:0021-9258.Most of the relatively large amts. of Cu in human fetal liver is bound to a low-mol.-wt. protein. By a combination of gel filtrations and covalent chromatog. on thiopropyl-Sepharose, the monomeric form of the Cu-binding protein and some polymers of it were prepd. in good yield. The protein was identified as a Cu-thionein by the criteria of mol. wt., shape, amino acid compn., and amino acid sequence homol. Gel filtration in 6M guanidine-HCl showed that the mol. was a single peptide chain of 58 residues, corresponding to a mol. wt. of 6000. The mol. appears to be asym. with an approx. frictional ratio of 1.40. The protein contained 2.4 g atoms of Cu/mol and traces of Mn and Zn, but no Cd. The Cu was not paramagnetic. Possible roles of this Cu-binding protein in the transport and storage of Cu are discussed.
- 9Piscator, M. Om kadmium i normala människornjurar samt redogörelse för isolering av metallthionein ur lever från kadmiumexponderade kaniner. Nordisk Hygienisk Tidskrift 1964, 65, 76– 82There is no corresponding record for this reference.
- 10Nordberg, G. F.; Nordberg, M.; Piscator, M.; Vesterberg, O. Separation of two forms of rabbit metallothionein by isoelectric focusing. Biochem. J. 1972, 126, 491– 498, DOI: 10.1042/bj126049110Separation of two forms of rabbit metallothionein by isoelectric focussingNordberg, Gunnar F.; Nordberg, Monica; Piscator, Magnus; Vesterberg, OlofBiochemical Journal (1972), 126 (3), 491-8CODEN: BIJOAK; ISSN:0264-6021.Rabbits were given repeated injections of CdCl2. Cd- and Zn-contg. protein fractions were obtained from the livers of these animals by pptn. procedures and Sephadex G-75 chromatog. The protein thus obtained showed several characteristics similar to those of the earlier described protein metallothionein. Further sepn. by isoelec. focusing showed two main protein peaks with isoelec. points at 3.9 and 4.5, resp. Amino acid anal. of these 2 forms showed similar content of most amino acids [residues percent.: cysteine (28%), aspartate (8%), threonine (5-6), serine (12%), glycine (7%), alanine (13%), methionine (2%), isoleucine (2%)] but with a small difference in content of lysine (12 and 13%, resp.), proline (9 and 5%, resp.) and glutamate (2 and 4% resp.). The 2 forms of the protein both contained Cd, but only the one with pI 4.5 contained also significant amts. of Zn.
- 11Hunziker, P. E.; Kägi, J. H. R. Isolation and characterization of six human hepatic isometallothioneins. Biochem. J. 1985, 231, 375– 382, DOI: 10.1042/bj231037511Isolation and characterization of six human hepatic isometallothioneinsHunziker, Peter E.; Kaegi, Jeremias H. R.Biochemical Journal (1985), 231 (2), 375-82CODEN: BIJOAK; ISSN:0264-6021.Human hepatic metallothionein (MT) was sepd. into 6 isoforms by using reversed-phase HPLC for anal. and prepn. By comparison with the HPLC elution profiles of the charge-separable species MT-1 and MT-2 isolated by earlier procedures, 5 of these isoproteins are identified as hitherto unresolved subforms of MT-1, and 1 is identical with MT-2. The 6 isoforms have distinct and reproducible retention times at neutral pH, where the metal remains bound to the protein, and at low pH, where the metal is removed. Their amino acid compns. display the high cysteine content and the lack of arom. amino acids and of histidine typical of mammalian metallothioneins, but they differ significantly with respect to all other amino acids. A survey of autopsy material indicates that in adult human liver, all 6 forms are usually expressed, albeit in somewhat variable relative proportions.
- 12Karin, M.; Richards, R. I. Human metallothionein genes – primary structure of the metallothionein-II gene and a related processed gene. Nature (London, U. K.) 1982, 299, 797– 802, DOI: 10.1038/299797a012Human metallothionein genes. Primary structure of the metallothionein-II gene and a related processed geneKarin, Michael; Richards, Robert I.Nature (London, United Kingdom) (1982), 299 (5886), 797-802CODEN: NATUAS; ISSN:0028-0836.The complete nucleotide sequences of 2 of the human metallothionein gene family were elucidated and compared. One was a functional metallothionein-II gene; the other was a pseudogene, which lacked introns and which terminated in a poly(A) tail and was flanked by 2 direct repeats. A size polymorphism was detected which was assocd. with the processed gene in the population examd., and a region of apparent secondary structure homol. was obsd. between a 5' flanking region of the functional metallothionein-II gene and that of a mouse metallothionein-I gene.
- 13Uchida, Y.; Takio, K.; Titani, K.; Ihara, Y.; Tomonaga, M. The growth inhibitory factor that is deficient in the Alzheimer’s disease brain is a 68 amino acid metallothionein-like protein. Neuron 1991, 7, 337– 347, DOI: 10.1016/0896-6273(91)90272-213The growth inhibitory factor that is deficient in the Alzheimer's disease brain is a 68 amino acid metallothionein-like proteinUchida, Yoko; Takio, Koji; Titani, Koiti; Ihara, Yasuo; Tomonaga, MasanoriNeuron (1991), 7 (2), 337-47CODEN: NERNET; ISSN:0896-6273.The authors purified and characterized the growth inhibitory factor (GIF) that is abundant in the normal human brain, but greatly reduced in the Alzheimer's disease (AD) brain. GIF inhibited survival and neurite formation of cortical neurons in vitro. Purified GIF is a 68 amino acid small protein, and its amino acid sequence is 70% identical to that of human metallothionein II with a 1 amino acid insert and a unique 6 amino acid insert in the NH2-terminal and the COOH-terminal portions, resp. The antibodies to the unique sequence of GIF revealed a distinct subset of astrocytes in the gray matter that appears to be closely assocd. with neuronal perikarya and dendrites. In the AD cortex, the no. of GIF-pos. astrocytes was drastically reduced, suggesting that GIF is down-regulated in the subset of astrocytes during AD.
- 14Sewell, A. K.; Jensen, L. T.; Erickson, J. C.; Palmiter, R. D.; Winge, D. R. Biocactivity of metallothionein-3 correlates with its novel β domain sequence rather than metal binding properties. Biochemistry 1995, 34, 4740– 4747, DOI: 10.1021/bi00014a03114Bioactivity of Metallothionein-3 Correlates with Its Novel β Domain Sequence Rather Than Metal Binding PropertiesSewell, Andrew K.; Jensen, Laran T.; Erickson, Jay C.; Palmiter, Richard D.; Winge, Dennis R.Biochemistry (1995), 34 (14), 4740-7CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)Human and mouse metallothionein-3 (MT-3) mols. exhibit the same metal binding stoichiometry with Zn(II), Cd(II), or Cu(I) as MT-1 or MT-2 mols., suggesting that MT-3 consists of two domains enfolding sep. polymetallic clusters. The kinetic reactivities of Zn(II) complexes of MT-3 with the chelator EDTA or the thiol reagent dithiobis(2-nitrobenzoic acid) (DTNB) resembles the reactivity of ZnMT-1. Furthermore, the candidate α and β domain peptides of human MT-3 are very similar to MT-1 domain peptides in the reactivity of Zn(II) complexes. Zn(II) complexes of human and mouse MT-3 inhibit the survival of rat cortical neurons cultured in the presence of an Alzheimer's disease brain ext. Inhibitory activity is unique to the MT-3 isoform and is a property of the N-terminal β domain. The inhibitory activity of the 32-residue MT-3 β domain is abolished by a double mutation within the β domain resulting in the conversion of the C-P-C-P sequence to either C-S-C-A or C-T-C-T. Thus, the bioactivity arises from a novel structure of the N-terminal β domain of MT-3 and not any unusual metal-binding properties.
- 15Palmiter, R. D.; Findley, S. D.; Whitmore, T. E.; Durnam, D. M. MT-III, a brain-specific member of the metallothionein gene family. Proc. Natl. Acad. Sci. U. S. A. 1992, 89, 6333– 6337, DOI: 10.1073/pnas.89.14.633315MT-III, a brain-specific member of the metallothionein gene familyPalmiter, Richard D.; Findley, Seth D.; Whitmore, Theodore E.; Durnam, Diane M.Proceedings of the National Academy of Sciences of the United States of America (1992), 89 (14), 6333-7CODEN: PNASA6; ISSN:0027-8424.A third member of the metallothionein (MT) gene family, designated MT-III, was cloned by virtue of its homol. to a human protein that was shown previously to inhibit neuronal survival in culture and to be deficient in the brain of people with Alzheimer disease. Human and mouse brains MT-IIIs have two insertions relative to all other known mammalian MTs: a threonine after the fourth amino acid and a block of six amino acids near the carboxyl terminus. The genes encoding MT-III resemble all other mammalian MT genes in their small size and exon/intron organization. The MT-III genes are closely linked to the other functional MT genes on human chromosome 16 and mouse chromosome 8. Mouse MT-III gene expression appears to be restricted to brain; in addn., it fails to respond to zinc, cadmium, dexamethasone, or bacterial endotoxin in vivo, thereby distinguishing MT-III from other known MTs.
- 16Quaife, C. J.; Findley, S. D.; Erickson, J. C.; Froelick, G. J.; Kelly, E. J.; Zambrowicz, B. P.; Palmiter, R. D. Induction of a new metallothionein isoform (MT-IV) occurs during differentiation of stratified squamous epithelia. Biochemistry 1994, 33, 7250– 7259, DOI: 10.1021/bi00189a02916Induction of a New Metallothionein Isoform (MT-IV) Occurs during Differentiation of Stratified Squamous EpitheliaQuaife, Carol J.; Findley, Seth D.; Erickson, Jay C.; Froelick, Glenda J.; Kelly, Edward J.; Zambrowicz, Brian P.; Palmiter, Richard D.Biochemistry (1994), 33 (23), 7250-9CODEN: BICHAW; ISSN:0006-2960.A new member of the metallothionein (MT) gene family was discovered that lies about 20 kb 5' of the MT-III genes in both mouse and human. The MT-IV proteins are highly conserved in both species and have a glutamate insertion at position 5 relative to the classical MT-I and MT-II proteins. Murine MT-IV mRNA appears to be expressed exclusively in stratified squamous epithelia assocd. with oral epithelia, esophagus, upper stomach, tail, footpads, and neonatal skin. The MT derived from tongue epithelium contains both zinc and copper. Many of these epithelia develop parakaratosis during zinc deficiency in the rat. In situ hybridization reveals intense labeling of MT-IV mRNA in the differentiating spinous layer of cornified epithelia, whereas MT-I is expressed predominantly in the basal, proliferative layer; thus, there is a switch in MT isoform synthesis during differentiation of these epithelia. The authors suggest that MT-IV plays a special role in regulating zinc metab. during the differentiation of stratified epithelia.
- 17Kojima, Y.; Berger, C.; Vallee, B. L.; Kägi, J. H. R. Amino-acid sequence of equine renal metallothionein-1B. Proc. Natl. Acad. Sci. U. S. A. 1976, 73, 3413– 3417, DOI: 10.1073/pnas.73.10.341317Amino-acid sequence of equine renal metallothionein-1BKojima, Yutaka; Berger, Christine; Vallee, Bert L.; Kaegi, Jeremias H. R.Proceedings of the National Academy of Sciences of the United States of America (1976), 73 (10), 3413-17CODEN: PNASA6; ISSN:0027-8424.The amino acid sequence of a metallothionein is reported. Metallothionein-1B is 1 of the 2 principal variants occurring in equine kidney cortex. The single-chain protein contains 61 amino acids. The N-terminal residue is N-acetylmethionine. The sequence shows distinct clustering of the 20 cysteinyl residues into 7 groups sepd. by stretches of ≥3 other residues. Within these groups the cysteines occur 7 times in alternating Cys-X-Cys sequences and 3 times each in Cys-Cys and Cys-X-X-Cys sequences where X is an amino acid other than cysteine. Another unique feature is the close assocn. of serine and of the basic amino acids with cysteine, as manifested by the occurrence of 7 Ser-Cys, 4 Cys-Lys, 7 Cys-Arg, and 3 Lys-Cys sequences. These findings are in agreement with the previous suggestion that metallothionein has structurally defined metal-binding sites, most of which contain 3 cysteinyl residues as the principal metal-binding ligands. The charge difference between the metal-free and the metal-contg. protein is consistent with the formation of neg. charged trimercaptide complexes with Cd2+ and(or) Zn2+. The possible addnl. involvement of serine and the basic amino acids in metal binding is discussed.
- 18Kojima, Y. Definitions and nomenclature of metallothioneins. Methods Enzymol. 1991, 205, 8– 10, DOI: 10.1016/0076-6879(91)05078-A18Definitions and nomenclature of metallothioneinsKojima, YutakaMethods in Enzymology (1991), 205 (Metallobiochem. Pt. B), 8-10CODEN: MENZAU; ISSN:0076-6879.Metallothioneins are defined and nomenclature of these proteins and the corresponding genes presented.
- 19Karin, M.; Eddy, R. L.; Henry, W. M.; Haley, L. L.; Byers, M. G.; Shows, T. B. Human metallothionein genes are clustered on chromosome 16. Proc. Natl. Acad. Sci. U. S. A. 1984, 81, 5494– 5498, DOI: 10.1073/pnas.81.17.549419Human metallothionein genes are clustered on chromosome 16Karin, Michael; Eddy, Roger L.; Henry, W. Michael; Haley, Linda L.; Byers, Mary G.; Shows, Thomas B.Proceedings of the National Academy of Sciences of the United States of America (1984), 81 (17), 5494-8CODEN: PNASA6; ISSN:0027-8424.In man, the metallothioneins are encoded by ≥10-12 genes sepd. into 2 groups, MT-I and MT-II. To understand the genomic organization of these genes and their involvement in hereditary disorders of trace metal metab., their chromosomal location was detd. By using human-mouse cell hybrids and hybridization probes derived from clones and functional human MT1 and MT2 genes, it was shown that the functional human genes are clustered on human chromosome 16. Anal. of RNA from somatic cell hybrids indicated that hybrids which contained human chromosome 16 expressed both human MT1 and MT2 mRNA, and this expression is regulated by both heavy metal ions and glucocorticoid hormones.
- 20Katoh, K.; Standley, D. M. MAFFT multiple sequence alignment software version 7: Improvements in performance and usability. Mol. Biol. Evol. 2013, 30, 772– 780, DOI: 10.1093/molbev/mst01020MAFFT Multiple Sequence Alignment Software Version 7: Improvements in Performance and UsabilityKatoh, Kazutaka; Standley, Daron M.Molecular Biology and Evolution (2013), 30 (4), 772-780CODEN: MBEVEO; ISSN:0737-4038. (Oxford University Press)We report a major update of the MAFFT multiple sequence alignment program. This version has several new features, including options for adding unaligned sequences into an existing alignment, adjustment of direction in nucleotide alignment, constrained alignment and parallel processing, which were implemented after the previous major update. This report shows actual examples to explain how these features work, alone and in combination. Some examples incorrectly aligned by MAFFT are also shown to clarify its limitations. We discuss how to avoid misalignments, and our ongoing efforts to overcome such limitations.
- 21Troshin, P. V.; Procter, J. B.; Barton, G. J. Java bioinformatics analysis web services for multiple sequence alignment – JABAWS:MSA. Bioinformatics 2011, 27, 2001– 2002, DOI: 10.1093/bioinformatics/btr30421Java bioinformatics analysis web services for multiple sequence alignment-JABAWS:MSATroshin, Peter V.; Procter, James B.; Barton, Geoffrey J.Bioinformatics (2011), 27 (14), 2001-2002CODEN: BOINFP; ISSN:1367-4803. (Oxford University Press)Summary: JABAWS is a web services framework that simplifies the deployment of web services for bioinformatics. JABAWS:MSA provides services for five multiple sequence alignment (MSA) methods (Probcons, T-coffee, Muscle, Mafft and ClustalW), and is the system employed by the Jalview multiple sequence anal. workbench since version 2.6. A fully functional, easy to set up server is provided as a Virtual Appliance (VA), which can be run on most operating systems that support a virtualization environment such as VMware or Oracle VirtualBox. JABAWS is also distributed as a Web Application aRchive (WAR) and can be configured to run on a single computer and/or a cluster managed by Grid Engine, LSF or other queuing systems that support DRMAA. JABAWS:MSA provides clients full access to each application's parameters, allows administrators to specify named parameter preset combinations and execution limits for each application through simple configuration files. The JABAWS command-line client allows integration of JABAWS services into conventional scripts. Availability and Implementation: JABAWS is made freely available under the Apache 2 license and can be obtained from: http://www.compbio.dundee.ac.uk/jabaws. Contact: [email protected].
- 22Trinchella, F.; Esposito, M. G.; Scudiero, R. Metallothionein primary structure in amphibians: Insights from comparative evolutionary analysis in vertebrates. C. R. Biol. 2012, 335, 480– 487, DOI: 10.1016/j.crvi.2012.05.00322Metallothionein primary structure in amphibians: Insights from comparative evolutionary analysis in vertebratesTrinchella, Francesca; Esposito, Maria Grazia; Scudiero, RosariaComptes Rendus Biologies (2012), 335 (7), 480-487CODEN: CRBOCM; ISSN:1631-0691. (Elsevier Masson SAS)Metallothioneins are cysteine-rich, low-mol. wt. metal-binding proteins ubiquitously expressed in living organisms. In the last past years, the increasing amt. of vertebrate non-mammalian metallothionein sequences available have disclosed for these proteins differences in the primary structure that have not been supposed before. To provide a more up-to-date view of the metallothioneins in non-mammalian tetrapods, we decided to increase the still scarce knowledge concerning the primary structure and the evolution of metallothioneins in amphibians. Our data demonstrate an unexpected diversity of metallothionein sequences among amphibians, accompanied by remarkable features in their phylogeny. Phylogenetic anal. also reveals the complexity of vertebrate metallothionein evolution, made by both ancient and more recent events of gene duplication and loss.
- 23Madeira, F.; Park, Y. M.; Lee, J.; Buso, N.; Gur, T.; Madhusoodanan, N.; Basutkar, P.; Tivey, A.; Potter, S. C.; Finn, R. D.; Lopez, R. The EMBL-EBI search and sequence analysis tools APIs in 2019. Nucleic Acids Res. 2019, 47 (W1), W636– W641, DOI: 10.1093/nar/gkz26823The EMBL-EBI search and sequence analysis tools APIs in 2019Madeira, Fabio; Park, Young Mi; Lee, Joon; Buso, Nicola; Gur, Tamer; Madhusoodanan, Nandana; Basutkar, Prasad; Tivey, Adrian R. N.; Potter, Simon C.; Finn, Robert D.; Lopez, RodrigoNucleic Acids Research (2019), 47 (W1), W636-W641CODEN: NARHAD; ISSN:1362-4962. (Oxford University Press)The EMBL-EBI provides free access to popular bioinformatics sequence anal. applications as well as to a full-featured text search engine with powerful cross-referencing and data retrieval capabilities. Access to these services is provided via user-friendly web interfaces and via established RESTful and SOAP Web Services APIs (https://www.ebi.ac.uk/seqdb/confluence/display/JDSAT/EMBL-EBI+Web+Services+APIs+-+Data+Retrieval). Both systems have been developed with the same core principles that allow them to integrate an ever-increasing vol. of biol. data, making them an integral part of many popular data resources provided at the EMBL-EBI. Here, we describe the latest improvements made to the frameworks which enhance the interconnectivity between public EMBL-EBI resources and ultimately enhance biol. data discoverability, accessibility, interoperability and reusability.
- 24Letunic, I.; Bork, P. Interactive Tree Of Life (iTOL): an online tool for phylogenetic tree display and annotation. Bioinformatics 2007, 23, 127– 128, DOI: 10.1093/bioinformatics/btl52924Interactive Tree Of Life: an online tool for phylogenetic tree display and annotationLetunic, Ivica; Bork, PeerBioinformatics (2007), 23 (1), 127-128CODEN: BOINFP; ISSN:1367-4803. (Oxford University Press)Summary: Interactive Tree Of Life (iTOL) is a web-based tool for the display, manipulation and annotation of phylogenetic trees. Trees can be interactively pruned and re-rooted. Various types of data such as genome sizes or protein domain repertoires can be mapped onto the tree. Export to several bitmap and vector graphics formats is supported.
- 25McCormick, C. C.; Lin, L.-Y. Quantification and identification of metallothioneins by gel electrophoresis and silver staining. Methods Enzymol. 1991, 205, 71– 76, DOI: 10.1016/0076-6879(91)05087-C25Quantification and identification of metallothioneins by gel electrophoresis and silver stainingMcCormick, Charles C.; Lin, Lih YuanMethods in Enzymology (1991), 205 (Metallobiochem. Pt. B), 71-8, 2 platesCODEN: MENZAU; ISSN:0076-6879.A nondenaturing PAGE for isolating and identifying metallothioneins has been presented. The method employs gradient PAGE and extended electrophoresis of heat-treated tissue exts. (cytosol). The salient feature of the procedure is the use of Coomassie Blue stain as an initial treatment followed by silver stain enhancement. The latter process appears to specifically identify (enhance) MTs among other heat-stable proteins.
- 26Vašák, M. Standard isolation procedure for metallothionein. Methods Enzymol. 1991, 205, 41– 44, DOI: 10.1016/0076-6879(91)05082-726Standard isolation procedure for metallothioneinVasak, MilanMethods in Enzymology (1991), 205 (Metallobiochem. Pt. B), 41-4CODEN: MENZAU; ISSN:0076-6879.Metallothionein (MT) isolation from rabbit liver included prepn. of crude fraction, gel-filtration chromatog., and ion-exchange chromatog. The purifn. of MT from human liver and horse liver and kidney is briefly described.
- 27Hunziker, P. E. Metal removal from mammalian metallothioneins. Methods Enzymol. 1991, 205, 451– 452, DOI: 10.1016/0076-6879(91)05129-J27Metal removal from mammalian metallothioneinsHunziker, Peter E.Methods in Enzymology (1991), 205 (Metallobiochem. Pt. B), 451-2CODEN: MENZAU; ISSN:0076-6879.Metal removal from metallothionein (MT) is a central step in the structural anal. of this protein. While zinc and cadmium are readily removed at low pH, copper remains partially bound and can be removed from MT only by using chelating agents. The methods for the prepn. of apo-MT that have been successfully used for the primary structure anal. of mammalian MTs are described.
- 28Vašák, M. Metal removal and substitution in vertebrate and invertebrate metallothioneins. Methods Enzymol. 1991, 205, 452– 458, DOI: 10.1016/0076-6879(91)05130-N28Metal removal and substitution in vertebrate and invertebrate metallothioneinsVasak, MilanMethods in Enzymology (1991), 205 (Metallobiochem. Pt. B), 452-8CODEN: MENZAU; ISSN:0076-6879.Prepn. of metal-free protein [mtallothionein(MT)] prepn. of metal-substituted MT, and examples of metal-substitution procedures are discussed.
- 29Winge, D. R.; Premakumar, R.; Rajagopalan, V. Metal-induced formation of metallothionein in rat liver. Arch. Biochem. Biophys. 1975, 170, 242– 252, DOI: 10.1016/0003-9861(75)90115-029Metal-induced formation of metallothionein in rat liverWinge, Dennis R.; Premakumar, R.; Rajagopalan, K. V.Archives of Biochemistry and Biophysics (1975), 170 (1), 242-52CODEN: ABBIA4; ISSN:0003-9861.The low mol. wt. proteins induced in rats exposed to zinc [7440-66-6], mercury [7439-97-6], or silver [7440-22-4] were purified by the same procedure as was used for Cd-contg. metallothionein (Cd-thionein). In each case the thionein was resolved into the same two fractions on DEAE-cellulose. The two forms of each metalloprotein exhibited mobilities identical to those of the corresponding Cd-thionein on polyacrylamide gel electrophoresis. The amino acid compns. of the more anionic forms of Hg-thionein and Zn-thionein were quite similar to that of the corresponding Cd-thionein. Thus, the identity of the proteins induced in rats by zinc, mercury, and silver with the previously known metallothionein induced by cadmium [7440-43-9] has been established.
- 30Vašák, M.; Hawkes, G. E.; Nicholson, J. K.; Sadler, P. J. 113Cd NMR studies of reconstituted seven-cadmium metallothionein: evidence for structural flexibility. Biochemistry 1985, 24, 740– 747, DOI: 10.1021/bi00324a03130Cadmium-113 NMR studies of reconstituted seven-cadmium metallothionein: evidence for structural flexibilityVasak, Milan; Hawkes, Geoffrey E.; Nicholson, Jeremy K.; Sadler, Peter J.Biochemistry (1985), 24 (3), 740-7CODEN: BICHAW; ISSN:0006-2960.A reproducible method for the reconstitution of rabbit liver metallothionein (MT) contg. 7 Cd atoms/mol of protein is described. This protein was studied in detail by 113Cd NMR at 88-, 55-, and 44-MHz frequencies, including the effects of pH, temp., and ionic strength on the spectra. The results differ significantly from previous reports of 113Cd NMR on similar samples. Thus, the spectra of both chromatog. distinguishable isoforms MT1 and MT2 were not identical, and neither could be interpreted in terms of a unique static model with the 7 Cd2+ ions forming 2 independent clusters of 4 and 3 Cd2+ ions. Large differential shifts of 113Cd resonances were obsd. with changes in temp. over the range 277-320 K and ionic strength (0.02-0.5M). At low temp. a slow structural change (half-life of several minutes) was detected. The structure was more rigid at high ionic strength. The frequency dependence and 2-dimensional J-resolved spectra revealed that 113Cd resonances were composed of several overlapping peaks, complicating the interpretation of fine structure in 1-dimensional spectra. A new flexible model of the Cd cluster in metallothionein is proposed. This model incorporates dynamic thiolate exchange reactions and involves several configurational substrates of the protein. The possible relation of such flexibility to the function of metallothionein is discussed.
- 31Sutherland, D. E. K.; Stillman, M. J. The “magic numbers” of metallothionein. Metallomics 2011, 3, 444– 463, DOI: 10.1039/c0mt00102c31The "magic numbers" of metallothioneinSutherland, Duncan E. K.; Stillman, Martin J.Metallomics (2011), 3 (5), 444-463CODEN: METAJS; ISSN:1756-591X. (Royal Society of Chemistry)A review. Metallothioneins (MT) are a family of small cysteine rich proteins, which since their discovery in 1957, have been implicated in a range of roles including toxic metal detoxification, protection against oxidative stress, and as a metallochaperone involved in the homeostasis of both zinc and copper. The most well studied member of the family is the mammalian metallothionein, which consists of two domains: a β-domain with 9 cysteine residues, which sequesters 3 Cd2+ or Zn2+ or 6 Cu+ ions, and an α-domain with 11 cysteine residues and, which sequesters 4 Cd2+ or Zn2+ or 6 Cu+ ions. Despite over half a century of research, the exact functions of MT are still unknown. Much of current research aims to elucidate the mechanism of metal binding, as well as to isolate intermediates in metal exchange reactions; reactions necessary to maintain homeostatic equil. These studies further our understanding of the role(s) of this remarkable and ubiquitous protein. Recently, supermetallated forms of the protein, where supermetallation describes metalation in excess of traditional levels, have been reported. These species may potentially be the metal exchange intermediates necessary to maintain homeostatic equil. This review focuses on recent advances in the understanding of the mechanistic properties of metal binding, the implications for the metal induced protein folding reactions proposed for metallothionein metalation, the value of "magic nos.", which we informally define as the commonly detd. metal-to-protein stoichiometric ratios and the significance of the new supermetallated states of the protein and the possible interpretation of the structural properties of this new metalation status. Together we provide a commentary on current exptl. and theor. advances and frame our consideration in terms of the possible functions of MT.
- 32Palumaa, P.; Mackay, E. A.; Vašák, M. Nonoxidative cadmium-dependent dimerization of Cd7-metallothionein from rabbit liver. Biochemistry 1992, 31, 2181– 2186, DOI: 10.1021/bi00122a04032Nonoxidative cadmium-dependent dimerization of cadmium-metallothionein from rabbit liverPalumaa, Peep; Mackay, Elaine A.; Vasak, MilanBiochemistry (1992), 31 (7), 2181-6CODEN: BICHAW; ISSN:0006-2960.The effect of free Cd(II) ions on monomeric Cd7-metallothionein-2 (MT) from rabbit liver has been studied. Slow, concn.-dependent dimerization of this protein was obsd. by gel filtration chromatog. studies. The dimeric MT form, isolated by gel filtration, contains approx. two addnl. and more weakly bound Cd(II) ions per monomer. The incubation of MT dimers with complexing agents EDTA and 2-mercaptoethanol leads to the dissocn. of dimers to monomers. The results of CD and electronic absorption studies indicate that the slow dimerization process is preceded by an initial rapid Cd-induced rearrangement of the monomeric Cd7-MT structure. The 113Cd NMR spectrum of the MT dimer revealed only four 113Cd resonances at chem. shift positions similar to those obsd. for the Cd4 cluster of the well-characterized monomeric 113Cd7-MT. This result suggests that on dimer formation major structural changes occur in the original three-metal cluster domain of Cd7-MT.
- 33Vašák, M.; Kägi, J. H.; Hill, H. A. Zinc(II), cadmium(II), and mercury(II) thiolate transitions in metallothionein. Biochemistry 1981, 20, 2852– 2856, DOI: 10.1021/bi00513a02233Zinc(II), cadmium(II), and mercury(II) thiolate transitions in metallothioneinVasak, Milan; Kaegi, Jeremias H. R.; Hill, H. Allen O.Biochemistry (1981), 20 (10), 2852-6CODEN: BICHAW; ISSN:0006-2960.The metal-specific absorption envelopes of Zn-, Cd-, and Hg-metallothioneins and of complexes of these metal ions with 2-mercaptoethanol were analyzed in terms of Joergensen's electronegativity theory for charge-transfer excitations by using the spectra of Zn(II), Cd(II), and Hg(II) tetrahalides as refs. By Gaussian anal. the difference absorption spectra of the various forms of metallothionein vs. thionein and of the corresponding 2-mercaptoethanol complexes vs. 2-mercaptoethanol were resolved into 3 components. For each metal deriv. the location of the lowest energy band is in good agreement with the position of the first ligand-metal charge-transfer (LMCT) transition (type t2 → a1) predicted from the optical electronegativity difference of the thiolate ligands and of the central metal ion by assuming tetrahedral coordination. There is also a correspondence between the effects of the metal ion on the position of the first LMCT band and the binding energy of the 2p electrons of the S ligands as found by x-ray photoelectron spectroscopic measurements. Due to the lack of exact structural information, the assignment of the 2 other resolved metal-dependent bands remains conjectural, but it is likely that they include a second LMCT transition (type t2 → a1) analogous to that occurring in tetrahalide complexes of group-2B metal ions. The simplicity of the resolved thiolate spectra and their correspondence to those of tetrahedral models support the view that the various metal-binding sites of metallothionein are chem. similar and that the coordination environment of the metal ion has a symmetry related to that of a tetrahedron.
- 34Krebs, B.; Henkel, G. Transition-metal thiolates: From molecular fragments of sulfidic solids to models for active centers of biomolecules. Angew. Chem., Int. Ed. Engl. 1991, 30, 769– 788, DOI: 10.1002/anie.199107691There is no corresponding record for this reference.
- 35Otvos, J. D.; Armitage, I. M. Structure of the metal clusters in rabbit liver metallothionein. Proc. Natl. Acad. Sci. U. S. A. 1980, 77, 7094– 7098, DOI: 10.1073/pnas.77.12.709435Structure of the metal clusters in rabbit liver metallothioneinOtvos, James D.; Armitage, Ian M.Proceedings of the National Academy of Sciences of the United States of America (1980), 77 (12), 7094-8CODEN: PNASA6; ISSN:0027-8424.Cd-113 NMR was used to det. the structures of the multiple Cd-binding sites in the 2 major isoproteins of rabbit liver metallothionein. Isotopically labeled metallothionein was sepd. from the livers of rabbits that had been subjected to repeated injections of 113CdCl2. The native protein isolated from these livers contains an appreciable amt. of Zn in addn. to Cd: 2-3 mol/mol protein out of a total metal content of 7 mol/mol protein. The 113Cd NMR spectrum of Cd, Zn-contg. metallothionein is quite complex, reflecting the fact that the native protein is a heterogeneous mixt. of species contg. different relative amts. of Zn and Cd. Replacement of the native Zn with 113Cd in vitro gave a protein whose 113Cd NMR spectrum was much simpler, contg. 8 distinct multiplets with chem. shifts ranging from 611 to 670 ppm. The multiplet structures were due to 113Cd-113Cd scalar coupling arising from 2-bond interactions between 113Cd ions linked to one another by bridging cysteine thiolate ligands. The sizes and structures of the metal clusters in the protein were detd. by the application of selective homonuclear 113Cd decoupling techniques. Rabbit liver metallothionein contains 2 sep. metal clusters, one contg. 4 Cd2+ ions and the other contg. 3. These 2 clusters, whose structures are the same in both isoproteins, were designated cluster A and cluster B, resp. Structures for the clusters are proposed that account for the 113Cd spin-coupling data and for the participation of all 20 of the cysteine residues in metal ligation, 11 in cluster A and 9 in cluster B. The appearance in the spectrum of 8 multiplets rather than the 7 that would be expected on the basis of the no. of metal-binding sites in the protein is an indication of some residual heterogeneity in the 113Cd-labeled metallothionein sample. The origin of this heterogeneity is suggested to be the presence of a protein species that lacks metal ions at its cluster B binding sites.
- 36Kägi, J. H. R. Overview of metallothionein. Methods Enzymol. 1991, 205, 613– 626, DOI: 10.1016/0076-6879(91)05145-L36Overview of metallothioneinKagi J HMethods in enzymology (1991), 205 (), 613-26 ISSN:0076-6879.There is no expanded citation for this reference.
- 37Winge, D. R.; Miklossy, K. A. Domain nature of metallothionein. J. Biol. Chem. 1982, 257, 3471– 3476, DOI: 10.1016/S0021-9258(18)34802-637Domain nature of metallothioneinWinge, Dennis R.; Miklossy, Kathy AnneJournal of Biological Chemistry (1982), 257 (7), 3471-6CODEN: JBCHA3; ISSN:0021-9258.Metallothionein purified from the livers of rats injected with CdCl2 was cleaved by proteolysis into a 32-residue polypeptide that contained 4 bound Cd ions. Appearance of this fragment designated α requires prior treatment of metallothionein with EDTA to remove Zn and destabilize the 3-metal cysteine cluster in the other domain. The half-mol. domain was not efficiently produced by proteolysis of native metallothionein. The Cd4-α fragment is asym. in shape, as is the parent mol. N-terminal sequence anal. revealed that the α fragment starts at lysine-30. Since the same amino acids are released from the C-terminus of intact thionein and the α fragment by carboxypeptidase Y, the α domain generated by digestion with subtilisin therefore comprises residues 30 through 61. The amino acid compn. of the α polypeptide is consistent with the structure of the 4-metal cysteine cluster proposed by J. D. Otvos and I. M. Armitage (1980). Metallothionein appears to consist of a 3-metal cysteine domain in the N-terminal half of the thionein mol. and the 4-metal cysteine domain in the C-terminal half.
- 38Boulanger, Y.; Armitage, I. M.; Miklossy, K. A.; Winge, D. R. 113Cd NMR study of a metallothionein fragment. Evidence for a two-domain structure. J. Biol. Chem. 1982, 257, 13717– 13719, DOI: 10.1016/S0021-9258(18)33506-338Cadmium-113 NMR study of a metallothionein fragment. Evidence for a two-domain structureBoulanger, Yvan; Armitage, Ian M.; Miklossy, Kathy Anne; Winge, Dennis R.Journal of Biological Chemistry (1982), 257 (22), 13717-19CODEN: JBCHA3; ISSN:0021-9258.A 32-residue polypeptide fragment, designated αI, of rat liver metallothionein obtained by subtilisin digestion was studied by 113Cd NMR. The amino acid compn. of the fragment corresponded to residues 30-61 of the metallothionein primary structure, and it contained 3.4 g atoms of Cd2+/mol of αI-fragment. Four 113Cd resonances were obsd., 3 of which had identical chem. shifts to those assigned to the 4-metal cluster in human liver metallothionein-2 under the same pH and buffer conditions. The 5-ppm chem. shift difference between the remaining resonance assigned to the 4-metal cluster in the intact protein can be explained to result from the removal of the N-terminal polypeptide fragment contg. the 3-metal cluster. These results provide unambiguous evidence for the 2-domain structure of metallothionein, contg. a sep. 3- and a 4-metal cluster.
- 39Romero-Isart, N.; Vašák, M. Advances in the structure and chemistry of metallothioneins. J. Inorg. Biochem. 2002, 88, 388– 396, DOI: 10.1016/S0162-0134(01)00347-639Advances in the structure and chemistry of metallothioneinsRomero-Isart, Nuria; Vasak, MilanJournal of Inorganic Biochemistry (2002), 88 (3-4), 388-396CODEN: JIBIDJ; ISSN:0162-0134. (Elsevier Science Inc.)A review. A low mol. wt. (6-7 kDa) class of metalloproteins, designated as metallothioneins (MTs), exhibit repeated sequence motifs of either CxC or CxxC through which mono or divalent d10 metal ions are bound in polymetallic-thiolate clusters. The preservation of metal-thiolate clusters in an increasing no. of three-dimensional structures of these proteins signifies the importance of this structural motif. This review focuses on the recent developments regarding the versatile and striking chem. reactivity of MTs as well as on the existence of conformational/configurational dynamics within their structure. Both properties and their interplay are likely to be essential for the still elusive biol. function of these proteins.
- 40Frey, M. H.; Wagner, G.; Vašák, M.; Soerensen, O. W.; Neuhaus, D.; Wörgötter, E.; Kägi, J. H. R.; Ernst, R. R.; Wüthrich, K. Polypeptide-metal cluster connectivities in metallothionein 2 by novel proton-cadmium-113 heteronuclear two-dimensional NMR experiments. J. Am. Chem. Soc. 1985, 107, 6847– 6851, DOI: 10.1021/ja00310a01740Polypeptide-metal cluster connectivities in metallothionein 2 by novel proton-cadmium-113 heteronuclear two-dimensional NMR experimentsFrey, Michael H.; Wagner, Gerhard; Vasak, Milan; Soerensen, Ole W.; Neuhaus, David; Woergoetter, Erich; Kaegi, Jeremias H. R.; Ernst, Richard R.; Wuethrich, KurtJournal of the American Chemical Society (1985), 107 (24), 6847-51CODEN: JACSAT; ISSN:0002-7863.Two-dimensional heteronuclear 1H-113Cd and homonuclear 113Cd-113Cd correlated NMR spectra were recorded for 113Cd-metallothionein 2 from rabbit liver. 1H detection was used for the heteronuclear expts. For 12 of the 20 cysteinyl residues, connectivities to one 113Cd could be identified; for the other 8 cysteinyl residues, connectivities to 2 113Cd nuclei were detected (bridging cysteines). In combination with the independently obtained sequence-specific NMR assignments of the cysteine 1H spin systems, the topol. and the locations of the metal-S clusters relative to the polypeptide structure could be established.
- 41Zhang, X.; Tamaru, H.; Khan, S. I.; Horton, J. R.; Keefe, L. J.; Selker, E. U.; Cheng, X. Structure of the Neurospora SET domain protein DIM-5, a histone H3 lysine methyltransferase. Cell 2002, 111, 117– 127, DOI: 10.1016/S0092-8674(02)00999-641Structure of the Neurospora SET domain protein DIM-5, a histone H3 lysine methyltransferaseZhang, Xing; Tamaru, Hisashi; Khan, Seema I.; Horton, John R.; Keefe, Lisa J.; Selker, Eric U.; Cheng, XiaodongCell (Cambridge, MA, United States) (2002), 111 (1), 117-127CODEN: CELLB5; ISSN:0092-8674. (Cell Press)AdoMet-dependent methylation of histones is part of the "histone code" that can profoundly influence gene expression. We describe the crystal structure of Neurospora DIM-5, a histone H3 lysine 9 methyltransferase (HKMT), detd. at 1.98 Å resoln., as well as results of biochem. characterization and site-directed mutagenesis of key residues. This SET domain protein bears no structural similarity to previously characterized AdoMet-dependent methyltransferases but includes notable features such as a triangular Zn3Cys9 zinc cluster in the pre-SET domain and a AdoMet binding site in the SET domain essential for Me transfer. The structure suggests a mechanism for the methylation reaction and provides the structural basis for functional characterization of the HKMT family and the SET domain.
- 42Zheng, S.; Wang, J.; Feng, Y.; Wang, J.; Ye, K. Solution structure of MSL2 CXC domain reveals an unusual Zn3Cys9 cluster and similarity to Pre-SET domains of histone lysine methyltransferases. PLoS One 2012, 7, e45437 DOI: 10.1371/journal.pone.004543742Solution structure of MSL2 CXC domain reveals an unusual Zn3Cys9 cluster and similarity to pre-SET domains of histone lysine methyltransferasesZheng, Sanduo; Wang, Jia; Feng, Yingang; Wang, Jinfeng; Ye, KeqiongPLoS One (2012), 7 (9), e45437CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)The dosage compensation complex (DCC) binds to single X chromosomes in Drosophila males and increases the transcription level of X-linked genes by approx. twofold. Male-specific lethal 2 (MSL2) together with MSL1 mediates the initial recruitment of the DCC to high-affinity sites in the X chromosome. MSL2 contains a DNA-binding cysteine-rich CXC domain that is important for X targeting. In this study, we detd. the soln. structure of MSL2 CXC domain by NMR spectroscopy. We identified three zinc ions in the CXC domain and detd. the metal-to-cysteine connectivities from 1H-113Cd correlation expts. The structure reveals an unusual zinc-cysteine cluster composed of three zinc ions coordinated by six terminal and three bridging cysteines. The CXC domain exhibits unexpected structural homol. to pre-SET motifs of histone lysine methyltransferases, expanding the distribution and structural diversity of the CXC domain superfamily. Our findings provide novel structural insight into the evolution and function of CXC domains.
- 43An, S.; Yeo, K. J.; Jeon, Y. H.; Song, J. J. Crystal structure of the human histone methyltransferase ASH1L catalytic domain and its implications for the regulatory mechanism. J. Biol. Chem. 2011, 286, 8369– 8374, DOI: 10.1074/jbc.M110.20338043Crystal Structure of the Human Histone Methyltransferase ASH1L Catalytic Domain and its Implications for the Regulatory MechanismAn, So-Jin; Yeo, Kwon-Joo; Jeon, Young-Ho; Song, Ji-JoonJournal of Biological Chemistry (2011), 286 (10), 8369-8374CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)Absent, small, or homeotic disk1 (Ash1) is a trithorax group histone methyltransferase that is involved in gene activation. Although there are many known histone methyltransferases, their regulatory mechanisms are poorly understood. Here, we present the crystal structure of the human ASH1L catalytic domain, showing its substrate binding pocket blocked by a loop from the post-SET domain. In this configuration, the loop limits substrate access to the active site. Mutagenesis of the loop stimulates ASH1L histone methyltransferase activity, suggesting that ASH1L activity may be regulated through the loop from the post-SET domain. In addn., we show that human ASH1L specifically methylates histone H3 Lys-36. Our data implicate that there may be a regulatory mechanism of ASH1L histone methyltransferases.
- 44Arseniev, A.; Schultze, P.; Wörgötter, E.; Braun, W.; Wagner, G.; Vašák, M.; Kägi, J. H.; Wüthrich, K. Three-dimensional structure of rabbit liver [Cd7]metallothionein-2a in aqueous solution determined by nuclear magnetic resonance. J. Mol. Biol. 1988, 201, 637– 657, DOI: 10.1016/0022-2836(88)90644-444Three-dimensional structure of rabbit liver [Cd7]metallothionein-2a in aqueous solution determined by nuclear magnetic resonanceArseniev, Alexandre; Schultze, Peter; Woergoetter, Erich; Braun, Werner; Wagner, Gerhard; Vasak, Milan; Kaegi, Jeremias H. R.; Wuthrich, KurtJournal of Molecular Biology (1988), 201 (3), 637-57CODEN: JMOBAK; ISSN:0022-2836.In previous work the metal-polypeptide coordinative bonds in the major protein species of a reconstituted [113Cd7]metallothionein-2 prepn. from rabbit liver in aq. soln. were detd., the secondary polypeptide structure was found to contain several half-turns and 310-helical segments, and a preliminary characterization of the overall polypeptide backbone fold in the β-domain contg. the 3-metal cluster, and the α-domain contg. the 4-metal cluster, was obtained. Using a new, more extensive set of NMR data these earlier structures were improved by new structure calcns. The new exptl. data consist of distance constraints from measurements of nuclear Overhauser effects, and dihedral angle constraints derived from both coupling consts. and nuclear Overhauser effects. The structure calcns. were performed with the program DISMAN. Since no information on the orientation of the 2 domains relative to each other could be obtained, the structure calcns. were performed sep. for the α-domain and the β-domain. The av. of the pairwise root-mean-square distances among the 20 structures with the least residual violations of input constraints was 2.9 Å for the β-domain and 1.4 Å for the α-domain. The overall chirality of the polypeptide fold is right-handed for the β-domain and left-handed for the α-domain. For each of the 7 metal ions, the local chirality of the coordination of the 4 cysteinyl Sγ atoms is clearly defined. The improved structures of both domains show the previously noted differences relative to the recently published crystal structure of metallothionein-2a from rat liver.
- 45Schultze, P.; Wörgötter, E.; Braun, W.; Wagner, G.; Vašák, M.; Kägi, J. H. R.; Wüthrich, K. Conformation of [Cd7]-metallothionein-2 from rat liver in aqueous solution determined by nuclear magnetic resonance spectroscopy. J. Mol. Biol. 1988, 203, 251– 268, DOI: 10.1016/0022-2836(88)90106-445Conformation of [Cd7]-metallothionein-2 from rat liver in aqueous solution determined by nuclear magnetic resonance spectroscopySchultze, Peter; Woergoetter, Erich; Braun, Werner; Wagner, Gerhard; Vasak, Milan; Kaegi, Jeremias H. R.; Wuethrich, KurtJournal of Molecular Biology (1988), 203 (1), 251-68CODEN: JMOBAK; ISSN:0022-2836.The 3-dimensional structure of [Cd7]-metallothionein-2 from rat liver was detd. in aq. soln., using NMR spectrometry and distance geometry calcns. The exptl. data provided proton-proton distance constraints from measurements of NOE, constraints on the geometry of the metal-cysteine clusters detd. by heteronuclear correlation spectroscopy, and dihedral angle constraints derived from both coupling consts. and NOE. The structure calcns. were performed with the program DISMAN. As in previous studies with rabbit liver metallothionein-2a, the structure calcns. were performed sep. for the α and β-domains contg. the 4 and 3-metal clusters, resp., since no interdomain constraints were found. For both domains, the global polypeptide fold, the location of polypeptide secondary structure elements, the architecture of the metal-S cluster, and the local chirality of the metal coordination are very similar to the soln. structure of rabbit metallothionein-2a, but show considerable difference relative to the crystal structure of rat metallothionein-2.
- 46Messerle, B. A.; Schäffer, A.; Vašák, M.; Kägi, J. H.; Wüthrich, K. Three-dimensional structure of human [113Cd7]metallothionein-2 in solution determined by nuclear magnetic resonance spectroscopy. J. Mol. Biol. 1990, 214, 765– 779, DOI: 10.1016/0022-2836(90)90291-S46Three-dimensional structure of human [113Cd7]metallothionein-2 in solution determined by nuclear magnetic resonance spectroscopyMesserle, Barbara A.; Schaeffer, Andreas; Vasak, Milan; Kaegi, Jeremias H. R.; Wuethrich, KurtJournal of Molecular Biology (1990), 214 (3), 765-79CODEN: JMOBAK; ISSN:0022-2836.The three-dimensional structure of human [113Cd7]metallothionein-2 was detd. by NMR spectroscopy in soln. Sequence-specific 1H resonance assignments were obtained using the sequential assignment method. The input for the structure calcns. consisted of the metal-cysteine coordinative bonds identified with heteronuclear correlation spectroscopy, 1H-1H distance constraints from nuclear Overhauser enhancement spectroscopy, and spin-spin coupling consts. 3JHNα and 3Jαβ. The mol. consists of 2 domains, the β-domain including amino acid residues 1-30 and 3 metal ions, and the α-domain including residues 31-61 and 4 metal ions. The NMR data present no evidence for a preferred relative orientation of the 2 domains. The polypeptide-to-metal coordinative bonds in human metallothionein-2 are identical to those in the previously detd. soln. structures of rat metallothionein-2 and rabbit metallothionein-2α, and the polypeptide conformations in the three proteins are also closely similar.
- 47Robbins, A. H.; McRhee, D. E.; Williamson, M.; Collett, S. A.; Xuong, N. H.; Furey, W. F.; Wang, B. C.; Stout, C. D. Refined Crystal Structure of Cd,Zn Metallothionein at 2.0 Å Resolution. J. Mol. Biol. 1991, 221, 1269– 1293, DOI: 10.1016/0022-2836(91)80126-F47Refined crystal structure of cadmium-zinc metallothionein at Å resolutionRobbins, A. H.; McRee, D. E.; Williamson, M.; Collett, S. A.; Xuong, N. H.; Furey, W. F.; Wang, B. C.; Stout, C. D.Journal of Molecular Biology (1991), 221 (4), 1269-93CODEN: JMOBAK; ISSN:0022-2836.The crystal structure of Cd5,Zn2-metallothionein from rat liver has been refined at 2.0 Å resoln. of a R-value of 0.176 for all obsd. data. The five Cd positions in the asym. unit of the crystal create a pseudo-centrosym. constellation about a crystallog. 2-fold axis. Consequently, the distribution of anomalous differences in almost ideally centrosym. Therefore, the previously reported metal positions and the protein model derived therefrom are incorrect. Direct methods were applied to the protein amplitudes to locate the Cd positions. The new positions were used to calc. a new electron d. map based on the Cd anomalous scattering and partial structure to model the metal clusters and the protein. Phases calcd. from this model predict the positions of three sites in a (NH4)2WS4 deriv. Single isomorphous replacement phases calcd. with these tungsten sites confirm the positions of the Cd sites from the new direct methods calcns. The refined metallothionein structure has a root-mean-square deviation of 0.016 Å from ideality of bonds and normal stereochem. of Φ, φ, and χ torsion angles. The metallothionein crystal structure is in agreement with the structures for the α and β domains in soln. derived by NMR methods. The overall chain folds and all metal to cysteine bonds are the same in the two structure detns. The handedness of a short helix in the α-domain (residues 41 to 45) is the same in both structures. The crystal structure provides information concerning the metal cluster geometry and cysteine solvent accessibility and side-chain stereochem. Short cysteine peptide sequences repeated in the structure adopt restricted conformations which favor the formation of amide to sulfur hydrogen bonds. The crystal packing reveals intimate assocn. of mols. about the diagonal 2-fold axes and trapped ions of crystn. (modeled as phosphate and sodium). Variation in the chem. and structural environments of the metal sites is in accord with data for metal exchange reactions in metallothioneins.
- 48Braun, W.; Vašák, M.; Robbins, A. H.; Stout, C. D.; Wagner, G.; Kägi, J. H. R.; Wüthrich, K. Comparison of the NMR solution and the x-ray crystal structure of rat metallothionein-2. Proc. Natl. Acad. Sci. U. S. A. 1992, 89, 10124– 10128, DOI: 10.1073/pnas.89.21.1012448Comparison of the NMR solution structure and the x-ray crystal structure of rat metallothionein-2Braun, W.; Vasak, M.; Robbins, A. H.; Stout, C. D.; Wagner, G.; Kaegi, J. H. R.; Wuethrich, K.Proceedings of the National Academy of Sciences of the United States of America (1992), 89 (21), 10124-8CODEN: PNASA6; ISSN:0027-8424.Metallothioneins are small cysteine-rich proteins capable of binding heavy metal ions such as Zn2+ and Cd2+. They are ubiquitous tissue components in higher organisms, which tentatively have been attributed both unspecific protective functions against toxic metal ions and highly specific roles in fundamental zinc-regulated cellular processes. In this paper a detailed comparison of the NMR soln. structure (Schultz, P., et al., 1988) and a recent x-ray crystal structure (Robbins, A. H., et al., 1991) of rat metallothionein-2 shows that the metal-lothionein structures in crystals and in soln. have identical mol. architectures. The structures obtained with both techniques now present a reliable basis for the discussions on structure-function correlations in this class of metalloproteins.
- 49Öz, G.; Zangger, K.; Armitage, I. M. Three-dimensional structure and dynamics of a brain specific growth inhibitory factor: Metallothionein 3. Biochemistry 2001, 40, 11433– 11441, DOI: 10.1021/bi010827l49Three-dimensional structure and dynamics of a brain specific growth inhibitory factor: metallothionein-3Oz G; Zangger K; Armitage I MBiochemistry (2001), 40 (38), 11433-41 ISSN:0006-2960.The brain specific member of the metallothionein (MT) family of proteins, metallothionein-3, inhibits the growth and survival of neurons, in contrast to the ubiquitous mammalian MT isoforms, MT-1 and MT-2, that are found in most tissues and are thought to function in metal ion homeostasis and detoxification. Solution NMR was utilized to determine the structural and dynamic differences of MT-3 from MT-1 and 2. The high-resolution solution structure of the C-terminal alpha-domain of recombinant mouse MT-3 revealed a tertiary fold very similar to MT-1 and 2, except for a loop that accommodates an acidic insertion relative to these isoforms. This loop was distinguished from the rest of the domain by dynamics of the backbone on the nano- to picosecond time-scale shown by (15)N relaxation studies and was identified as a possible interaction site with other proteins. The N-terminal beta-domain contains the region responsible for the growth inhibitory activity, a CPCP tetrapeptide close to the N-terminus. Because of exchange broadening of a large number of the NMR signals from this domain, homology modeling was utilized to calculate models for the beta-domain and suggested that while the backbone fold of the MT-3 beta-domain is identical to MT-1 and 2, the second proline responsible for the activity, Pro9, may show structural heterogeneity. (15)N relaxation analyses implied fast internal motions for the beta-domain. On the basis of these observations, we conclude that the growth inhibitory activity exhibited by MT-3 is a result of a combination of local structural differences and global dynamics in the beta-domain.
- 50Wang, H.; Zhang, Q.; Cai, B.; Li, H. Y.; Sze, K. H.; Huang, Z. X.; Wu, H. M.; Sun, H. Z. Solution structure and dynamics of human metallothionein-3 (MT-3). FEBS Lett. 2006, 580, 795– 800, DOI: 10.1016/j.febslet.2005.12.09950Solution structure and dynamics of human metallothionein-3 (MT-3)Wang, Hui; Zhang, Qi; Cai, Bin; Li, Hongyan; Sze, Kong-Hung; Huang, Zhong-Xian; Wu, Hou-Ming; Sun, HongzheFEBS Letters (2006), 580 (3), 795-800CODEN: FEBLAL; ISSN:0014-5793. (Elsevier B.V.)Alzheimer's disease is characterized by progressive loss of neurons accompanied by the formation of intraneural neurofibrillary tangles and extracellular amyloid plaques. Human neuronal growth inhibitory factor, classified as metallothionein-3 (MT-3), was found to be related to the neurotrophic activity promoting cortical neuron survival and dendrite outgrowth in the cell culture studies. We have detd. the soln. structure of the α-domain of human MT-3 (residues 32-68) by multinuclear and multidimensional NMR spectroscopy in combination with the mol. dynamic simulated annealing approach. The human MT-3 shows two metal-thiolate clusters, one in the N-terminus (β-domain) and one in the C-terminus (α-domain). The overall fold of the α-domain is similar to that of mouse MT-3. However, human MT-3 has a longer loop in the acidic hexapeptide insertion than that of mouse MT-3. Surprisingly, the backbone dynamics of the protein revealed that the β-domain exhibits similar internal motion to the α-domain, although the N-terminal residues are more flexible. Our results may provide useful information for understanding the structure-function relationship of human MT-3.
- 51Dolderer, B.; Echner, H.; Beck, A.; Hartmann, H.-J.; Weser, U.; Luchinat, C.; Del Bianco, C. Coordination of three and four Cu(I) to the α- and β-domain of vertebrate Zn-metallothionein-1, respectively, induces significant structural changes. FEBS J. 2007, 274, 2349– 2362, DOI: 10.1111/j.1742-4658.2007.05770.x51Coordination of three and four Cu(I) to the α- and β-domain of vertebrate Zn-metallothionein-1, respectively, induces significant structural changesDolderer, Benedikt; Echner, Hartmut; Beck, Alexander; Hartmann, Hans-Juergen; Weser, Ulrich; Luchinat, Claudio; Del Bianco, CristinaFEBS Journal (2007), 274 (9), 2349-2362CODEN: FJEOAC; ISSN:1742-464X. (Blackwell Publishing Ltd.)Vertebrate metallothioneins are found to contain Zn(II) and variable amts. of Cu(I), in vivo, and are believed to be important for d10-metal control. To date, structural information is available for the Zn(II) and Cd(II) forms, but not for the Cu(I) or mixed metal forms. Cu(I) binding to metallothionein-1 has been investigated by CD, luminescence and 1H NMR using two synthetic fragments representing the α- and the β-domain. The 1H NMR data and thus the structures of Zn4α metallothionein (MT)-1 and Zn3βMT-1 were essentially the same as those already published for the corresponding domains of native Cd7MT-1. Cu(I) titrn. of the Zn(II)-reconstituted domains provided clear evidence of stable polypeptide folds of the three Cu(I)-contg. α- and the four Cu(I)-contg. β-domains. The soln. structures of these two species are grossly different from the structures of the starting Zn(II) complexes. Further addn. of Cu(I) to the two single domains led to the loss of defined domain structures. Upon mixing of the sep. prepd. aq. three and four Cu(I) loaded α- and β-domains, no interaction was seen between the two species. There was neither any indication for a net transfer of Cu(I) between the two domains nor for the formation of one large single Cu(I) cluster involving both domains.
- 52Rupp, H.; Weser, U. Conversion of metallothionein into Cu-thionein, the possible low molecular weight form of neonatal hepatic mitochondrocuprein. FEBS Lett. 1974, 44, 293– 297, DOI: 10.1016/0014-5793(74)81161-052Conversion of metallothionein into copper-thionein, the possible low molecular weight form of neonatal hepatic mitochondrocupreinRupp, Heinz; Weser, UlrichFEBS Letters (1974), 44 (3), 293-7CODEN: FEBLAL; ISSN:0014-5793.When chicken metallothionein was titrated using Cu(CH3CN)4ClO4, absorbance did not occur in the visible region. A plot of the concn. of added Cu vs. absorbance at const. wavelengths revealed that at 250 μ the linear response decreased and at 280, 300, and 320 μ it increased. All curves showed a sharp bending at the same point corresponding to 15.2 g atoms of Cu/12,000 g of protein. Increased Cu concns. resulted in 2 new Cotton effects with pos. and neg. extremes at 359 and 302 μ, resp. The neg. Cotton effect at 238 μ leveled off. The 2 new Cotton effects were assigned to electronic transitions of Cu chelates where the free functional groups of lysine, histidine, and cysteine participated as ligands. Displacement of Zn2+ and Cd2+ by H+, using partially loaded Cu-thionein, gave a highly polymeric form which was similar to neonatal hepatic mitochondrocuprein. The binding energy of the S core electrons of this polymer, 162.5 eV, was between that of the resp. S-binding energies of Cu-thionein and the fully oxidized cystine-thionein. Polymeric Cu-thionen is probably composed of either Cu-thionein or cysteine-thionein residues.
- 53Presta, A.; Green, A. R.; Zelazowski, A.; Stillman, M. J. Formation of a continuum of copper(I) thiolate stoichiometric species. Eur. J. Biochem. 1995, 227, 226– 240, DOI: 10.1111/j.1432-1033.1995.tb20380.x53Copper binding to rabbit liver metallothionein. Formation of a continuum of copper(I)-thiolate stoichiometric speciesPresta, Anthony; Green, Anna Rae; Zelazowski, Andrzej; Stillman, Martin J.European Journal of Biochemistry (1995), 227 (1/2), 226-40CODEN: EJBCAI; ISSN:0014-2956. (Springer)CD and UV absorption spectral data have been used to probe the binding mechanism for formation and the structure of the copper(I)-thiolate binding clusters in rabbit liver metallothionein during addn. of Cu+ to aq. solns. of Zn7-metallothionein 2 and Cd5Zn2-metallothionein 2. Mammalian metallothionein binds metals in two binding sites, namely the α and β domains. Spectral data which probe the distribution of Cu(I) between the two binding domains clearly show that both the site of binding (α or β), and the structures of the specific metal-thiolate clusters formed, are dependent on temp. and on the nature of the starting protein (either Zn7-metallothionein or Cd5Zn2-metallothionein). CD spectra acquired during the addn. of Cu+ to Zn7-metallothionein show that Cu+ replace the bound Zn(II) in a domain-distributed manner with complete removal of the Zn(II) after addn. of 12 Cu+. Spectral and metal analyses prove that a series of Cu(I)-metallothionein species are formed by a non-cooperative metal-binding mechanism with a continuum of Cu(I):metallothionein stoichiometries. Observation of a series of spectral satn. points signal the formation of distinct optically active Cu(I)-thiolate structures for the Cu9Zn2-metallothionein, Cu12-metallothionein, and the Cu15-metallothionein species. These data very clearly show that for Cu(I) binding to Zn7-metallothionein, there are several key Cu(I):metallothionein stoichiometric ratios, and not just the single value of 12. The CD spectra up to the Cu12-metallothionein species are defined by bands located at 255(+) nm and 280(-) nm. Interpretation of the changes in the CD and UV absorption spectral data recorded between 3 °C and 52 °C as Cu+ is added to Zn-metallothionein show that copper-thiolate cluster formation is strongly temp. dependent. These changes in spectral properties are interpreted in terms of kinetic vs. thermodn. control of the metal-binding pathways as Cu+ binds to the protein. At low temps. (3°C and 10°C) the spectral data indicate a kinetically controlled mechanism whereby an activation barrier inhibits formation of ordered copper-thiolate structures until formation of Cu12-metallothionein. At higher temps. (>30°C) the activation barrier is overcome, allowing formation of new Cu(I)-thiolate clusters with unique spectral properties, esp. at the Cu9Zn2-metallothionein point. The CD spectra also show that a Cu15-metallothionein species with a well-defined, three-dimensional structure forms at all temps., characterized by a band near 335 nm, indicating the presence of digonal Cu(I). Complicated CD spectral changes are obsd. when Cu+ is added to Cd5Zn2-metallothionein. The spectral data are interpreted in terms of domain-distributed binding followed by rearrangement to form the domain-specific product. In the Cu6Cd4-metallothionein species, the Cu+ are ultimately bound specifically to the β domain of the protein. This complex is characterized by the CD spectrum of the Cd4S'Cys'11 in the α domain. The domain-specific product arises from the result of two interdependent driving forces, leading to formation of the Cu6S'Cys'9, β-domain cluster and the Cd4S'Cys'11 α-domain cluster. These findings imply physiol. roles for the individual domains of this protein. Further Cu+ addn. yields the mixed Cu12Cd4-metallothionein species which exhibits a unique CD spectrum with bands at 240, 268, 293 and 332 nm. Mol. modeling calcns. were used to create a structure for the Cu12-metallothionein 2 species, based on domain stoichiometries identified by the spectroscopic data of Cu6S'Cys'11 (α domain) and Cu6S'Cys'9 (β domain). In accord with the CD spectral data, this structure involves exclusive trigonal coordination of all 12 bound Cu+ to the 20 cysteinyl thiolates. All cysteinyl thiolates in the β domain adopt bridging geometry, while cysteinyl thiolates in the α domain adopt both bridging and terminal geometries.
- 54Melenbacher, A.; Korkola, N. C.; Stillman, M. J. The pathways and domain specificity of Cu(I) binding to human metallothionein 1A. Metallomics 2020, 12, 1951– 1964, DOI: 10.1039/D0MT00215A54The pathways and domain specificity of Cu(I) binding to human metallothionein 1AMelenbacher, Adyn; Korkola, Natalie C.; Stillman, Martin J.Metallomics (2020), 12 (12), 1951-1964CODEN: METAJS; ISSN:1756-591X. (Royal Society of Chemistry)Copper is an essential element, but as a result of numerous adverse reactions, it is also a cellular toxin. Nature protects itself from these toxic reactions by binding cuprous copper to chaperones and other metalloproteins. Metallothionein has been proposed as a storage location for Cu(I) and potentially as the donor of Cu(I) to copper-dependent enzymes. We report that the addn. of Cu(I) to apo recombinant human metallothionein 1a cooperatively forms a sequential series of Cu(I)-cysteinyl thiolate complexes that have specific Cu(I) : MT stoichiometries of 6 : 1, 10 : 1, and finally 13 : 1. The individual domain Cu : SCys stoichiometries were detd. as Cu6S9 (for 6 : 1), Cu6S9 + Cu4S6 (for 10 : 1), and Cu6S9 + Cu7S9 (for 13 : 1) based on the no. of modified free cysteines not involved in Cu(I) binding. The stoichiometries are assocd. with Cu-SCys cluster formation involving bridging thiols in the manner similar to the clusters formed with Cd(II) and Zn(II). The locations of these clustered species within the 20 cysteine full protein were detd. from the unique speciation profiles of Cu(I) binding to the β and α domain fragments of recombinant human metallothionein 1a with 9 and 11 cysteines, resp. Competition reactions using these domain fragments challenged Cu(I) metalation of the βα protein, allowing the sequence of cluster formation in the full protein to be detd. Relative binding consts. for each Cu(I) bound are reported. The emission spectra of the Cu4S6, Cu6S9, and Cu7S9 clusters have unique λmax and phosphorescent lifetime properties. These phosphorescent data provide unambiguous supporting evidence for the presence of solvent shielded clusters reported concurrently by ESI-MS. Simulated emission spectra based on the cluster specific emission profiles matched the exptl. spectra and are used to confirm that the relative concns. seen by ESI-MS are representative of the soln. Our results suggest that the availability of a series of sequential Cu(I)-thiolate clusters provides flexibility as a means of protecting the cell from toxicity while still allowing for homeostatic control of the total copper content in the cell. This mechanism provides a dynamic and reactive method of reducing the cellular free copper concns.
- 55Calderone, V.; Dolderer, B.; Hartmann, H.-J.; Echner, H.; Luchinat, C.; Del Bianco, C.; Mangani, S.; Weser, U. The crystal structure of yeast copper thionein: The solution of a long-lasting enigma. Proc. Natl. Acad. Sci. U. S. A. 2005, 102, 51– 56, DOI: 10.1073/pnas.040825410155The crystal structure of yeast copper thionein: The solution of a long-lasting enigmaCalderone, Vito; Dolderer, Benedikt; Hartmann, Hans-Juergen; Echner, Hartmut; Luchinat, Claudio; Del Bianco, Cristina; Mangani, Stefano; Weser, UlrichProceedings of the National Academy of Sciences of the United States of America (2005), 102 (1), 51-56CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)We report here the crystal structure of yeast copper thionein (Cu-MT), detd. at 1.44-Å resoln. The Cu-MT structure shows the largest known oligonuclear Cu(I) thiolate cluster in biol., consisting of six trigonally and two digonally coordinated Cu(I) ions. This is at variance with the results from previous spectroscopic detns., which were performed on MT samples contg. seven rather than eight metal ions. The protein backbone has a random coil structure with the loops enfolding the copper cluster, which is located in a cleft where it is bound to 10 cysteine residues. The protein structure is somewhat different from that of Ag7-MT and similar, but not identical, to that of Cu7-MT. Besides the different structure of the metal cluster, the main differences lie in the cysteine topol. and in the conformation of some portions of the backbone. The present structure suggests that Cu-MT, in addn. to its role as a safe depository for copper ions in the cell, may play an active role in the delivery of copper to metal-free chaperones.
- 56Davis, J. J.; Hill, H. A. O.; Kurz, A.; Jacob, C.; Maret, W.; Vallee, B. L. A scanning tunnelling microscopy study of rabbit metallothionein. PhysChemComm 1998, 1, 12– 22, DOI: 10.1039/a806057fThere is no corresponding record for this reference.
- 57Maret, W.; Heffron, G.; Hill, H. A. O.; Djuricic, D.; Jiang, L.-J.; Vallee, B. L. The ATP/metallothionein interaction: NMR and STM. Biochemistry 2002, 41, 1689– 1694, DOI: 10.1021/bi011608357The ATP/Metallothionein Interaction: NMR and STMMaret, Wolfgang; Heffron, Gregory; Hill, H. Allen O.; Djuricic, Dejana; Jiang, Li-Juan; Vallee, Bert L.Biochemistry (2002), 41 (5), 1689-1694CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)We have previously established that ATP binds to mammalian metallothionein-2 (MT). The interaction between ATP and MT and the assocd. conformational change of the protein affect the sulfhydryl reactivity and zinc transfer potential of MT [Jiang, L.-J., Maret, W., and Vallee, B. L. (1998) The ATP-metallothionein complex. Proc. Natl. Acad. Sci. U.S.A. 95, 9146-9149]. NMR spectroscopic investigations have now provided further evidence for the interaction. 35Cl NMR spectroscopy has further identified chloride as an addnl. biol. MT ligand, which can interfere with the interaction of ATP with MT. 1H NMR/TOCSY spectra demonstrate that ATP binding affects the N- and C-terminal amino acids of the MT mol. Scanning tunneling microscopy recorded images of single MT mols. in buffered solns. Moreover, this technique demonstrates that the otherwise nearly linear MT mol. bends by about 20° at its central hinge region between the domains in the presence of ATP. These results may bear on the development of mild obesity in MT null mice and the role of MT in the regulation of energy balance. The interaction suggests a mechanism for the cellular translocation, retention, and reactivity of the ATP·MT complex in the mitochondrial intermembrane space. Both MT and ATP are localized there, and MT and thionein alternately bind and release zinc, thereby affecting mitochondrial respiration.
- 58Otvos, J. D.; Liu, X.; Li, H.; Shen, G.; Basti, M. Dynamic aspects of metallothionein structure. In Metallothionein III; Suzuki, K. T., Imura, N., Kimura, M., Eds.; Birkhäuser: Basel, Switzerland, 1993; pp 57– 74.There is no corresponding record for this reference.
- 59Maret, W.; Larsen, K. S.; Vallee, B. L. Coordination dynamics of biological zinc “clusters” in metallothioneins and in the DNA-binding domain of the transcription factor Gal4. Proc. Natl. Acad. Sci. U. S. A. 1997, 94, 2233– 2237, DOI: 10.1073/pnas.94.6.223359Coordination dynamics of biological zinc "clusters" in metallothioneins and in the DNA-binding domain of the transcription factor Gal4Maret, Wolfgang; Larsen, Kjeld S.; Vallee, Bert L.Proceedings of the National Academy of Sciences of the United States of America (1997), 94 (6), 2233-2237CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)The almost universal appreciation for the importance of zinc in metab. has been offset by the considerable uncertainty regarding the proteins that store and distribute cellular zinc. We propose that some zinc proteins with so-called zinc cluster motifs have a central role in zinc distribution, since they exhibit the rather exquisite properties of binding zinc tightly while remaining remarkably reactive as zinc donors. We have used zinc isotope exchange both to probe the coordination dynamics of zinc clusters in metallothionein, the small protein that has the highest known zinc content, and to investigate the potential function of zinc clusters in cellular zinc distribution. When mixed and incubated, metallothionein isoproteins-1 and -2 rapidly exchange zinc, as demonstrated by fast chromatog. sepn. and radiometric anal. Exchange kinetics exhibit two distinct phases (kfast ≃ 5000 min-1·M-1; kslow ≃ 200 min-1·M-1, pH 8.6, 25°C) that are thought to reflect exchange between the three-zinc clusters and between the four-zinc clusters, resp. Moreover, we have obsd. and examd. zinc exchange between metallothionein-2 and the Gal4 protein (k ≃ 800 min-1·M-1, pH 8.0, 25°C), which is a prototype of transcription factors with a two-zinc cluster. This reaction constitutes the first exptl. example of intermol. zinc exchange between heterologous proteins. Such kinetic reactivity distinguishes zinc in biol. clusters from zinc in the coordination environment of zinc enzymes, where the metal does not exchange over several days with free zinc in soln. The mol. organization of these clusters allows zinc exchange to proceed through a ligand exchange mechanism, involving mol. contact between the reactants.
- 60Hamer, D. H. Metallothionein. Annu. Rev. Biochem. 1986, 55, 913– 951, DOI: 10.1146/annurev.bi.55.070186.00440560MetallothioneinHamer, Dean H.Annual Review of Biochemistry (1986), 55 (), 913-51CODEN: ARBOAW; ISSN:0066-4154.A review, with 217 refs., on metallothionein. Nomenclature, occurrence, and detection of the proteins are described, and their structure and metal-binding properties are reviewed. In addn., consideration is given to the genetics of metallothioneins and to the regulation of expression of metallothionein genes. Possible functions of metallothioneins are also discussed.
- 61Westin, G.; Schaffner, W. A zinc-responsive factor interacts with a metal-regulated enhancer element (MRE) of the mouse metallothionein-I gene. EMBO J. 1988, 7, 3763– 3770, DOI: 10.1002/j.1460-2075.1988.tb03260.x61A zinc-responsive factor interacts with a metal-regulated enhancer element (MRE) of the mouse metallothionein-I geneWestin, Gunnar; Schaffner, WalterEMBO Journal (1988), 7 (12), 3763-70CODEN: EMJODG; ISSN:0261-4189.Heavy metal ions are effective inducers of metallothionein gene transcription. The metal response is dependent on short DNA motifs, so-called MREs (metal responsive elements) that occur in multiple copies in the promoter region of these genes. An MRE of the mouse metallothionein-I gene (MREd) was analyzed, and it was demonstrated that this can function over long distances as a bona fide metal ion-inducible enhancer. The transcription factor Sp1 and a zinc-inducible factor, designated MTF-1, bind to the MREd enhancer in vitro. The combined use of MREd mutants in a transient assay in HeLa cells and a competition band shift assay show that the zinc-inducible formation of the MTF-1/DNA complex in vitro correlates with zinc-inducible transcription in vivo. A chem. methylation interference assay revealed remarkably similar but non-identical guanine interference patterns for the MTF-1 and Sp1 complexes, which may mean that MTF-1 is related to the Sp1 factor.
- 62Miles, A. T.; Hawksworth, G. M.; Beattie, J.; Rodilla, V. Induction, regulation, degradation, and biological significance of mammalian metallothioneins. Crit. Rev. Biochem. Mol. Biol. 2000, 35, 35– 70, DOI: 10.1080/1040923009116916862Induction, regulation, degradation, and biological significance of mammalian metallothioneinsMiles, A. T.; Hawksworth, G. M.; Beattie, J. H.; Rodilla, V.Critical Reviews in Biochemistry and Molecular Biology (2000), 35 (1), 35-70CODEN: CRBBEJ; ISSN:1040-9238. (CRC Press LLC)A review with 328 refs. Metallothioneins (MTs) are small cysteine-rich metal-binding proteins found in many species and, although there are differences between them, it is of note that they have a great deal of sequence and structural homol. Mammalian MTs are 61 or 62 amino acid polypeptides contg. 20 conserved cysteine residues that underpin the binding of metals. The existence of MT across species is indicative of its biol. demand, while the conservation of cysteines indicates that these are undoubtedly central to the function of this protein. Four MT isoforms have been found so far, MT-1, MT-2, MT-3, and MT-4, but these also have subtypes with 17 MT genes identified in man, of which 10 are known to be functional. Different cells express different MT isoforms with varying levels of expression perhaps as a result of the different function of each isoform. Even different metals induce and bind to MTs to different extents. Over 40 yr of research into MT have yielded much information on this protein, but have failed to assign to it a definitive biol. role. The fact that multiple MT isoforms exist, and the great variety of substances and agents that act as inducers, further complicates the search for the biol. role of MTs. This article reviews the current knowledge on the biochem., induction, regulation, and degrdn. of this protein in mammals, with a particular emphasis on human MTs. It also considers the possible biol. roles of this protein, which include participation in cell proliferation and apoptosis, homeostasis of essential metals, cellular free radical scavenging, and metal detoxification.
- 63Beyersmann, D.; Haase, H. Functions of zinc in signaling, proliferation and differentiatioin of mammalian cells. BioMetals 2001, 14, 331– 341, DOI: 10.1023/A:101290540654863Functions of zinc in signaling, proliferation and differentiation of mammalian cellsBeyersmann, Detmar; Haase, HajoBioMetals (2001), 14 (3-4), 331-341CODEN: BOMEEH; ISSN:0966-0844. (Kluwer Academic Publishers)A review and discussion with 88 refs. Zn is essential for cell proliferation and differentiation, esp. for the regulation of DNA synthesis and mitosis. On the mol. level, it is a structural constituent of a great no. of proteins, including enzymes of cellular signaling pathways and transcription factors. Zn homeostasis in eukaryotic cells is controlled on the levels of uptake, intracellular sequestration in Zn-storing vesicles ("zincosomes"), nucleocytoplasmic distribution, and elimination. These processes involve the major Zn-binding protein, metallothionein, as a tool for the regulation of the cellular Zn level and the nuclear translocation of Zn in the course of the cell cycle and differentiation. In addn., there is also increasing evidence for a direct signaling function for Zn on all levels of signal transduction. Zn can modulate cellular signal recognition, 2nd messenger metab., protein kinase and protein phosphatase activities, and it may stimulate or inhibit activities of transcription factors, depending on the exptl. systems studied. Zn has been shown to modify specifically the metab. of cGMP, the activities of protein kinase C and MAP kinases, and the activity of transcription factor MTF-1 which controls the transcription of the genes for metallothionein and Zn transporter ZnT-1. As a conclusion of these observations, new hypotheses regarding regulatory functions of Zn2+ ions in cellular signaling pathways are proposed.
- 64Masters, B. A.; Kelly, E. J.; Quaife, C. J.; Brinster, R. L.; Palmiter, R. D. Targeted disruption of MTI and MTII genes increases sensitivity to cadmium. Proc. Natl. Acad. Sci. U. S. A. 1994, 91, 584– 588, DOI: 10.1073/pnas.91.2.58464Targeted disruption of metallothionein I and II genes increases sensitivity to cadmiumMasters, Brian A.; Kelly, Edward J.; Quaife, Carol J.; Brinster, Ralph L.; Palmiter, Richard D.Proceedings of the National Academy of Sciences of the United States of America (1994), 91 (2), 584-8CODEN: PNASA6; ISSN:0027-8424.The authors inactivated the mouse metallothionein (MT)-I and MT-II genes in embryonic stem cells and generated mice homozygous for these mutant alleles. These mice were viable and reproduced normally when reared under normal lab. conditions. They were, however, more susceptible to hepatic poisoning by cadmium. This proves that these widely expressed MTs are not essential for development but that they do protect against cadmium toxicity. These mice provide a means for testing other proposed functions of MT in vivo.
- 65Michalska, A. E.; Choo, A. K. H. Targeting and germ-line transmission of a null mutation at the metallothionein I and II loci in mouse. Proc. Natl. Acad. Sci. U. S. A. 1993, 90, 8088– 8092, DOI: 10.1073/pnas.90.17.808865Targeting and germ-line transmission of a null mutation at the metallothionein I and II loci in mouseMichalska, Anna E.; Choo, K. H. AndyProceedings of the National Academy of Sciences of the United States of America (1993), 90 (17), 8088-92CODEN: PNASA6; ISSN:0027-8424.The authors report the generation of transgenic mice deficient in the metallothionein MT-I and MT-II genes. The mutations were introduced into embryonic stem cells by homologous recombination. Chimeric mice resulting from the targeted embryonic stem cells transmitted the disrupted alleles through their germ line. Homozygous animals were born alive and appeared phenotypically normal and fertile. Absence of MT proteins was confirmed by direct measurement in liver exts. Challenging the mutant animals with moderate levels of CdSO4 indicated their greater susceptibility to cadmium toxicity than wild-type animals. These mice should provide a useful model to allow detailed study of the physiol. roles of MT-I and MT-II.
- 66Karin, M. Metallothioneins: Proteins in search of function. Cell 1985, 41, 9– 10, DOI: 10.1016/0092-8674(85)90051-066Metallothioneins: proteins in search of functionKarin, MichaelCell (Cambridge, MA, United States) (1985), 41 (1), 9-10CODEN: CELLB5; ISSN:0092-8674.A review, with 20 refs., of induction of metallothioneins (MT) by heavy metal ions and protection against toxicity, regulation of MT by hormones and MT expression in development, MT and metab. regulation, MT and differentiation and proliferation regulation, MT and free radicals, and MT and the UV response.
- 67Thornalley, P. J.; Vašák, M. Possible role for metallothionein in protection against radiation-induced oxidative stress. Kinetics and mechanism of its reaction with superoxide and hydroxyl radicals. Biochim. Biophys. Acta, Protein Struct. Mol. Enzymol. 1985, 827, 36– 44, DOI: 10.1016/0167-4838(85)90098-667Possible role for metallothionein in protection against radiation-induced oxidative stress. Kinetics and mechanism of its reaction with superoxide and hydroxyl radicalsThornalley, Paul J.; Vasak, MilanBiochimica et Biophysica Acta, Protein Structure and Molecular Enzymology (1985), 827 (1), 36-44CODEN: BBAEDZ; ISSN:0167-4838.Rabbit liver metallothionein-1 (MT) [mol. wt. (Mr) 6500], which contains Zn and(or) Cd ions, apparently scavenges free •OH and O-2 produced by the xanthine/xanthine oxidase reaction much more effectively than the control, bovine serum albumin (Mr 65,000). Kinetic competition studies between MT and either a spin trap for •OH or ferricytochrome c for O2- gave bimol. rate consts. on the order of k•OH/MT ≈1012 M-1/s and kO2-/MT ≈5 × 105 M-1/s, resp. The former value suggests that all 20 cysteine S atoms are involved in this quenching process and that they all act in the diffusion control limit. Aerobic radiolysis of an aq. soln. of MT, which generated O2- and •OH, induced metal ion loss and thiolate oxidn. These effects were reversed by incubation of the irradiated protein with GSH and the appropriate divalent metal ion. MT is apparently an extraordinarily efficient •OH scavenger, even compared with proteins 10-50-fold its mol. wt. •OH damage to MT evidently occurs at the metal ion-thiolate clusters, which may be repaired in the cell by GSH. MT has characteristics of a sacrificial but renewable cellular target for •OH-mediated cellular damage.
- 68Sato, M.; Bremner, I. Oxygen free radicals and metallothionein. Free Radical Biol. Med. 1993, 14, 325– 337, DOI: 10.1016/0891-5849(93)90029-T68Oxygen free radicals and metallothioneinSato, Masao; Bremner, IanFree Radical Biology & Medicine (1993), 14 (3), 325-37CODEN: FRBMEH; ISSN:0891-5849.A review, with 128 refs., on the evidence supporting a physiol. role as a free radical scavenger and describing induction of metallothionein synthesis by oxidative stress, possible mediators for this induction, and the radical scavenging capability of metallothionein in tissues and cells. The relationship between metallothionein and other antioxidant defense systems and the medical implications of the free radical scavenging properties of metallothionein are also discussed.
- 69Fliss, H.; Ménard, M. Oxidant-induced mobilization of zinc from metallothionein. Arch. Biochem. Biophys. 1992, 293, 195– 199, DOI: 10.1016/0003-9861(92)90384-969Oxidant-induced mobilization of zinc from metallothioneinFliss, Henry; Menard, MichelArchives of Biochemistry and Biophysics (1992), 293 (1), 195-9CODEN: ABBIA4; ISSN:0003-9861.Neutrophils which accumulate at sites of inflammation secrete a no. of injurious oxidants which are highly reactive with protein sulfhydryls. The present study examd. the possibility that this reactivity with thiols may cause protein damage by mobilizing zinc from cellular metalloproteins in which the metal is bound to cysteine. The ability of the 3 principal neutrophil oxidants, HOCl, O2-, and H2O2, to cleave thiolate bonds and mobilize complexed zinc was compared using 2 model compds. (2,3-dimercaptopropanol and metallothionein peptide fragment 56-61), as well as metallothionein. With all compds., 50 μM HOCl caused high rates of Zn2+ mobilization as measured spectrophotometrically with the metallochromic indicator 4-(2-pyridylazo)resorcinol. Xanthine (500 μM) plus xanthine oxidase (30 mU), which produced a similar concn. of O2-, also effected a rapid rate of Zn2+ mobilization which was inhibited by superoxide dismutase but not catalase, indicating that O2- is also highly reactive with thiolate bonds. In contrast, H2O2 alone was much less reactive at comparable concns. Thus, HOCl and O2- can cause damage to cellular metalloproteins through the mobilization of complexed zinc. In view of the essential role played by zinc in numerous cellular processes, Zn2+ mobilization by neutrophil oxidants may cause significant cellular injury at sites of inflammation.
- 70Otvos, J. D.; Petering, D. H.; Shaw, C. F. Structure-reactivity relationships of metallothionein, a unique metal-binding protein. Comments Inorg. Chem. 1989, 9, 1– 35, DOI: 10.1080/0260359890803580170Structure-reactivity relationships of metallothionein, a unique metal-binding proteinOtvos, James D.; Petering, David H.; Shaw, C. FrankComments on Inorganic Chemistry (1989), 9 (1), 1-35CODEN: COICDZ; ISSN:0260-3594.A review, with 89 refs., on the structure of metallothioneins and relating the structure to its metal-binding properties and kinetics. Particular emphasis is placed on its binding of Cd, Au, Pt, and Zn.
- 71Li, T. Y.; Minkel, D. T.; Shaw, C. F., 3rd; Petering, D. H. On the reactivity of metallothioneins with 5,5′-dithiobis(2-nitrobenzoic acid). Biochem. J. 1981, 193, 441– 446, DOI: 10.1042/bj193044171On the reactivity of metallothioneins with 5,5'-dithiobis-(2-nitrobenzoic acid)Li, Ta-Yuen; Minkel, Daniel T.; Shaw, C. Frank, III; Petering, David H.Biochemical Journal (1981), 193 (2), 441-6CODEN: BIJOAK; ISSN:0264-6021.Rat liver and horse kidney metallothioneins reacted with 5,5'-dithiobis(2-nitrobenzoic acid) (I), releasing 5-thio-2-nitrobenzoate and metal ions. The reactions were slow and showed biphasic kinetics, each process having an empirical rate law of similar form. The pseudo-1st-order kinetics were insensitive to pH, but were modified in guanidine-HCl soln. Rat liver metallothioneins of variable Zn, Cu, and Cd compn. reacted similarly, giving observable thiol/total metal ratios in good agreement with stoichiometries of SH/(Cd + Zn) and SH/Cu of 3 and 1, resp. A model complex, Cd-2,3-dimercaptopropanol, resembled the protein in its reaction with I.
- 72Jacob, C.; Maret, W.; Vallee, B. L. Control of zinc transfer between thionein, metallothionein and zinc proteins. Proc. Natl. Acad. Sci. U. S. A. 1998, 95, 3489– 3494, DOI: 10.1073/pnas.95.7.348972Control of zinc transfer between thionein, metallothionein, and zinc proteinsJacob, Claus; Maret, Wolfgang; Vallee, Bert L.Proceedings of the National Academy of Sciences of the United States of America (1998), 95 (7), 3489-3494CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Metallothionein (MT), despite its high metal binding const. (KZn = 3.2 × 1013 M-1 at pH 7.4), can transfer zinc to the apoforms of zinc enzymes that have inherently lower stability consts. To gain insight into this paradox, we have studied zinc transfer between zinc enzymes and MT. Zinc can be transferred in both directions-i.e., from the enzymes to thionein (the apoform of MT) and from MT to the apoenzymes. Agents that mediate or enhance zinc transfer have been identified that provide kinetic pathways in either direction. MT does not transfer all of its seven zinc atoms to an apoenzyme, but apparently contains at least one that is more prone to transfer than the others. Modification of thiol ligands in MT zinc clusters increases the total no. of zinc ions released and, hence, the extent of transfer. Aside from disulfide reagents, we show that selenium compds. are potential cellular enhancers of zinc transfer from MT to apoenzymes. Zinc transfer from zinc enzymes to thionein, on the other hand, is mediated by zinc-chelating agents such as Tris buffer, citrate, or glutathione. Redox agents are asym. involved in both directions of zinc transfer. For example, reduced glutathione mediates zinc transfer from enzymes to thionein, whereas glutathione disulfide oxidizes MT with enhanced release of zinc and transfer of zinc to apoenzymes. Therefore, the cellular redox state as well as the concn. of other biol. chelating agents might well det. the direction of zinc transfer and ultimately affect zinc distribution.
- 73Krężel, A.; Maret, W. Dual nanomolar and picomolar Zn(II) binding properties of metallothionein. J. Am. Chem. Soc. 2007, 129, 10911– 10921, DOI: 10.1021/ja071979s73Dual Nanomolar and Picomolar Zn(II) Binding Properties of MetallothioneinKrezel, Artur; Maret, WolfgangJournal of the American Chemical Society (2007), 129 (35), 10911-10921CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Each of the seven Zn(II) ions in the Zn3S9 and Zn4S11 clusters of human metallothionein is in a tetrathiolate coordination environment. Yet anal. of Zn(II) assocn. with thionein, the apoprotein, and anal. of Zn(II) dissocn. from metallothionein using the fluorescent chelating agents FluoZin-3 and RhodZin-3 reveal at least three classes of sites with affinities that differ by 4 orders of magnitude. Four Zn(II) ions are bound with an apparent av. log K of 11.8, and with the methods employed, their binding is indistinguishable. This binding property makes thionein a strong chelating agent. One Zn(II) ion is relatively weakly bound, with a log K of 7.7, making metallothionein a zinc donor in the absence of thionein. The binding data demonstrate that Zn(II) binds with at least four species: Zn4T, Zn5T, Zn6T, and Zn7T. Zn5T and Zn6T bind Zn(II) with a log K of ∼10 and are the predominant species at micromolar concns. of metallothionein in cells. Central to the function of the protein is the reactivity of its cysteine side chains in the absence and presence of Zn(II). Chelating agents, such as physiol. ligands with moderate affinities for Zn(II), cause dissocn. of Zn(II) ions from metallothionein at pH 7.4 (Zn7T (symbol) Zn7-nT + nZn2+), thereby affecting the reactivity of its thiols. Thus, the rate of thiol oxidn. increases in the presence of Zn(II) acceptors but decreases if more free Zn(II) becomes available. Thionein is such an acceptor. It regulates the reactivity and availability of free Zn(II) from metallothionein. At thionein/metallothionein ratios > 0.75, free Zn(II) ions are below a pZn (-log[Zn2+]free) of 11.8, and at ratios < 0.75, relatively large fluctuations of free Zn(II) ions are possible (pZn between 7 and 11). These chem. characteristics match cellular requirements for Zn(II) and suggest how the mol. structures and redox chemistries of metallothionein and thionein det. Zn(II) availability for biol. processes.
- 74Savas, M. M.; Petering, D. H.; Shaw, C. F., III On the rapid, monophasic reaction of rabbit liver metallothionein α-domain with 5,5′-dithiobis(2-nitrobenzoic acid)(DTNB). Inorg. Chem. 1991, 30, 581– 583, DOI: 10.1021/ic00003a04974On the rapid, monophasic reaction of the rabbit liver metallothionein α-domain with 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB)Savas, M. Meral; Petering, David H.; Shaw, C. Frank, IIIInorganic Chemistry (1991), 30 (3), 581-3CODEN: INOCAJ; ISSN:0020-1669.The first kinetic study of an isolated metallothionein domain, in order to obtain insight into the origin of the biphasic reactions of the holoprotein, is reported. The reactions of the α-Cd4 cluster from rabbit liver metallothionein-II with DTNB were monophasic and 1st-order in the presence of excess DTNB at pH 7.4 and 25°. This result also holds at pH 6.8 (25°) and at temps. 5 and 50° (pH 7.4). The obsd. rate consts. are linearly dependent upon DTNB concn. over the range studied, yielding a complex rate law, rate=k1+k2[DTNB]. For the DTNB-independent process, the 1st-order rate const. (intercept) is k1 = 6.4 × 10-4 s-1, and for the DTNB-dependent processes, the 2nd-order rate const. (slope) is k2 = 1.12 s-1 M-1. The significance of this result for the biphasic reaction of the holoprotein with DTNB is discussed briefly.
- 75Jiang, L.-J.; Vašák, M.; Vallee, B. L.; Maret, W. Zinc transfer potentials of the α- and β-clusters of metallothionein are affected by domain interactions in the whole molecule. Proc. Natl. Acad. Sci. U. S. A. 2000, 97, 2503– 2508, DOI: 10.1073/pnas.97.6.250375Zinc transfer potentials of the α- and β-clusters of metallothionein are affected by domain interactions in the whole moleculeJiang, Li-Juan; Vasak, Milan; Vallee, Bert L.; Maret, WolfgangProceedings of the National Academy of Sciences of the United States of America (2000), 97 (6), 2503-2508CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)The α- and β-polypeptides of human metallothionein (isoform 2), obtained by chem. synthesis, were converted into their resp. zinc/thiolate clusters, and each domain was investigated sep. Proton titrn. data for the N-terminal β-domain fit a simple model with three ionizations of the same apparent pKa value of 4.9 and a collective binding const. for zinc of 5×10-12 M at pH 7.0. The zinc cluster in the C-terminal α-domain is more stable than that in the β-domain. Its pH titrn. is also more complex, indicating at least two classes of zinc sites with different affinities. The whole mol. is stabilized with regard to the individual domains. Chem. modification implicates lysine side chains in both the stabilization of the β-domain cluster and the mutual stabilization of the domains in the whole mol. The two zinc clusters also differ in the reactivity of their cysteine sulfurs and their potential to donate zinc to an acceptor mol. dependent on its type and characteristics. The isolated β-domain cluster reacts faster with Ellman's reagent and is a better zinc donor toward zinc-depleted sorbitol dehydrogenase than is the isolated α-domain cluster, whereas the reverse is obsd. when a chelating agent is the zinc acceptor. Thus, although each cluster assembles independently of the other, the cumulative properties of the individual domains do not suffice to describe metallothionein either structurally or functionally. The two-domain structure of the whole mol. is important for its interaction with ligands and for control of its reactivity and overall conformation.
- 76Savas, M. M.; Shaw, C. F., III; Petering, D. H. The oxidation of rabbit liver metallothionein-II by 5,5′-dithiobis(2-nitrobenzoic acid) and glutathione disulfide. J. Inorg. Biochem. 1993, 52, 235– 249, DOI: 10.1016/0162-0134(93)80028-876The oxidation of rabbit liver metallothionein-II by 5,5'-dithiobis(2-nitrobenzoic acid) and glutathione disulfideSavas, M. Meral; Shaw, C. Frank, III; Petering, David H.Journal of Inorganic Biochemistry (1993), 52 (4), 235-49CODEN: JIBIDJ; ISSN:0162-0134.Because metallothionein (MT) may undergo thiol-disulfide or other redox reactions under certain cellular conditions, the partially and completely oxidized products of the reactions of Cd7MT-II with the electrophile 5,5'-dithiobis(2-nitrobenzoic acid), ESSE, and oxidized glutathione, GSSG, were characterized. Reaction with the stoichiometric quantity of ESSE (1 ESSE per MT thiolate) generates monomeric and polymeric MTs with three types of disulfide bonds: intra- and intermol. CyS-SCy linkages and a small no. (2-3/MT) of mixed disulfides, CyS-SE, involving thionitrobenzoate (ES-). Reaction with substoichiometric quantities of ESSE (0.02 or 0.1 per MT thiolate) causes the formation of intra- and intermol. CyS-SCy disulfides, but no mixed disulfides. In the latter reactions, two equiv. of ES- are released per mol of ESSE, but the release is described by a single first-order rate const. (k = 3.0 ± 0.5 s-1). Substantial amts. of cadmium remained bound to the MT monomers and polymers after reaction with the substoichiometric quantities. Despite the Cd bound to the MT after reaction with 0.1 ESSE per MT thiolate, no 111Cd NMR signals were detected, indicating rapid equilibration of the remaining metal ions among the disrupted binding sites. Large excesses of the endogenous aliph. disulfide, GSSG, displace Zn+2 from Zn7-MT slowly. The reaction is complete after 24 h with 5000 μM GSSG, but only 25% complete after 72 h with 250 μM GSSG. Approx. one Cd+2 is displaced rapidly from Cd7MT by 5000 μM GSSG and half as much by 250 μM GSSG, but no further reaction occurs. It is unlikely that GSSG oxidn. of MTs would be physiol. significant.
- 77Maret, W. Oxidative metal release from metallothionein via zinc-thiol/disulfide interchange. Proc. Natl. Acad. Sci. U. S. A. 1994, 91, 237– 241, DOI: 10.1073/pnas.91.1.23777Oxidative metal release from metallothionein via zinc-thiol/disulfide interchangeMaret, WolfgangProceedings of the National Academy of Sciences of the United States of America (1994), 91 (1), 237-41CODEN: PNASA6; ISSN:0027-8424.Mammalian metallothionein has been postulated to play a pivotal role in cellular zinc distribution. All seven of its metal atoms are bound with high thermodn. stability in two clusters buried deeply in the mol. If the protein is to function in metal delivery, there must be a biol. mechanism to facilitate metal release. One means to achieve this would be a labilization of the cluster by interaction of metallothionein with an appropriate cellular ligand. To search for such a mediator, the authors have designed a rapid radiochromatog. method that can detect changes in the zinc content of 65Zn-labeled metallothionein in response to other biomols. Using this methodol., the authors have established that rabbit liver metallothionein 2 interacts with glutathione disulfide with concomitant release of zinc. Under conditions of pseudo-first-order kinetics, the monophasic reaction depends linearly on the concn. of glutathione disulfide in the range from 5 to 30 mM with a second-order rate const. k = 4.9 × 10-3 s-1·M-1 (pH 8.6; 25°C). Apparently, zinc release does not involve direct access of glutathione disulfide to the inner coordination sphere of the metals. Rather it appears that the solvent-accessible zinc-bound thiolates in two clefts of each domain of metallothionein [Robbins, A. H., McRee, D. E., Williamson, M., Collett, S. A., Xuong, N. H., Furey, W. F. Wang, B. C. & Stout, C. D. (1991) J. Mol. Biol. 221, 1269-1293] participate in a thiol/disulfide interchange with glutathione disulfide. This rate-limiting initial S-thiolation, which occurs with indistinguishable rates in both clusters, then causes the clusters to collapse and release their zinc. Such a mechanism of metal release would link the control of the metal content of metallothionein to the cellular glutathione redox status and raises important questions about the physiol. implications of this observation with regard to a role of glutathione in zinc metab. and in making zinc available for other biomols.
- 78Maret, W.; Krężel, A. Cellular zinc and redox buffering capacity of metallothionein/thionein in health and disease. Mol. Med. 2007, 13, 371– 375, DOI: 10.2119/2007-00036.Maret78Cellular zinc and redox buffering capacity of metallothionein/thionein in health and diseaseMaret, Wolfgang; Krezel, ArturMolecular Medicine (Manhasset, NY, United States) (2007), 13 (7-8), 371-375CODEN: MOMEF3; ISSN:1076-1551. (Feinstein Institute for Medical Research)A review. Zinc is involved in virtually all aspects of cellular and mol. biol. as a catalytic, structural, and regulatory cofactor in over 1000 proteins. Zinc binding to proteins requires an adequate supply of zinc and intact mol. mechanisms for redistributing zinc ions to make them available at the right time and location. Several dozen gene products participate in this process, in which interactions between zinc and sulfur donors det. the mobility of zinc and establish coupling between cellular redox state and zinc availability. Specifically, the redox properties of metallothionein and its apoprotein thionein are crit. for buffering zinc ions and for controlling fluctuations in the range of picomolar concns. of "free" zinc ions in cellular signaling. Metallothionein and other proteins with sulfur coordination environments are sensitive to redox perturbations and can render cells susceptible to injury when oxidative stress compromises the cellular redox and zinc buffering capacity in chronic diseases. The implications of these fundamental principles for zinc metab. in type 2 diabetes are briefly discussed.
- 79Maret, W.; Vallee, B. L. Thiolate ligands in metallothionein confer redox activity on zinc clusters. Proc. Natl. Acad. Sci. U. S. A. 1998, 95, 3478– 3482, DOI: 10.1073/pnas.95.7.347879Thiolate ligands in metallothionein confer redox activity on zinc clustersMaret, Wolfgang; Vallee, Bert L.Proceedings of the National Academy of Sciences of the United States of America (1998), 95 (7), 3478-3482CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)We postulate a novel and general mechanism in which the redox-active sulfur donor group of cyst(e)ine confers oxidoreductive characteristics on stable zinc sites in proteins. Thus, the present, an earlier, and accompanying manuscripts [Maret, W., Larsen, K. S. & Vallee, B. L. (1997) Proc. Natl. Acad. Sci. USA 94, 2233-2237; Jiang, L.-J., Maret, W. & Vallee, B. L. (1998) Proc. Natl. Acad. Sci. USA 95, 3483-3488; and Jacob, C., Maret, W. & Vallee, B. L. (1998) Proc. Natl. Acad. Sci. USA 95, 3489-3494] demonstrate that the interactive network featuring multiple zinc/sulfur bonds as found in the clusters of metallothionein (MT) constitutes a coordination unit crit. for the concurrent oxidn. of cysteine ligands and the ensuing release of zinc. The low position of MT (<-366 mV) on a scale of redox reagents allows its effective oxidn. by relatively mild cellular oxidants, in particular disulfides. When MT is exposed to an excess of dithiodipyridine, all of its 20 cysteines are oxidized within 1 h with the concomitant release of all 7 zinc atoms; similarly, the thiol/disulfide oxidoreductase DsbA reacts stoichiometrically with MT to release zinc. Zinc and sulfur ligands in the clusters are in a spatial arrangement that seemingly favors disulfide bond formation. Jointly, this and the above-mentioned manuscripts conclude that the control of cellular zinc distribution as a function of the energy state of the cell is the long sought role of MT. This specific MT function renders dubious the widely held belief that MT primarily scavenges radicals or detoxifies metals and is consistent with the frequent use of cysteine as a zinc ligand in proteins as a means of both tight and weak zinc binding of thiols and disulfides, resp. Thus, we relate changes in the reducing power of the cell to the stability of the zinc/sulfur network in MT and the relative mobility of zinc and its control.
- 80Maret, W. The function of zinc metallothionein: A link between cellular zinc and redox state. J. Nutr. 2000, 130, 1455S– 1458S, DOI: 10.1093/jn/130.5.1455S80The function of zinc metallothionein: a link between cellular zinc and redox stateMaret, WolfgangJournal of Nutrition (2000), 130 (5S), 1455S-1458SCODEN: JONUAI; ISSN:0022-3166. (American Society for Nutritional Sciences)A review with 42 refs. A chem. and biochem. mechanism of action of the metallothionein (MT)/thionein (T) couple has been proposed. The mechanism emphasizes the importance of zinc/sulfur cluster bonding in MT and the significance of the two cluster networks as redox units that confer mobility on otherwise tightly bound and redox-inert zinc in MT. In this article, it is further explored how this redox mechanism controls the metabolically active cellular zinc pool. The low redox potential of the sulfur donor atoms in the clusters readily allows oxidn. by mild cellular oxidants with concomitant release of zinc. Such a release by oxidants and the preservation of zinc binding by antioxidants place MT under the control of the cellular redox state and, consequently, energy metab. The binding of effectors, e.g., ATP, elicits conformational changes and alters zinc binding in MT. The glutathione/glutathione disulfide redox couple as well as selenium compds. effect zinc delivery from MT to the apoforms of zinc enzymes. This novel action of selenium on zinc/sulfur coordination sites has significant implications for the interaction between these essential elements. Tight binding and kinetic lability, modulation of MT by cellular ligands and the redox state, control of MT gene expression by zinc and many other inducers all support a crit. function of the MT/T system in cellular homeostasis and distribution of zinc.
- 81Maret, W. Cellular zinc and redox stress converge in the metallothionein/thionein pair. J. Nutr. 2003, 133, 1460S– 1462S, DOI: 10.1093/jn/133.5.1460S81Cellular zinc and redox states converge in the metallothionein/thionein pairMaret, WolfgangJournal of Nutrition (2003), 133 (5S-1), 1460S-1462SCODEN: JONUAI; ISSN:0022-3166. (American Society for Nutritional Sciences)A review. The paramount importance of zinc for a wide range of biol. functions is based on its occurrence in thousands of known zinc proteins. To regulate the availability of zinc dynamically, eukaryotes have compartmentalized zinc and the metallothionein/thionein pair, which controls the pico- to nanomolar concns. of metabolically active cellular zinc. Interactions of zinc with sulfur ligands of cysteines turn out to be crit. both for tight binding and creation of a redox-active coordination environment from which the redox-inert zinc can be distributed. Biol. oxidants such as disulfides and S-nitrosothiols oxidize the zinc/thiolate clusters in metallothionein with concomitant zinc release. In addn., selenium compds. that have the capacity to form selenol(ate)s catalytically couple with the glutathione/glutathione disulfide and metallothionein/thionein redox pairs to either release or bind zinc. In this pathway, selenium expresses its antioxidant effects through redox catalysis in zinc metab. Selenium affects the redox state of thionein, an endogenous chelating agent. With its 20 cysteines, thionein contributes significantly to the zinc- and thiol-redox-buffering capacity of the cell. Thus, hitherto unknown interactions between the essential micronutrients zinc and selenium on the one hand and zinc and redox metab. on the other are key features of the cellular homeostatic zinc system.
- 82IUPAC. Compendium of Chemical Terminology, 2nd ed.; McNaught, A. D., Wilkinson, A., Eds.; Blackwell Scientific Publications: Oxford, England,1997. DOI: 10.1351/goldbook .There is no corresponding record for this reference.
- 83Maret, W. Zinc and sulfur: A critical biological partnership. Biochemistry 2004, 43, 3301– 3309, DOI: 10.1021/bi036340p83Zinc and Sulfur: A Critical Biological PartnershipMaret, WolfgangBiochemistry (2004), 43 (12), 3301-3309CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)A review. The Zn-S interaction offers specific mechanisms for enzyme catalysis, establishes reactivities of zinc sites that hitherto were believed to have only a structural role, allows zinc to be tightly bound and yet to be available, and, importantly, generates redox-active coordination environments for the redox-inert zinc ion. These activities are critically involved in the regulation of protein structure and function, and in mobility, transfer, redistribution, and sensing of cellular zinc, as is becoming evident from the continuing exploration of the protein metallothionein and other proteins involved in cellular zinc homeostasis.
- 84Lee, S.-H.; Maret, W. Redox control of zinc finger proteins: Mechanisms and role in gene regulation. Antioxid. Redox Signaling 2001, 3, 531– 534, DOI: 10.1089/1523086015254290784Redox control of zinc finger proteins: mechanism and role in gene regulationLee, Suk-Hee; Maret, WolfgangAntioxidants & Redox Signaling (2001), 3 (4), 531-534CODEN: ARSIF2; ISSN:1523-0864. (Mary Ann Liebert, Inc.)A review, with 10 refs., on the redox control of zinc finger proteins (ZFPs) and its effect on gene expression and DNA metab.
- 85Maret, W. Metallothionein/disulfide interactions, oxidative stress, and the mobilization of cellular zinc. Neurochem. Int. 1995, 27, 111– 117, DOI: 10.1016/0197-0186(94)00173-R85Metallothionein/disulfide interactions, oxidative stress, and the mobilization of cellular zincMaret, WolfgangNeurochemistry International (1995), 27 (1), 111-17CODEN: NEUIDS; ISSN:0197-0186. (Elsevier)Glutathione disulfide, the major cell. disulfide, releases zinc from metallothionein (MT). Here, the interaction of rabbit liver MT-II with other selected biol. disulfides (CoA/glutathione mixed disulfide, CoA disulfide, and cystamine) was investigated by measuring concomitant release of radioactive 65-zinc from MT. These disulfides react more rapidly than glutathione disulfide, thus underscoring the reactivity of zinc sulfur bonds in the clusters of MT and the importance of the MT/disulfide interactions as a chem. mechanism for mobilizing zinc from a thermodynamically stable zinc complex. Two implications of these in vitro findings are discussed. (I) Apparently, in the case of zinc which is redox inert, Nature has availed itself of the redox activity of the cysteine ligand to mobilize the metal, and, presumably to permit redox-control of cellular zinc distribution. The mobilization of zinc from MT suggests a possible function of MT as a physiol. zinc donor. (Ii) A shift of the glutathione redox balance under conditions of oxidative stress will accelerate metal release from MT. Such a disturbance of metal metab. has important consequences for the progression of diseases such as Alzheimer's and Parkinson's disease where oxidative stress occurs in affected brain tissue.
- 86Maret, W. Zinc coordination environments in proteins as redox sensors and signal transducers. Antioxid. Redox Signaling 2006, 8, 1419– 1441, DOI: 10.1089/ars.2006.8.141986Zinc coordination environments in proteins as redox sensors and signal transducersMaret, WolfgangAntioxidants & Redox Signaling (2006), 8 (9 & 10), 1419-1441CODEN: ARSIF2; ISSN:1523-0864. (Mary Ann Liebert, Inc.)A review. Zn/Cys coordination environments in proteins are redox-active. Oxidn. of the S ligands mobilizes Zn, while redn. of the oxidized ligands enhances Zn binding, providing redox control over the availability of Zn2+ ions. Some Zn-proteins are redox sensors, in which Zn release is coupled to conformational changes that control varied functions such as enzymic activity, binding interactions, and mol. chaperone activity. Whereas the released Zn2+ ion in redox sensors has no known function, the redox signal is transduced to specific and sensitive Zn signals in redox transducers. Released Zn can bind to sites on other proteins and modulate signal transduction, generation of metabolic energy, mitochondrial function, and gene expression. The paradigm of such redox transducers is the Zn-protein, metallothionein, which, together with its apoprotein, thionein, functions at a central node in cellular signaling by redistributing cellular Zn, presiding over the availability of Zn, and interconverting redox and Zn signals. In this regard, the transduction of NO signals into Zn signals by metallothionein has received particular attention. It appears that redox-inert Zn has been chosen to control some aspects of cellular thiol/disulfide redox metab. Tight control of Zn is essential for redox homeostasis because both increases and decreases of cellular Zn elicit oxidative stress. Depending on its availability, Zn can be cytoprotective as a pro-antioxidant or cytotoxic as a pro-oxidant. Any condition with acute or chronic oxidative stress is expected to perturb Zn homeostasis.
- 87Pattanaik, A.; Shaw, C. F., III; Petering, D. H.; Garvey, J.; Kraker, A. J. Basal metallothionein in tumours: Widespread presence of apoprotein. J. Inorg. Biochem. 1994, 54, 91– 105, DOI: 10.1016/0162-0134(94)80023-587Basal metallothionein in tumors: widespread presence of apoproteinPattanaik, Asima; Shaw, C. Frank; Petering, David H.; Garvey, Justine; Kraker, Alan J.Journal of Inorganic Biochemistry (1994), 54 (2), 91-105CODEN: JIBIDJ; ISSN:0162-0134. (Elsevier)A survey has been conducted of solid and ascites tumors from mice and solid tumors in rats for the presence of metallothionein or metallothionein-like protein. In most tumors, a pos. identification was made on the basis of Sephadex G-75 and HPLC-DEAE chromatog. followed by competitive RIA for metallothionein. Apometallothionein was revealed in a no. of tumor for the first time by comparing in Sephadex G-75 chromatog. profiles of Zn in native cytosol and Cd in cytosol incubated briefly with CdCl2 to sat. free binding sites on the protein before Sephadex G-75 chromatog. In two cases unsatn. of metallothionein was correlated with a lack of zinc in the ascites fluid which supplies the tumor with zinc.
- 88Yang, Y.; Maret, W.; Vallee, B. L. Differential fluorescence labeling of cysteinyl clusters uncovers high tissue levels of thionein. Proc. Natl. Acad. Sci. U. S. A. 2001, 98, 5556– 5559, DOI: 10.1073/pnas.10112329888Differential fluorescence labeling of cysteinyl clusters uncovers high tissue levels of thioneinYang, Yi; Maret, Wolfgang; Vallee, Bert L.Proceedings of the National Academy of Sciences of the United States of America (2001), 98 (10), 5556-5559CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)The isolation of thionein (T) from tissues has not been reported heretofore. T contains 20 cysteinyl residues that react with 7-fluorobenz-2-oxa-1,3-diazole-4-sulfonamide to form fluorescent adducts. In metallothionein (MT), the cysteinyl residues, which are bound to zinc, do not react. However, they do react in the presence of a chelating agent such as EDTA. The resultant difference in chem. reactivity provides a means to measure T in the absence of EDTA, (MT + T) in its presence, and, of course, MT by difference. The 7-fluorobenz-2-oxa-1,3-diazole-4-sulfonamide deriv. of T can be isolated from tissue homogenates by HPLC and quantified fluorimetrically with a detection limit in the femtomolar range and a linear response over 3 orders of magnitude. Anal. of liver, kidney, and brain of rats reveals almost as much T as MT. Moreover, in contrast to earlier views, MT in tissue exts. appears to be less stable than T. The existence of T in tissues under normal physiol. conditions has important implications for its function both in zinc metab. and the redox balance of the cell.
- 89Krężel, A.; Maret, W. Zinc-buffering capacity of a eukaryotic cell at physiological pZn. JBIC, J. Biol. Inorg. Chem. 2006, 11, 1049– 1062, DOI: 10.1007/s00775-006-0150-589Zinc-buffering capacity of a eukaryotic cell at physiological pZnKrezel, Artur; Maret, WolfgangJBIC, Journal of Biological Inorganic Chemistry (2006), 11 (8), 1049-1062CODEN: JJBCFA; ISSN:0949-8257. (Springer GmbH)In spite of the paramount importance of zinc in biol., dynamic aspects of cellular zinc metab. remain poorly defined at the mol. level. Investigations with human colon cancer (HT-29) cells establish a total cellular zinc concn. of 264 μM. Remarkably, about 10% of the potential high-affinity zinc-binding sites are not occupied by zinc, resulting in a surplus of 28 μM ligand (av. Kcd = 83 pM) that ascertain cellular zinc-buffering capacity and maintain the "free" zinc concn. in proliferating cells at picomolar levels (784 pM, pZn = 9.1). This zinc-buffering capacity allows zinc to fluctuate only with relatively small amplitudes (ΔpZn = 0.3; below 1 nM) without significantly perturbing physiol. pZn. Thus, the "free" zinc concns. in resting and differentiated HT-29 cells are 614 pM and 1.25 nM, resp. The calcn. of these "free" zinc concns. is based on measurements at different concns. of the fluorogenic zinc-chelating agent and extrapolation to a zero concn. of the agent. It depends on the state of the cell, its buffering capacity, and the zinc dissocn. const. of the chelating agent. Zinc induction of thionein (apometallothionein) ensures a surplus of unbound ligands, increases zinc-buffering capacity and the availability of zinc (ΔpZn = 0.8), but preserves the zinc-buffering capacity of the unoccupied high-affinity zinc-binding sites, perhaps for crucial physiol. functions. Jointly, metallothionein and thionein function as the major zinc buffer under conditions of increased cellular zinc.
- 90Krężel, A.; Maret, W. Different redox states of metallothionein/thionein in biological tissue. Biochem. J. 2007, 402, 551– 558, DOI: 10.1042/BJ2006104490Different redox states of metallothionein/thionein in biological tissueKrezel, Artur; Maret, WolfgangBiochemical Journal (2007), 402 (3), 551-558CODEN: BIJOAK; ISSN:0264-6021. (Portland Press Ltd.)Mammalian metallothioneins are redox-active metalloproteins. In the case of zinc metallothioneins, the redox activity resides in the cysteine sulfur ligands of zinc. Oxidn. releases zinc, whereas redn. re-generates zinc-binding capacity. Attempts to demonstrate the presence of the apoprotein (thionein) and the oxidized protein (thionin) in tissues posed tremendous anal. challenges. One emerging strategy is differential chem. modification of cysteine residues in the protein. Chem. modification distinguishes three states of the cysteine ligands (reduced, oxidized and metal-bound) based on (i) quenched reactivity of the thiolates when bound to metal ions and restoration of thiol reactivity in the presence of metal-ion-chelating agents, and (ii) modification of free thiols with alkylating agents and subsequent redn. of disulfides to yield reactive thiols. Under normal physiol. conditions, metallothionein exists in three states in rat liver and in cell lines. Ras-mediated oncogenic transformation of normal HOSE (human ovarian surface epithelial) cells induces oxidative stress and increases the amt. of thionin and the availability of cellular zinc. These expts. support the notion that metallothionein is a dynamic protein in terms of its redox state and metal content and functions at a juncture of redox and zinc metab. Thus redox control of zinc availability from this protein establishes multiple methods of zinc-dependent cellular regulation, while the presence of both oxidized and reduced states of the apoprotein suggest that they serve as a redox couple, the generation of which is controlled by metal ion release from metallothionein.
- 91Haase, H.; Maret, W. A differential assay for metallothionein and the reduced and oxidized states of thionein. Anal. Biochem. 2004, 333, 19– 26, DOI: 10.1016/j.ab.2004.04.03991A differential assay for the reduced and oxidized states of metallothionein and thioneinHaase, Hajo; Maret, WolfgangAnalytical Biochemistry (2004), 333 (1), 19-26CODEN: ANBCA2; ISSN:0003-2697. (Elsevier)In the cellular environment, the sulfur ligands in zinc/thiolate coordination sites of proteins can be oxidized with concomitant mobilization of zinc. The characterization of such "redox zinc switches" requires the detn. of three species, i.e., the zinc-contg. complex and the zinc-free complex with the thiolate ligands either reduced or oxidized. Differential chem. modification of thiol groups in the presence and absence of either reducing or chelating agents allows the anal. speciation of such systems as demonstrated here for the characterization of the redox and metal-binding states of mammalian metallothionein. Thiol derivatization with 6-iodoacetamidofluorescein in the presence and absence of the reducing agent tris(2-carboxyethyl)phosphine, high-performance liq. chromatog. sepn., and photometric detection are employed to det. the reduced and oxidized protein. Because the holoprotein reacts only in the presence of a chelating agent such as ethylenediaminetetraacetate (EDTA) its amt. can be detd. as the difference between measurements in the presence and the absence of EDTA. This method is applied to the study of the chem. and enzymic oxidn. of metallothionein/thionein. It should also greatly facilitate the characterization of the redox and metal-binding properties of zinc/thiolate coordination environments of other proteins such as zinc finger proteins.
- 92Feng, W.; Benz, F. W.; Cai, J.; Pierce, W. M.; Kang, Y. J. Metallothionein disulfides are present in metallothionein-overexpressing transgenic mouse heart and increase under conditions of oxidative stress. J. Biol. Chem. 2006, 281, 681– 687, DOI: 10.1074/jbc.M50695620092Metallothionein Disulfides Are Present in Metallothionein-overexpressing Transgenic Mouse Heart and Increase under Conditions of Oxidative StressFeng, Wenke; Benz, Frederick W.; Cai, Jian; Pierce, William M.; Kang, Y. JamesJournal of Biological Chemistry (2006), 281 (2), 681-687CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)Metallothionein (MT) releases zinc under oxidative stress conditions in cultured cells. The change in the MT mol. after zinc release in vivo is unknown although in vitro studies have identified MT disulfide bond formation. The present study was undertaken to test the hypothesis that MT disulfide bond formation occurs in vivo. A cardiac-specific MT-overexpressing transgenic mouse model was used. Mice were administered saline as a control or doxorubicin (20 mg/kg), which is an effective anticancer drug but with severe cardiac toxicity at least partially because of the generation of reactive oxygen species. A differential alkylation of cysteine residues in MT of the heart exts. was performed. Free and metal-bound cysteines were first trapped by N-ethylmaleimide and the disulfide bonds were reduced by dithiothreitol followed by alkylation with radiolabeled iodoacetamide. Analyses of the differentially alkylated MTs in the heart ext. by high performance liq. chromatog., SDS-PAGE, Western blot, and mass spectrometry revealed that disulfide bonds were present in MT in vivo under both physiol. and oxidative stress conditions. More disulfide bonds were found in MT under the oxidative stress conditions. The MT disulfide bonds were likely intramol. and both α- and β-domains were involved in the disulfide bond formation, although the α-domain appeared to be more easily oxidized than the β-domain. The results suggest that under physiol. conditions, the formation of MT disulfide bonds is involved in the regulation of zinc homeostasis. Addnl. zinc release from MT under oxidative stress conditions is accompanied by more MT disulfide bond formation.
- 93Hathout, Y.; Fabris, D.; Fenselau, C. Stoichiometry in zinc ion transfer from metallothionein to zinc finger peptides. Int. J. Mass Spectrom. 2001, 204, 1– 6, DOI: 10.1016/S1387-3806(00)00343-293Stoichiometry in zinc ion transfer from metallothionein to zinc finger peptidesHathout, Y.; Fabris, D.; Fenselau, C.International Journal of Mass Spectrometry (2001), 204 (1-3), 1-6CODEN: IMSPF8; ISSN:1387-3806. (Elsevier Science B.V.)Electrospray and nanospray ionizations are used to study the transfer of zinc ions between Zn7-metallothionein and apo peptides that are models for several kinds of zinc finger proteins. A membrane expt. is reported here which demonstrates that interprotein contact is required for these transfers. Anal. on a quadrupole ion trap has allowed all reactants and all products to be monitored simultaneously. Evidence is provided for the preferential transfer of a single zinc ion from each Zn7-metallothionein complex.
- 94Haase, H.; Maret, W. Partial oxidation and oxidative polymerization of metallothionein. Electrophoresis 2008, 29, 4169– 4176, DOI: 10.1002/elps.20070092294Partial oxidation and oxidative polymerization of metallothioneinHaase, Hajo; Maret, WolfgangElectrophoresis (2008), 29 (20), 4169-4176CODEN: ELCTDN; ISSN:0173-0835. (Wiley-VCH Verlag GmbH & Co. KGaA)One mechanism for regulation of metal binding to metallothionein (MT) involves the non-enzymic or enzymic oxidn. of its thiols to disulfides. Formation and speciation of oxidized MT have not been investigated in detail despite the biol. significance of this redox biochem. While metal ion-bound thiols in MT are rather resistant towards oxidn., free thiols are readily oxidized. MT can be partially oxidized to a state in which some of its thiols remain reduced and bound to metal ions. Anal. of the oxidn. products with SDS-PAGE and a thiol-specific labeling technique, employing eosin-5-iodoacetamide, demonstrates higher-order aggregates of MT with intermol. disulfide linkages. The polymn. follows either non-enzymic or enzymic oxidn., indicating that it is a general property of oxidized MT. Supramol. assemblies of MT add new perspectives to the complex redox and metal equil. of this protein.
- 95Hou, T.; An, Y.; Ru, B.; Bi, R.; Xu, X. Cysteine-independent polymerization of metallothioneins in solutions and in crystals. Protein Sci. 2000, 9, 2302– 2312, DOI: 10.1110/ps.9.12.230295Cysteine-independent polymerization of metallothioneins in solutions and in crystalsHou, Tingjun; An, Yu; Ru, Binggen; Bi, Ruchang; Xu, XiaojieProtein Science (2000), 9 (12), 2302-2312CODEN: PRCIEI; ISSN:0961-8368. (Cambridge University Press)Polymn. of metallothioneins complicates the research of metallothioneins' structure and function. Our work focuses on the cysteine-independent polymn. of metallothionein monomers in different milieus. After the purifn. of metallothionein monomers, a dynamic light-scattering technique is used to detect the polymd. states of rabbit liver metallothionein I and II in different buffers. This is the first systematical detection of polymd. states of metallothioneins in solns. The effects of buffer compn. are discussed in detail. Steric complementarity, hydrophobic, and electrostatic interaction characteristics are studied, following the modeling of monomers and relevant polymers of rat metallothionein II, rabbit liver metallothionein I and II. These theor. calcns. are the first complete computer simulations on different factors affecting metallothioneins' polymn. A mol. recognition mechanism of metallothioneins' polymn. in solns. is proposed on the bases of exptl. results and theor. calcns. Preliminary X-ray studies of two crystal forms of rabbit liver metallothionein II are compared with the crystal structure of rat metallothionein II, and the polymd. states in crystal packing are discussed with the knowledge of polymn. of metallothioneins in solns. The hypothesis, which is consistent with theor. calcns. and exptl. results, is expected to construct a connection between the biochem. characteristics and physiol. functions of metallothioneins, and this research may give some enlightenment to the topics of protein polymns.
- 96Vašák, M. Large-scale preparation of metallothionein: Biological sources. Methods Enzymol. 1991, 205, 39– 41, DOI: 10.1016/0076-6879(91)05081-696Large-scale preparation of metallothionein: biological sourcesVasak, MilanMethods in Enzymology (1991), 205 (Metallobiochem. Pt. B), 39-41CODEN: MENZAU; ISSN:0076-6879.The title process included metallothionein induction by Cd and Zn (the latter metal preferred). Some comments are included related to large-scale protein isolation.
- 97Hong, S.-H.; Toyama, M.; Maret, W.; Murooka, Y. High yield expression and single step purification of human thionein/metallothionein. Protein Expression Purif. 2001, 21, 243– 250, DOI: 10.1006/prep.2000.137297High Yield Expression and Single Step Purification of Human Thionein/MetallothioneinHong, Sung-Hye; Toyama, Mitsutoshi; Maret, Wolfgang; Murooka, YoshikatsuProtein Expression and Purification (2001), 21 (1), 243-250CODEN: PEXPEJ; ISSN:1046-5928. (Academic Press)Human metallothionein (MT), isoform 2, was expressed in Escherichia coli as an intein (protein splicing element) fusion protein in the absence of added metals and purified by intein-mediated purifn. with an affinity chitin-binding tag (IMPACT system). This procedure constitutes a novel and simple strategy to prep. thionein (T), the metal-free form, or MT when reconstituting T with metals in vitro. The yield was 8 mg of T or 6 mg of pure Cd7- or Zn7-MT from a 1-L culture, significantly higher than yields from any other expression system. Purified recombinant protein is indistinguishable from the native protein on the basis of its metal-binding ability, titrn. of its sulfhydryls, and UV and CD spectra. The MALDI-TOF mass spectrum is consistent with that of T with a free N-terminus. (c) 2001 Academic Press.
- 98Peris-Díaz, M. D.; Guran, R.; Zitka, O.; Adam, V.; Krężel, A. Metal- and affinity-specific dual labeling of cysteine-rich proteins for identification of metal-binding sites. Anal. Chem. 2020, 92, 12950– 12958, DOI: 10.1021/acs.analchem.0c0160498Metal- and Affinity-Specific Dual Labeling of Cysteine-Rich Proteins for Identification of Metal-Binding SitesPeris-Diaz, Manuel David; Guran, Roman; Zitka, Ondrej; Adam, Vojtech; Krezel, ArturAnalytical Chemistry (Washington, DC, United States) (2020), 92 (19), 12950-12958CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)Here, using human metallothionein (MT2) as an example, the authors describe an improved strategy based on differential alkylation coupled to MS, assisted by zinc probe monitoring, for identification of cysteine-rich binding sites with nanomolar and picomolar metal affinity using iodoacetamide (IAM) and N-ethylmaleimide reagents. An SN2 reaction provided by IAM is more suitable to label free Cys residues, avoiding nonspecific metal dissocn. Afterward, metal-bound Cys can be easily labeled in a nucleophilic addn. reaction after sepn. by reverse-phase C18 at acidic pH. Finally, the authors evaluated the efficiency of the method by mapping metal-binding sites of Zn7-xMT species using a bottom-up MS approach with respect to metal-to-protein affinity and element(al) resoln. The methodol. presented might be applied not only for MT2 but to identify metal-binding sites in other Cys-contg. proteins.
- 99Jiang, L.-J.; Maret, W.; Vallee, B. L. The glutathione redox couple modulates zinc transfer from metallothionein to zinc-depleted sorbitol dehydrogenase. Proc. Natl. Acad. Sci. U. S. A. 1998, 95, 3483– 3488, DOI: 10.1073/pnas.95.7.348399The glutathione redox couple modulates zinc transfer from metallothionein to zinc-depleted sorbitol dehydrogenaseJiang, Li-Juan; Maret, Wolfgang; Vallee, Bert L.Proceedings of the National Academy of Sciences of the United States of America (1998), 95 (7), 3483-3488CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)The release and transfer of zinc from metallothionein (MT) to zinc-depleted sorbitol dehydrogenase (EC 1.1.1.14) in vitro has been used to explore the role of MT in cellular zinc distribution. A 1:1 molar ratio of MT to sorbitol dehydrogenase is required for full reactivation, indicating that only one of the seven zinc atoms of MT is transferred in this process. Reduced glutathione (GSH) and glutathione disulfide (GSSG) are crit. modulators of both the rate of zinc transfer and the ultimate no. of zinc atoms transferred. GSSG increases the rate of zinc transfer 3-fold, and its concn. is the major determinant for efficient zinc transfer. GSH has a dual function. In the absence of GSSG, it inhibits zinc transfer from MT, indicating that MT is in a latent state under the relatively high cellular concns. of GSH. In addn., it primes MT for the reaction with GSSG by enhancing the rate of zinc transfer 10-fold and by increasing the no. of zinc atoms transferred to four. 65Zn-labeling expts. confirm the release of one zinc from MT in the absence of glutathione and the more effective release of zinc in the presence of GSH and GSSG. In vivo, MT may keep the cellular concns. of free zinc very low and, acting as a temporary cellular reservoir, release zinc in a process that is dynamically controlled by its interactions with both GSH and GSSG. These results suggest that a change of the redox state of the cell could serve as a driving force and signal for zinc distribution from MT.
- 100Brouwer, M.; Brouwer-Hoexum, T.; Cashon, R. Crustaceans as models for metal metabolism. III. Interaction of lobster and mammalian metallothionein with glutathione. Mar. Environ. Res. 1993, 35, 13– 17, DOI: 10.1016/0141-1136(93)90006-LThere is no corresponding record for this reference.
- 101Brouwer, M.; Hoexum-Brouwer, T.; Cashon, R. E. A putative glutathione-binding site in CdZn metallothionein identified by equilibrium binding and molecular-modelling studies. Biochem. J. 1993, 294, 219– 225, DOI: 10.1042/bj2940219101A putative glutathione-binding site in cadmium-zinc-metallothionein identified by equilibrium binding and molecular-modeling studiesBrouwer, Marius; Hoexum-Brouwer, Thea; Cashon, Robert E.Biochemical Journal (1993), 294 (1), 219-25CODEN: BIJOAK; ISSN:0264-6021.Glutathione (GSH) has been found to form a complex with both vertebrate and invertebrate copper-metallothionein (CuMT) [Freedman, Ciriolo and Peisach (1989) J. Biol. Chem. 264, 5598-5605; Brouwer and Brouwer-Hoexum (1991) Arch. Biochem. Biophys. 290, 207-213]. In this paper the authors report on the interaction of GSH with CdZnMT-I and CdZnMT-II from rabbit liver and with CdMT-I from Blue crab hepatopancreas. Ultrafiltration expts. showed that all three MTs combined with GSH. The measured binding data for the three MTs could be described by a single binding isotherm. The GSH/MT stoichiometry was 1.4 ± 0.3 and Kdiss. = 14 ± 6 μM. Partially Zn-depleted MT does not significantly bind GSH, indicating that the GSH-binding site is located on MT's Zn-contg. N-terminal domain. The putative GSH-binding site on rabbit liver MT was investigated using mol.-graphics anal. A cleft on the MTs N-terminal domain, which has the labile Zn-2 at its base, could easily accommodate GSH. Cysteine-ligand exchange between the terminal (non-bridging) Cys-26, bound to Zn-2, and the cysteine in GSH is stereochem. possible. Based on these considerations a model of MT-GSH was built in which GSH's cysteine replaces Cys-26 as a terminal Zn-2 ligand. This complex was energy-minimized by mol.-mechanics calcns., taking into account computed partial electrostatic charges on all atoms, including Cd and Zn. These calcns. showed that the MT-GSH complex was thermodynamically more stable than MT, due to favorable non-bonded, electrostatic and van der Waals interactions. Six hydrogen bonds can form between GSH and MT. The av. pairwise root-mean-square deviations (RMSD) of the metals in energy-minimized MT and MT-GSH, compared with the metals in the crystal structure, were 0.0087 ± 0.0028 nm (0.087 ± 0.028 Å) and 0.0168 ± 0.0087 nm (0.168 ± 0.087 Å) resp. The RMSD values for the polypeptide-backbone α carbons were 0.0136 ± 0.0060 nm (0.136 ± 0.060 Å) and 0.0491 ± 0.0380 nm (0.491 ± 0.380 Å) resp. No other docking sites for GSH were found. The energy-minimized structure of an MT-2-mercaptoethanol complex was somewhat less stable than the native MT domain, attesting to the specificity of the MT-GSH interaction. The possible physiol. significance of the MT-GSH interaction is discussed.
- 102Freedman, J. H.; Ciriolo, M.; Peisach, J. The role of glutathione in copper metabolism and toxicity. J. Biol. Chem. 1989, 264, 5598– 5605, DOI: 10.1016/S0021-9258(18)83589-X102The role of glutathione in copper metabolism and toxicityFreedman, Jonathan H.; Ciriolo, Maria Rosa; Peisach, JackJournal of Biological Chemistry (1989), 264 (10), 5598-605CODEN: JBCHA3; ISSN:0021-9258.Cellular copper metab. and the mechanism of resistance to copper toxicity were investigated using a wild type hepatoma cell line (HAC) and a copper-resistant cell line (HAC600) that accumulates copper and has a highly elevated level of metallothionein (MT). Of the enzymes involved in reactive oxygen metab., only glutathionine peroxidase was elevated (3-4-fold) in resistant cells, suggestive of an increase in the cellular flux of hydrogen peroxide. A majority of the cytoplasmic copper (>60%) was isolated from both cell lines as a GSH complex. Kinetic studies of 67Cu uptake showed that GSH bound 67Cu before the metal was complexed by MT. Depletion of cellular GSH with buthionine sulfoximine inhibited the incorporation of 67Cu into MT by >50%. Apparently, copper is complexed by GSH soon after entering the cell. The complexed metal is then transferred to MT where it is stored. This study also indicates that resistance to metal toxicity in copper-resistant hepatoma cells is due to increases in both cellular GSH and MT. Furthermore, it is suggested that elevated levels of GSH peroxidase allows cells to more efficiently accommodate an increased cellular hydrogen peroxide flux that may occur as a consequence of elevated levels of cytoplasmic copper.
- 103Krężel, A.; Wójcik, J.; Maciejczyk, M.; Bal, W. May GSH and L-His contribute to intracellular binding of zinc? Thermodynamic and solution structural study of a ternary complex. Chem. Commun. 2003, 704– 705, DOI: 10.1039/b300632h103May GSH and L-His contribute to intracellular binding of zinc? Thermodynamic and solution structural study of a ternary complexKrezel, Artur; Wojcik, Jacek; Maciejczyk, Maciej; Bal, WojciechChemical Communications (Cambridge, United Kingdom) (2003), (6), 704-705CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)GSH and L-His are abundant biomols. and likely biol. ligands for Zn(II) under certain conditions. Potentiometric titrns. provide evidence of formation of ternary Zn(II) complexes with GSH and L-His or D-His with slight stereoselectivity in favor of L-His (ca. 1 log unit of stability const.). The soln. structure of the ZnH(GSH)(L-His)(H2O) complex at pH 6.8, detd. by NMR, includes tridentate L-His, monodentate (sulfur) GSH, and weak interligand interactions. Calcns. of competitiveness of this complex for Zn(II) binding at pH 7.4 indicate that it is likely to be formed in vivo under conditions of GSH depletion. Otherwise, GSH alone emerges as a likely Zn(I) carrier.
- 104Krężel, A.; Bal, W. Studies of zinc(II) and nickel(II) complexes of GSH, GSSG and their analogs shed more light on their biological relevance. Bioinorg. Chem. Appl. 2004, 2, 293– 305, DOI: 10.1155/S1565363304000172104Studies of zinc(II) and nickel(II) complexes of GSH, GSSG and their analogs shed more light on their biological relevanceKrezel, Artur; Bal, WojciechBioinorganic Chemistry and Applications (2004), 2 (3-4), 293-305CODEN: BCAIAH; ISSN:1565-3633. (Freund Publishing House)A review. Glutathione, γ-Glu-Cys-Gly, is one of the most abundant small mols. in biosphere. Its main form is the reduced monomer (GSH), serving to detoxicate xenobiotics and heavy metals, reduce protein thiols, maintain cellular membranes and deactivate free radicals. Its oxidized dimer (GSSG) controls metal content of metallothionein. The results presented provided a quant. and structural description of Zn(II)-glutathione complexes, including a novel ternary Zn(II)-GSH-His complex. A soln. structure for this complex was obtained using 2D-NMR. The complexes studied may contribute to both zinc and glutathione physiol. In the case of Ni(II) complexes an interesting dependence of coordination modes on the ratios of reactants was found. At high GSH excess a Ni(GSH)2 complex is formed, with Ni(II) bonded through S and N and/or O donor atoms. This complex may exist as a high- or low-spin species. Another goal of the studies presented was to describe the catalytic properties of Ni(II) ions towards GSH oxidn., which appeared to be an important step in nickel carcinogenesis. The pH dependence of oxidn. rates allowed to det. the Ni(GSH)2 complex as the most active among the toxicol. relevant species. Protonation and oxidn. of metal-free GSH and its analogs were also studied in detail. The monoprotonated form HL2- of GSH is the one most susceptible to oxidn., due to a salt bridge between S- and NH3+ groups, which activates the thiol.
- 105Casadei, M.; Persichini, T.; Polticelli, F.; Musci, G.; Colasanti, M. S-glutathionylation of metallothioneins by nitrosative/oxidative stress. Exp. Gerontol. 2008, 43, 415– 422, DOI: 10.1016/j.exger.2007.11.004105S-Glutathionylation of metallothioneins by nitrosative/oxidative stressCasadei, Manuela; Persichini, Tiziana; Polticelli, Fabio; Musci, Giovanni; Colasanti, MarcoExperimental Gerontology (2008), 43 (5), 415-422CODEN: EXGEAB; ISSN:0531-5565. (Elsevier)Cysteine residues within metallothionein (MT) structure have been shown to be particularly prone to S-nitrosylation. The objective of this study was to examine the possibility that MTs undergo S-glutathionylation under nitrosative/oxidative stress. MT from rabbit liver was treated with different concns. of GSNO, diamide plus GSH or H2O2 plus GSH. Parallel sets of samples were treated with 10 mM DTT for 30 min at 37 °C to reduce mixed disulfides. Incubations were then processed for Western blot or dot-immunobinding assay. Western blot with anti-MT or anti-GSH were also performed on peripheral blood mononuclear cell exts. Structural aspects of S-glutathionylation of MTs were also examd. Treatment with GSNO, diamide/GSH or H2O2/GSH induced a dose-dependent increase in the levels of MT S-glutathionylation. This effect was completely reversed by treatment with the reducing agent DTT, indicating that S-glutathionylation of MT protein was related to formation of protein-mixed disulfides. Structural anal. of rat MT indicated that Cys residues located in the N-terminal domain of the protein are the likely targets for S-glutathionylation, both for their solvent accessibility and electrostatics induced reactivity. S-Glutathionylation of MT, given its reversibility, would provide protection from irreversible oxidn. of Cys residues, thus representing a mechanism of high potential biol. relevance.
- 106Zangger, K.; Öz, G.; Haslinger, E.; Kunert, O.; Armitage, I. M. Nitric oxide selectively releases metals from the amino-terminal domain of metallothioneins: potential role at inflammatory sites. FASEB J. 2001, 15, 1303– 1305, DOI: 10.1096/fj.00-0641fje106Nitric oxide selectively releases metals from the N-terminal domain of metallothioneins: potential role at inflammatory sitesZangger, Klaus; Oz, Guilin; Haslinger, Ernst; Kunert, Olaf; Armitage, Ian M.FASEB Journal (2001), 15 (7), 1303-1305, 10.1096/fj/00-0641/fjeCODEN: FAJOEC; ISSN:0892-6638. (Federation of American Societies for Experimental Biology)Metallothioneins (MTs) and various other metal binding proteins release metals when exposed to nitric oxide (NO). We investigated the structural consequences of the interaction between MTs and NO by using 1H- and 113Cd-NMR spectroscopy and found that only the three metals from the N-terminal β-domain were selectively released whereas the C-terminal α-domain remains intact. Since it has been proposed that the β-domain is responsible for the postulated role of MTs in zinc homeostasis, whereas the tight binding of metals in the α-domain appears to play a role in heavy metal detoxification, our results suggest a potential regulatory role of NO in zinc distribution. Specifically, we present a mechanism whereby MT counteracts the cytotoxic effects of NO at inflammatory sites.
- 107Barbato, J. C.; Catanescu, O.; Murray, K.; DiBello, P. M.; Jacobsen, D. W. Targeting of metallothionein by L-homocysteine. A novel mechanism for disruption of zinc and redox homeostasis. Arterioscler., Thromb., Vasc. Biol. 2007, 27, 49– 54, DOI: 10.1161/01.ATV.0000251536.49581.8a107Targeting of metallothionein by L-homocysteine: a novel mechanism for disruption of zinc and redox homeostasisBarbato John C; Catanescu Otilia; Murray Kelsey; DiBello Patricia M; Jacobsen Donald WArteriosclerosis, thrombosis, and vascular biology (2007), 27 (1), 49-54 ISSN:.OBJECTIVE: L-homocysteine and/or L-homocystine interact in vivo with albumin and other extracellular proteins by forming mixed-disulfide conjugates. Because of its extremely rich cysteine content, we hypothesized that metallothionein, a ubiquitous intracellular zinc-chaperone and superoxide anion radical scavenger, reacts with L-homocysteine and that homocysteinylated-metallothionein suffers loss of function. METHODS AND RESULTS: 35S-homocysteinylated-metallothionein was resolved in lysates of cultured human aortic endothelial cells in the absence and presence of reduced glutathione by SDS-PAGE and identified by Western blotting and phosphorimaging. Using zinc-Sepharose chromatography, L-homocysteine was shown to impair the zinc-binding capacity of metallothionein even in the presence of reduced glutathione. L-Homocysteine induced a dose-dependent increase in intracellular free zinc in zinquin-loaded human aortic endothelial cells within 30 minutes, followed by the appearance of early growth response protein-1 within 60 minutes. In addition, intracellular reactive oxygen species dramatically increased 6 hours after L-homocysteine treatment. In vitro studies demonstrated that L-homocysteine is a potent inhibitor of the superoxide anion radical scavenging ability of metallothionein. CONCLUSIONS: These studies provide the first evidence that L-homocysteine targets intracellular metallothionein by forming a mixed-disulfide conjugate and that loss of function occurs after homocysteinylation. The data support a novel mechanism for disruption of zinc and redox homeostasis.
- 108Aizenman, E.; Stout, A. K.; Hartnett, K. A.; Dineley, K. E.; McLaughlin, B.; Reynolds, I. J. Induction of neuronal apoptosis by thiol oxidation: Putative role of intracellular zinc release. J. Neurochem. 2000, 75, 1878– 1888, DOI: 10.1046/j.1471-4159.2000.0751878.x108Induction of neuronal apoptosis by thiol oxidation: putative role of intracellular zinc releaseAizenman, Elias; Stout, Amy K.; Hartnett, Karen A.; Dineley, Kirk E.; McLaughlin, BethAnn; Reynolds, Ian J.Journal of Neurochemistry (2000), 75 (5), 1878-1888CODEN: JONRA9; ISSN:0022-3042. (Lippincott Williams & Wilkins)The membrane-permeant oxidizing agent 2,2'-dithiodipyridine (DTDP) can induce Zn2+ release from metalloproteins in cell-free systems. Here, we report that brief exposure to DTDP triggers apoptotic cell death in cultured neurons, detected by the presence of both DNA laddering and asym. chromatin formation. Neuronal death was blocked by increased extracellular potassium levels, by tetraethylammonium, and by the broad-spectrum cysteine protease inhibitor butoxy-carbonyl-aspartate-fluoromethylketone. N,N,N',N'-Tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN) and other cell-permeant metal chelators also effectively blocked DTDP-induced toxicity in neurons. Cell death, however, was not abolished by the NMDA receptor blocker MK-801, by the intracellular calcium release antagonist dantrolene, or by high concns. of ryanodine. DTDP generated increases in fluorescence signals in cultured neurons loaded with the zinc-selective dye Newport Green. The fluorescence signals following DTDP treatment also increased in fura-2- and magfura-2-loaded neurons. These responses were completely reversed by TPEN, consistent with a DTDP-mediated increase in intracellular free Zn2+ concns. Our studies suggest that under conditions of oxidative stress, Zn2+ released from intracellular stores may contribute to the initiation of neuronal apoptosis.
- 109Fass, D.; Thorpe, C. Chemistry and enzymology of disulfide cross-linking in proteins. Chem. Rev. 2018, 118, 1169– 1198, DOI: 10.1021/acs.chemrev.7b00123109Chemistry and Enzymology of Disulfide Cross-Linking in ProteinsFass, Deborah; Thorpe, ColinChemical Reviews (Washington, DC, United States) (2018), 118 (3), 1169-1198CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)A review. Cysteine thiols are among the most reactive functional groups in proteins, and their pairing in disulfide linkages is a common post-translational modification in proteins entering the secretory pathway. This modest amino acid alteration, the mere removal of a pair of hydrogen atoms from juxtaposed cysteine residues, contrasts with the substantial changes that characterize most other post-translational reactions. However, the wide variety of proteins that contain disulfides, the profound impact of crosslinking on the behavior of the protein polymer, the numerous and diverse players in intracellular pathways for disulfide formation, and the distinct biol. settings in which disulfide bond formation can take place belie the simplicity of the process. Here we lay the groundwork for appreciating the mechanisms and consequences of disulfide bond formation in vivo by reviewing chem. principles underlying cysteine pairing and oxidn. We then show how enzymes tune redox-active cofactors and recruit oxidants to improve the specificity and efficiency of disulfide formation. Finally, we discuss disulfide bond formation in a cellular context and identify important principles that contribute to productive thiol oxidn. in complex, crowded, dynamic environments.
- 110Hu, H. Y.; Cheng, H. Q.; Li, Q.; Zou, Y. S.; Xu, G. J. Study of the redox properties of metallothionein in vitro by reacting with DsbA protein. J. Protein Chem. 1999, 18, 665– 670, DOI: 10.1023/A:1020654206878110Study of the redox properties of metallothionein in vitro by reacting with DsbA proteinHu, H. Y.; Cheng, H. Q.; Li, Q.; Zou, Y. S.; Xu, G. J.Journal of Protein Chemistry (1999), 18 (6), 665-670CODEN: JPCHD2; ISSN:0277-8033. (Kluwer Academic/Plenum Publishers)Mammalian metallothionein (MT) contains 20 cysteine residues involved in the two metal clusters without a disulfide bond. The redox reaction of the Cys thiols was proposed to be assocd. with the metal distribution of MT. The E. coli DsbA protein is extremely active in facilitating thiol/disulfide exchange both in vivo and in vitro. To further investigate the redox properties of MT, reaction between MT and DsbA was carried out in vitro by fluorescence detection. Equil. characterization indicates that the reaction is stoichiometric (1:1) under certain conditions. Kinetic study gives a rate const. of the redox reaction of 4.42 × 105 sec-1 M-1, which is 103-fold larger than that of glutathione reacting with DsbA. Metal-free MT (apo-MT) shows a higher equil. redn. potential than MT, but exhibits an indistinguishable kinetic rate. Oxidn. of MT by DsbA leads to metal release from the clusters. The characteristic fluorescence increase during redn. of DsbA may provide a sensitive probe for exploring the redox properties of some reductants of biol. interest. The result also implies that oxidn. of Cys thiols may influence the metal release or delivery from MT.
- 111Jacob, C.; Maret, W.; Vallee, B. L. Selenium redox biochemistry of zinc/sulfur coordination sites in proteins and enzymes. Proc. Natl. Acad. Sci. U. S. A. 1999, 96, 1910– 1914, DOI: 10.1073/pnas.96.5.1910111Selenium redox biochemistry of zinc-sulfur coordination sites in proteins and enzymesJacob, Claus; Maret, Wolfgang; Vallee, Bert L.Proceedings of the National Academy of Sciences of the United States of America (1999), 96 (5), 1910-1914CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Selenium has been increasingly recognized as an essential element in biol. and medicine. Its biochem. resembles that of sulfur, yet differs from it by virtue of both redox potentials and stabilities of its oxidn. states. Selenium can substitute for the more ubiquitous sulfur of cysteine and as such plays an important role in more than a dozen selenoproteins. We have chosen to examine zinc-sulfur centers as possible targets of selenium redox biochem. Selenium compds. release zinc from zinc/thiolate-coordination environments, thereby affecting the cellular thiol redox state and the distribution of zinc and likely of other metal ions. Arom. selenium compds. are excellent spectroscopic probes of the otherwise relatively unstable functional selenium groups. Zinc-coordinated thiolates, e.g., metallothionein (MT), and uncoordinated thiolates, e.g., glutathione, react with benzeneseleninic acid (oxidn. state +2), benzeneselenenyl chloride (oxidn. state 0) and selenocystamine (oxidn. state -1). Benzeneseleninic acid and benzeneselenenyl chloride react very rapidly with MT and titrate substoichiometrically and with a 1:1 stoichiometry, resp. Selenium compds. also catalyze the release of zinc from MT in peroxidn. and thiol/disulfide-interchange reactions. The selenoenzyme glutathione peroxidase catalytically oxidizes MT and releases zinc in the presence of t-Bu hydroperoxide, suggesting that this type of redox chem. may be employed in biol. for the control of metal metab. Moreover, selenium compds. are likely targets for zinc/thiolate coordination centers in vivo, because the reactions are only partially suppressed by excess glutathione. This specificity and the potential to undergo catalytic reactions at low concns. suggests that zinc release is a significant aspect of the therapeutic antioxidant actions of selenium compds. in antiinflammatory and anticarcinogenic agents.
- 112Chen, Y.; Maret, W. Catalytic oxidation of zinc/sulfur coordination sites in proteins by selenium compounds. Antioxid. Redox Signaling 2001, 3, 651– 656, DOI: 10.1089/15230860152542998112Catalytic oxidation of zinc/sulfur coordination sites in proteins by selenium compoundsChen, Yu; Maret, WolfgangAntioxidants & Redox Signaling (2001), 3 (4), 651-656CODEN: ARSIF2; ISSN:1523-0864. (Mary Ann Liebert, Inc.)Zinc/thiolate (cysteine) coordination occurs in a very large no. of proteins. These coordination sites are thermodynamically quite stable. Yet the redox chem. of thiolate ligands confers extraordinary reactivities on these sites. The significance of such ligand-centered reactions is that they affect the binding and release of zinc, thus helping to distribute zinc, and perhaps controlling zinc-dependent cellular events. One new aspect focuses on the thiolate ligands of zinc as targets for the redox action of selenium compds. A distinctive feature of this chem. is the capacity of selenols to catalyze the oxidn. of zinc/thiolate sites. We here use a chromophoric compd., 2-nitrophenylselenocyanate, to investigate its reaction mechanism with the zinc/thiolate clusters of metallothionein, a protein that is a cellular reservoir for zinc and together with its apoprotein, thionein, is involved in zinc distribution as a zinc donor/acceptor pair. The reaction is particularly revealing as it occurs in two steps. A selenenylsulfide intermediate is formed in the fast oxidative step, followed by the generation of 2-nitrophenylselenol that initiates the second, catalytic step. The findings demonstrate the high reactivity of selenium compds. with zinc/thiolate coordination sites and the potent catalytic roles that selenoproteins and selenium redox drugs may have in affecting gene expression via modulation of the zinc content of zinc finger proteins.
- 113Chen, Y.; Maret, W. Catalytic selenols couple the redox cycles of metallothionein and glutathione. Eur. J. Biochem. 2001, 268, 3346– 3353, DOI: 10.1046/j.1432-1327.2001.02250.x113Catalytic selenols couple the redox cycles of metallothionein and glutathioneChen, Yu; Maret, WolfgangEuropean Journal of Biochemistry (2001), 268 (11), 3346-3353CODEN: EJBCAI; ISSN:0014-2956. (Blackwell Science Ltd.)Co-ordination of zinc to the thiol group of cysteine allows mobilization of zinc through oxidn. of its ligand. This mol. property links the binding and release of zinc in metallothionein (MT) to the cellular redox state. Biol. disulfides such as glutathione disulfide (GSSG) oxidize MT with concomitant release of zinc, while glutathione (GSH) reduces the oxidized protein to thionein, which then binds to available zinc. Neither of these two redox processes is very efficient, even at high concns. of GSSG or GSH. However, the GSH/GSSG redox pair can efficiently couple with the MT/thionein system in the presence of a selenium compd. that has the capacity to form a catalytic selenol(ate). This coupling provides a very effective means of modulating oxidn. and redn. Remarkably, selenium compds. catalyze the oxidn. of MT even under overall reducing conditions such as those prevailing in the cytosol. In this manner, the binding and release of zinc from zinc-thiolate co-ordination sites is linked to redox catalysis by selenium compds., changes in the glutathione redox state, and the availability of either a zinc donor or a zinc acceptor. The results also suggest that the pharmacol. actions of selenium compds. in cancer prevention and other antiviral and anti-inflammatory therapeutic applications, as well as unknown functions of selenium-contg. proteins, may relate to coupling between the thiol redox state and the zinc state.
- 114Zhang, S.; Li, J.; Wang, C.-C.; Tsou, C.-L. Metal regulation of metallothionein participation in redox reactions. FEBS Lett. 1999, 462, 383– 386, DOI: 10.1016/S0014-5793(99)01562-8114Metal regulation of metallothionein participation in redox reactionsZhang, S.; Li, J.; Wang, C.-C.; Tsou, C.-L.FEBS Letters (1999), 462 (3), 383-386CODEN: FEBLAL; ISSN:0014-5793. (Elsevier Science B.V.)Like glutathione or dithiothreitol, metallothionein effects the formation of pancreatic RNase A from its S-sulfonated deriv. catalyzed by protein disulfide isomerase. EDTA increases the yield of RNase A activity recovery with metallothionein but does not affect the reaction with glutathione or dithiothreitol. EDTA also increases the reactivity of thiol groups in metallothionein with 5,5'-dithiobis-(2-nitrobenzoic acid) by chelation of zinc ions. It is suggested that the thiol groups in metallothionein form a part of the pool of cellular thiols in the regulation of cellular redox reactions and their availability is modulated by zinc chelation.
- 115Sagher, D.; Brunell, D.; Hejtmancik, J. F.; Kantorow, M.; Brot, N.; Weissbach, H. Thionein can serve as a reducing agent for the methionine sulfoxide reductases. Proc. Natl. Acad. Sci. U. S. A. 2006, 103, 8656– 8661, DOI: 10.1073/pnas.0602826103115Thionein can serve as a reducing agent for the methionine sulfoxide reductasesSagher, Daphna; Brunell, David; Hejtmancik, J. Fielding; Kantorow, Marc; Brot, Nathan; Weissbach, HerbertProceedings of the National Academy of Sciences of the United States of America (2006), 103 (23), 8656-8661CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)It has been generally accepted, primarily from studies on methionine sulfoxide reductase (Msr) A, that the biol. reducing agent for the members of the Msr family is reduced thioredoxin (Trx), although high levels of DTT can be used as the reductant in vitro. Preliminary expts. using both human recombinant MsrB2 (hMsrB2) and MsrB3 (hMsrB3) showed that although DTT can function in vitro as the reducing agent, Trx works very poorly, prompting a more careful comparison of the ability of DTT and Trx to function as reducing agents with the various members of the Msr family. Escherichia coli MsrA and MsrB and bovine MsrA efficiently use either Trx or DTT as reducing agents. In contrast, hMsrB2 and hMsrB3 show <10% of the activity with Trx as compared with DTT, raising the possibility that, in animal cells, Trx may not be the direct hydrogen donor or that there may be a Trx-independent reducing system required for MsrB2 and MsrB3 activity. A heat-stable protein has been detected in bovine liver that, in the presence of EDTA, can support the Msr reaction in the absence of either Trx or DTT. This protein has been identified as a zinc-contg. metallothionein (Zn-MT). The results indicate that thionein (T), which is formed when the zinc is removed from Zn-MT, can function as a reducing system for the Msr proteins because of its high content of cysteine residues and that Trx can reduce oxidized T.
- 116Sagher, D.; Brunell, D.; Brot, N.; Vallee, B. L.; Weissbach, H. Selenocompounds can serve as oxidoreductants with the methionine sulfoxide reductase enzymes. J. Biol. Chem. 2006, 281, 31184– 31187, DOI: 10.1074/jbc.M606962200116Selenocompounds Can Serve as Oxidoreductants with the Methionine Sulfoxide Reductase EnzymesSagher, Daphna; Brunell, David; Brot, Nathan; Vallee, Bert L.; Weissbach, HerbertJournal of Biological Chemistry (2006), 281 (42), 31184-31187CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)In a recent study on the reducing requirement for the methionine sulfoxide reductases (Msr) (Sagher, D., Brunell, D., Hejtmancik, J. F., Kantorow, M., Brot, N. & Weissbach, H. (2006) PROC: Natl. Acad. Sci. U. S. A. 103, 8656-8661), we have shown that thioredoxin, although an excellent reducing system for Escherichia coli MsrA and MsrB and bovine MsrA, is not an efficient reducing agent for either human MsrB2 (hMsrB2) or human MsrB3 (hMsrB3). In a search for another reducing agent for hMsrB2 and hMsrB3, it was recently found that thionein, the reduced, metal-free form of metallothionein, could function as a reducing system for hMsrB3, with weaker activity using hMsrB2. In the present study, we provide evidence that some selenium compds. are potent reducing agents for both hMsrB2 and hMsrB3.
- 117Maret, W. Regulation of cellular zinc ions and their signalling functions. In Zinc signalling; Fukada, T., Kambe, T., Eds.; 2019, Springer: Singapore; pp 5– 22.There is no corresponding record for this reference.
- 118Kröncke, K. D.; Fehsel, K.; Schmidt, T.; Zenke, F. T.; Dasting, I.; Wesener, J. R.; Bettermann, H.; Breunig, K. D.; Kolb-Bachofen, V. Nitric oxide destroys zinc-sulfur clusters inducing zinc release from metallothionein and inhibition of the zinc-finger type yeast transcription activator LAC9. Biochem. Biophys. Res. Commun. 1994, 200, 1105– 1110, DOI: 10.1006/bbrc.1994.1564118Nitric oxide destroys zinc-sulfur clusters inducing zinc release from metallothionein and inhibition of the zinc finger-type yeast transcription activator LAC9Kroncke K D; Fehsel K; Schmidt T; Zenke F T; Dasting I; Wesener J R; Bettermann H; Breunig K D; Kolb-Bachofen VBiochemical and biophysical research communications (1994), 200 (2), 1105-10 ISSN:0006-291X.Nitric oxide, generated from S-nitrosocysteine or applied as gas mediates metal ion release from the Zn2+/Cd(2+)-complexing protein metallothionein via oxidation of SH-groups. Time-dependent S-nitrosylation and subsequent disulfide formation of metallothionein are demonstrated. Furthermore, nitric oxide inhibits DNA binding activity of the yeast transcription factor LAC9 containing a zinc finger like DNA binding domain. These results show that nitric oxide interacts with and destroys zinc-sulfur clusters in proteins.
- 119St Croix, C. M.; Wasserloos, K. J.; Dineley, K. E.; Reynolds, I. J.; Levitan, E. S.; Pitt, B. R. Nitric oxide-induced changes in intracellular zinc homeostasis are mediated by metallothionein/thionein. Am. J. Physiol. Lung Cell. Mol. Physiol. 2002, 282, L185– L192, DOI: 10.1152/ajplung.00267.2001119Nitric oxide-induced changes in intracellular zinc homeostasis are mediated by metallothionein/thioneinSt. Croix, Claudette M.; Wasserloos, K. J.; Dineley, K. E.; Reynolds, I. J.; Levitan, E. S.; Pitt, B. R.American Journal of Physiology (2002), 282 (2, Pt. 1), L185-L192CODEN: AJPHAP; ISSN:0002-9513. (American Physiological Society)We hypothesized that metallothionein (MT), a cysteine-rich protein with a strong affinity for Zn2+, plays a role in nitric oxide (NO) signaling events via sequestration or release of Zn2+ by the unique thiolate clusters of the protein. Exposing mouse lung fibroblasts (MLF) to the NO donor S-nitrosocysteine resulted in 20-30% increases in fluorescence of the Zn2+-specific fluorophore Zinquin that were rapidly reversed by the Zn2+ chelator N,N,N'N'-tetrakis-(2-pyridylmethyl)ethylenediamine. The absence of a NO-mediated increase in labile Zn2+ in MLF from MT knockouts and its restoration after MT complementation by adenoviral gene transfer inferred a crit. role for MT in the regulation of Zn2+ homeostasis by NO. Addnl. data obtained in sheep pulmonary artery endothelial cells suggested a role for the apo form of MT, thionein (T), as a Zn2+-binding protein in intact cells, as overexpression of MT caused inhibition of NO-induced changes in labile Zn2+ that were reversed by Zn2+ supplementation. Furthermore, fluorescence-resonance energy-transfer data showed that overexpression of green fluorescent protein-modified MT prevented NO-induced conformational changes, which are indicative of Zn2+ release from thiolate clusters. This effect was restored by Zn2+ supplementation. Collectively, these data show that MT mediates NO-induced changes in intracellular Zn2+ and suggest that the ratio of MT to T can regulate Zn2+ homeostasis in response to nitrosative stress.
- 120Spahl, D. U.; Berendji-Grün, D.; Suschek, C. V.; Kolb-Bachofen, V.; Kröncke, K. D. (2003) Regulation of zinc homeostasis by inducible NO synthase-derived NO: nuclear metallothionein translocation and intranuclear Zn2+ release. Proc. Natl. Acad. Sci. U. S. A. 2003, 100, 13952– 13957, DOI: 10.1073/pnas.2335190100120Regulation of zinc homeostasis by inducible NO synthase-derived NO: Nuclear metallothionein translocation and intranuclear Zn2+ releaseSpahl, Daniela U.; Berendji-gruen, Denise; Suschek, Christoph V.; Kolb-bachofen, Victoria; Kroencke, Klaus -D.Proceedings of the National Academy of Sciences of the United States of America (2003), 100 (24), 13952-13957CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Zn2+ is crit. for the functional and structural integrity of cells and contributes to a no. of important processes including gene expression. It has been shown that NO exogenously applied via NO donors resulting in nitrosative stress leads to cytoplasmic Zn2+ release from the zinc storing protein metallothionein (MT) and probably other proteins that complex Zn2+ via cysteine thiols. We show here that, in cytokine-activated murine aortic endothelial cells, NO derived from the inducible NO synthase (iNOS) induces a transient nuclear release of Zn2+. This nuclear Zn2+ release depends on the presence of MT as shown by the lack of this effect in activated endothelial cells from MT-deficient mice and temporally correlates with nuclear MT translocation. Data also show that NO is an essential but not sufficient signal for MT-mediated Zn2+ trafficking from the cytoplasm into the nucleus. In addn., we found that, endogenously via iNOS, synthesized NO increases the constitutive mRNA expression of both MT-1 and MT-2 genes and that nitrosative stress exogenously applied via an NO donor increases constitutive MT mRNA expression via intracellular Zn2+ release. In conclusion, we here provide evidence for a signaling mechanism based on iNOS-derived NO through the regulation of intracellular Zn2+ trafficking and homeostasis.
- 121Chen, Y.; Irie, Y.; Keung, W. M.; Maret, W. S-Nitrosothiols react preferentially with zinc thiolate clusters of metallothionein III through transnitrosation. Biochemistry 2002, 41, 8360– 8367, DOI: 10.1021/bi020030+121S-Nitrosothiols React Preferentially with Zinc Thiolate Clusters of Metallothionein III through TransnitrosationChen, Yu; Irie, Yoshifumi; Keung, Wing Ming; Maret, WolfgangBiochemistry (2002), 41 (26), 8360-8367CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)Metallothionein (MT) is a two-domain protein with zinc thiolate clusters that bind and release zinc depending on the redox states of the sulfur ligands. Since S-nitrosylation of cysteine is considered a prototypic cellular redox signaling mechanism, we here investigate the reactions of S-nitrosothiols with different isoforms of MT. MT-III is significantly more reactive than MT-I/II toward S-nitrosothiols, whereas the reactivity of all three isoforms toward reactive oxygen species is comparable. A cellular system, in which all three MTs are similarly effective in protecting rat embryonic cortical neurons in primary culture against hydrogen peroxide but where MT-III has a much more pronounced effect of protecting against S-nitrosothiols, confirms this finding. MT-III is the only isoform with consensus acid-base sequence motifs for S-nitrosylation in both domains. Studies with synthetic and zinc-reconstituted domain peptides demonstrate that S-nitrosothiols indeed release zinc from both the α- and the β-domain of MT-III. S-Nitrosylation occurs via transnitrosation, a mechanism that differs fundamentally from that of previous studies of reactions of MT with NO•. Our data demonstrate that zinc thiolate bonds are targets of S-nitrosothiol signaling and further indicate that MT-III is biol. specific in converting NO signals to zinc signals. This could bear importantly on the physiol. action of MT-III, whose biol. activity as a neuronal growth inhibitory factor is unique, and for brain diseases that have been related to oxidative or nitrosative stress.
- 122Zhang, L.-M.; Croix, C. S.; Cao, R.; Wasserloos, K.; Watkins, S. C.; Stevens, T.; Li, S.; Tyurin, V.; Kagan, V. E.; Pitt, B. R. Cell-surface protein disulfide isomerase is required for transnitrosation of metallothionein by S-nitroso-albumin in intact rat pulmonary vascular endothelial cells. Exp. Biol. Med. 2006, 231, 1507– 1515, DOI: 10.1177/153537020623100909122Cell-surface protein disulfide isomerase is required for transnitrosation of metallothionein by S-nitroso-albumin in intact rat pulmonary vascular endothelial cellsZhang, Li-Ming; St. Croix, Claudette; Cao, Rong; Wasserloos, Karla; Watkins, Simon C.; Stevens, Troy; Li, Song; Tyurin, Vladimir; Kagan, Valerian E.; Pitt, Bruce R.Experimental Biology and Medicine (Maywood, NJ, United States) (2006), 231 (9), 1507-1515CODEN: EBMMBE; ISSN:1535-3702. (Society for Experimental Biology and Medicine)S-nitrosation of the metal binding protein, metallothionein (MT) appears to be a crit. link in affecting endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS)-derived nitric oxide (NO)-induced changes in cytoplasmic and nuclear labile zinc, resp. Although low mol. wt. S-nitrosothiols also appear to affect this signaling system, less is known about the ability of extracellular protein nitrosothiols to transnitrosate MT. Accordingly, we synthesized fluorescently labeled S-nitroso-albumin (SNO-albumin, a major protein S-nitrosothiol in plasma) and detd., via confocal microscopy in fixed tissue, that it is transported into cultured rat pulmonary vascular endothelial cells in a temp. sensitive fashion. The cells were transfected with an expression vector that encodes human MT-IIa cDNA sandwiched between enhanced cyan (donor) and yellow (acceptor) fluorescent proteins (FRET-MT) that can detect conformational changes in MT through fluorescence resonance energy transfer (FRET). SNO-albumin and the membrane-permeant low mol. wt. S-nitroso-L-cysteine Et ester (L-SNCEE) caused a conformational change in FRET-MT as ascertained by full spectral laser scanning confocal microscopy in live rat pulmonary vascular endothelial cells, a result which is consistent with transnitrosation of the reporter mol. Transnitrosation of FRET-MT by SNO-albumin, but not L-SNCEE, was sensitive to antisense oligonucleotide-mediated inhibition of the expression of cell surface protein disulfide isomerase (csPDI). These results extend the original observations of Ramachandran et al. and suggest that csPDI-mediated denitrosation helps to regulate the ability of the major plasma NO carrier (SNO-albumin) to transnitrosate endothelial cell mol. targets (e.g. MT).
- 123Stoyanovsky, D. M.; Tyurina, Y. Y.; Tyurin, V. A.; Anand, D.; Mandavia, D. N.; Gius, D.; Ivanova, J.; Pitt, B.; Billiar, T. R.; Kagan, V. E. Thioredoxin and lipoic acid catalyse the denitrosation of low molecular weight and protein S-nitrosothiols. J. Am. Chem. Soc. 2005, 127, 15815– 15823, DOI: 10.1021/ja0529135123Thioredoxin and Lipoic Acid Catalyze the Denitrosation of Low Molecular Weight and Protein S-NitrosothiolsStoyanovsky, Detcho A.; Tyurina, Yulia Y.; Tyurin, Vladimir A.; Anand, Deepthi; Mandavia, Dhara N.; Gius, David; Ivanova, Juliana; Pitt, Bruce; Billiar, Timothy R.; Kagan, Valerian E.Journal of the American Chemical Society (2005), 127 (45), 15815-15823CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)The nitrosation of cellular thiols has attracted much interest as a regulatory mechanism that mediates some of the pathophysiol. effects of nitric oxide (NO). In cells, virtually all enzymes contain cysteine residues that can be subjected to S-nitrosation, whereby this process often acts as an activity switch. Nitrosation of biol. thiols is believed to be mediated by N2O3, metal-nitrosyl complexes, and peroxynitrite. To date, however, enzymic pathways for S-denitrosation of proteins have not been identified. Herein, we present exptl. evidence that two ubiquitous cellular dithiols, thioredoxin and dihydrolipoic acid, catalyze the denitrosation of S-nitrosoglutathione, S-nitrosocaspase 3, S-nitrosoalbumin, and S-nitrosometallothionenin to their reduced state with concomitant generation of nitroxyl (HNO), the one-electron redn. product of NO. In these reactions, formation of NO and HNO was assessed by ESR spectrometry, potentiometric measurements, and quantification of hydroxylamine and sodium nitrite as end reaction products. Nitrosation and denitrosation of caspase 3 was correlated with its proteolytic activity. We also report that thioredoxin-deficient HeLa cells with mutated thioredoxin reductase denitrosate S-nitrosothiols less efficiently. We conclude that both thioredoxin and dihydrolipoic acid may be involved in the regulation of cellular S-nitrosothiols.
- 124Aravindakumar, C. T.; Ceulemans, J.; De Ley, M. Nitric oxide induces Zn2+ release from metallothionein by destroying zinc-sulphur clusters without concomitant formation of S-nitrosothiol. Biochem. J. 1999, 344, 253– 258, DOI: 10.1042/bj3440253124Nitric oxide induces Zn2+ release from metallothionein by destroying zinc-sulphur clusters without concomitant formation of S-nitrosothiolAravindakumar, Charuvila T.; Ceulemans, Jan; De Ley, MarcBiochemical Journal (1999), 344 (1), 253-258CODEN: BIJOAK; ISSN:0264-6021. (Portland Press Ltd.)The reaction of nitric oxide (NO) with metallothionein (MT) has been investigated at neutral pH under strictly anaerobic conditions. It is obsd. that NO mediates zinc release from MT by destroying zinc-sulfur clusters, but that it does not by itself S-nitrosylate MT in contrast to common belief. Zinc release and loss of thiolate groups under anaerobic conditions is found to be much slower than under aerobic conditions. The obsd. percentage loss of Zn2+ and thiolate groups after 3 h of NO treatment are 62 and 39%, resp. The reaction of NO with cysteine is reinvestigated and it is found that cysteine is quant. converted to cystine after 5 min of NO treatment at pH 7.3. At lower pH, a much lower rate of conversion is obsd. confirming the base-catalyzed nature of the reaction of NO with thiols. On the basis of these results, a reaction mechanism involving electrophilic attack of NO on thiolate groups and subsequent formation of a nitrogen-centered radical, MTSNOH, as intermediate is proposed for the reaction of NO with MT that leads to zinc release.
- 125Misra, R. R.; Hochadel, J. F.; Smith, G. T.; Cook, J. C.; Waalkes, M. P.; Wink, D. A. Evidence that nitric oxide enhances cadmium toxicity by displacing the metal from metallothionein. Chem. Res. Toxicol. 1996, 9, 326– 332, DOI: 10.1021/tx950109y125Evidence That Nitric Oxide Enhances Cadmium Toxicity by Displacing the Metal from MetallothioneinMisra, R. Rita; Hochadel, James F.; Smith, George T.; Cook, John C.; Waalkes, Michael P.; Wink, David A.Chemical Research in Toxicology (1996), 9 (1), 326-32CODEN: CRTOEC; ISSN:0893-228X. (American Chemical Society)When growing Chinese hamster ovary cells were treated for 24 h with 0.5, 0.75, or 1.0 mM CdCl2 followed by a 1-h exposure to 1.0, 1.5, or 2.0 mM 1,1-diethyl-2-hydroxy-2-nitrosohydrazine (DEA/NO), an NO-generating sodium salt, NO enhanced Cd-induced inhibition of colony forming ability without affecting Cd-induced cytolethality. In expts. designed to det. whether NO acts by displacing Cd from cellular metallothionein (MT), cells treated with 2.0 mM CdCl2 followed by 1.5 or 3.0 mM DEA/NO exhibited 29 and 38% redns., resp., in the amt. of Cd bound to MT. When purified rat liver MT was used to further characterize NO-induced release of Cd from MT, dose-related increases in Cd displacement were obsd. at DEA/NO concns. between 0.1 and 0.5 mM, and a plateau was reached at 3 mol of Cd displaced/mol of MT at higher DEA/NO concns. Compared to cells exposed to Cd or DEA/NO alone, cells treated with Cd followed by DEA/NO also exhibited a transient 2-3-fold decrease in c-myc proto-oncogene expression. Taken together, our results support the hypothesis that NO mediates Cd release from MT in vivo and suggest that intracellular generation of free Cd may induce DNA damage and force cells into a period of growth arrest. Such findings may have particular relevance with regard to the etiol. of Cd-induced carcinogenesis in human populations.
- 126Fabisiak, J. P.; Tyurin, V. A.; Tyurina, Y. Y.; Borisenko, G. G.; Korotaeva, A.; Pitt, B. R.; Lazo, J. S.; Kagan, V. E. Redox regulation of copper-metallothionein. Arch. Biochem. Biophys. 1999, 363, 171– 181, DOI: 10.1006/abbi.1998.1077126Redox Regulation of Copper-MetallothioneinFabisiak, James P.; Tyurin, Vladimir A.; Tyurina, Yulia Y.; Borisenko, Grigory G.; Korotaeva, Alexandra; Pitt, Bruce R.; Lazo, John S.; Kagan, Valerian E.Archives of Biochemistry and Biophysics (1999), 363 (1), 171-181CODEN: ABBIA4; ISSN:0003-9861. (Academic Press)Copper (Cu) is an essential element whose localization within cells must be carefully controlled to avoid Cu-dependent redox cycling. Metallothioneins (MTs) are cysteine-rich metal-binding proteins that exert cytoprotective effects during metal exposure and oxidative stress. The specific role of MTs, however, in modulating Cu-dependent redox cycling remains unresolved. Our studies utilized a chem. defined model system to study MT modulation of Cu-dependent redox cycling under reducing (Cu/ascorbate) and mild oxidizing (Cu/ascorbate + H2O2) conditions. In the presence of Cu and ascorbate, MT blocked Cu-dependent lipid oxidn. and ascorbyl radical formation with a stoichiometry corresponding to Cu/MT ratios ≤12. In the presence of H2O2 the degree of protection by MT was less and biol. oxidns. and radical formation were inhibited only up to Cu/MT ratios of 6. Phys. interaction of MT and Cu was measured by using low-temp. EPR of free Cu2+ in soln. The maximal amt. of EPR-silent Cu1+ (presumably in complex with MT) corresponded to 12 molar equivalents of Cu/MT under reducing conditions, but only 9 in the presence of H2O2. H2O2 modulated the ability of MT to protect HL-60 cells from Cu-induced cell death in a manner that correlated with the ability of MT to mitigate Cu-redox cycling in cell-free systems. Thus, optimal binding of Cu to MT is achieved under reducing conditions; however, a portion of this Cu appears releasable under oxidizing conditions. Release of free Cu from MT during oxidative stress could enhance the formation of reactive oxygen species and potentiate cellular damage. (c) 1999 Academic Press.
- 127Hao, Q.; Maret, W. Aldehydes release zinc from proteins. A pathway from oxidative stress/lipid peroxidation to cellular functions of zinc. FEBS J. 2006, 273, 4300– 4310, DOI: 10.1111/j.1742-4658.2006.05428.x127Aldehydes release zinc from proteins. A pathway from oxidative stress/lipid peroxidation to cellular functions of zincHao, Qiang; Maret, WolfgangFEBS Journal (2006), 273 (18), 4300-4310CODEN: FJEOAC; ISSN:1742-464X. (Blackwell Publishing Ltd.)Oxidative stress, lipid peroxidn., hyperglycemia-induced glycations and environmental exposures increase the cellular concns. of aldehydes. A novel aspect of the mol. actions of aldehydes, e.g. acetaldehyde and acrolein, is their reaction with the cysteine ligands of zinc sites in proteins and concomitant zinc release. Stoichiometric amts. of acrolein release zinc from zinc-thiolate coordination sites in proteins such as metallothionein and alc. dehydrogenase. Aldehydes also release zinc intracellularly in cultured human hepatoma (HepG2) cells and interfere with zinc-dependent signaling processes such as gene expression and phosphorylation. Thus both acetaldehyde and acrolein induce the expression of metallothionein and modulate protein tyrosine phosphatase activity in a zinc-dependent way. Since minute changes in the availability of cellular zinc have potent effects, zinc release is a mechanism of amplification that may account for many of the biol. effects of aldehydes. The zinc-releasing activity of aldehydes establishes relationships among cellular zinc, the functions of endogenous and xenobiotic aldehydes, and redox stress, with implications for pathobiochem. and toxicol. mechanisms.
- 128Miyazaki, I.; Asanuma, M.; Hozumi, H.; Miyoshi, K.; Sogawa, N. Protective effects of metallothionein against dopamine quinone-induced dopaminergic neurotoxicity. FEBS Lett. 2007, 581, 5003– 5008, DOI: 10.1016/j.febslet.2007.09.046128Protective effects of metallothionein against dopamine quinone-induced dopaminergic neurotoxicityMiyazaki, Ikuko; Asanuma, Masato; Hozumi, Hiroaki; Miyoshi, Ko; Sogawa, NorioFEBS Letters (2007), 581 (25), 5003-5008CODEN: FEBLAL; ISSN:0014-5793. (Elsevier B.V.)Dopamine (DA) quinone as DA neuron-specific oxidative stress conjugates with cysteine residues in functional proteins to form quinoproteins. Here, we examd. the effects of cysteine-rich metal-binding proteins, metallothionein (MT)-1 and -2, on DA quinone-induced neurotoxicity. MT quenched DA semiquinones in vitro. In dopaminergic cells, DA exposure increased quinoproteins and decreased cell viability; these were ameliorated by pretreatment with MT-inducer zinc. Repeated L-DOPA administration markedly elevated striatal quinoprotein levels and reduced the DA nerve terminals specifically on the lesioned side in MT-knockout parkinsonian mice, but not in wild-type mice. Our results suggested that intrinsic MT protects against L-DOPA-induced DA quinone neurotoxicity in parkinsonian mice by its quinone-quenching property.
- 129Gauthier, M. A.; Eibl, J. K.; Crispo, J. A.; Ross, G. M. Covalent arylation of metallothionein by oxidized dopamine products: a possible mechanism for zinc-mediated enhancement of dopaminergic neuron survival. Neurotoxic. Res. 2008, 14, 317– 328, DOI: 10.1007/BF03033856129Covalent arylation of metallothionein by oxidized dopamine products: a possible mechanism for zinc-mediated enhancement of dopaminergic neuron survivalGauthier, Michelle A.; Eibl, Joseph K.; Crispo, James A. G.; Ross, Gregory M.Neurotoxicity Research (2008), 14 (4), 317-328CODEN: NURRFI; ISSN:1029-8428. (F. P. Graham Publishing Co.)Metallothioneins are a group of low mol. wt. proteins which can be induced upon exposure to metal ions, including Zn2+. These cysteine-rich proteins are believed to have anti-oxidant-like properties due to their ability to scavenge free radicals with their multiple sulfhydryl groups. Dopamine is a neurotransmitter that can form toxic quinone and semi-quinone products in an oxidative environment. While Zn2+ is known to be toxic to some neuron subtypes, here we report a beneficial effect of Zn2+ on dopaminergic neurons and identify a mechanism through which metallothionein may scavenge toxic dopamine oxidn. products. Cultured embryonic neurons were treated with Zn2+, and the no. of dopaminergic neurons surviving after two or three weeks in culture was detd. We demonstrate that under these conditions metallothionein is upregulated and is able to form covalent arylation products with dopamine and 6-hydroxydopamine both in vitro and in culture. These expts. suggest that Zn2+ enhances the survival of dopaminergic neurons, and we propose that as a mechanism, upregulated metallothioneins form covalent adducts with both dopamine and 6-hydroxydopamine, resulting in the obsd. neuroprotective effect of Zn2+ on these cells. As Zn2+ homeostasis and modulation of metallothionein expression are often markers of neurodegeneration, these studies may have significant implications for understanding the underlying basis of degenerative diseases involving dopaminergic neurons, including Parkinson's disease.
- 130Maret, W. Metallothionein redox biology in the cytoprotective and cytotoxic functions of zinc. Exp. Gerontol. 2008, 43, 363– 369, DOI: 10.1016/j.exger.2007.11.005130Metallothionein redox biology in the cytoprotective and cytotoxic functions of zincMaret, WolfgangExperimental Gerontology (2008), 43 (5), 363-369CODEN: EXGEAB; ISSN:0531-5565. (Elsevier)A review. A crit. aspect of cellular zinc metab. is the tight control of the picomolar concns. of free zinc ions and their fluctuations to balance folding and misfolding of proteins, supply of thousands of zinc-requiring proteins with zinc, and dual functions of zinc as either a pro-oxidant or a pro-antioxidant. Zinc/sulfur (cysteine) bonds in proteins have a key role in this control because they generate redox-active coordination environments. Metallothionein (MT) is such a redox-active zinc protein, which couples biochem. to the cellular redox state. The coordination dynamics and redox state of its zinc/thiolate clusters det. cellular zinc availability. A fraction of MT in tissues and cells contains free thiols and disulfides. Thus, MT with seven zinc ions and twenty reduced thiols as characterized by high-resoln. 3D structures does not represent its biol. active form. Redox stress affects the zinc and redox buffering capacity of MT and elicits fluctuations of zinc ions that are potent effectors of multiple metabolic and signaling pathways. We are beginning to appreciate the sensitivity of cellular zinc homeostasis to perturbations, the clin. importance of linked zinc and redox imbalances for aging and the development of chronic diseases, and the tangible benefits of preventive and therapeutic nutritional interventions.
- 131Xiao, Z.; Wedd, A. G. The challenges of determining metal-protein affinities. Nat. Prod. Rep. 2010, 27, 768– 789, DOI: 10.1039/b906690j131The challenges of determining metal-protein affinitiesXiao, Zhi-Guang; Wedd, Anthony G.Natural Product Reports (2010), 27 (5), 768-789CODEN: NPRRDF; ISSN:0265-0568. (Royal Society of Chemistry)A review. A key property of metallo-proteins and -enzymes is the affinity of metal ion M for protein ligand P as defined by the dissocn. const. KD = [M][P]/[MP]. Its accurate detn. is essential for a quant. understanding of metal selection and speciation. However, the surfaces of proteins are defined by the sidechains of amino acids and so abound in good metal ligands (e.g., imidazole of histidine, thiol of cysteine, carboxylate of aspartic and glutamic acids, etc.). Consequently, adventitious binding of metal ions to protein surfaces is common with KD values ≥ 10-6 M. On the other hand, transport proteins responsible for chaperoning' essential metals to their cellular destinations appear to bind the metal ions selectively (KD < 10-7 M), both for speciation and to minimize the toxic effects of free' metal ions. These ions are normally bound with still higher affinities at their ultimate destinations (the active sites of metallo-proteins and -enzymes). This review surveys possible approaches to estn. of these dissocn. consts. and pinpoints the various problems assocd. with each approach.
- 132Pearson, R. G. Hard and soft acids and bases. J. Am. Chem. Soc. 1963, 85, 3533– 3539, DOI: 10.1021/ja00905a001132Hard and soft acids and basesPearson, Ralph G.Journal of the American Chemical Society (1963), 85 (22), 3533-9CODEN: JACSAT; ISSN:0002-7863.A number of Lewis acids of diverse types are classified as (a) or (b) following the criterion of Ahrland, et al. (CA 53, 960c). Other, auxiliary criteria are proposed. Class (a) acids prefer to bind to "hard" or nonpolarizable bases. Class (b) acids prefer to bind to "soft" or polarizable bases. Since class (a) acids are themselves "hard" and since class (b) acids are "soft" a simple, useful rule is proposed: hard acids bind strongly to hard bases and soft acids bind strongly to soft bases. The explanations for such behavior include: (1) various degrees of ionic and covalent σ-bonding; (2) π-bonding; (3) electron correlation phenomena; (4) solvation effects.
- 133Ejnik, J.; Robinson, J.; Zhu, J.; Forsterling, H.; Shaw, C. F.; Petering, D. H. Folding pathway of apo-metallothionein induced by Zn2+, Cd2+ and Co2+. J. Inorg. Biochem. 2002, 88, 144– 152, DOI: 10.1016/S0162-0134(01)00393-2133Folding pathway of apo-metallothionein induced by Zn2+, Cd2+ and Co2+Ejnik, John; Robinson, James; Zhu, Jianyu; Forsterling, Holger; Shaw, C. Frank; Petering, David H.Journal of Inorganic Biochemistry (2002), 88 (2), 144-152CODEN: JIBIDJ; ISSN:0162-0134. (Elsevier Science Inc.)Metal ion binding to the sulfhydryl groups of apometallothionein (apo-MT) causes both the formation of native metal-thiolate clusters and the folding of the polypeptide chain of each domain. Cd2+ and Zn2+ react with apo-MT to form metal-thiolate bonds in reactions that are complete within milliseconds and which are pH-dependent. Dual mixing expts. were conducted that involve the initial reaction of metal ion and apo-MT followed by mixing with 5,5'-N-dithio-bis(2-nitrobenzoate) or EDTA after 26 ms. They showed that structures had formed within the brief reaction period which were resistant to rapid reaction with reagents that interact with sulfhydryl groups or metal ions, resp. It was concluded that native metallothionein domains had been constituted within this brief period. Apo-MT was also titrated with Co2+ to yield Con-MT (n=1-7). Initially, Co2+ bound to independent, tetrahedral thiolate sites. Spectrophotometric anal. of the titrn. suggested that the independent Co(II) sites began to coalesce into clusters at n=4 (pH 7.2) or n=5 (pH 8.4). Back titrn. of free sulfhydryl groups (S) in Con-MT (n=1-7) with iodoacetamide at pH 7.2 confirmed that clustering began at n=4. Upon conversion of these alkylated structures to the corresponding 113Cd2+ species 113Cd NMR spectroscopy established that the location of Co(II) in Con-MT (n=1-3) was non-specific and that at n=4, the only observable structure was Co(II)4S11. The results suggest possible kinetic pathways of folding that are conceptually similar to those hypothesized for other small proteins.
- 134Bertini, I.; Luchinat, C.; Messori, L.; Vašák, M. Proton NMR studies of the cobalt(II)-metallothionein system. J. Am. Chem. Soc. 1989, 111, 7296– 7300, DOI: 10.1021/ja00201a002134Proton NMR studies of the cobalt(II)-metallothionein systemBertini, Ivano; Luchinat, Claudio; Messori, Luigi; Vasak, MilanJournal of the American Chemical Society (1989), 111 (19), 7296-300CODEN: JACSAT; ISSN:0002-7863.The binding of Co2+ to metallothionein (MT) from rabbit liver was followed through 1H NMR spectroscopy of cysteine side-chain protons and magnetic susceptibility measurements at room temp. in soln. In the course of the titrn. of apo-MT with Co2+, no isotropically shifted 1H NMR signals are obsd. up to ∼3 equiv of metal. A few broad signals in the 300-50 ppm region are obsd. when 4 equiv are added, whereas >30 sharp signals in the same region are obsd. when ≥5 equiv are added, up to the full metal complement of 7 equiv. Parallel magnetic susceptibility measurements show that little magnetic coupling among metal ions occurs at ≤3 equiv. Strong coupling is obsd. when the 4th equiv. is added, and a sizeable coupling is maintained, although with a smaller redn. of susceptibility value per metal ion, at ≤7 equiv. The results are consistent with a metal binding scheme in which the 1st 3 metals do not share any cysteine S donor, 2 metals possibly occupying the four-metal site and 1 the three-metal site. The 4th metal binds in the four-metal site, giving rise to a strongly coupled Co3 cluster, and the 5th completes the four-metal cluster, yielding a well-resolved 1H NMR spectrum. The 6th and 7th equiv complete the three-metal cluster, where they are probably strongly coupled as well. It is proposed that the well-resolved 1H NMR spectrum of the Co7MT deriv. essentially arises from the four-metal cluster.
- 135Kluska, K.; Adamczyk, J.; Krężel, A. Metal binding properties, stability and reactivity of zinc fingers. Coord. Chem. Rev. 2018, 367, 18– 64, DOI: 10.1016/j.ccr.2018.04.009135Metal binding properties, stability and reactivity of zinc fingersKluska, Katarzyna; Adamczyk, Justyna; Krezel, ArturCoordination Chemistry Reviews (2018), 367 (), 18-64CODEN: CCHRAM; ISSN:0010-8545. (Elsevier B.V.)A review. Zinc fingers (ZFs) are among the most structurally diverse protein domains. They interact with nucleic acids, other proteins and lipids to facilitate a multitude of biol. processes. Currently, there are more than 10 known classes of ZFs, with various architectures, metal binding modes, functions and reactivity. The versatility, selectivity and stability of these short amino acid sequences is achieved mainly by (i) residues participating in Zn(II) coordination (mostly Cys and His), (ii) hydrophobic core and ZF structure formation, and (iii) variable residues responsible for inter- and intramol. interactions. Since their discovery, ZFs have been extensively studied in terms of their structure, stability and recognition targets by the application of various methodologies. Studies based on interactions with other metal ions and their complexes have contributed to the understanding of their chem. properties and the discovery of new types of ZF complexes, such as gold fingers or lead fingers. Moreover, due to the presence of nucleophilic thiolates, ZFs are targets for reactive oxygen and nitrogen species as well as alkylating agents. Interactions with many reactive mols. lead to disturb the native Zn(II) coordination site which further result in structural and functional damage of the ZFs. The post-translational modifications including phosphorylation, acetylation, methylation or nitrosylation frequently affect ZFs function via changes in the protein structure and dynamics. Even though the literature is replete with structural and stability data regarding classical (ββα) ZFs, there is still a huge gap in the knowledge on physicochem. properties and reactivity of other ZF types. In this review, metal binding properties of ZFs and stability factors that modulate their functions are reviewed. These include interactions of ZFs with biogenic and toxic metal ions as well as damage occurring upon reaction with reactive oxygen and nitrogen species, the methodol. used for ZFs characterization, and aspects related to coordination chem.
- 136Kluska, K.; Peris-Díaz, M. D.; Płonka, D.; Moysa, A.; Dadlez, M.; Deniaud, A.; Bal, W.; Krężel, A. Formation of highly stable multinuclear AgnSn clusters in zinc fingers disrupts their structure and function. Chem. Commun. (Cambridge, U. K.) 2020, 56, 1329– 1332, DOI: 10.1039/C9CC09418K136Formation of highly stable multinuclear AgnSn clusters in zinc fingers disrupts their structure and functionKluska, Katarzyna; Peris-Diaz, Manuel D.; Plonka, Dawid; Moysa, Alexander; Dadlez, Michal; Deniaud, Aurelien; Bal, Wojciech; Krezel, ArturChemical Communications (Cambridge, United Kingdom) (2020), 56 (9), 1329-1332CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)Silver (Ag(I)) binding to consensus zinc fingers (ZFs) causes Zn(II) release inducing a gradual disruption of the hydrophobic core, followed by an overall conformational change and formation of highly stable AgnSn clusters. A compact eight-membered Ag4S4 structure formed by a CCCC ZF is the first cluster example reported for a single biol. mol. Ag(I)-induced conformational changes of ZFs can, as a consequence, affect transcriptional regulation and other cellular processes.
- 137Carpenter, M. C.; Shami Shah, A.; DeSilva, S.; Gleaton, A.; Su, A.; Goundie, B.; Croteau, M. L.; Stevenson, M. J.; Wilcox, D. E.; Austin, R. N. Thermodynamics of Pb(II) and Zn(II) binding to MT-3, a neurologically important metallothionein. Metallomics 2016, 8, 605– 617, DOI: 10.1039/C5MT00209E137Thermodynamics of Pb(II) and Zn(II) binding to MT-3, a neurologically important metallothioneinCarpenter, M. C.; Shami Shah, A.; DeSilva, S.; Gleaton, A.; Su, A.; Goundie, B.; Croteau, M. L.; Stevenson, M. J.; Wilcox, D. E.; Austin, R. N.Metallomics (2016), 8 (6), 605-617CODEN: METAJS; ISSN:1756-591X. (Royal Society of Chemistry)Isothermal titrn. calorimetry (ITC) was used to quantify the thermodn. of Pb2+ and Zn2+ binding to metallothionein-3 (MT-3). Pb2+ binds to zinc-replete Zn7MT-3 displacing each zinc ion with a similar change in free energy (G) and enthalpy (H). EDTA chelation measurements of Zn7MT-3 and Pb7MT-3 reveal that both metal ions are extd. in a tri-phasic process, indicating that they bind to the protein in three populations with different binding thermodn. Metal binding is entropically favored, with an enthalpic penalty that reflects the enthalpic cost of cysteine deprotonation accompanying thiolate ligation of the metal ions. These data indicate that Pb2+ binding to both apo MT-3 and Zn7MT-3 is thermodynamically favorable, and implicate MT-3 in neuronal lead biochem.
- 138Melenbacher, A.; Korkola, N. C.; Stillman, M. J. The pathways and domain specificity of Cu(I) binding to human metallothionein 1A. Metallomics 2020, 12, 1951– 1964, DOI: 10.1039/D0MT00215A138The pathways and domain specificity of Cu(I) binding to human metallothionein 1AMelenbacher, Adyn; Korkola, Natalie C.; Stillman, Martin J.Metallomics (2020), 12 (12), 1951-1964CODEN: METAJS; ISSN:1756-591X. (Royal Society of Chemistry)Copper is an essential element, but as a result of numerous adverse reactions, it is also a cellular toxin. Nature protects itself from these toxic reactions by binding cuprous copper to chaperones and other metalloproteins. Metallothionein has been proposed as a storage location for Cu(I) and potentially as the donor of Cu(I) to copper-dependent enzymes. We report that the addn. of Cu(I) to apo recombinant human metallothionein 1a cooperatively forms a sequential series of Cu(I)-cysteinyl thiolate complexes that have specific Cu(I) : MT stoichiometries of 6 : 1, 10 : 1, and finally 13 : 1. The individual domain Cu : SCys stoichiometries were detd. as Cu6S9 (for 6 : 1), Cu6S9 + Cu4S6 (for 10 : 1), and Cu6S9 + Cu7S9 (for 13 : 1) based on the no. of modified free cysteines not involved in Cu(I) binding. The stoichiometries are assocd. with Cu-SCys cluster formation involving bridging thiols in the manner similar to the clusters formed with Cd(II) and Zn(II). The locations of these clustered species within the 20 cysteine full protein were detd. from the unique speciation profiles of Cu(I) binding to the β and α domain fragments of recombinant human metallothionein 1a with 9 and 11 cysteines, resp. Competition reactions using these domain fragments challenged Cu(I) metalation of the βα protein, allowing the sequence of cluster formation in the full protein to be detd. Relative binding consts. for each Cu(I) bound are reported. The emission spectra of the Cu4S6, Cu6S9, and Cu7S9 clusters have unique λmax and phosphorescent lifetime properties. These phosphorescent data provide unambiguous supporting evidence for the presence of solvent shielded clusters reported concurrently by ESI-MS. Simulated emission spectra based on the cluster specific emission profiles matched the exptl. spectra and are used to confirm that the relative concns. seen by ESI-MS are representative of the soln. Our results suggest that the availability of a series of sequential Cu(I)-thiolate clusters provides flexibility as a means of protecting the cell from toxicity while still allowing for homeostatic control of the total copper content in the cell. This mechanism provides a dynamic and reactive method of reducing the cellular free copper concns.
- 139Payne, J. C.; ter Horst, M. A.; Godwin, H. A. Lead fingers: Pb2+ binding to structural zinc-binding domains determined directly by monitoring lead-thiolate charge-transfer bands. J. Am. Chem. Soc. 1999, 121, 6850– 6855, DOI: 10.1021/ja990899o139Lead Fingers: Pb2+ Binding to Structural Zinc-Binding Domains Determined Directly by Monitoring Lead-Thiolate Charge-Transfer BandsPayne, John C.; Horst, Marc A. ter; Godwin, Hilary ArnoldJournal of the American Chemical Society (1999), 121 (29), 6850-6855CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Here, we report that lead-thiolate charge-transfer bands (250-400 nm) can be used to monitor lead binding to cysteine-rich sites in proteins and report the application of this technique to det. the thermodn. of lead binding to a series of structural zinc-binding domains. These studies reveal that Pb2+ binds tightly to structural zinc-binding domains with dissocn. consts. that range from KdPb = 10-9 to 10-14 M, depending on the no. of cysteine residues in the metal-binding site. Competition expts. with Zn2+ lead to two striking conclusions: first, the two metals rapidly equilibrate, and second, the ratio of Pb2+ to Zn2+ bound to a particular site is detd. by the relative affinities of the two metals for that site, rather than being under kinetic control. We conclude that Pb2+ should be able to compete effectively with Zn2+ for Cys4 sites under physiol. conditions. Despite the fact that Pb2+ binds tightly to cysteine-rich structural zinc sites, CD and 1H NMR studies reveal that Pb2+ does not stabilize the correct fold of the peptides.
- 140Mesterházy, E.; Lebrun, C.; Crouzy, S.; Jancsó, A.; Delangle, P. Short oligopeptides with three cysteine residues as models of sulphur-rich Cu(I)- and Hg(II)-binding sites in proteins. Metallomics 2018, 10, 1232– 1244, DOI: 10.1039/C8MT00113H140Short oligopeptides with three cysteine residues as models of sulphur-rich Cu(I)- and Hg(II)-binding sites in proteinsMesterhazy, Edit; Lebrun, Colette; Crouzy, Serge; Jancso, Attila; Delangle, PascaleMetallomics (2018), 10 (9), 1232-1244CODEN: METAJS; ISSN:1756-591X. (Royal Society of Chemistry)The essential Cu(I) and the toxic Hg(II) ions possess similar coordination properties, and therefore, similar cysteine rich proteins participate in the control of their intracellular concn. In this work we present the metal binding properties of linear and cyclic model peptides incorporating the three-cysteine motifs, CxCxxC or CxCxC, found in metallothioneins. Cu(I) binding to the series of peptides at physiol. pH revealed to be rather complicated, with the formation of mixts. of polymetallic species. In contrast, the Hg(II) complexes display well-defined structures with spectroscopic features characteristic for a HgS2 and HgS3 coordination mode at pH = 2.0 and 7.4, resp. Stability data reflect a ca. 20 orders of magnitude larger affinity of the peptides for Hg(II) (log βpH7.4HgP ≈41) than for Cu(I) (log βpH7.4CuP ≈ 18). The different behavior with the two metal ions demonstrates that the use of Hg(II) as a probe for Cu(I), coordinated by thiolate ligands in water, may not always be fully appropriate.
- 141Vašák, M.; Kägi, J. H. R. Metallothioneins. In Encyclopedia of Inorganic Chemistry; King, R. B., Ed.; John Wiley & Sons: New York, NY, 1994; pp 2229– 2241.There is no corresponding record for this reference.
- 142Leiva-Presa, A.; Capdevila, M.; Gonzàlez-Duarte, P. Mercury(II) binding to metallothioneins. Variables governing the formation and structural features of the mammalian HgMT species. Eur. J. Biochem. 2004, 271, 4872– 4880, DOI: 10.1111/j.1432-1033.2004.04456.x142Mercury(II) binding to metallothioneins. Variables governing the formation and structural features of the mammalian Hg-MT speciesLeiva-Presa, Angels; Capdevila, Merce; Gonzalez-Duarte, PilarEuropean Journal of Biochemistry (2004), 271 (23/24), 4872-4880CODEN: EJBCAI; ISSN:0014-2956. (Blackwell Publishing Ltd.)With the aim of extending our knowledge on the reaction pathways of Zn-metallothionein (MT) and apo-MT species in the presence of Hg(II), we monitored the titrn. of Zn7-MT, Zn4-αMT and Zn3-βMT proteins, at pH 7 and 3, with either HgCl2 or Hg(ClO4)2 by CD and UV-vis spectroscopy. Detailed anal. of the optical data revealed that std. variables, such as the pH of the soln., the binding ability of the counterion (chloride or perchlorate), and the time elapsed between subsequent addns. of Hg(II) to the protein, play a determinant role in the stoichiometry, stereochem. and degree of folding of the Hg-MT species. Despite the fact that the effect of these variables is clear, it is difficult to generalize. Overall, it can be concluded that the reaction conditions [pH, time elapsed between subsequent addns. of Hg(II) to the protein] affect the structural properties more substantially than the stoichiometry of the Hg-MT species, and that the role of the counterion becomes particularly apparent on the structure of overloaded Hg-MT.
- 143Carter, K. P.; Young, A. M.; Palmer, A. E. Fluorescent sensors for measuring metal ions in living systems. Chem. Rev. 2014, 114, 4564– 4601, DOI: 10.1021/cr400546e143Fluorescent Sensors for Measuring Metal Ions in Living SystemsCarter, Kyle P.; Young, Alexandra M.; Palmer, Amy E.Chemical Reviews (Washington, DC, United States) (2014), 114 (8), 4564-4601CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)This review focuses on fluorescent sensors for transition metals commonly found in biol. organisms. The authors start this review by giving a basic overview of fluorescence imaging and sensor design, followed by a crit. anal. of parameters and properties to consider when using sensors in biol. systems. The authors then present a historical perspective of how the field has evolved. While this review focuses on transition metals, the authors discuss some of the key advances/milestones achieved in the development of fluorescent Ca2+ indicators as these helped lay the groundwork for much of the subsequent work developing sensors for transition metals. Finally, the authors highlight progress in sensor development for biol. metals, emphasizing recent advances, while including a discussion of the most widely used sensors. To demonstrate what kind of measurements can be made and what kind of information can be learned from using fluorescent sensors, the authors review several applications of sensors for defining metal homeostasis and dynamics in cells or organisms. The authors would also like to call readers' attention to several excellent prior reviews that focus on different aspects of sensor development.
- 144Hessels, A. M.; Merkx, M. Genetically-encoded FRET-based sensors for monitoring Zn2+ in living cells. Metallomics 2015, 7, 258– 266, DOI: 10.1039/C4MT00179F144Genetically-encoded FRET-based sensors for monitoring Zn2+ in living cellsHessels, Anne M.; Merkx, MaartenMetallomics (2015), 7 (2), 258-266CODEN: METAJS; ISSN:1756-591X. (Royal Society of Chemistry)A review. Genetically-encoded fluorescent sensor proteins are attractive tools for studying intracellular Zn2+ homeostasis and signaling. Here we provide an overview of recently developed sensors based on F.ovrddot.orster Resonance Energy Transfer (FRET). The pros and cons of the various sensors are discussed with respect to Zn2+ affinity, dynamic range, intracellular targeting and multicolor imaging. Recent applications of these sensors are described, as well as some of the challenges that remain to be addressed in future research.
- 145Morgan, M. T.; Bourassa, D.; Harankhedkar, S.; McCallum, A. M.; Zlatic, S. A.; Calvo, J. S.; Meloni, G.; Faundez, V.; Fahrni, C. J. Ratiometric two-photon microscopy reveals attomolar copper buffering in normal and Menkes mutant cells. Proc. Natl. Acad. Sci. U. S. A. 2019, 116, 12167– 12172, DOI: 10.1073/pnas.1900172116145Ratiometric two-photon microscopy reveals attomolar copper buffering in normal and Menkes mutant cellsMorgan, M. Thomas; Bourassa, Daisy; Harankhedkar, Shefali; McCallum, Adam M.; Zlatic, Stephanie A.; Calvo, Jenifer S.; Meloni, Gabriele; Faundez, Victor; Fahrni, Christoph J.Proceedings of the National Academy of Sciences of the United States of America (2019), 116 (25), 12167-12172CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Copper is controlled by a sophisticated network of transport and storage proteins within mammalian cells, yet its uptake and efflux occur with rapid kinetics. Present as Cu(I) within the reducing intracellular environment, the nature of this labile copper pool remains elusive. While glutathione is involved in copper homeostasis and has been assumed to buffer intracellular copper, the authors demonstrate with a ratiometric fluorescent indicator, crisp-17, that cytosolic Cu(I) levels are buffered to the vicinity of 1 aM, where negligible complexation by glutathione is expected. Enabled by the phosphine sulfide-stabilized phosphine (PSP) ligand design strategy, crisp-17 offers a Cu(I) dissocn. const. of 8 aM, thus exceeding the binding affinities of previous synthetic Cu(I) probes by four to six orders of magnitude. Two-photon excitation microscopy with crisp-17 revealed rapid, reversible increases in intracellular Cu(I) availability upon addn. of the ionophoric complex CuGTSM or the thiol-selective oxidant 2,2'-dithiodipyridine (DTDP). While the latter effect was dramatically enhanced in 3T3 cells grown in the presence of supplemental copper and in cultured Menkes mutant fibroblasts exhibiting impaired copper efflux, basal Cu(I) availability in these cells showed little difference from controls, despite large increases in total copper content. Intracellular copper is thus tightly buffered by endogenous thiol ligands with significantly higher affinity than glutathione. The dual utility of crisp-17 to detect normal intracellular buffered Cu(I) levels as well as to probe the depth of the labile copper pool in conjunction with DTDP provides a promising strategy to characterize perturbations of cellular copper homeostasis.
- 146Berg, J. M.; Merkle, D. L. On the metal ion specificity of zinc finger proteins. J. Am. Chem. Soc. 1989, 111, 3759– 3761, DOI: 10.1021/ja00192a050146On the metal ion specificity of zinc finger proteinsBerg, Jeremy M.; Merkle, Denise L.Journal of the American Chemical Society (1989), 111 (10), 3759-61CODEN: JACSAT; ISSN:0002-7863.The affinities of a "zinc finger" peptide, H-Pro-Phe-Pro-Cys-Lys-Glu-Glu-Gly-Cys-Glu-Lys-Gly-Phe-Thr-Ser-Leu-His-His-Leu-Thr-Arg-His-Ser-Leu-Thr-His-Thr-Gly-Glu-Lys-OH, for Co2+ and Zn2+ have been detd. by spectrophotometrically monitored titrns. The dissocn. consts. are 3.8 ± 0.5 × 10-6M and 2.8 ± 0.9 × 10-9M for Co2+ and Zn2+, resp. The preference for Zn2+ over Co2+ is interpreted in terms of changes in ligand field stabilization energy for Co2+ in leaving an octahedral environment in Co(OH2)62+ and entering a tetrahedral environment when bound by the peptide. This model predicts an enthalpy difference of -4.5 kcal/mol favoring Zn2+ over Co2+ for binding to the peptide. This compares favorably to the obsd. free energy difference of -4.3 kcal/mol. These results suggest that the loss in ligand field stabilization energy incumbent in the transition from an octahedral site in aq. soln. to a tetrahedral site in a protein may be responsible for the obsd. specificity of the "zinc finger" proteins (and other proteins with tetrahedral binding sites) for zinc.
- 147Lachenmann, M. J.; Ladbury, J. E.; Dong, J.; Huang, K.; Carey, P.; Weiss, M. A. Why zinc fingers prefer zinc: ligand-field symmetry and the hidden thermodynamics of metal ion selectivity. Biochemistry 2004, 43, 13910– 13925, DOI: 10.1021/bi0491999147Why Zinc Fingers Prefer Zinc: Ligand-Field Symmetry and the Hidden Thermodynamics of Metal Ion SelectivityLachenmann, Marcel J.; Ladbury, John E.; Dong, Jian; Huang, Kun; Carey, Paul; Weiss, Michael A.Biochemistry (2004), 43 (44), 13910-13925CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)The zinc finger, a motif of protein-nucleic acid recognition broadly conserved among eukaryotes, is a globular minidomain contg. a tetrahedral metal-binding site. Preferential coordination of Zn2+ (relative to Co2+) is proposed to reflect differences in ligand-field stabilization energies (LFSEs) due to complete or incomplete occupancy of d orbitals. LFSE predicts that the preference for Zn2+ should be purely enthalpic in accord with calorimetric studies of a high-affinity consensus peptide. Despite its elegance, the general predominance of LFSE is unclear as (i) the magnitude by which CP-1 prefers Zn2+ is greater than that expected and (ii) the analogous metal ion selectivity of a zinc metalloenzyme (carbonic anhydrase) is driven by changes in entropy rather than enthalpy. Because CP-1 was designed to optimize zinc binding, the authors have investigated the NMR structure and metal ion selectivity of a natural finger of lower stability derived from human tumor-suppressor protein WT1. Raman spectroscopy suggests that the structure of the WT1 domain is unaffected by interchange of Zn2+ and Co2+. As in CP-1, preferential binding of Zn2+ (relative to Co2+) is driven predominantly by differences in enthalpy, but in this case the enthalpic advantage is less than that predicted by LFSE. A theor. framework is presented to define the relationship between LFSE and other thermodn. factors, such as metal ion electroaffinities, enthalpies of hydration, and the topog. of the underlying folding landscape. The contribution of environmental coupling to entropy-enthalpy compensation is delineated in a formal thermodn. cycle. Together, these considerations indicate that LFSE provides an important but incomplete description of the stringency and thermodn. origin of metal-ion selectivity.
- 148Posewitz, M. C.; Wilcox, D. E. Properties of the Sp1 zinc finger 3 peptide: coordination chemistry, redox reactions, and metal binding competition with metallothionein. Chem. Res. Toxicol. 1995, 8, 1020– 1028, DOI: 10.1021/tx00050a005148Properties of the Sp1 Zinc Finger 3 Peptide: Coordination Chemistry, Redox Reactions, and Metal Binding Competition with MetallothioneinPosewitz, Matthew C.; Wilcox, Dean E.Chemical Research in Toxicology (1995), 8 (8), 1020-8CODEN: CRTOEC; ISSN:0893-228X. (American Chemical Society)Toxic and/or carcinogenic consequences may result from metal ion substitution for the Zn(II) in transcription factors contg. zinc fingers, and the small Cys-rich metal-binding protein metallothionein (MT) may play a role in this metal substitution. To begin to evaluate this hypothesis, with regard to the carcinogenic metal ion Ni(II), a peptide corresponding to the third finger of the transcription factor Sp1 (Sp1-3) has been synthesized and its metal binding and redox reactions have been studied. The peptide binds Zn(II), Co(II), and Ni(II), with spectroscopic data indicating a tetrahedral coordination for the latter two; metal ion affinities have been quantified (Kd = 6 × 10-10, 3 × 10-7, and 4 × 10-6, resp.) and less than those of an optimized zinc finger peptide (B. A. Krizek et al. (1993)) but greater than those of the second finger of transcription factor IIIA (J. M. Berg and D. L. Merkle (1989)). Reactions of the peptide and its metal-bound forms with dioxygen or hydrogen peroxide did not produce oxygen radical species; however, oxidn. of the two Sp1-3 cysteines was modulated by metal ions (Zn < Co = apo < Ni), suggesting a protective role for Zn(II) but an enhancing role for Ni(II). Metal binding competition between Sp1-3 and the α domain of human liver MT-2 (α-hMT2) indicates a similar affinity for Zn(II). However, α-hMT2 has a higher affinity for Ni(II), suggesting that MT may play a protective role by ensuring Zn(II), rather than Ni(II), coordination to zinc finger sequences of transcription factors.
- 149Romero-Isart, N.; Oliva, B.; Vašák, M. Influence of NH-Sγ bonding interactions on the structure and dynamics of metallothioneins. J. Mol. Model. 2010, 16, 387– 394, DOI: 10.1007/s00894-009-0542-x149Influence of NH-Sγ bonding interactions on the structure and dynamics of metallothioneinsRomero-Isart, Nuria; Oliva, Baldo; Vasak, MilanJournal of Molecular Modeling (2010), 16 (3), 387-394CODEN: JMMOFK; ISSN:0948-5023. (Springer GmbH)Mammalian metallothioneins (MII7MTs) show a clustered arrangement of the metal ions and a nonregular protein structure. The soln. structures of Cd3-thiolate cluster contg. β-domain of mouse β-MT-1 and rat β-MT-2 show high structural similarities, but widely differing structure dynamics. Mol. dynamics simulations revealed a substantially increased no. of NH-Sγ hydrogen bonds in β-MT-2, features likely responsible for the increased stability of the Cd3-thiolate cluster and the enfolding protein domain. Alterations in the NH-Sγ hydrogen-bonding network may provide a rationale for the differences in dynamic properties encountered in the β-domains of MT-1, -2, and -3 isoforms, believed to be essential for their different biol. function.
- 150Kochańczyk, T.; Nowakowski, M.; Wojewska, D.; Kocyła, A.; Ejchart, A.; Koźmiński, W.; Krężel, A. Metal-coupled folding as the driving force for the extreme stability of Rad50 zinc hook dimer assembly. Sci. Rep. 2016, 6, 36346, DOI: 10.1038/srep36346150Metal-coupled folding as the driving force for the extreme stability of Rad50 zinc hook dimer assemblyKochanczyk, Tomasz; Nowakowski, Michal; Wojewska, Dominika; Kocyla, Anna; Ejchart, Andrzej; Kozminski, Wiktor; Krezel, ArturScientific Reports (2016), 6 (), 36346CODEN: SRCEC3; ISSN:2045-2322. (Nature Publishing Group)The binding of metal ions at the interface of protein complexes presents a unique and poorly understood mechanism of mol. assembly. A remarkable example is the Rad50 zinc hook domain, which is highly conserved and facilitates the Zn2+-mediated homodimerization of Rad50 proteins. Here, we present a detailed anal. of the structural and thermodn. effects governing the formation and stability (logK12 = 20.74) of this evolutionarily conserved protein assembly. We have dissected the determinants of the stability contributed by the small β-hairpin of the domain surrounding the zinc binding motif and the coiled-coiled regions using peptides of various lengths from 4 to 45 amino acid residues, alanine substitutions and peptide bond-to-ester perturbations. In the studied series of peptides, an >650 000-fold increase of the formation const. of the dimeric complex arises from favorable enthalpy because of the increased acidity of the cysteine thiols in metal-free form and the structural properties of the dimer. The dependence of the enthalpy on the domain fragment length is partially compensated by the entropic penalty of domain folding, indicating enthalpy-entropy compensation. This study facilitates understanding of the metal-mediated protein-protein interactions in which the metal ion is crit. for the tight assocn. of protein subunits.
- 151Hong, S.-H.; Hao, Q.; Maret, W. Domain-specific fluorescence resonance energy transfer (FRET) sensors of metallothionein/thionein. Protein Eng., Des. Sel. 2005, 18, 255– 263, DOI: 10.1093/protein/gzi031151Domain-specific fluorescence resonance energy transfer (FRET) sensors of metallothionein/thioneinHong, S.-H.; Hao, Q.; Maret, W.Protein Engineering, Design & Selection (2005), 18 (6), 255-263CODEN: PEDSBR; ISSN:1741-0126. (Oxford University Press)Each of the two domains of mammalian metallothioneins contains a zinc-thiolate cluster. Employing site-directed mutagenesis and chem. modification, fluorescent probes were introduced into human metallothionein (isoform 2) with minimal perturbations of the structures of these clusters. The resulting FRET (fluorescence resonance energy transfer) sensors are specific for each domain. The design and construction of a sensor for the α-domain cluster is based on a FRET pair where a C-terminally added tryptophan serves as the donor for a fluorescence acceptor attached to a free cysteine in the linker region between the two domains. Mol. modeling studies and steady-state fluorescence polarization anisotropy measurements suggest unrestricted motion of the tryptophan donor, but limited motion of the AEDANS ({[(amino)ethyl]amino}naphthalene-1-sulfonic acid) acceptor, putting constraints on the use of the α-domain sensor with this FRET pair as a spectroscopic ruler. The fluorescent metallothioneins allow distance measurements during binding and removal of metals in the individual domains. The overall dimensions of the apoprotein, thionein, for which no structural information is available, do not seem to be significantly different from those of the holoprotein. The single- and double-labeled fluorescent metallothioneins overcome a longstanding impediment in studies of the function of this protein, namely its lack of intrinsic probe characteristics.
- 152Summers, K. L.; Mahrok, A. K.; Dryden, M. D. M; Stillman, M. J. Structural properties of metal-free apometallothioneins. Biochem. Biophys. Res. Commun. 2012, 425, 485– 492, DOI: 10.1016/j.bbrc.2012.07.141152Structural properties of metal-free apometallothioneinsSummers, Kelly L.; Mahrok, AnjanPreet K.; Dryden, Michael D. M.; Stillman, Martin J.Biochemical and Biophysical Research Communications (2012), 425 (2), 485-492CODEN: BBRCA9; ISSN:0006-291X. (Elsevier B.V.)The metalated forms of metallothionein (MT) are well studied (particularly Zn-MT, Cu-MT, and Cd-MT), but almost nothing is known about the chem. and structural properties of apo-MTs despite their importance in initial metalation and subsequent demetalation. Electrospray ionization mass spectrometry was used to provide a detailed view of the structural properties of the metal-free protein. Mass spectra of Zn7-MT and apo-MT at pH 7 exhibited the same charge state distribution, indicating that apo-MT was tightly folded like the metalated protein, whereas apo-MT at pH 3 exhibited a charge state spectrum assocd. with unfolding or denaturation. Benzoquinone was used to modify the Cys residues in the β-MT (9 Bq), and α-MT (11 Bq) fragments, and the full βα-MT (20 Bq) protein. ESI-MS showed that the overall vol. and, therefore, the extent of folding for the modified proteins was similar to that of Zn-MT. Mol. modeling using MM3-MD methods provided the vol. of each modified protein. The vols. of the partially modified proteins followed the same trend as the charge states, showing that ESI-MS is an excellent method with which to follow small changes in protein folding as a function of applied chem. stress. The data suggested that the structure of apo-βα-MT was more organized than previously considered.
- 153Drozd, A.; Wojewska, D.; Peris-Díaz, M. D.; Jakimowicz, P.; Krężel, A. Crosstalk of the structural and zinc buffering properties of mammalian metallothionein-2. Metallomics 2018, 10, 595– 613, DOI: 10.1039/C7MT00332C153Crosstalk of the structural and zinc buffering properties of mammalian metallothionein-2Drozd, Agnieszka; Wojewska, Dominika; Peris-Diaz, Manuel David; Jakimowicz, Piotr; Krezel, ArturMetallomics (2018), 10 (4), 595-613CODEN: METAJS; ISSN:1756-591X. (Royal Society of Chemistry)Metallothioneins (MTs), small cysteine-rich proteins, present in four major isoforms, are key proteins involved in zinc and copper homeostasis in mammals. To date, only one X-ray crystal structure of a MT has been solved. It demonstrates seven bivalent metal ions bound in two structurally independent domains with M4S11 (a) and M3S9 (β) clusters. Recent discoveries indicate that Zn(II) ions are bound with MT2 with the range from nano- to picomolar affinity, which dets. its cellular zinc buffering properties that are demonstrated by the presence of partially Zn(II)-depleted MT2 species. These forms serve as Zn(II) donors or acceptors and are formed under varying cellular free Zn(II) concns. Due to the lack of appropriate methods, knowledge regarding the structure of partially-depleted metallothionein is lacking. Here, we describe the Zn(II) binding mechanism in human MT2 with high resoln. with respect to particular Zn(II) binding sites, and provide structural insights into Zn(II)-depleted MT species. The results were obtained by the labeling of metal-free cysteine residues with iodoacetamide and subsequent top-down electrospray ionization anal., MALDI MS, bottom-up nanoLC-MALDI-MS/MS approaches and mol. dynamics (MD) simulations. The results show that the a-domain is formed sequentially in the first stages, followed by the formation of the β-domain, although both processes overlap, which is in contrast to the widely investigated cadmium MT. Independent ZnS4 cores are characteristic for early stages of domain formation and are clustered in later stages. However, Zn-S network rearrangement in the β-domain upon applying the seventh Zn(II) ion explains its lower affinity. Detailed anal. showed that the weakest Zn(II) ion assocs. with the β-domain by coordination to Cys21, which was also found to dissoc. first in the presence of the apo-form of sorbitol dehydrogenase. We found that Zn(II) binding to the isolated β-domain differs significantly from the whole protein, which explains its previously obsd. different Zn(II)-binding properties. MD results obtained for Zn(II) binding to the whole protein and isolated β-domain are highly convergent with mass spectrometry data. This study provides a comprehensive overview of the crosstalk of structural and zinc buffering related-to-thermodn. properties of partially metal-satd. mammalian MT2 and sheds more light on other MT proteins and zinc homeostasis.
- 154Wątły, J.; Łuczkowski, M.; Padjasek, M.; Krężel, A. Phytochelatins as a dynamic system for Cd(II) buffering from micro- to femtomolar range. Inorg. Chem. 2021, 60, 4657– 4675, DOI: 10.1021/acs.inorgchem.0c03639154Phytochelatins as a Dynamic System for Cd(II) Buffering from the Micro- to Femtomolar RangeWatly, Joanna; Luczkowski, Marek; Padjasek, Michal; Krezel, ArturInorganic Chemistry (2021), 60 (7), 4657-4675CODEN: INOCAJ; ISSN:0020-1669. (American Chemical Society)Phytochelatins (PCs) are short Cys-rich peptides with repeating γ-Glu-Cys motifs found in plants, algae, certain fungi, and worms. Their biosynthesis has been found to be induced by heavy metals-both biogenic and toxic. Among all metal inducers, Cd(II) has been the most explored from a biol. and chem. point of view. Although Cd(II)-induced PC biosynthesis has been widely examd., still little is known about the structure of Cd(II) complexes and their thermodn. stability. Here, we systematically investigated glutathione (GSH) and PC2-PC6 systems, with regard to their complex stoichiometries and spectroscopic and thermodn. properties. We paid special attention to the detn. of stability consts. using several complementary techniques. All peptides form CdL complexes, but CdL2 was found for GSH, PC2, and partially for PC3. Moreover, binuclear species CdxLy were identified for the series PC3-PC6 in an excess of Cd(II). Potentiometric and competition spectroscopic studies showed that the affinity of Cd(II) complexes increases from GSH to PC4 almost linearly from micromolar (log K7.4GSH = 5.93) to the femtomolar range (log K7.4PC4 = 13.39) and addnl. chain elongation does not increase the stability significantly. Data show that PCs form an efficient system which buffers free Cd(II) ions in the pico- to femtomolar range under cellular conditions, avoiding significant interference with Zn(II) complexes. Our study confirms that the favorable entropy change is the factor governing the elevation of phytochelatins' stability and illuminates the importance of the chelate effect in shifting the free Gibbs energy.
- 155Chen, S. H.; Russell, D. H. How closely related are conformations of protein ions sampled by IM-MS to native solution strcutures?. J. Am. Soc. Mass Spectrom. 2015, 26, 1433– 1443, DOI: 10.1007/s13361-015-1191-1155How Closely Related Are Conformations of Protein Ions Sampled by IM-MS to Native Solution Structures?Chen, Shu-Hua; Russell, David H.Journal of the American Society for Mass Spectrometry (2015), 26 (9), 1433-1443CODEN: JAMSEF; ISSN:1044-0305. (Springer)Here, we critically evaluate the effects of changes in the ion internal energy (Eint) on ion-neutral collision cross sections (CCS) of ions of two structurally diverse proteins, specifically the [M + 6H]6+ ion of ubiquitin (ubq6+), the [M + 5H]5+ ion of the intrinsically disordered protein (IDP) apo-metallothionein-2A (MT), and its partially- and fully-metalated isoform, the [CdiMT]5+ ion. The ion-neutral CCS for ions formed by "native-state" ESI show a strong dependence on Eint. Collisional activation is used to increase Eint prior to the ions entering and within the traveling wave (TW) ion mobility analyzer. Comparisons of exptl. CCSs with those generated by mol. dynamics (MD) simulation for soln.-phase ions and solvent-free ions as a function of temp. provide new insights about conformational preferences and retention of soln. conformations. The Eint-dependent CCSs, which reveal increased conformational diversity of the ion population, are discussed in terms of folding/unfolding of solvent-free ions. For example, ubiquitin ions that have low internal energies retain native-like conformations, whereas ions that are heated by collisional activation possess higher internal energies and yield a broader range of CCS owing to increased conformational diversity due to losses of secondary and tertiary structures. In contrast, the CCS profile for the IDP apoMT is consistent with kinetic trapping of an ion population composed of a wide range of conformers, and as the Eint is increased, these structurally labile conformers unfold to an elongated conformation.
- 156Chen, S. H.; Chen, L.; Russell, D. H. Metal-induced conformational changes of human metallothionien-2a: A combined theortical and experimental study of metal-free and partially metalated intermediates. J. Am. Chem. Soc. 2014, 136, 9499– 9508, DOI: 10.1021/ja5047878156Metal-Induced Conformational Changes of Human Metallothionein-2A: A Combined Theoretical and Experimental Study of Metal-Free and Partially Metalated IntermediatesChen, Shu-Hua; Chen, Liuxi; Russell, David H.Journal of the American Chemical Society (2014), 136 (26), 9499-9508CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Electrospray ionization ion mobility mass spectrometry (ESI IM-MS) and mol. dynamics (MD) simulations reveal new insights into metal-induced conformational changes and the mechanism for metalation of human metallothionein-2A (MT), an intrinsically disordered protein. ESI of solns. contg. apoMT yields multiple charge states of apoMT; following addn. of Cd2+ to the soln., ESI yields a range of CdiMT (i = 1-7) product ions. Ion mobility arrival-time distributions (ATDs) for the CdiMT (i = 0-7) ions reveal a diverse population of ion conformations. The ion mobility data clearly show that the conformational diversity for apoMT and partially metalated ions converges toward ordered, compact conformations as the no. of bound Cd2+ ions increase. MD simulations provide addnl. information on conformation candidates of CdiMT (i = 0-7) that supports the convergence of distinct conformational populations upon metal binding. Integrating the IM-MS and MD data provides a global view that shows stepwise conformational transition of an ensemble as a function of metal ion bound. ApoMT is comprised of a wide range of conformational states that populate between globular-like compact and coil-rich extended conformations. During the initial stepwise metal addn. (no. of metal ions bound i = 1-3), the metal ions bind to different sites to yield distinct conformations, whereas for i > 4, the conformational changes appear to be domain-specific, attributed to different degrees of disorder of the β domain; the β domain becomes more ordered as addnl. metal ions are added, promoting convergences to the dumbbell-shaped conformation.
- 157Chen, S. H.; Russell, W. K.; Russell, D. H. Combining chemical labeling, bottom-up and top-down ion-mobility mass spectrometry to identify metal-binding sites of partially metalated metallothionein. Anal. Chem. 2013, 85, 3229– 3237, DOI: 10.1021/ac303522h157Combining Chemical Labeling, Bottom-Up and Top-Down Ion-Mobility Mass Spectrometry To Identify Metal-Binding Sites of Partially Metalated MetallothioneinChen, Shu-Hua; Russell, William K.; Russell, David H.Analytical Chemistry (Washington, DC, United States) (2013), 85 (6), 3229-3237CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)Metalation and demetalation of human metallothionein-2A (MT) with Cd2+ is investigated by using chem. labeling and "bottom-up" and "top-down" proteomics approaches. Both metalation and demetalation of MT-2A by Cd2+ are shown to be domain specific and occur as two distinct processes. Metalation involves sequential addn. of Cd2+ to the α-domain resulting in formation of an intermediate, Cd4MT. Chem. labeling with N-ethylmaleimide (NEM) and tandem mass spectrometry expts. clearly show that the four metal ions are located in the α-domain. In the presence of excess Cd2+, the Cd4MT intermediate reacts to add Cd2+ to the β-domain to yield the fully metalated Cd7MT. Demetalation occurs in the reverse order, i.e., Cd2+ is removed (by EDTA) first from the β-domain followed by Cd2+ removal from the α-domain. Metalation of human MT-2A is shown to be metal ion specific by comparing relative metal ion binding consts. for Cd2+ and Zn2+.
- 158Dong, S.; Wagner, N. D.; Russell, D. H. Collision-induced unfolding of partially metalated metallothionein-2A: Tracking unfolding reactions of gas-phase ions. Anal. Chem. 2018, 90, 11856– 11862, DOI: 10.1021/acs.analchem.8b01622158Collision-Induced Unfolding of Partially Metalated Metallothionein-2A: Tracking Unfolding Reactions of Gas-Phase IonsDong, Shiyu; Wagner, Nicole D.; Russell, David H.Analytical Chemistry (Washington, DC, United States) (2018), 90 (20), 11856-11862CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)Metallothioneins (MTs) constitute a group of intrinsically disordered proteins that exhibit extreme diversity in structure, biol. functionality, and metal ion specificity. Structures of coordinatively satd. metalated MTs have been extensively studied, but very limited structural information for the partially metalated MTs exists. Here, the conformational preferences from partial metalation of rabbit metallothionein-2A (MT) by Cd2+, Zn2+, and Ag+ are studied using nanoelectrospray ionization ion mobility mass spectrometry. We also employ collision-induced unfolding to probe differences in the gas-phase stabilities of these partially metalated MTs. Our results show that despite their similar ion mobility profiles, Cd4-MT, Zn4-MT, Ag4-MT, and Ag6-MT differ dramatically in their gas-phase stabilities. Furthermore, the sequential addn. of each Cd2+ and Zn2+ ion results in the incremental stabilization of unique unfolding intermediates.
- 159Reddi, A. R.; Guzman, T. R.; Breece, R. M.; Tierney, D. L.; Gibney, B. R. Deducing the energetic cost of protein folding in zinc finger proteins using designed metallopeptides. J. Am. Chem. Soc. 2007, 129, 12815– 12827, DOI: 10.1021/ja073902+159Deducing the Energetic Cost of Protein Folding in Zinc Finger Proteins Using Designed MetallopeptidesReddi, Amit R.; Guzman, Tabitha R.; Breece, Robert M.; Tierney, David L.; Gibney, Brian R.Journal of the American Chemical Society (2007), 129 (42), 12815-12827CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Zinc finger transcription factors represent the largest single class of metalloproteins in the human genome. Binding of Zn(II) to their canonical Cys4, Cys3His1, or Cys2His2 sites results in metal-induced protein folding events required to achieve their proper structure for biol. activity. The thermodn. contribution of Zn(II) in each of these coordination spheres toward protein folding is poorly understood because of the coupled nature of the metal-ligand and protein-protein interactions. Using an unstructured peptide scaffold, GGG, we have employed fluorometry, potentiometry, and calorimetry to det. the thermodn. of Zn(II) binding to the Cys4, Cys3His1, and Cys2His2 ligand sets with minimal interference from protein folding effects. The data show that Zn(II) complexation is entropy driven and modulated by proton release. The formation consts. for Zn(II)-GGG with a Cys4, Cys3His1, or Cys2His2 site are 5.6×1016, 1.5×1015, or 2.5×1013 M-1, resp. Thus, the Zn(II)-Cys4, Zn(II)-Cys3His1, and Zn(II)-Cys2His2 interactions can provide up to 22.8, 20.7, and 18.3 kcal/mol, resp., in driving force for protein stabilization, folding, and/or assembly at pH values above the ligand pKa values. While the contributions from the three coordination motifs differ by 4.5 kcal/mol in Zn(II) affinity at pH 9.0, they are equiv. at physiol. pH, ΔG = -16.8 kcal/mol or a Ka = 2.0×1012 M-1. Calorimetric data show that this is due to proton-based enthalpy-entropy compensation between the favorable entropic term from proton release and the unfavorable enthalpic term due to thiol deprotonation. Since protein folding effects have been minimized in the GGG scaffold, these peptides possess nearly the tightest Zn(II) affinities possible for their coordination motifs. The Zn(II) affinities in each coordination motif are compared between the GGG scaffold and natural zinc finger proteins to det. the free energy required to fold the latter. Several proteins have identical Zn(II) affinities to GGG. That is, little, if any, of their Zn(II) binding energy is required to fold the protein, whereas some have affinities weakened by up to 5.7 kcal/mol; i.e., the Zn(II) binding energy is being used to fold the protein.
- 160Sénèque, O.; Latour, J. M. Coordination properties of zinc finger peptides revisited: ligand competition studies reveal higher affinities for zinc and cobalt. J. Am. Chem. Soc. 2010, 132, 17760– 17774, DOI: 10.1021/ja104992h160Coordination Properties of Zinc Finger Peptides Revisited: Ligand Competition Studies Reveal Higher Affinities for Zinc and CobaltSeneque, Olivier; Latour, Jean-MarcJournal of the American Chemical Society (2010), 132 (50), 17760-17774CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Zinc fingers are ubiquitous small protein domains which have a Zn(Cys)4-x(His)x site. They possess great diversity in their structure and amino acid compn. Using a family of six peptides, it was possible to assess the influence of hydrophobic amino acids on the metal-peptide affinities and on the rates of metal assocn. and dissocn. A model of a treble-clef zinc finger, a model of the zinc finger site of a redox-switch protein, and four variants of the classical ββα zinc finger were used. They differ in their coordination set, their sequence length, and their hydrophobic amino acid content. The speciation, metal binding consts., and structure of these peptides have been investigated as a function of pH. The zinc binding consts. of peptides, which adopt a well-defined structure, were found to be around 1015 at pH 7.0. The rates of zinc exchange between EDTA and the peptides were also assessed. We evidenced that the packing of hydrophobic amino acids into a well-defined hydrophobic core can have a drastic influence on both the binding const. and the kinetics of metal exchange. Notably, well-packed hydrophobic amino acids can increase the stability const. by 4 orders of magnitude. The half-life of zinc exchange was also seen to vary significantly depending on the sequence of the zinc finger. The possible causes for this behavior are discussed. This work will help in understanding the dynamics of zinc exchange in zinc-contg. proteins.
- 161Miłoch, A.; Krężel, A. Metal binding properties of the zinc finger metallome--insights into variations in stability. Metallomics 2014, 6, 2015– 2024, DOI: 10.1039/C4MT00149D161Metal binding properties of the zinc finger metallome - insights into variations in stabilityMiloch, Anna; Krezel, ArturMetallomics (2014), 6 (11), 2015-2024CODEN: METAJS; ISSN:1756-591X. (Royal Society of Chemistry)Zinc is one of the most widespread metal ions found in biol. systems. Of the expected 3000 zinc proteins in the human proteome, most contain zinc in structural sites. Among these structures, the most important are zinc fingers, which are well suited to facilitate interactions with DNA, RNA, proteins and lipid mols. Knowledge regarding their stability is a crit. issue in understanding the function of zinc fingers and their reactivity under fluxing cellular Zn(II) availability and different redox states. Zinc stability consts. that have been detd. using a variety of methods demonstrate wide diversity. Recent studies on the stability of consensus zinc fingers have demonstrated that the known metal-ion affinities for zinc fingers may have been underestimated by as much as three or more orders of magnitude. Here, using four natural ββα zinc fingers, we compare in detail several different methods that have been used for the detn. of zinc finger stability consts., such as common reverse-titrn., potentiometry, competition with metal chelators, and a new approach based on a three-step spectrophotometric titrn. We discuss why the stabilities of zinc fingers that are detd. spectrophotometrically are frequently underestimated due to the lack of effective equil. competition, which leads to large errors during the processing of the titrn. data. The literature stability consts. of many natural zinc fingers have been underestimated, and they are significantly lower when compared with the consensus peptides. Our data show that in the cell, some naturally occurring zinc fingers may potentially be unoccupied and are instead loaded transiently with Zn(II). Large variations in stability within the same class of zinc fingers have demonstrated that the thermodn. effects hidden in the sequence and structure are the key elements responsible for the differentiation of the stability of the zinc finger metallome.
- 162Alderighi, L.; Gans, P.; Ienco, A.; Peters, D.; Sabatini, A.; Vacca, A. Hyperquad simulation and speciation (HySS): a utility program for the investigation of equilibria involving soluble and partially soluble species. Coord. Chem. Rev. 1999, 184, 311– 318, DOI: 10.1016/S0010-8545(98)00260-4162Hyperquad simulation and speciation (HySS): a utility program for the investigation of equilibria involving soluble and partially soluble speciesAlderighi, Lucia; Gans, Peter; Ienco, Andrea; Peters, Daniel; Sabatini, Antonio; Vacca, AlbertoCoordination Chemistry Reviews (1999), 184 (), 311-318CODEN: CCHRAM; ISSN:0010-8545. (Elsevier Science S.A.)A review is presented with 23 refs. describing hyperquad simulation and speciation (HySS) that is a computer program written for the Windows operating system on personal computers which provides (a) a system for simulating titrn. curves and (b) a system for providing speciation diagrams. The calcns. relate to equil. in soln. and also include the possibility of formation of a partially sol. ppt. There are no restrictions on the no. of reagents that may be present or on the no. of complexes that may be formed.
- 163Irvine, G. W.; Pinter, T. B. J.; Stillman, M. J. Defining the metal binding pathways of human metallothionein 1a: balancing zinc availability and cadmium seclusion. Metallomics 2016, 8, 71– 81, DOI: 10.1039/C5MT00225G163Defining the metal binding pathways of human metallothionein 1a: balancing zinc availability and cadmium seclusionIrvine, Gordon W.; Pinter, Tyler B. J.; Stillman, Martin J.Metallomics (2016), 8 (1), 71-81CODEN: METAJS; ISSN:1756-591X. (Royal Society of Chemistry)Metallothioneins (MTs) are cysteine-rich, metal-binding proteins that are found throughout Nature. This ubiquity highlights their importance in essential metal regulation, heavy metal detoxification and cellular redox chem. Missing from the current description of MT function is the underlying mechanism by which MTs achieve their proposed biol. functions. To date, there have been conflicting reports on the mechanism of metal binding and the structures of the metal binding intermediates formed during metalation of apoMTs. The form of the metal-bound intermediates dictates the metal sequestering and metal-donating properties of the protein. Through a detailed anal. of spectral data from electrospray ionization mass spectrometric and CD methods we report that Zn(II) and Cd(II) metalation of the human MT1a takes place through two distinct pathways. The first pathway involves formation of beaded structures with up to five metals bound terminally to the 20 cysteines of the protein via a noncooperative mechanism. The second pathway is dominated by the formation of the four-metal domain cluster structure M4SCYS11via a cooperative mechanism. We report that there are different pathway preferences for Zn(II) and Cd(II) metalation of apo-hMT1a. Cd(II) binding follows the beaded pathway above pH 7.1 but beginning below pH 7.1 the clustered (Cd4Scys11) pathway begins to dominate. In contrast, Zn(II) binding follows the terminal, "beaded", pathway at all physiol. relevant pH (pH ≥ 5.2) only following the clustered pathway below pH 5.1. The results presented here allow us to reconcile the conflicting reports concerning the presence of different metalation intermediates of MTs. The conflict regarding cooperative vs. noncooperative binding mechanisms is also reconciled with the exptl. results described here. These two metal-specific pathways and the presence of radically different intermediate structures provide insight into the multi-functional nature of MT: binding Zn(II) terminally for donation to metalloenzymes and sequestering toxic Cd(II) in a cluster structure.
- 164Vazquez, F.; Vašák, M. Comparative 113Cd-n.m.r. studies on rabbit 113Cd7-, (Zn1,Cd6)- and partially metal-depleted 113Cd6-metallothionein-2a. Biochem. J. 1988, 253, 611– 614, DOI: 10.1042/bj2530611164Comparative cadmium-113 NMR studies on rabbit 113Cd7-, (Zn1,Cd6)- and partially metal-depleted 113Cd6-metallothionein-2aVazquez, Francisco; Vasak, MilanBiochemical Journal (1988), 253 (2), 611-14CODEN: BIJOAK; ISSN:0264-6021.Rabbit 113Cd7-metallothionein-2a (MT) contains 2 metal-thiolate clusters of 3 (cluster B) and 4 (cluster A) metal ions. The 113Cd NMR spectrum of 113Cd6-MT, isolated from 113Cd7-MT upon treatment with EDTA, is similar to that of 113Cd7-MT, but the cluster B resonances are lower in intensity, suggesting its cooperative metal depletion. Zn1,113Cd6-MT, formed by addn. of the Zn(II) ions to 113Cd6-MT, shows 113Cd NMR features characteristic of cluster B populations contg. both Cd(II) and Zn(II) ions. The overall intensity gain of the mixed cluster B resonances per Cd as to those in 113Cd6- and 113Cd7-MT suggests a stabilizing effect of the bound Zn(II) ions upon the previously established intramol. 113Cd exchange within this cluster.
- 165Li, T. Y.; Kraker, A. J.; Shaw, C. F., III; Petering, D. H. Ligand substitution reactions of metallothioneins with EDTA and apo-carbonic anhydrase. Proc. Natl. Acad. Sci. U. S. A. 1980, 77, 6334– 6338, DOI: 10.1073/pnas.77.11.6334165Ligand substitution reactions of metallothioneins with EDTA and apo-carbonic anhydraseLi, Ta-Yuen; Kraker, Alan J.; Shaw, C. Frank, III; Petering, David H.Proceedings of the National Academy of Sciences of the United States of America (1980), 77 (11), 6334-8CODEN: PNASA6; ISSN:0027-8424.The reactions of Zn-, Cd-, and Zn,Cd-thioneins with EDTA and apo-carbonic anhydrase were studied. The ligand substitution reaction of Zn with EDTA is multiphasic having both associative and dissociative components in the rate expression. The Cd sites are ∼2 orders of magnitude less reactive. In contrast, apo-carbonic anhydrase abstrs. Zn from Zn-thionein and Zn,Cd-thionein in 2nd-order processes that are 2-3 orders of magnitude more rapid than those involving EDTA and approach the rate for unligated Zn2+ with the apo-protein. In comparison with other Zn proteins, Zn-thionein contains unusually reactive metal sites, suggesting that this protein may be a physiol. Zn transfer protein, able to donate Zn to Zn-requiring apo macromols.
- 166Otvos, J. D.; Petering, D. H.; Shaw, C. F. Structure-reactivity relationships of metallothionein, a unique metal-binding protein. Comments Inorg. Chem. 1989, 9, 1– 35, DOI: 10.1080/02603598908035801166Structure-reactivity relationships of metallothionein, a unique metal-binding proteinOtvos, James D.; Petering, David H.; Shaw, C. FrankComments on Inorganic Chemistry (1989), 9 (1), 1-35CODEN: COICDZ; ISSN:0260-3594.A review, with 89 refs., on the structure of metallothioneins and relating the structure to its metal-binding properties and kinetics. Particular emphasis is placed on its binding of Cd, Au, Pt, and Zn.
- 167Namdarghanbari, M. A.; Meeusen, J.; Bachowski, G.; Giebel, N.; Johnson, J.; Petering, D. H. Reaction of the zinc sensor FluoZin-3 with Zn7-metallothionein. Inquiry into the existence of a proposed weak binding site. J. Inorg. Biochem. 2010, 104, 224– 231, DOI: 10.1016/j.jinorgbio.2009.11.003167Reaction of the zinc sensor FluoZin-3 with Zn7-metallothionein: Inquiry into the existence of a proposed weak binding siteNamdarghanbari, Mohammad Ali; Meeusen, Jeffrey; Bachowski, Gary; Giebel, Nicholas; Johnson, Jeremiah; Petering, David H.Journal of Inorganic Biochemistry (2010), 104 (3), 224-231CODEN: JIBIDJ; ISSN:0162-0134. (Elsevier B.V.)It has been reported that Zn7-metallothionein (MT), contains one weak binding site for Zn2+. To test this conclusion, rabbit liver MT isolated at pH 7 was reacted with chelating agents of modest affinity for Zn2+. Contrary to the previous study, no evidence was found for Zn2+ stoichiometrically bound to the protein with an apparent stability const. of about 108. Indeed, stability const. measurements based upon competition between Zn7-MT and ligands of known stability with Zn2+ showed that all of the protein bound Zn2+ displayed the same stability const. at pH 7.4 and 25 °C of (1.7 ± 0.6) × 1011. Brief reaction of Zn7-MT with strong acid converted it into MT* and upon re-neutralization into Zn7-MT*, which demonstrated reactivity of about 1 Zn2+/mol MT with competing ligands. Acid titrn. of Zn7-MT to pH 2 or below rapidly resulted in the formation of Zn7-MT* that displayed biphasic titrn. with base, revealing the re-binding of lower affinity Zn2+ between pH 5 and 7. Since MT is commonly acidified during prepn., care must be taken to document which form of the protein is present in subsequent expts. at pH 7.
- 168Hasler, D. W.; Jensen, L. T.; Zerbe, O.; Winge, D. R.; Vašák, M. Effect of the two conserved prolines of human growth inhibitory factor (metallothionein-3) on its biological activity and structure fluctuation: comparison with a mutant protein. Biochemistry 2000, 39, 14567– 14575, DOI: 10.1021/bi001569f168Effect of the Two Conserved Prolines of Human Growth Inhibitory Factor (Metallothionein-3) on Its Biological Activity and Structure Fluctuation: Comparison with a Mutant ProteinHasler, Daniel W.; Jensen, Laran T.; Zerbe, Oliver; Winge, Dennis R.; Vasak, MilanBiochemistry (2000), 39 (47), 14567-14575CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)Human neuronal growth inhibitory factor, a metalloprotein classified as metallothionein-3 (MT-3), impairs the survival and the neurite formation of cultured neurons. In these studies the double P7S/P9A mutant (mutMT-3) and single mutants P7S and P9A of human Zn7-MT-3 were generated, and their effects on the biol. activity and the structure of the protein were examd. The biol. results clearly established the necessity of both proline residues for the inhibitory activity, as even single mutants were found to be inactive. Using electronic absorption, CD (CD), magnetic CD (MCD), and 113Cd NMR spectroscopy, the structural features of the metal-thiolate clusters in the double mutant Cd7-mutMT-3 were investigated and compared with those of wild-type Cd7-MT-3 [Faller, P., Hasler, D. W., Zerbe, O., Klauser, S., Winge, D. R., and Vasak, M. (1999) Biochem. 38, 10158] and the well characterized Cd7-MT-2a from rabbit liver. Similarly to 113Cd7-MT-3 the 113Cd NMR spectrum of 113Cd7-mutMT-3 at 298 K revealed four major and three minor resonances (approx. 20% of the major ones) between 590 and 680 ppm, originating from a Cd4S11 cluster in the α-domain and a Cd3S9 cluster in the β-domain, resp. Due to the presence of dynamic processes in the structure of MT-3 and mutMT-3, all resonances showed the absence of resolved homonuclear [113Cd-113Cd] couplings and large apparent line widths (between 140 and 350 Hz). However, whereas in 113Cd7-mutMT-3 the temp. rise to 323 K resulted in a major recovery of the originally NMR nondetectable population of the Cd3S9 cluster resonances, no such temp. effect was obsd. in 113Cd7-MT-3. To account for the obsd. NMR features, a dynamic structural model for the β-domain is proposed, which involves a folded and a partially unfolded state. It is suggested that in the partially unfolded state a slow cis/trans isomerization of Cys-Pro(7) or Cys-Pro(9) amide bonds in 113Cd7-MT-3 takes place and that this process represents a rate-limiting step in a correct domain refolding. In addn., closely similar apparent stability consts. of human MT-3, mutMT-3, and rabbit MT-2a with Cd(II) and Zn(II) ions were found. These results suggest that specific structural features dictated by the repetitive (Cys-Pro)2 sequence in the β-domain of MT-3 and not its altered metal binding affinity compared to MT-1/MT-2 isoforms are responsible for the biol. activity of this protein.
- 169Peroza, E. A.; dos Santos Cabral, A.; Wan, X.; Freisinger, E. Metal ion release from metallothioneins: proteolysis as an alternative to oxidation. Metallomics 2013, 5, 1204– 1214, DOI: 10.1039/c3mt00079f169Metal ion release from metallothioneins: proteolysis as an alternative to oxidationPeroza, Estevao A.; dos Santos Cabral, Augusto; Wan, Xiaoqiong; Freisinger, EvaMetallomics (2013), 5 (9), 1204-1214CODEN: METAJS; ISSN:1756-591X. (Royal Society of Chemistry)Metallothioneins (MTs) are among others involved in the cellular regulation of essential ZnII and CuI ions. However, the high binding affinity of these proteins requires addnl. factors to promote metal ion release under physiol. conditions. The mechanisms and efficiencies of these processes leave many open questions. We report here a comprehensive anal. of the ZnII-release properties of various MTs with special focus on members of the four main subfamilies of plant MTs. ZnII competition expts. with the metal ion chelator 4-(2-pyridylazo)resorcinol (PAR) in the presence of the cellular redox pair glutathione (GSH)/glutathione disulfide (GSSG) show that plant MTs from the subfamilies MT1, MT2, and MT3 are remarkably more affected by oxidative stress than those from the Ec subfamily and the well-characterized human MT2 form. In addn., we evaluated proteolytic digestion with trypsin and proteinase K as an alternative mechanism for selective promotion of metal ion release from MTs. Also here the obsd. percentage of liberated metal ions depends strongly on the MT form evaluated. Closer evaluation of the data addnl. allowed deducing the thermodn. and kinetic properties of the ZnII release processes. The CuI-form of chickpea MT2 was used to exemplify that both oxidn. and proteolysis are also effective ways to increase the transfer of copper ions to other mols. ZnII release expts. with the individual metal-binding domains of Ec-1 from wheat grain reveal distinct differences from the full-length protein. This triggers the question about the roles of the long cysteine-free peptide stretches typical for plant MTs.
- 170Calvo, J. S.; Lopez, V. M.; Meloni, G. Non-coordinative metal selectivity bias in human metallothioneins metal-thiolate clusters. Metallomics 2018, 10, 1777– 1791, DOI: 10.1039/C8MT00264A170Non-coordinative metal selectivity bias in human metallothioneins metal-thiolate clustersCalvo, Jenifer S.; Lopez, Victor M.; Meloni, GabrieleMetallomics (2018), 10 (12), 1777-1791CODEN: METAJS; ISSN:1756-591X. (Royal Society of Chemistry)Mammalian metallothioneins (MT-1 through MT-4) are a class of metal binding proteins contg. two metal-thiolate clusters formed through the preferential coordination of d10 metals, Cu(I) and Zn(II), by 20 conserved cysteine residues located in two protein domains. MT metalation (homometallic or heterometallic Zn(II)/Cu(I) species) appears to be isoform specific and controlling zinc and copper concns. to perform specific and distinct biol. functions. Structural and functional relationships, and in vivo metalation studies, identified evolutionary features defining the metal-selectivity nature for MTs. Metallothionein-3 (MT-3) has been shown to possess the most pronounced Cu-thionein character forming Cu(I)-contg. species more favorably than metallothionein-2 (MT-2), which possesses the strongest Zn-thionein character. In this work, we identify isoform-specific determinants which control metal binding selectivity bias in different MTs isoforms. By studying the reactivity of Zn7MT-2, Zn7MT-3 and Zn7MT-3 mutants towards Cu(II) to form Cu(I)4Zn4MTs, we have identified isoform-specific key non-coordinating residues governing folding/outer sphere control of metal selectivity bias in MTs metal clusters. By mutating selected residues and motifs in MT-3 to the corresponding MT-2 amino acids, we dissected key roles in modulating cluster dynamic and metal exchange rates, in increasing the Cu(I)-affinity in MT-3 N-terminal β-domain and/or modulating the higher stability of the Zn(II)-thiolate cluster in MT-2 β-domain. We thus engineered MT-3 variants in which the copper-thionein character is converted into a zinc-thionein. These results provide new insights into the mol. determinants governing metal selectivity in metal-thiolate clusters.
- 171Kocyła, A.; Tran, J. B.; Krężel, A. Galvanization of protein-protein interactions in a dynamic zinc interactome. Trends Biochem. Sci. 2021, 46, 64– 79, DOI: 10.1016/j.tibs.2020.08.011171Galvanization of Protein-Protein Interactions in a Dynamic Zinc InteractomeKocyla, Anna; Tran, Jozef Ba; Krezel, ArturTrends in Biochemical Sciences (2021), 46 (1), 64-79CODEN: TBSCDB; ISSN:0968-0004. (Elsevier Ltd.)A review The presence of Zn2+ at protein-protein interfaces modulates complex function, stability, and introduces structural flexibility/complexity, chem. selectivity, and reversibility driven in a Zn2+-dependent manner. Recent studies have demonstrated that dynamically changing Zn2+ affects numerous cellular processes, including protein-protein communication and protein complex assembly. How Zn2+-involved protein-protein interactions (ZPPIs) are formed and dissoc. and how their stability and reactivity are driven in a zinc interactome remain poorly understood, mostly due to exptl. obstacles. Here, we review recent research advances on the role of Zn2+ in the formation of interprotein sites, their architecture, function, and stability. Moreover, we underline the importance of zinc networks in intersystemic communication and highlight bioinformatic and exptl. challenges required for the identification and investigation of ZPPIs.
- 172Pinter, T. B.; Stillman, M. J. The zinc balance: competitive zinc metalation of carbonic anhydrase and metallothionein 1A. Biochemistry 2014, 53, 6276– 6285, DOI: 10.1021/bi5008673172The Zinc Balance: Competitive Zinc Metalation of Carbonic Anhydrase and Metallothionein 1APinter, Tyler B. J.; Stillman, Martin J.Biochemistry (2014), 53 (39), 6276-6285CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)The small, cysteine-rich metallothionein family of proteins is currently considered to play a crit. role in the provision of metals to metalloenzymes. However, there is limited information available on the mechanisms of these fundamentally important interactions. We report on the competitive zinc metalation of apocarbonic anhydrase in the presence of apometallothionein 1A using electrospray-ionization mass spectrometry. These expts. revealed the relative affinities of zinc to all species in soln. The carbonic anhydrase is shown to compete efficiently only against Zn5-7MT. The calcd. equil. zinc binding consts. of each of the 7 zinc metallothionein 1A species ranged from a high of (log(KF)) 12.5 to a low of 11.8. The 8 equil. consts. connecting the 10 active species in competition for the zinc were modeled by fitting the KF values of the 8 competitive bimol. reactions to the ESI-mass spectral data. These modeled K values are shown to be exptl. connected to the metalation efficiency of the carbonic anhydrase. The series of 7 metallothionein binding affinities for zinc highlight the buffering role of zinc metallothioneins that permit simultaneously zinc storage and zinc sensing. Finally, the significance of the multiple zinc binding affinities of zinc metallothionein is discussed in relation to zinc homeostasis.
- 173Maret, W. Analyzing free zinc(II) ion concentrations in cell biology with fluorescent chelating molecules. Metallomics 2015, 7, 202– 211, DOI: 10.1039/C4MT00230J173Analyzing free zinc(ii) ion concentrations in cell biology with fluorescent chelating moleculesMaret, WolfgangMetallomics (2015), 7 (2), 202-211CODEN: METAJS; ISSN:1756-591X. (Royal Society of Chemistry)A review. Essential metal ions are tightly controlled in biol. systems. An understanding of metal metab. and homeostasis is being developed from quant. information of the sizes, concns., and dynamics of cellular and subcellular metal ion pools. In the case of human zinc metab., minimally 24 proteins of two zinc transporter families and a dozen metallothioneins participate in cellular uptake, extrusion, and re-distribution among cellular compartments. Significantly, zinc(II) ions are now considered signaling ions in intra- and intercellular communication. Such functions require transients of free zinc ions. It is exptl. quite challenging to distinguish zinc that is protein-bound from zinc that is not bound to proteins. Measurement of total zinc is relatively straightforward with anal. techniques such as at. absorption/emission spectroscopy or inductively coupled plasma mass spectrometry. Total zinc concns. of human cells are 200-300 μM. In contrast, the pool of non-protein bound zinc is mostly examd. with fluorescence microscopy/spectroscopy. There are two widely applied fluorescence approaches, one employing low mol. wt. chelating agents ("probes") and the other metal-binding proteins ("sensors"). The protein sensors, such as the CALWY, Zap/ZifCY, and carbonic anhydrase-based sensors, can be genetically encoded and have certain advantages in terms of controlling intracellular concn., localization, and calibration. When employed correctly, both probes and sensors can establish qual. differences in free zinc ion concns. However, when quant. information is sought, the assumptions underlying the applications of probes and sensors must be carefully examd. and even then measured pools of free zinc ions remain methodol. defined. A consensus is building that the steady-state free zinc ion concns. in the cytosol are in the picomolar range but there is no consensus on their concns. in subcellular compartments. Applying the extensive toolbox of available probes/sensors in biol. systems requires an understanding of the principles of cellular zinc homeostasis and the chem. biol. of the probes and sensors. Regardless of limitations in specificity (for a particular metal ion), selectivity (for a particular metal pool), and sensitivity (detection limit), the technol. is making remarkable contributions to imaging zinc with high spatiotemporal resoln. in single cells and to defining the biochem. functions of zinc ions in cellular regulation.
- 174Kochańczyk, T.; Drozd, A.; Krężel, A. Relationship between the architecture of zinc coordination and zinc binding affinity in proteins-insights into zinc regulation. Metallomics 2015, 7, 244– 257, DOI: 10.1039/C4MT00094C174Relationship between the architecture of zinc coordination and zinc binding affinity in proteins - insights into zinc regulationKochanczyk, Tomasz; Drozd, Agnieszka; Krezel, ArturMetallomics (2015), 7 (2), 244-257CODEN: METAJS; ISSN:1756-591X. (Royal Society of Chemistry)A review. Zinc proteins are an integral component of the proteome of all domains of life. Zn(II), one of the most widespread transition elements, serves multiple functions in proteins, such as a catalytic co-factor, a structural center and a signaling component. The mechanism by which proteins assoc. with and dissoc. from Zn(II) and the factors that modulate their affinity and stability remain incompletely understood. In this article, we aim to address how zinc binding sites present in proteins differ in their architecture and how their structural arrangement is assocd. with protein function, thermodn. and kinetic stability, reactivity, as well as zinc-dependent regulation. Here, we emphasize that the concn.-dependent functionality of the interprotein zinc binding site may serve as another factor regulating the relationship between cellular Zn(II) availability and protein function.
- 175Maret, W. Zinc in cellular regulation: The nature and significance of ″zinc signals″. Int. J. Mol. Sci. 2017, 18, 2285, DOI: 10.3390/ijms18112285175Zinc in cellular regulation: the nature and significance of "zinc signals"Maret, WolfgangInternational Journal of Molecular Sciences (2017), 18 (11), 2285/1-2285/12CODEN: IJMCFK; ISSN:1422-0067. (MDPI AG)In the last decade, we witnessed discoveries that established Zn2+ as a second major signalling metal ion in the transmission of information within cells and in communication between cells. Together with Ca2+ and Mg2+, Zn2+ covers biol. regulation with redox-inert metal ions over many orders of magnitude in concns. The regulatory functions of zinc ions, together with their functions as a cofactor in about three thousand zinc metalloproteins, impact virtually all aspects of cell biol. This article attempts to define the regulatory functions of zinc ions, and focuses on the nature of zinc signals and zinc signalling in pathways where zinc ions are either extracellular stimuli or intracellular messengers. These pathways interact with Ca2+, redox, and phosphorylation signalling. The regulatory functions of zinc require a complex system of precise homeostatic control for transients, subcellular distribution and traffic, organellar homeostasis, and vesicular storage and exocytosis of zinc ions.
- 176Maret, W.; Li, Y. Coordination dynamics of zinc in proteins. Chem. Rev. 2009, 109, 4682– 4707, DOI: 10.1021/cr800556u176Coordination dynamics of zinc in proteinsMaret, Wolfgang; Li, YuanChemical Reviews (Washington, DC, United States) (2009), 109 (10), 4682-4707CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)A review. In thousands of proteins, Zn participates in enzymic catalysis, structural organization, and/or regulation of function. The great no. and variety of Zn-contg. proteins has stimulated research on Zn2+ regulatory and chem. mechanisms that safeguard the distribution of Zn to proteins within the cell in a timely and spatially coordinated manner. The chem. properties of Zn in enzymes is largely attributed to its function as a strong Lewis acid. Generally, fast ligand exchange, stereochem. flexibility, and redox-inertness are addnl. characteristics for the selection of Zn2+ ions in the function of so many proteins. Here, the authors address the dynamics of Zn2+ coordination and the cellular distribution of Zn, namely, how proteins control Zn2+ (Zn2+ metalloregulation), how Zn2+ controls proteins (Zn2+ signaling), and how Zn2+ concns. are regulated and buffered intracellularly.
- 177Mattapalli, H.; Monteith, W. B.; Burns, C. S.; Danell, A. S. Zinc deposition during ESI-MS analysis of peptide-zinc-complexes. J. Am. Soc. Mass Spectrom. 2009, 20, 2199– 2205, DOI: 10.1016/j.jasms.2009.08.007177Zinc Deposition During ESI-MS Analysis of Peptide-Zinc ComplexesMattapalli, Haritha; Monteith, William B.; Burns, Colin S.; Danell, Allison S.Journal of the American Society for Mass Spectrometry (2009), 20 (12), 2199-2205CODEN: JAMSEF; ISSN:1044-0305. (Elsevier Inc.)Electrospray ionization (ESI) mass spectrometry (MS) has proven to be an extremely powerful technique for studying the stoichiometry and binding strength of peptide-metal complexes. We have found a significant new problem in the ESI-MS of zinc-peptide systems involving the deposition of zinc in the ESI emitter. This deposition of zinc during the expt. removes a significant amt. of zinc ions from the soln., impacting the resulting mass spectral intensities used to quantify the amt. of the zinc-bound species. Anal. of infused zinc-peptide samples with at. absorption spectrometry and with a custom-built nanoflow ESI source confirms the alteration of the analyte solns. with pos. or neg. or no potential applied to the emitter. Ultimately, the location of the zinc deposition was detd. to be the stainless steel emitter. The use of a custom-built nanoESI interface using glass emitters was found to mitigate the zinc deposition problem. The phenomenon of metal deposition warrants further investigation as it may not be limited to just zinc and may represent a significant obstacle in the ESI-MS anal. of all protein-metal systems.
- 178Kostyukevich, Y.; Kononikhin, A.; Popov, I.; Indeykina, M.; Kozin, S. A.; Makarov, A. A.; Nikolaev, E. Supermetallization of peptides and proteins during electrospray ionization. J. Mass Spectrom. 2015, 50, 1079– 1087, DOI: 10.1002/jms.3622178Supermetallization of peptides and proteins during electrospray ionizationKostyukevich, Yury; Kononikhin, Alexey; Popov, Igor; Indeykina, Maria; Kozin, Sergey A.; Makarov, Alexander A.; Nikolaev, EugeneJournal of Mass Spectrometry (2015), 50 (9), 1079-1087CODEN: JMSPFJ; ISSN:1076-5174. (John Wiley & Sons Ltd.)The formation of metal-peptide complexes during electrospray ionization (ESI) is a widely known phenomenon and is often considered to be undesirable. Such effect considerably limits the use of ESI mass spectrometry for the study of biol. relevant metal-peptide compds. that are present in the soln. and play crit. roles in many bioprocesses such as progression of neurodegenerative diseases. Under specific conditions such as high temp. of the desolvating capillary, an interesting effect, which can be called supermetallization, occurs. Using a model peptide Aβ amyloid domain 1-16, an increase in the temp. of the desolvating capillary results in multiple substitutions of hydrogen atoms by Zn atoms in this peptide. At high temps. (T ∼ 400°), up to 11 zinc atoms can be covalently bound to (1-16) Aβ. Supermetallization of (1-16) Aβ depends on the solvent compn. and pH. Supermetallization was also demonstrated for proteins, such as ubiquitin and cytochrome C. That proves that the supermetallization is a general phenomenon for peptides and proteins. For the structural study of supermetallized complexes, electron-capture dissocn. (ECD) fragmentation was applied. The effect of hydrogen rearranging during ECD was obsd. In addn., quantum chem. calcns. were used to est. the possible structures of different supermetallized complexes. These results allow a more deep understanding of the limitations of the use of ESI mass spectrometry for the study of biol. relevant metal-peptide complexes.
- 179Blindauer, C. A.; Leszczyszyn, O. I. Metallothioneins: unparalleled diversity in structures and functions for metal ion homeostasis and more. Nat. Prod. Rep. 2010, 27, 720– 741, DOI: 10.1039/b906685n179Metallothioneins: Unparalleled diversity in structures and functions for metal ion homeostasis and moreBlindauer, Claudia A.; Leszczyszyn, Oksana I.Natural Product Reports (2010), 27 (5), 720-741CODEN: NPRRDF; ISSN:0265-0568. (Royal Society of Chemistry)A review. Metallothioneins have been the subject of intense study for five decades, and have greatly inspired the development of bio-anal. methodologies including multi-dimensional and multi-nuclear NMR. With further advancements in mol. biol., protein science, and instrumental techniques, recent years have seen a renaissance of research into metallothioneins. The current report focuses on in vitro studies of so-called class II metallothioneins from a variety of phyla, highlighting the diversity of metallothioneins in terms of structure, biol. functions, and mol. functions such as metal ion specificity, thermodn. stabilities, and kinetic reactivity. We are still far from being able to predict any of these properties, and further efforts will be required to generate the knowledge that will enable a better understanding of what governs the biol. and chem. properties of these unusual and intriguing small proteins.
- 180Chan, J.; Huang, Z.; Merrifield, M. E.; Salgado, M. T.; Stillman, M. J. Studies of metal binding reactions in metallothioneins by spectroscopic, molecular biology, and molecular modeling techniques. Coord. Chem. Rev. 2002, 233, 319– 339, DOI: 10.1016/S0010-8545(02)00176-5180Studies of metal binding reactions in metallothioneins by spectroscopic, molecular biology, and molecular modeling techniquesChan, Jayna; Huang, Zuyun; Merrifield, Maureen E.; Salgado, Maria T.; Stillman, Martin J.Coordination Chemistry Reviews (2002), 233-234 (), 319-339CODEN: CCHRAM; ISSN:0010-8545. (Elsevier Science B.V.)A review with refs. Metallothioneins (MTs) are a class of metal-binding proteins characterized by a high cysteine content (up to 30% of the amino acid residues), low mol. wt., and lack of arom. amino acid residues. Remarkable metal-binding properties have been reported both in vivo and in vitro by a no. of different anal. techniques. Chem. and spectroscopic studies have shown that an unusually wide range of metals bind to MT. 113Cd and 1H-NMR techniques have been used successfully to det. the structures in a no. of different proteins. Together with X-ray diffraction results, analyses of these NMR data have established that in mammalian Cd7-MT and Zn7-MT, the metals are tetrahedrally coordinated in two isolated domains with stoichiometries of M4S11 and M3S9. Recently, Stillman, collaborators, and coworkers have characterized the Zn(II), Cd(II), Cu(I), Ag(I), Au(I) and Hg(II) contg. forms of MTs from mammalian, yeast, and recombinant human sources using a no. of different spectroscopic techniques. Optical spectroscopy, and in particular CD and luminescence, have provided rich details of a complicated metal binding chem., whether metals are added directly to the metal free, apo-MT, or to the Zn-contg. protein. Electrospray ionization mass spectrometry is a powerful technique for the characterization of proteins in general directly from soln. For MTs, both from mammalian and recombinant sources, use of this technique allows study of the details of metal binding to the protein as a function of metal loading and pH. Kinetic studies allow detn. of the metal binding mechanism when Cu(I) binds to both the metal-free and Zn(II) protein. Scanning tunneling microscopy is a surface anal. technique capable of producing nanoscale images with at. resoln. and is being applied to obtain images of MT on Au(111) surfaces. Mol. modeling, together with XANES, XAFS, and structural information from 2D-1H-NMR data allow the detn. of 3D structures of a range of MTs. Finally, mol. dynamics (MD) calcns. have been carried out to investigate the motion of the metal-contg. and metal free peptides, with special interest in the region of the metal binding site.
- 181Krężel, A.; Maret, W. The functions of metamorphic metallothioneins in zinc and copper metabolism. Int. J. Mol. Sci. 2017, 18, 1237, DOI: 10.3390/ijms18061237181The functions of metamorphic metallothioneins in zinc and copper metabolismKrezel, Artur; Maret, WolfgangInternational Journal of Molecular Sciences (2017), 18 (6), 1237/1-1237/20CODEN: IJMCFK; ISSN:1422-0067. (MDPI AG)A REVIEW. Recent discoveries in zinc biol. provide a new platform for discussing the primary physiol. functions of mammalian metallothioneins (MTs) and their exquisite zinc-dependent regulation. It is now understood that the control of cellular zinc homeostasis includes buffering of Zn2+ ions at picomolar concns., extensive subcellular re-distribution of Zn2+, the loading of exocytotic vesicles with zinc species, and the control of Zn2+ ion signalling. In parallel, characteristic features of human MTs became known: their graded affinities for Zn2+ and the redox activity of their thiolate coordination environments. Unlike the single species that structural models of mammalian MTs describe with a set of seven divalent or eight to twelve monovalent metal ions, MTs are metamorphic. In vivo, they exist as many species differing in redox state and load with different metal ions. The functions of mammalian MTs should no longer be considered elusive or enigmatic because it is now evident that the reactivity and coordination dynamics of MTs with Zn2+ and Cu+ match the biol. requirements for controlling-binding and delivering-these cellular metal ions, thus completing a 60-yr search for their functions. MT represents a unique biol. principle for buffering the most competitive essential metal ions Zn2+ and Cu+. How this knowledge translates to the function of other families of MTs awaits further insights into the specifics of how their properties relate to zinc and copper metab. in other organisms.
- 182Li, Y.; Maret, W. Human metallothionein metallomics. J. Anal. At. Spectrom. 2008, 23, 1055– 1062, DOI: 10.1039/b802220h182Human metallothionein metallomicsLi, Yuan; Maret, WolfgangJournal of Analytical Atomic Spectrometry (2008), 23 (8), 1055-1062CODEN: JASPE2; ISSN:0267-9477. (Royal Society of Chemistry)A review. Research on metallothionein (MT), unlike studies on any other metalloprotein, captures best the difficulties that investigators faced, and continue to face, when relating protein structure to protein function. This article discusses the structural complexity of the family of human MTs with the goal of providing a background for future investigations of their functions in health and disease by mol. and at. spectroscopies. The anal. (bio)chemist needs to overcome formidable challenges in terms of sample prepn. and speciation. MTs are expressed tissue- and isoprotein-specifically; their expression responds to a variety of physiol. and environmental stimuli; they are present as closely related products of at least ten different genes, several of which can be expressed simultaneously at considerably different levels in a given tissue; they have addnl. polymorphic forms, variable metal load, oxidn. states, and d.p.; they react with different metals and thiol-modifying agents; they are localized in the extracellular and intracellular space and re-distribute subcellularly. Results from the application of highly sensitive fluorimetric techniques suggest that the active form of the zinc/thiolate clusters in MT is not the one described by x-ray crystallog. or NMR spectroscopy and that the thiol chem. is the central aspect of MT's mechanism of action. MT is not satd. with zinc ions under normal physiol. conditions, and it exists to variable extent in the apoform (thionein) and in partially oxidized forms (thionin). The complexity of human MTs calls for metallomics and metalloproteomics approaches that integrate the knowledge from several scientific disciplines. Any interpretation of MT functions is based on the linkage between the metal ions and their sulfur ligands from the cysteines. Studies on the antioxidant or reactive species-scavenging properties of the cysteine sulfurs of MT need to take the effects on zinc metab. into account, while any studies with transition metal ions need to consider the effect of metal binding on the reactivity of the cysteine sulfurs.
- 183Maret, W.; Vallee, B. L. Cobalt as probe and label of proteins. Methods Enzymol. 1993, 226, 52– 71, DOI: 10.1016/0076-6879(93)26005-T183Cobalt as probe and label of proteinsMaret, Wolfgang; Vallee, Bert L.Methods in Enzymology (1993), 226 (Metallobiochemistry, Pt. C), 52-71CODEN: MENZAU; ISSN:0076-6879.A review with 68 refs. Crystallog. studies of 2 cobalt-substituted zinc enzymes that have now proved that the isomorphous replacements of the native zinc atoms provide a firm structural basis for this metal substitution. Whether cobalt could also serve as an isostructural probe in metalloenzymes other than zinc enzymes has not yet been ascertained. Cobalt derivs. of zinc enzymes have, for the first time, visualized dynamic events that occur in the coordination environment of zinc active sites during catalysis. The cobalt enzymes are almost as efficient catalysts as are the native zinc enzymes. Despite these characteristics, however, zinc and cobalt enzymes are not strictly isofunctional. They differ in substrate specificity and in some kinetic steps of the catalytic cycle.
- 184Freisinger, E.; Vašák, M. Cadmium in metallothioneins. Met. Ions Life Sci. 2013, 11, 339– 371, DOI: 10.1007/978-94-007-5179-8_11184Cadmium in metallothioneinsFreisinger, Eva; Vasak, MilanMetal Ions in Life Sciences (2013), 11 (Cadmium: From Toxicity to Essentiality), 339-371CODEN: MILSCT; ISSN:1559-0836. (Springer)Metallothioneins (MTs) are low-mol.-mass cysteine-rich proteins with the ability to bind mono- and divalent metal ions with the electron configuration d10 in form of metal-thiolate clusters. MTs are thought, among others, to play a role in the homeostasis of essential Zn(II) and Cu(I) ions. Besides these metal ions also Cd(II) can be bound to certain MTs in vivo, giving rise to the perception that another physiol. role of MTs is in the detoxification of heavy metal ions. Substitution of the spectroscopically silent Zn(II) ions in metalloproteins by Cd(II) proved to be an indispensable tool to probe the Zn(II) sites in vitro. In this review, methods applied in the studies of structural and chem. properties of Cd-MTs are presented. The first section focuses on the phys. basis of spectroscopic techniques such as electronic absorption, CD (CD), magnetic CD, X-ray absorption, and perturbed angular correlation of γ-rays spectroscopy, as well as mass spectrometry, and their applications to Cd-MTs from different organisms. The following is devoted to the discussion of metal binding affinities of Cd-MTs, cluster dynamics, the reactivity of bound Cd(II) ions with metal ion chelators and of thiolate ligands with alkylating and oxidizing agents. Finally, a brief summary of the known three-dimensional structures of Cd-MTs, detd. almost exclusively by multinuclear NMR techniques, is presented. Besides Cd-MTs, the described methods can also be applied to the study of metal binding sites in other metalloproteins.
- 185Stillman, M. J.; Cai, W.; Zelazowski, A. J. Cadmium binding to metallothioneins. Domain specificity in reactions of alpha and beta fragments, apometallothionein, and zinc metallothionein with Cd2+. J. Biol. Chem. 1987, 262, 4538– 4548, DOI: 10.1016/S0021-9258(18)61226-8185Cadmium binding to metallothioneins. Domain specificity in reactions of α and β fragments, apometallothionein, and zinc metallothionein with cadmium(2+)Stillman, Martin J.; Cai, Wuhua; Zelazowski, Andrzej J.Journal of Biological Chemistry (1987), 262 (10), 4538-48CODEN: JBCHA3; ISSN:0021-9258.The Cd-binding properties of rabbit liver Zn7-metallothionein (MT) 2 and apo-MT, rat liver apo-α MT and Zn4-α MT, and calf liver apo-β MT, were studied using CD and magnetic CD spectroscopies. Both sets of spectra recorded during the titrn. of Zn7-MT 2 with Cd exhibit a complicated pattern that is quite unexpected. Such behavior is not found at all in sets of spectra recorded during titrns. of the apo-species (apo-MT, apo-α MT, and apo-β MT), and is obsd. to a much lesser extent in the titrn. of Zn-α MT. Comparison between the band centers of the Cd-α MT and Cd-β MT indicates that the CD spectrum of Cd7-MT is dominated by intensity from transitions that originate on Cd-S chromophores in the α domain, with little direct contribution from the β domain. Anal. of the spectra recorded during titrns. of Zn7-MT 2 with Cd suggests: (i) that Cd replaces Zn in Zn7-MT isomorphously; (ii) that Cd binds in a nonspecific, distributed manner across both domains; (iii) that cluster formation in the α domain only occurs after 4 mol eq of Cd were added and is indicated by the presence of a cluster-sensitive, CD spectral feature; (iv) that the characteristic deriv. CD spectrum of native Cd4,Zn3-Met is only obtained from synthetic Cd4,Zn3-MT following a treatment cycle that allows the redistribution of Cd into the α domain; warming the synthetic native, Cd4,Zn3-MT, to 65° results in Cd being preferentially bound in the α domain; and (v) Zn7-MT will bind Cd quite normally at up to 65° but with greater specificity for the α domain compared with titrns. carried out at 25°. Apparently, the initial presence of Zn in both domains is an important factor in the lack of any domain specificity during Cd binding to Zn-MT which contrasts the domain specific manner obsd. for Cd binding to apo-MT.
- 186Vašák, M.; Kägi, J. H. R.; Holmquist, B.; Vallee, B. L. Spectral studies of cobalt(II)- and nickel(II)-metallothionein. Biochemistry 1981, 20, 6659– 6664, DOI: 10.1021/bi00526a021186Spectral studies of cobalt(II)- and nickel(II)-metallothioneinVasak, Milan; Kaegi, Jeremias H. R.; Holmquist, Barton; Vallee, Bert L.Biochemistry (1981), 20 (23), 6659-64CODEN: BICHAW; ISSN:0006-2960.The Zn and Cd of native rabbit metallothionein-1 were replaced stoichiometrically with either Co(II) or Ni(II). The electronic, MCD, and ESR spectra of Co(II)-metallothionein reflect distorted tetrahedral coordination of the Co atoms. Both the d-d and charge-transfer spectral regions closely resemble those of simple Co-tetrathiolate complexes, implying that their coordination chem. is analogous. Ni(II) complex ions and Ni(II)-metallothionein similarly exhibit analogous MCD bands in the d-d region. The CD bands assocd. with ligand-metal charge-transfer transitions in the non-d-d region of Co(II)- and Ni(II)-metallothionein afford addnl. evidence for the similarity in tetrahedral microsymmetry of the 2 metal derivs. The known ratio of 20 thiolate ligands to 7 metal ions, in conjunction with the spectral evidence for tetrathiolate coordination in metallothionein, represents good evidence that these metal thiolates are organized in clusters.
- 187Bertini, I.; Luchinat, C.; Messori, L.; Vašák, M. A two-dimensional NMR study of Co(II)7 rabbit liver metallothionein. Eur. J. Biochem. 1993, 211, 235– 240, DOI: 10.1111/j.1432-1033.1993.tb19891.x187A two-dimensional NMR study of cobalt(II)7 rabbit liver metallothioneinBertini, Ivano; Luchinat, Claudio; Messori, Luigi; Vasak, MilanEuropean Journal of Biochemistry (1993), 211 (1-2), 235-40CODEN: EJBCAI; ISSN:0014-2956.The 600-MHz 1H-NMR NOESY spectra on Co(II)7-reconstituted metallothionein (Co7MT), exhibiting hyperfine signals in the range 350 ppm to -50 ppm, with nuclear relaxation times of the order of a few milliseconds, have been measured and several interproton connectivities have been detected. This is the largest spectral window ever reported for a two-dimensional 1H-NMR spectrum in the case of a paramagnetic metalloprotein. No scalar connectivities could be detected. The hyperfine-shifted signals belong to the cysteine-ligand protons of the Co4S11 cluster of Co7MT. Together with results from one-dimensional NOE expts., the two-dimensional expts. permitted the pairwise assignment of the isotropically shifted signals of the CβH2 groups of the metal-coordinated cysteines. With the aid of computer-graphics inspection of the four-metal-cluster domain, based on the NMR soln. structure of Cd7MT, it is possible to propose sequence-specific assignments of a few hyperfine-shifted 1H-NMR signals. In particular, a tentative assignment is given for the six signals whose shifts exhibit an antiCurie temp. dependence. The assignment relies on the theor. model that qual. rationalizes the isotropic-shift pattern and its temp. dependence. Inferences on the soln. structure of the Co4S11 cluster are drawn.
- 188Pearce, L. L.; Gandley, R. E.; Han, W.; Wasserloos, K.; Stitt, M.; Kanai, A. J.; McLaughlin, M. K.; Pitt, B. R.; Levitan, E. S. Role of metallothionein in nitric oxide signaling as revealed by a green fluorescent fusion protein. Proc. Natl. Acad. Sci. U. S. A. 2000, 97, 477– 482, DOI: 10.1073/pnas.97.1.477188Role of metallothionein in nitric oxide signaling as revealed by a green fluorescent fusion proteinPearce, Linda L.; Gandley, Robin E.; Han, Weiping; Wasserloos, Karla; Stitt, Molly; Kanai, Anthony J.; McLaughlin, Margaret K.; Pitt, Bruce R.; Levitan, Edwin S.Proceedings of the National Academy of Sciences of the United States of America (2000), 97 (1), 477-482CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Although the function of metallothionein (MT), a 6- to 7-kDa cysteine-rich metal binding protein, remains unclear, it has been suggested from in vitro studies that MT is an important component of intracellular redox signaling, including being a target for nitric oxide (NO). To directly study the interaction between MT and NO in live cells, we generated a fusion protein consisting of MT sandwiched between two mutant green fluorescent proteins (GFPs). In vitro studies with this chimera (FRET-MT) demonstrate that fluorescent resonance energy transfer (FRET) can be used to follow conformational changes indicative of metal release from MT. Imaging expts. with live endothelial cells show that agents that increase cytoplasmic Ca2+ act via endogenously generated NO to rapidly and persistently release metal from MT. A role for this interaction in intact tissue is supported by the finding that the myogenic reflex of mesenteric arteries is absent in MT knockout mice (MT-/-) unless endogenous NO synthesis is blocked. These results are the first application of intramol. green fluorescent protein (GFP)-based FRET in a native protein and demonstrate the utility of FRET-MT as an intracellular surrogate indicator of NO prodn. In addn., an important role of metal thiolate clusters of MT in NO signaling in vascular tissue is revealed.
- 189Hong, S. H.; Maret, W. A fluorescence resonance energy transfer sensor for the beta-domain of metallothionein. Proc. Natl. Acad. Sci. U. S. A. 2003, 100, 2255– 2260, DOI: 10.1073/pnas.0438005100189A fluorescence resonance energy transfer sensor for the β-domain of metallothioneinHong, Sung-Hye; Maret, WolfgangProceedings of the National Academy of Sciences of the United States of America (2003), 100 (5), 2255-2260CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)We have designed a nanosensor to study the potential function of metallothionein (MT) in metal transfer and its interactions with redox partners and ligands by attaching two fluorescent probes to recombinant human MT. The specific labeling takes advantage of two different modification reactions. One is based on the fact that recombinant MT has a free N-terminal amino group when produced by the IMPACT T7 expression and purifn. system, the other on the observation that one human MT isoform (1b) contains an addnl. cysteine at position 32. It is located in the linker region of the mol., allowing the introduction of a probe between the two domains. An S32C mutation was introduced into hMT-2. Its thiol reactivity, metal binding capacity, and CD and UV spectra all demonstrate that the addnl. cysteine contains a free thiol(ate); it perturbs neither the overall structure of the protein nor the formation of the metal/thiolate clusters. MT contg. only cadmium was labeled stoichiometrically with Alexa 488 succinimidyl ester at the N terminus and with Alexa 546 maleimide at the free thiol group, followed by conversion to MT contg. only zinc. Energy transfer between Alexa 488 (donor) and Alexa 546 (acceptor) in double-labeled MT allows the monitoring of metal binding and conformational changes in the N-terminal β-domain of the protein.
- 190Otvos, J. D.; Armitage, I. M. Structure of the metal clusters in rabbit liver metallothionein. Proc. Natl. Acad. Sci. U. S. A. 1980, 77, 7094– 7098, DOI: 10.1073/pnas.77.12.7094190Structure of the metal clusters in rabbit liver metallothioneinOtvos, James D.; Armitage, Ian M.Proceedings of the National Academy of Sciences of the United States of America (1980), 77 (12), 7094-8CODEN: PNASA6; ISSN:0027-8424.Cd-113 NMR was used to det. the structures of the multiple Cd-binding sites in the 2 major isoproteins of rabbit liver metallothionein. Isotopically labeled metallothionein was sepd. from the livers of rabbits that had been subjected to repeated injections of 113CdCl2. The native protein isolated from these livers contains an appreciable amt. of Zn in addn. to Cd: 2-3 mol/mol protein out of a total metal content of 7 mol/mol protein. The 113Cd NMR spectrum of Cd, Zn-contg. metallothionein is quite complex, reflecting the fact that the native protein is a heterogeneous mixt. of species contg. different relative amts. of Zn and Cd. Replacement of the native Zn with 113Cd in vitro gave a protein whose 113Cd NMR spectrum was much simpler, contg. 8 distinct multiplets with chem. shifts ranging from 611 to 670 ppm. The multiplet structures were due to 113Cd-113Cd scalar coupling arising from 2-bond interactions between 113Cd ions linked to one another by bridging cysteine thiolate ligands. The sizes and structures of the metal clusters in the protein were detd. by the application of selective homonuclear 113Cd decoupling techniques. Rabbit liver metallothionein contains 2 sep. metal clusters, one contg. 4 Cd2+ ions and the other contg. 3. These 2 clusters, whose structures are the same in both isoproteins, were designated cluster A and cluster B, resp. Structures for the clusters are proposed that account for the 113Cd spin-coupling data and for the participation of all 20 of the cysteine residues in metal ligation, 11 in cluster A and 9 in cluster B. The appearance in the spectrum of 8 multiplets rather than the 7 that would be expected on the basis of the no. of metal-binding sites in the protein is an indication of some residual heterogeneity in the 113Cd-labeled metallothionein sample. The origin of this heterogeneity is suggested to be the presence of a protein species that lacks metal ions at its cluster B binding sites.
- 191Boulanger, Y.; Armitage, I. M.; Miklossy, K. A.; Winge, D. R. 113Cd NMR study of a metallothionein fragment. Evidence for a two-domain structure. J. Biol. Chem. 1982, 257, 13717– 13719, DOI: 10.1016/S0021-9258(18)33506-3191Cadmium-113 NMR study of a metallothionein fragment. Evidence for a two-domain structureBoulanger, Yvan; Armitage, Ian M.; Miklossy, Kathy Anne; Winge, Dennis R.Journal of Biological Chemistry (1982), 257 (22), 13717-19CODEN: JBCHA3; ISSN:0021-9258.A 32-residue polypeptide fragment, designated αI, of rat liver metallothionein obtained by subtilisin digestion was studied by 113Cd NMR. The amino acid compn. of the fragment corresponded to residues 30-61 of the metallothionein primary structure, and it contained 3.4 g atoms of Cd2+/mol of αI-fragment. Four 113Cd resonances were obsd., 3 of which had identical chem. shifts to those assigned to the 4-metal cluster in human liver metallothionein-2 under the same pH and buffer conditions. The 5-ppm chem. shift difference between the remaining resonance assigned to the 4-metal cluster in the intact protein can be explained to result from the removal of the N-terminal polypeptide fragment contg. the 3-metal cluster. These results provide unambiguous evidence for the 2-domain structure of metallothionein, contg. a sep. 3- and a 4-metal cluster.
- 192Zangger, K.; Shen, G.; Öz, G.; Otvos, J. D.; Armitage, I. M. Oxidative dimerization in metallothionein is a result of intermolecular disulphide bonds between cysteines in the alpha-domain. Biochem. J. 2001, 359, 353– 360, DOI: 10.1042/bj3590353192Oxidative dimerization in metallothionein is a result of intermolecular disulphide bonds between cysteines in the α-domainZangger, Klaus; Shen, Gong; Oz, Gulin; Otvos, James D.; Armitage, Ian M.Biochemical Journal (2001), 359 (2), 353-360CODEN: BIJOAK; ISSN:0264-6021. (Portland Press Ltd.)Upon storage under aerobic conditions metallothioneins (MTs) form a new species, which is characterized by a mol. mass approx. twice the size of monomeric MT and shifted 113/111Cd- and 1H-NMR resonances. The investigation of this oxidative dimerization process by NMR spectroscopy allowed us to structurally characterize this MT species that has been described to occur in vivo and might be synthesized under conditions of oxidative stress. The oxidative dimer was characterized by the formation of an intermol. cysteine disulfide bond involving the α-domain, and a detailed anal. of chem. shift changes and intermol. nuclear Overhauser effects points towards a disulfide bond involving Cys36. In contrast to the metal-bridged (non-oxidative) dimerization, the metal-cysteine cluster structures in both MT domains remain intact and no conformational exchange or metal-metal exchange was obsd. Also in contrast to the many recently reported oxidative processes which involve the β-domain cystcine groups and result in the increased dynamics of the bound metal ions in this N-terminal domain, we found no evidence for any increased dynamics in the α-domain metals following this oxidn. Therefore these findings provide addnl. corroboration that metal binding in the C-terminal α-domain is rather tight, even under conditions of a changing cellular oxidn. potential; compared with the more labile/dynamic nature of the metals in the N-terminal β-domain cluster under similar conditions.
- 193Gan, T.; Munoz, A.; Shaw, C. F., 3rd; Petering, D. H. Reaction of 111Cd7-metallothionein with EDTA. A reappraisal. J. Biol. Chem. 1995, 270, 5339– 5345, DOI: 10.1074/jbc.270.10.5339193Reaction of 111Cd7-metallothionein with EDTA. A reappraisalGan, Tong; Munoz, Amalia; Shaw, C. Frank, III; Petering, David H.Journal of Biological Chemistry (1995), 270 (10), 5339-45CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)The ligand substitution reaction of EDTA with Cd7-metallothionein (Cd7-MT) has been reinvestigated. NMR titrn. of the 111Cd-protein with EDTA showed that the ligand interacts preferentially and cooperatively with Cd2+ ions in the β-domain cluster. NMR and ultrafiltration kinetic anal. of this reaction using 5.6 mM Cd2+ as 111Cd7-MT and 56 mM EDTA indicated that cadmium-EDTA formed less rapidly than 111Cd peak intensity declined. Spectrophotometric and gel filtration studies of the reaction with 20 μM Cd2+ as Cd7-MT with various concns. of EDTA revealed biphasic kinetics with much larger rate consts. than obsd. in the NMR expts. The fraction of total ligand substitution occurring in each kinetic step varied with EDTA concn. The EDTA concn. dependence of both kinetic steps was consistent with the initial formation of protein·EDTA adducts, followed by their breakdown into products. Kinetic measurements were also made for the reactions of the isolated Cd4-α- and Cd3-β-domains with EDTA. The Cd4 domain reacted with EDTA with biphasic kinetics, in which one Cd2+ was removed rapidly with first-order kinetics, which were zero-order in EDTA. The other three reacted with kinetics like those for the slower step of the holoprotein. Cd3-β reacted with EDTA like the faster rate process assocd. with the Cd7-protein. The obsd. rate consts. for the reaction of Cd7-metallothionein with EDTA and the fraction of reaction in the faster rate process were sensitive to protein concn. These results are consistent with the hypothesis that the monomer-dimer equil. of the protein controls its kinetic reactivity with EDTA.
- 194Yu, X.; Wu, Z.; Fenselau, C. Covalent sequestration of melphalan by metallothionein and selective alkylation of cysteines. Biochemistry 1995, 34, 3377– 3385, DOI: 10.1021/bi00010a029194Covalent Sequestration of Melphalan by Metallothionein and Selective Alkylation of CysteinesYu, Xiaolan; Wu, Zhuchun; Fenselau, CatherineBiochemistry (1995), 34 (10), 3377-85CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)Rabbit liver metallothionein-2 is shown to form covalent bonds with the anticancer agent melphalan, in support of the hypothesis that covalent sequestration by metallothionein constitutes one mechanism for the cross-resistance acquired by cancer patients to therapeutic alkylating agents. Among 20 cysteines in the 2-domain protein, 89% of the first alkylation reaction occurs with 2 that co-chelate a zinc cation in the carboxy domain. Computer-supported docking studies indicate a favorable binding site for melphalan near these cysteine sulfhydryl groups. Although folded metallothionein-2 is resistant to trypsin cleavage, alkylation by 1 mol of melphalan allows cleavage by trypsin between the two globular domains.
- 195Zaia, J.; Jiang, L.; Han, M. S.; Tabb, J. R.; Wu, Z.; Fabris, D.; Fenselau, C. A binding site for chlorambucil on metallothionein. Biochemistry 1996, 35, 2830– 2835, DOI: 10.1021/bi952243n195A Binding Site for Chlorambucil on MetallothioneinZaia, Joseph; Jiang, Licong; Han, Mark S.; Tabb, Jeremiah R.; Wu, Zuchun; Fabris, Daniele; Fenselau, CatherineBiochemistry (1996), 35 (9), 2830-5CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)It is of interest to test the hypothesis that induced metallothionein (MT) acts in acquired drug resistance by covalent sequestration. In this study MT was incubated in vitro with chlorambucil (CHB) under conditions where only 1:1 covalent adducts were formed. The proteolytic products of these adducts were analyzed by HPLC and mass spectrometry to reveal two major sites of modification. These were the sulfur atoms of cysteines 33 and 48, which cochelate the same metal atom in native MT. The time course of the reaction was followed using online electrospray ionization with a double-focusing mass spectrometer. These expts. showed that drug-modified MT binds seven metal ions, as does the unmodified protein. Mol. docking expts. showed that the selectivity of drug binding is influenced by the presence of the aziridinium ion in the drug structure and complementary charge densities in the protein structure.
- 196Yu, X.; Wojciechowski, M.; Fenselau, C. Assessment of metals in reconstituted metallothioneins by electrospray mass spectrometry. Anal. Chem. 1993, 65, 1355– 1359, DOI: 10.1021/ac00058a010196Assessment of metals in reconstituted metallothioneins by electrospray mass spectrometryYu, Xiaolan; Wojciechowski, Marek; Fenselau, CatherineAnalytical Chemistry (1993), 65 (10), 1355-9CODEN: ANCHAM; ISSN:0003-2700.A method was developed that combines electrospray-ionization mass spectrometry with pH control to provide anal. of metals in native or reconstituted metallothioneins. These metalloproteins cooperatively bind 7 divalent metal ions, most commonly Zn2+ and Cd2+. Since the protein is denatured and metal ions are lost below pH 3, the pH of the electrospray soln. is crit. to successful results. The metal-free apoprotein was detected with its most abundant ions in a charge state of 6+, while the folded metallothionein-metal complexes were obsd. with lower charge states. The retention of 7 metals in the mol. ions detected is consistent with the hypothesis that metallothionein retains its conformation in the gas phase. This mass spectrometric technique can be used to det. rapidly and accurately how many and what cations are incorporated per mol. of protein. Information about molar distributions and ests. of relative abundances of various complexes in the sample can be acquired in a single measurement.
- 197Zaia, J.; Fabris, D.; Wei, D.; Karpel, R. L.; Fenselau, C. Monitoring metal ion flux in reactions of metallothionein and drug-modified metallothionein by electrospray mass spectrometry. Protein Sci. 1998, 7, 2398– 2404, DOI: 10.1002/pro.5560071117197Monitoring metal ion flux in reactions of metallothionein and drug-modified metallothionein by electrospray mass spectrometryZaia, Joseph; Fabris, Daniele; Wei, Dong; Karpel, Richard L.; Fenselau, CatherineProtein Science (1998), 7 (11), 2398-2404CODEN: PRCIEI; ISSN:0961-8368. (Cambridge University Press)The capabilities of electrospray ionization mass spectrometry are demonstrated for monitoring the flux of metal ions out of and into the metalloprotein rabbit liver metallothionein and, in one example, chlorambucil-alkylated metallothionein. Metal ion transfers may be followed as the reactions proceed in situ to provide kinetic information. More uniquely to this technique, metal ion stoichiometries may be detd. for reaction intermediates and products. Partners used in these studies include EDTA, carbonic anhydrase, a zinc-bound hexamer of insulin, and the core domain of bacteriophage T4 gene 32 protein, a binding protein for single-stranded DNA.
- 198Gehrig, P. M.; You, C.; Dallinger, R.; Gruber, C.; Brouwer, M.; Kägi, J. H.; Hunziker, P. E. Electrospray ionization mass spectrometry of zinc, cadmium, and copper metallothioneins: evidence for metal-binding cooperativity. Protein Sci. 2000, 9, 395– 402, DOI: 10.1110/ps.9.2.395198Electrospray ionization mass spectrometry of zinc, cadmium, and copper metallothioneins: evidence for metal-binding cooperativityGehrig, Peter M.; You, Chunhui; Dallinger, Reinhard; Gruber, Christine; Brouwer, Marius; Kagi, Jeremias H. R.; Hunziker, Peter E.Protein Science (2000), 9 (2), 395-402CODEN: PRCIEI; ISSN:0961-8368. (Cambridge University Press)Electrospray ionization (ESI) mass spectra of both well-characterized and novel metallothioneins (MTs) from various species were recorded to explore their metal-ion-binding modes and stoichiometries. The ESI mass spectra of the zinc- and cadmium-binding MTs showed a single main peak corresponding to metal-to-protein ratios of 4, 6, or 7. These findings combined with data obtained by other methods suggest that these MTs bind zinc or cadmium in a single predominant form and are consistent with the presence of three- and four-metal clusters. An unstable copper-specific MT isoform from Roman snails (Helix pomatia) could be isolated intact and was shown to preferentially bind 12 copper ions. To obtain addnl. information on the formation and relative stability of metal-thiolate clusters in MTs, a mass spectrometric titrn. study was conducted. One to seven molar equivalents of zinc or of cadmium were added to metal-free human MT-2 at neutral pH, and the resulting complexes were measured by ESI mass spectrometry. These expts. revealed that the formation of the four-metal cluster and of the thermodynamically less stable three-metal cluster is sequential and largely cooperative for both zinc and cadmium. Minor intermediate forms between metal-free MT, Me4MT, and fully reconstituted Me7MT were also obsd. The addn. of increasing amts. of cadmium to metal-free blue crab MT-I resulted in prominent peaks whose masses were consistent with apoMT, Cd3MT, and Cd6MT, reflecting the known structure of this MT with two Me3Cys9 centers. In a similar reconstitution expt. performed with Caenorhabditis elegans MT-II, a series of signals corresponding to apoMT and Cd3MT to Cd6MT species were obsd.
- 199Meloni, G.; Zovo, K.; Kazantseva, J.; Palumaa, P.; Vašák, M. Organization and assembly of metal-thiolate clusters in epithelium specific metallothionein-4. J. Biol. Chem. 2006, 281, 14588– 14595, DOI: 10.1074/jbc.M601724200199Organization and Assembly of Metal-Thiolate Clusters in Epithelium-specific Metallothionein-4Meloni, Gabriele; Zovo, Kairit; Kazantseva, Jekaterina; Palumaa, Peep; Vasak, MilanJournal of Biological Chemistry (2006), 281 (21), 14588-14595CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)Mammalian metallothionein-4 (MT-4) was found to be specifically expressed in stratified squamous epithelia where it plays an essential but poorly defined role in regulating zinc or copper metab. Here we report on the organization, stability, and the pathway of metal-thiolate cluster assembly in MT-4 reconstituted with Cd2+ and Co2+ ions. Both the 113Cd NMR studies of 113Cd7MT-4 and the spectroscopic characterization of Co7MT-4 showed that, similar to the classical MT-1 and MT-2 proteins, metal ions are organized in two independent Cd4Cys11 and Cd3Cys9 clusters with each metal ion tetrahedrally coordinated by terminal and bridging cysteine ligands. Moreover, we have demonstrated that the cluster formation in Cd7MT-4 is cooperative and sequential, with the Cd4Cys11 cluster being formed first, and that a distinct single-metal nucleation intermediate Cd1MT-4 is required in the cluster formation process. Conversely, the absorption and CD features of metal-thiolate clusters in Cd7MT-4 indicate that marked differences in the cluster geometry exist when compared with those in Cd7MT-1/2. The biol. implication of our studies as to the role of MT-4 in zinc metab. of stratified epithelia is discussed.
- 200Wan, X.; Freisinger, E. The plant metallothionein 2 from Cicer arietinum forms a single metal-thiolate cluster. Metallomics 2009, 1, 489– 500, DOI: 10.1039/b906428a200The plant metallothionein 2 from Cicer arietinum forms a single metal-thiolate clusterWan, Xiaoqiong; Freisinger, EvaMetallomics (2009), 1 (6), 489-500CODEN: METAJS; ISSN:1756-591X. (Royal Society of Chemistry)The plant metallothionein 2 from Cicer arietinum (chickpea; cicMT2) is a typical member of this subfamily and features two cysteine-rich regions contg. eight and six cysteine residues, resp., sepd. by a linker region 41 amino acids in length. This metallothionein thus differs significantly from the well-studied vertebrate forms. A synthetic gene encoding cicMT2 was designed, cloned into a suitable vector, and the protein was over-expressed in Escherichia coli. For the first time, an in-depth spectroscopic characterization of cicMT2 in the presence of divalent metal ions is performed showing a binding capacity for five ZnII, CdII, or CoII ions and the typical features of metal-thiolate clusters. Based on proteolytic digestion expts., the cluster arrangement formed by the divalent metal ions and the cysteine thiolate groups connects the amino-terminal with the carboxy-terminal cysteine-rich region. The cluster formation process, put into effect with the addn. of the fourth metal ion to the apo protein, was investigated using the characteristic shift of absorption bands obsd. in the UV/Vis spectra upon titrn. with CoII. The pH-dependent ZnII- and CdII-thiolate cluster stability is one of the highest obsd. for plant MTs so far, but lower than that usually found in vertebrate metallothioneins. The dependence of the pH stability on the ionic strength of the soln. is more pronounced for the CdII- than for the ZnII-form of the protein.
- 201Artells, E.; Palacios, Ò.; Capdevila, M.; Atrian, S. In vivo-folded metal-metallothionein 3 complexes reveal the Cu-thionein rather than Zn-thionein character of this brain-specific mammalian metallothionein. FEBS J. 2014, 281, 1659– 1678, DOI: 10.1111/febs.12731201In vivo-folded metal-metallothionein 3 complexes reveal the Cu-thionein rather than Zn-thionein character of this brain-specific mammalian metallothioneinArtells, Ester; Palacios, Oscar; Capdevila, Merce; Atrian, SilviaFEBS Journal (2014), 281 (6), 1659-1678CODEN: FJEOAC; ISSN:1742-464X. (Wiley-Blackwell)Metallothionein-3 (MT3) is one of the 4 mammalian metallothioneins (MT), and is constitutively synthesized in the brain. MT3 acts both intracellularly and extracellularly in this organ, performing functions related to neuronal growth and physiol. metal (Zn and Cu) handling. It appears to be involved in the prevention of neurodegenerative disorders caused by insol. Cu-peptide aggregates, as it triggers a Zn-Cu swap that may counteract the deleterious presence of Cu in neural tissues. The literature data on MT3 coordination come from studies either on apo-MT3 reconstitution or the reaction of Zn-MT3 with Cu2+, an ion that is hardly present inside cells. To ascertain the MT3 metal-binding features in a scenario closer to the reductive cell cytoplasm, a study of the recombinant Zn2+, Cd2+ and Cu+ complexes of MT3, βMT3, and αMT3, as well as the in vitro Zn2+-Cd2+ and Zn2+-Cu+ replacement processes, is presented here. The authors conclude that MT3 has a Cu-thionein character that is stronger than that of the MT1 and MT2 isoforms, which are also present in the mammalian brain, which is mainly contributed by its β domain. In contrast, the α domain retains a high capacity to bind Zn2+ ions, and, consequently, the entire MT3 peptide shows a peculiar dual ability to handle both metal ions. The nature of the formed Cu+-MT3 complexes oscillates from heterometallic Cu6Zn4-MT3 to homometallic Cu10-MT3 major species, in a narrow Cu concn. range. Therefore, the entire MT3 peptide shows a high capacity to bind Cu+, provided that this occurs in a nonoxidative milieux. This reflects a peculiar property of this MT isoform, which accurately senses different Cu contents in the environment in which it is synthesized.
- 202Breuker, K.; McLafferty, F. W. Stepwise evolution of protein native structure with electrospray into the gas phase, 10–12 to 102 s. Proc. Natl. Acad. Sci. U. S. A. 2008, 105, 18145– 18152, DOI: 10.1073/pnas.0807005105202Stepwise evolution of protein native structure with electrospray into the gas phase, 10-12 to 102 sBreuker, Kathrin; McLafferty, Fred W.Proceedings of the National Academy of Sciences of the United States of America (2008), 105 (47), 18145-18152CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)A review. Mass spectrometry (MS) has been revolutionized by electrospray ionization (ESI), which is sufficiently "gentle" to introduce nonvolatile biomols. such as proteins and nucleic acids (RNA or DNA) into the gas phase without breaking covalent bonds. Although in some cases noncovalent bonding can be maintained sufficiently for ESI/MS characterization of the soln. structure of large protein complexes and native enzyme/substrate binding, the new gaseous environment can ultimately cause dramatic structural alterations. The temporal (picoseconds to minutes) evolution of native protein structure during and after transfer into the gas phase, as proposed here based on a variety of studies, can involve side-chain collapse, unfolding, and refolding into new, non-native structures. Control of individual exptl. factors allows optimization for specific research objectives.
- 203Konermann, L.; Ahadi, E.; Rodriguez, A. D.; Vahidi, S. Unraveling the mechanism of electrospray ionization. Anal. Chem. 2013, 85, 2– 9, DOI: 10.1021/ac302789c203Unraveling the Mechanism of Electrospray IonizationKonermann, Lars; Ahadi, Elias; Rodriguez, Antony D.; Vahidi, SiavashAnalytical Chemistry (Washington, DC, United States) (2013), 85 (1), 2-9CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)A review. Electrospray ionization (ESI) generates intact gas phase ions from analytes in soln. for mass spectrometric studies. ESI can proceed via different mechanisms. Low mol. wt. analytes follow the ion evapn. model (IEM), whereas the charged residue model (CRM) applies to large globular species. A chain ejection model (CEM) was proposed for disordered polymers.
- 204Jayawardena, D. P.; Heinemann, I. U.; Stillman, M. J. Zinc binds non-cooperatively to human liver metallothionein 2a at physiological pH. Biochem. Biophys. Res. Commun. 2017, 493, 650– 653, DOI: 10.1016/j.bbrc.2017.08.137204Zinc binds non-cooperatively to human liver metallothionein 2a at physiological pHJayawardena, Devika P.; Heinemann, Ilka U.; Stillman, Martin J.Biochemical and Biophysical Research Communications (2017), 493 (1), 650-653CODEN: BBRCA9; ISSN:0006-291X. (Elsevier B.V.)Maintenance of the homeostasis of zinc is very important in regulating bodily functions. There are over 300 Zn-dependent enzymes identified where Zn(II) plays a structural or catalytic role. However, an excess of Zn(II) in a cell is toxic and free Zn(II) is tightly controlled. Metallothioneins (MTs) are small cysteine rich proteins that can bind up to seven Zn(II) and act as a Zn(II) reservoir. The MT2a isoform is predominantly found in the liver. This study focused on designing an MT2a construct of recombinant human MT2a to det. the Zn(II) binding profile of MT2a in vitro. We analyzed the pH dependence of Zn-MT2a speciation from electrospray ionization mass spectral data. At physiol. pH, Zn(II) is terminally bound to the cysteine thiols of MT2a, making bead-like structures (non-cooperative metal binding), while at low pH, Zn(II) formed Zn4S11-MT2a clusters involving bridged cysteinyl thiols to the Zn(II) (cooperative metal binding). The Zn(II) binding profile of MT2a was compared to Zn(II) binding profile of human kidney MT1a, which was reported in literature, and found that the Zn(II) binding profile of MT2a is similar to that of MT1a. The facility of forming bead-like structures at physiol. pH for Zn5-MT2a means that Zn7-MT2a can donate up to two Zn(II) to Zn-dependent enzymes.
- 205Scheller, J. S.; Irvine, G. W.; Stillman, M. Unraveling the mechanistic details of metal binding to mammalian metallothioneins from stoichiometric, kinetic, and binding affinity data. Dalton Trans. 2018, 47, 3599– 4002, DOI: 10.1039/C7DT03319BThere is no corresponding record for this reference.
- 206Korkola, N. C.; Scarrow, P. M.; Stillman, M. J. pH dependence of the non-cooperative binding of Bi3+ to human apo-metallothionein 1A: kinetics, speciation, and stoichiometry. Metallomics 2020, 12, 435– 448, DOI: 10.1039/C9MT00285E206pH dependence of the non-cooperative binding of Bi3+ to human apo-metallothionein 1A: kinetics, speciation, and stoichiometryKorkola, Natalie C.; Scarrow, Patti M.; Stillman, Martin J.Metallomics (2020), 12 (3), 435-448CODEN: METAJS; ISSN:1756-591X. (Royal Society of Chemistry)In this paper, we explored the binding of [Bi(cit)]- and [Bi(EDTA)]- to apo-MT 1a as the most basic of binding motifs. It was found that both Bi3+ salts bound in a non-cooperative stepwise manner to terminal cysteinal thiolates at pH 2.6, 5.0, and 7.4. We report that [Bi(EDTA)]- only binds stepwise up to Bi6MT, whereas [Bi(cit)]- forms up to Bi8MT, where the 7th and 8th Bi3+ appear to be adducts. Stepwise speciation anal. provided the 7 binding consts. that decreased systematically from K1 to K7 indicating a non-cooperative binding profile. Cysteine modifications with benzoquinone and iodoacetamide revealed that when apoMT is fully metalated with Bi3+ there are two free cysteines, meaning 18 cysteines are used in binding the 6 Bi3+. Kinetic studies showed that [Bi(EDTA)]- binds very slowly at pH 2.6 (k = 0.0290 x 106 M-1 s-1) and approx. 2000 times faster at pH 7.4 (k = 66.5 x 106 M-1 s-1). [Bi(cit)]- binding at pH 2.6 was faster than [Bi(EDTA)]- (k = 672 x 106 M-1 s-1) at either pH level. The data strongly support a non-clustered binding motif, emphasizing the non-traditional pathway reported previously for As3+.
- 207Peris-Díaz, M. D.; Guran, R.; Zitka, O.; Adam, V.; Krężel, A. Mass spectrometry-based structural analysis of cysteine-rich metal-binding sites in proteins with MetaOdysseus R software. J. Proteome Res. 2021, 20, 776– 785, DOI: 10.1021/acs.jproteome.0c00651207Mass Spectrometry-Based Structural Analysis of Cysteine-Rich Metal-Binding Sites in Proteins with MetaOdysseus R SoftwarePeris-Diaz, Manuel David; Guran, Roman; Zitka, Ondrej; Adam, Vojtech; Krezel, ArturJournal of Proteome Research (2021), 20 (1), 776-785CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)Identification of metal-binding sites in proteins and understanding metal-coupled protein folding mechanisms are aspects of high importance for the structure-to-function relationship. Mass spectrometry (MS) has brought a powerful adjunct perspective to structural biol., obtaining from metal-to-protein stoichiometry to quaternary structure information. Currently, the different exptl. and/or instrumental setups usually require the use of multiple data anal. software, and in some cases, they lack some of the main data anal. steps (MS processing, scoring, identification). Here, we present a comprehensive data anal. pipeline that addresses charge-state deconvolution, statistical scoring, and mass assignment for native MS, bottom-up, and native top-down with emphasis on metal-protein complexes. We have evaluated all of the approaches using assemblies of increasing complexity, including free and chem. labeled proteins, from low- to high-resoln. MS. In all cases, the results have been compared with common software and proved how MetaOdysseus outperformed them.
- 208Laganowsky, A.; Reading, E.; Allison, T. M.; Ulmschneider, M. B.; Degiacomi, M. T.; Baldwin, A. J.; Robinson, C. V. Membrane proteins bind lipids selectively to modulate their structure and function. Nature 2014, 510, 172– 175, DOI: 10.1038/nature13419208Membrane proteins bind lipids selectively to modulate their structure and functionLaganowsky, Arthur; Reading, Eamonn; Allison, Timothy M.; Ulmschneider, Martin B.; Degiacomi, Matteo T.; Baldwin, Andrew J.; Robinson, Carol V.Nature (London, United Kingdom) (2014), 510 (7503), 172-175CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)Previous studies have established that the folding, structure and function of membrane proteins are influenced by their lipid environments and that lipids can bind to specific sites, for example, in potassium channels. Fundamental questions remain however regarding the extent of membrane protein selectivity towards lipids. Here we report a mass spectrometry approach designed to det. the selectivity of lipid binding to membrane protein complexes. We investigate the mechanosensitive channel of large conductance (MscL) from Mycobacterium tuberculosis and aquaporin Z (AqpZ) and the ammonia channel (AmtB) from Escherichia coli, using ion mobility mass spectrometry (IM-MS), which reports gas-phase collision cross-sections. We demonstrate that folded conformations of membrane protein complexes can exist in the gas phase. By resolving lipid-bound states, we then rank bound lipids on the basis of their ability to resist gas phase unfolding and thereby stabilize membrane protein structure. Lipids bind non-selectively and with high avidity to MscL, all imparting comparable stability; however, the highest-ranking lipid is phosphatidylinositol phosphate (PI), in line with its proposed functional role in mechanosensation. AqpZ is also stabilized by many lipids, with cardiolipin (CDL) imparting the most significant resistance to unfolding. Subsequently, through functional assays we show that cardiolipin modulates AqpZ function. Similar expts. identify AmtB as being highly selective for phosphatidylglycerol (PG), prompting us to obtain an X-ray structure in this lipid membrane-like environment. The 2.3 Å resoln. structure, when compared with others obtained without lipid bound, reveals distinct conformational changes that re-position AmtB residues to interact with the lipid bilayer. Our results demonstrate that resistance to unfolding correlates with specific lipid-binding events, enabling a distinction to be made between lipids that merely bind from those that modulate membrane protein structure and/or function. We anticipate that these findings will be important not only for defining the selectivity of membrane proteins towards lipids, but also for understanding the role of lipids in modulating protein function or drug binding.
- 209Przygońska, K.; Poznański, J.; Mistarz, U. H.; Rand, K. D.; Dadlez, M. Side-chain moieties from the N-terminal region of Aβ are involved in an oligomer-stabilizing network of interactions. PLoS One 2018, 13, e0201761 DOI: 10.1371/journal.pone.0201761209Side-chain moieties from the N-terminal region of Aβ are Involved in an oligomer stabilizing network of interactionsPrzygonska, Kaja; Poznanski, Jarosoaw; Mistarz, Ulrik H.; Rand, Kasper D.; Dadlez, MichaoPLoS One (2018), 13 (8), e0201761/1-e0201761/25CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)Oligomeric forms of the Aβ peptide represent the most probable neurotoxic agent in Alzheimer's disease. The dynamic and heterogeneous character of these oligomers makes their structural characterization by classic methods difficult. Native mass spectrometry, when supported by addnl. gas phase techniques, like ion mobility sepn. and hydrogendeuterium exchange (IM-HDX-MS), enable anal. of different oligomers coexisting in the sample and may provide species-specific structural information for each oligomeric form populated in the gas phase. Here, we have combined these three techniques to obtain insight into the structural properties of oligomers of Aβ1-40 and two variants with scrambled sequences. Gas-phase HDX-MS revealed a sequence-specific engagement of the sidechains of residues located at the N-terminal part of the peptide in a network of oligomer-stabilizing interactions. Oligomer-specific interactions were no longer obsd. in the case of the fully scrambled sequence. Also, the ability to form alternative structures, obsd. for WT Aβ peptide, was lost upon scrambling. Our data underscore a role for the N-terminal residues in shaping the equil. of oligomeric forms. Although the peptide lacking the N-terminal 1-16 residues (p3 peptide) is thought to be benign, the role of the N-terminus has not been sufficiently characterized yet. We speculate that the interaction networks revealed here may be crucial for enabling structural transitions necessary to obtain mature parallel cross-β structures from smaller antiparallel oligomers. We provide a hypothetical mol. model of the trajectory that allows a gradual conversion from antiparallel to parallel oligomers without decompn. of oligomers. Oligomer-defining interactions involving the Aβ peptide N-terminus may be important in prodn. of the neurotoxic forms and thus should not be neglected.
- 210Guo, Y.; Ling, Y.; Thomson, B. A.; Siu, K. W. Combined ion-mobility and mass-spectrometry investigations of metallothionein complexes using a tandem mass spectrometer with a segmented second quadrupole. J. Am. Soc. Mass Spectrom. 2005, 16, 1787– 1794, DOI: 10.1016/j.jasms.2005.07.011210Combined Ion-Mobility and Mass-Spectrometry Investigations of Metallothionein Complexes Using a Tandem Mass Spectrometer with a Segmented Second QuadrupoleGuo, Yuzhu; Ling, Yun; Thomson, Bruce A.; Siu, K. W. MichaelJournal of the American Society for Mass Spectrometry (2005), 16 (11), 1787-1794CODEN: JAMSEF; ISSN:1044-0305. (Elsevier Inc.)Rabbit metallothionein (MT) 2A complexes with Cd(II), Zn(II), Ag(I), Cu(I), Hg(II), arsenite, monomethylarsonous acid (MMA), and dimethylarsinous acid (DMA) have been examd. using ion-mobility measurements and mass spectrometry in a triple-quadrupole mass spectrometer equipped with a segmented second quadrupole that doubled as an ion-mobility cell (2005). The metal ions confer conformational rigidity on the MT complexes, which counteracts Coulombic repulsion among protons added as a result of electrospray. Triply and quadruply protonated Cd7MT2A have smaller cross-sections than the Cd7MT2A structure deduced from published NMR data. For the 6+ ions, the As6MT2A complex has a cross-section of 790 Å2; the MMA10MT2A complex, 920 Å2; and the DMA20MT2A complex, 1220 Å2. This increase in cross-section of the As(III) species, from As3+ to MMA to DMA, is interpreted as a consequence of decreasing multiple coordination and increasing no. of Me groups.
- 211Dupuis, N. F.; Wu, C.; Shea, J. E.; Bowers, M. T. The amyloid formation mechanism in human IAPP: dimers have β-strand monomer-monomer interfaces. J. Am. Chem. Soc. 2011, 133, 7240– 7243, DOI: 10.1021/ja1081537211The Amyloid Formation Mechanism in Human IAPP: Dimers Have β-Strand Monomer-Monomer InterfacesDupuis, Nicholas F.; Wu, Chun; Shea, Joan-Emma; Bowers, Michael T.Journal of the American Chemical Society (2011), 133 (19), 7240-7243CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Early oligomerization of human IAPP (hIAPP) is responsible for β-cell death in the pancreas and is increasingly considered a primary pathol. process linked to Type II Diabetes (T2D). Yet, the assembly mechanism remains poorly understood, largely due to the inability of conventional techniques to probe distributions or detailed structures of early oligomeric species. Here, we describe the first exptl. data on the isolated and unmodified dimers of human (hIAPP) and nonamyloidogenic rat IAPP (rIAPP). The expts. reveal that the human IAPP dimers are more extended than those formed by rat IAPP and likely descend from extended monomers. Independent all-atom mol. dynamics simulations show that rIAPP forms compact helix and coil rich dimers, whereas hIAPP forms β-strand rich dimers that are generally more extended. Also, the simulations reveal that the monomer-monomer interfaces of the hIAPP dimers are dominated by β-strands and that β-strands can recruit coil or helix structured regions during the dimerization process. Our β-rich interface contrasts with an N-terminal helix-to-helix interface proposed in the literature but is consistent with existing exptl. data on the self-interaction pattern of hIAPP, mutation effects, and inhibition effects of the N-methylation in the mutation region.
- 212Pagel, K.; Natan, E.; Hall, Z.; Fersht, A. R.; Robinson, C. V. Intrinsically disordered p53 and its complexes populate compact conformations in the gas phase. Angew. Chem., Int. Ed. 2013, 52, 361– 365, DOI: 10.1002/anie.201203047212Intrinsically Disordered p53 and Its Complexes Populate Compact Conformations in the Gas PhasePagel, Kevin; Natan, Eviatar; Hall, Zoe; Fersht, Alan R.; Robinson, Carol V.Angewandte Chemie, International Edition (2013), 52 (1), 361-365CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)The use of ion mobility mass spectrometry (IM-MS) to study the topol. of proteins and their complexes is attracting considerable attention. Under the appropriate conditions results have shown that unique insight can be gained for intractable systems such as protein aggregates, viral capsids, and membrane complexes. Here we applied IM-MS to one of the most studied natively disordered tetrameric complexes, that of the tumor suppressor protein p53. This protein complex, discovered more than 30 years ago, is a major chemotherapeutic target, and has continued to thwart structural biologists largely because disordered regions comprise ca. 40 % of the protein. For our study we systematically introduced intrinsically disordered regions (flexible linker, N and C termini) to the two folded domains. This enabled us to probe the effects of disordered regions on the various constructs as well as full-length tetrameric p53 bound to cognate DNA.
- 213Jurneczko, E.; Cruickshank, F.; Porrini, M.; Clarke, D. J.; Campuzano, I. D. G.; Morris, M.; Nikolova, P. V.; Barran, P. E. Probing the conformational diversity of cancer-associated mutations in p53 with ion-mobility mass spectrometry. Angew. Chem., Int. Ed. 2013, 52, 4370– 4374, DOI: 10.1002/anie.201210015213Probing the Conformational Diversity of Cancer-Associated Mutations in p53 with Ion-Mobility Mass SpectrometryJurneczko, Ewa; Cruickshank, Faye; Porrini, Massimiliano; Clarke, David J.; Campuzano, Iain D. G.; Morris, Michael; Nikolova, Penka V.; Barran, Perdita E.Angewandte Chemie, International Edition (2013), 52 (16), 4370-4374CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)Suppressor p53 is the most mutated protein in human cancers. Herein ion mobility mass spectrometry studies are reported on the conformational diversity of wild type p53 and its cancer-assocd. mutants.
- 214Ruotolo, B. T.; Giles, K.; Campuzano, I.; Sandercock, A. M.; Bateman, R. H.; Robinson, C. V. Evidence for macromolecular protein rings in the absence of bulk water. Science 2005, 310, 1658– 1661, DOI: 10.1126/science.1120177214Evidence for Macromolecular Protein Rings in the Absence of Bulk WaterRuotolo, Brandon T.; Giles, Kevin; Campuzano, Iain; Sandercock, Alan M.; Bateman, Robert H.; Robinson, Carol V.Science (Washington, DC, United States) (2005), 310 (5754), 1658-1661CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)We have examd. the architecture of a protein complex in the absence of bulk water. By detg. collision cross sections of assemblies of the trp RNA binding protein, TRAP, we established that the 11-membered ring topol. of the complex can be maintained within a mass spectrometer. We also found that the binding of tryptophan enhances the stability of the ring structure and that addn. of a specific RNA mol. increases the size of the complex and prevents structural collapse. These results provide definitive evidence that protein quaternary structure can be maintained in the absence of bulk water and highlight the potential of ion mobility sepn. for defining shapes of heterogeneous macromol. assemblies.
- 215Dong, S.; Shirzadeh, M.; Fan, L.; Laganowsky, A.; Russell, D. H. Ag+ ion binding to human metallothionein-2A is cooperative and domain specific. Anal. Chem. 2020, 92, 8923– 8932, DOI: 10.1021/acs.analchem.0c00829215Ag+ Ion Binding to Human Metallothionein-2A Is Cooperative and Domain SpecificDong, Shiyu; Shirzadeh, Mehdi; Fan, Liqi; Laganowsky, Arthur; Russell, David H.Analytical Chemistry (Washington, DC, United States) (2020), 92 (13), 8923-8932CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)Metallothioneins (MTs) constitute a family of cysteine-rich proteins that play key biol. roles for a wide range of metal ions, but unlike many other metalloproteins, the structures of apo- and partially metalated MTs are not well understood. Here, we combine nano-electrospray ionization-mass spectrometry (ESI-MS) and nano-ESI-ion mobility (IM)-MS with collision-induced unfolding (CIU), chem. labeling using N-ethylmaleimide (NEM), and both bottom-up and top-down proteomics in an effort to better understand the metal binding sites of the partially metalated forms of human MT-2A, viz., Ag4-MT. The results for Ag4-MT are then compared to similar results obtained for Cd4-MT. The results show that Ag4-MT is a cooperative product, and data from top-down and bottom-up proteomics mass spectrometry anal. combined with NEM labeling revealed that all four Ag+ ions of Ag4-MT are bound to the β-domain. The binding sites are identified as Cys13, Cys15, Cys19, Cys21, Cys24, and Cys26. While both Ag+ and Cd2+ react with MT to yield cooperative products, i.e., Ag4-MT and Cd4-MT, these products are very different; Ag+ ions of Ag4-MT are located in the β-domain, whereas Cd2+ ions of Cd4-MT are located in the α-domain. Ag6-MT has been reported to be fully metalated in the β-domain, but our data suggest the two addnl. Ag+ ions are more weakly bound than are the other four. Higher order Agi-MT complexes (i = 7-17) are formed in solns. that contain excess Ag+ ions, and these are assumed to be bound to the α-domain or shared between the two domains. Interestingly, the excess Ag+ ions are displaced upon addn. of NEM to this soln. to yield predominantly Ag4NEM14-MT. Results from CIU suggest that Agi-MT complexes are structurally more ordered and that the energy required to unfold these complexes increases as the no. of coordinated Ag+ increases.
- 216Peris-Díaz, M. D.; Guran, R.; Domene, C.; de los Rios, V.; Zitka, O.; Adam, V.; Krężel, A. An integrated mass spectrometry and molecular dynamics simulations approach reveals the spatial organization impact of metal-binding sites on the stability of metal-depleted metallothionein-2 species. J. Am. Chem. Soc. 2021, DOI: 10.1021/jacs.1c05495There is no corresponding record for this reference.
- 217Riordan, J. R.; Richards, V. Human foetal liver contains both zinc- and copper-rich forms of metallothionein. J. Biol. Chem. 1980, 255, 5380– 5383, DOI: 10.1016/S0021-9258(19)70797-2217Human fetal liver contains both zinc- and copper-rich forms of metallothioneinRiordan, John R.; Richards, VivienneJournal of Biological Chemistry (1980), 255 (11), 5380-3CODEN: JBCHA3; ISSN:0021-9258.Metallothionein was purified under anaerobic conditions from livers of human fetuses ranging from 19 wk gestational age to term. Homogeneous metallothionein obtained in the absence of reducing agents from the sol. fraction of the tissue which contained 24% and 85% of the total liver Cu and Zn, resp., had <1 g atom of Cu and ∼3.1 g atoms of Zn/mol. Extn. with 1% 2-mercaptoethanol of the insol. fraction of the tissue (contg. 76% and 15% of the liver Cu and Zn, resp.) yielded a metallothionein with ∼2.5 g atoms of Cu and 1.3 g atoms of Zn. When the whole tissue was extd. similarly by the procedure of L. Ryden and H. F. Deutsch (1978), nearly equal proportions of the 2 metals were obtained in the sol. fraction from which metallothionein, with ∼3.0 g atoms of Zn, and 1.5 g atoms of Cu, was purified. Thus, Zn- and Cu-rich forms of metallothionein are differently distributed between the sol. and insol. fractions of fetal liver. Nevertheless, the predominant metal in metallothionein of human fetal liver is Zn as is the case in the adult; significant amts. of Cu are also present, consistent with the elevated quantity of this metal in the fetal tissue relative to the adult. Cd was not detected. Zn is much more easily removed from the protein than is Cu.
- 218Klein, D.; Scholz, G. A.; Drasch, G. A.; Müller-Höcker, J.; Summer, K. H. Metallothionein, copper and zinc in fetal and neonatal human liver: changes during development. Toxicol. Lett. 1991, 56, 61– 67, DOI: 10.1016/0378-4274(91)90090-S218Metallothionein, copper and zinc in fetal and neonatal human liver: changes during developmentKlein, D.; Scholz, P.; Drasch, G. A.; Mueller-Hoecker, J.; Summer, Karl H.Toxicology Letters (1991), 56 (1-2), 61-7CODEN: TOLED5; ISSN:0378-4274.Total and cytosolic Zn and Cu, cytosolic metallothionein (MT) and the Cu-load of MT were investigated in fetal (22, 24 and 32 gestational weeks) and neonatal (2-15 mo) human liver. Whereas the fraction of cytosolic Zn remained const. at 66% of the total independent of the stage of development, the fraction of cytosolic Cu increased from 26% in preterm liver to about 100% within 12 mo postnatally. The MT content was higher in fetal than in neonatal liver. There was a linear correlation between cytosolic MT and Zn in both fetal and neonatal liver but not between MT and Cu. In contrast to fetal liver, the Cu-load of MT in neonatal liver seems to be detd. by the Zn/Cu ratio in the cytosol. The results suggest that MT is involved in the regulation of Cu and Zn metab. during fetal and neonatal development.
- 219Kägi, J. H. R.; Vašák, M.; Lerch, K.; Gilg, D. E. O.; Hunziker, P.; Bernhard, W. R.; Good, M. Structure of mammalian metallothionein. Environ. Health Persp. 1984, 54, 93– 103, DOI: 10.2307/3429795219Structure of mammalian metallothioneinKagi J H; Vasak M; Lerch K; Gilg D E; Hunziker P; Bernhard W R; Good MEnvironmental health perspectives (1984), 54 (), 93-103 ISSN:0091-6765.All mammalian metallothioneins characterized contain a single polypeptide chain of 61 amino acid residues, among them 20 cysteines providing the ligands for seven metal-binding sites. Native metallothioneins are usually heterogeneous in metal composition, with Zn, Cd, and Cu occurring in varying proportions. However, forms containing only a single metal species, i.e., Zn, Cd, Ni, Co, Hg, Pb, Bi, have now been prepared by in vitro reconstitution from the metal-free apoprotein. By spectroscopic analysis of such derivatives it was established that all cysteine residues participate in metal binding, that each metal ion is bound to four thiolate ligands, and that the symmetry of each complex is close to that of a tetrahedron. To satisfy the requirements of the overall Me7(Cys-)20 stoichiometry, the complexes must be combined to form metal-thiolate cluster structures. Experimental proof for the occurrence of such clusters comes from the demonstration of metal-metal interactions by spectroscopic and magnetic means. Thus, in Co(II)7-metallothionein, the Co(II)-specific ESR signals are effectively suppressed by antiferromagnetic coupling of juxtaposed paramagnetic metal ions. By monitoring changes in ESR signal size occurring on stepwise incorporation of Co(II) into the protein, it is possible to follow the building up of the clusters. This process is biphasic. Up to binding of four equivalents of Co(II), the ESR amplitude increases in proportion to the metal content, indicating generation of magnetically noninteracting high-spin complexes. However, upon addition of the remaining three equivalents of Co(II), these features are progressively suppressed, signaling the formation of clusters. The same mode of cluster formation has also been documented for Cd and Hg. The actual spatial organization of the clusters and the polypeptide chain remains to be established. An attractive possibility is the arrangement of the tetrahedral metal-thiolates in adamantane-like structures surrounded by properly folded segments of the chain providing the ligands. 1H-NMR data and infrared absorption measurements are consistent with a tightly folded structure rich in beta-type conformation.
- 220Alvarez, L.; Gonzalez-Iglesias, H.; Garcia, M.; Ghosh, S.; Sanz-Medel, A.; Coca-Prados, M. The stoichiometric transition from Zn6Cu1-metallothionein to Zn7-metallothionein underlies the up-regulation of metallothionein (MT) expression: Quantitative analysis of MT-metal load in eye cells. J. Biol. Chem. 2012, 287, 28456– 28469, DOI: 10.1074/jbc.M112.365015220The Stoichiometric Transition from Zn6Cu1-Metallothionein to Zn7-Metallothionein Underlies the Up-regulation of Metallothionein (MT) Expression. Quantitative Analysis of MT-Metal Load in Eye Cells.Alvarez, Lydia; Gonzalez-Iglesias, Hector; Garcia, Montserrat; Ghosh, Sikha; Sanz-Medel, Alfredo; Coca-Prados, MiguelJournal of Biological Chemistry (2012), 287 (34), 28456-28469CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)We examd. the profiling of gene expression of metallothioneins (MTs) in human tissues from cadaver eyes with microarray-based anal. All MT1 isoforms, with the exception of MT1B, were abundantly expressed in lens and corneal tissue. Along with MT1B, MT4 was not detected in any tissues. Antibodies to MT1/2 labeled the corneal epithelial and endothelial cells, whereas MT3 label the retinal ganglion cells. We studied the effects of zinc and cytokines on the gene expression of MT isoforms in a corneal epithelial cell line (HCEsv). Zinc exerted an up-regulation of the expression of MT isoforms, and this effect was further potentiated in the presence of IL1α or TNFα. Zinc also elicited a strong down-regulation of the expression of inflammatory cytokines, and this effect was blocked in the presence of TNFα or IL1α. The concn. of MTs, bound zinc, and the metal stoichiometry of MTs in cultured HCEsv were detd. by mass spectrometry. The total concn. of MTs was 0.24 ± 0.03 μm and, after 24 h of zinc exposure, increased to 0.96 ± 0.01 μm. The combination of zinc and IL1α further enhanced the level of MTs to 1.13 ± 0.03 μm. The av. metal stoichiometry of MTs was Zn6Cu1-MT, and after exposure to the different treatments, it changed to Zn7-MT. Actinomycin D blocked transcription, and cycloheximide attenuated synthesis of MTs in the presence or absence of zinc, suggesting transcriptional regulation. Overall the data provide mol. and anal. evidence on the interplay between zinc, MTs, and proinflammatory cytokines in HCEsv cells, with potential implications on cell-based inflammatory eye diseases.
- 221Jara-Biedma, R.; González-Dominguez, R.; García-Barrera, T.; Lopez-Barea, J.; Pueyo, C.; Gómez-Ariza, J. L. Evolution of metallothionein isoforms complexes in hepatic cells of Mus musculus along cadmium exposure. BioMetals 2013, 26, 639– 650, DOI: 10.1007/s10534-013-9636-0221Evolution of metallotionein isoforms complexes in hepatic cells of Mus musculus along cadmium exposureJara-Biedma, R.; Gonzalez-Dominguez, R.; Garcia-Barrera, T.; Lopez-Barea, J.; Pueyo, C.; Gomez-Ariza, J. L.BioMetals (2013), 26 (4), 639-650CODEN: BOMEEH; ISSN:0966-0844. (Springer)Characterization of Cd-binding proteins has great anal. interest due to the high toxicity of Cd to living organisms. Metallothioneins (MTs), as Cd(II)-binding proteins are of increasing interest, since they form very stable Cd chelates and are involved in many detoxification processes. In this work, inductively coupled plasma octopole reaction cell mass spectrometry and nanospray ionization time-of-flight mass spectrometry were used in parallel and combined with two-dimensional chromatog.: size exclusion followed by reversed-phase high performance liq. chromatog., to study metal complexes of MT isoforms produced in hepatic cytosols of Mus musculus during exposure expts. to Cd. Exposure expts. were carried out by s.c. injection of a growing dose of the toxic element ranging from 0.1 to 1.0 mg of Cd per kg of body wt. per day during 10 days. A control group and three exposure groups at days 2, 6 and 10 of exposure were studied, and different cadmium, copper and zinc complexes with MTs isoforms were isolated and characterized from the two most exposed groups. The results allow gaining insight into the mechanisms involved in metal detoxification by MTs, showing the changes in the stoichiometry of metal complexes-MTs along cadmium exposure.
- 222Suzuki, Y. Metal-binding properties of metallothionein in extracellular fluids and its role in cadmium-exposed rats. In Biological Roles of Metallothionein; Foulkes, E. C., Ed.; Elsevier: Amsterdam, The Netherlands, 1982; pp 27– 35.There is no corresponding record for this reference.
- 223Tio, L.; Villarreal, L.; Atrian, S.; Capdevila, M. Functional differentiation in the mammalian metallothionein gene family: Metal-binding features of mouse MT-4 and comparison with its paralog MT-1. J. Biol. Chem. 2004, 279, 24403– 24413, DOI: 10.1074/jbc.M401346200223Functional Differentiation in the Mammalian Metallothionein Gene Family: Metal binding features of mouse MT4 and comparison with its paralog MT1Tio, Laura; Villarreal, Laura; Atrian, Silvia; Capdevila, MerceJournal of Biological Chemistry (2004), 279 (23), 24403-24413CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)This paper reports on the characterization of the metal binding abilities of mammalian MT4 and their comparison with those of the well known MT1. Heterologous Escherichia coli expression in cultures supplemented with zinc, cadmium, or copper was achieved for MT4 and for its sep. αMT4 and βMT4 domains as well as for MT1 and its αMT1 domain in cadmium-enriched medium. The in vivo conformed metal complexes and the in vitro substituted zinc/cadmium and zinc/copper MT4 aggregates were characterized. Biosynthesis of MT4 and βMT4 in Cd(II)-supplemented medium revealed that these peptides failed to form the same homometallic species as MT1, thus appearing less effective for cadmium coordination. Conversely, the entire MT4 and both of its domains showed better Cu(I) binding properties than MT1, affording Cu10-MT4, Cu5-αMT4 and Cu7-βMT4, stoichiometries that make the domain dependence toward Cu(I) clear. Overall results allow consideration of MT4 as a novel copper-thionein, made up of two copper-thionein domains, the first of this class reported in mammals, and by extension in vertebrates. Furthermore, the in silico protein sequence analyses corroborated the copper-thionein nature of the MT4 peptides. As a consequence, there is the suggestion of a possible physiol. role played by MT4 related with copper requirements in epithelial differentiating tissues, where MT4 is expressed.
- 224Włostowski, T. Postnatal changes in subcellular distribution of copper, zinc and metallothionein in the liver of bank vole (Clethrionomys glareolus): a possible involvement of metallothionein and copper in cell proliferation. Comp. Biochem. Physiol., C: Comp. Pharmacol. 1992, 103, 285– 290, DOI: 10.1016/0742-8413(92)90009-V224Postnatal changes in subcellular distribution of copper, zinc and metallothionein in the liver of bank vole (Clethrionomys glareolus): a possible involvement of metallothionein and copper in cell proliferationWlostowski, T.Comparative Biochemistry and Physiology, Part C: Pharmacology, Toxicology & Endocrinology (1992), 103C (2), 285-90CODEN: CBPCEE; ISSN:0742-8413.Dramatic interdependent changes in the intracellular concns. of Cu, Zn and metallothionein (MT) in the liver of bank voles during the first 30 days of their life were obsd. The post-mitochondrial Cu, Zn and MT (Zn-MT) abruptly decreased between 1 and 3 days following birth but the nuclear MT (Cu-MT) and Cu increased at the same time, suggesting that Cu displaced Zn already bound to MT in the cytoplasm and subsequently the complex Cu-MT was translocated to the nuclei. The nuclear Cu concn. reached the highest level (62-71% of the total tissue Cu) in the period from day 3 to 20 post-partum, just prior to and during a rapid growth of the liver. The data indicate that MT and Cu may be involved in the hepatocyte proliferation.
- 225Tottey, S.; Waldron, K. J.; Firbank, S. J.; Reale, B.; Bessant, C.; Sato, K.; Cheek, T. R.; Gray, J.; Banfield, M. J.; Dennison, C.; Robinson, N. J. Protein-folding location can regulate manganese-binding versus copper- or zinc-binding. Nature 2008, 455, 1138– 1145, DOI: 10.1038/nature07340225Protein-folding location can regulate manganese-binding versus copper- or zinc-bindingTottey, Steve; Waldron, Kevin J.; Firbank, Susan J.; Reale, Brian; Bessant, Conrad; Sato, Katsuko; Cheek, Timothy R.; Gray, Joe; Banfield, Mark J.; Dennison, Christopher; Robinson, Nigel J.Nature (London, United Kingdom) (2008), 455 (7216), 1138-1142CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)Metals are needed by at least 25% of all proteins. Although metallochaperones insert the correct metal into some proteins, they have not been found for the vast majority, and the view is that most metalloproteins acquire their metals directly from cellular pools. However, some metals form more stable complexes with proteins than do others. For instance, as described in the Irving-Williams series, Cu2+ and Zn2+ typically form more stable complexes than Mn2+. Thus, it is unclear what cellular mechanisms manage metal acquisition by most nascent proteins. To investigate this question, the authors identified the most abundant Cu2+-protein, CucA (Cu2+-cupin A), and the most abundant Mn2+-protein, MncA (Mn2+-cupin A), in the periplasm of the cyanobacterium, Synechocystis PCC 6803. Each of these newly identified proteins binds its resp. metal via identical ligands within a cupin fold. Consistent with the Irving-Williams series, MncA only binds Mn2+ after folding in solns. contg. at least a 104-fold molar excess of Mn2+ over Cu2+ or Zn2+. However, once MncA has bound Mn2+, the metal does not exchange with Cu2+. MncA and CucA had signal peptides for different export pathways into the periplasm, Tat and Sec, resp. Export by the Tat pathway allows MncA to fold in the cytoplasm, which contains only tightly bound Cu2+ or Zn2+, but micromolar Mn2+. In contrast, CucA folds in the periplasm to acquire Cu2+. These results reveal a mechanism whereby the compartment in which a protein folds overrides its binding preference to control its metal content. They explain why the cytoplasm must contain only tightly bound and buffered Cu2+ and Zn2+.
- 226Pountney, D. L.; Schauwecker, I.; Zarn, J.; Vašák, M. Formation of mammalian Cu8-metallothionein in vitro: Evidence for the existence of two Cu(I)4-thiolate clusters. Biochemistry 1994, 33, 9699– 9705, DOI: 10.1021/bi00198a040226Formation of Mammalian Cu8-Metallothionein in vitro: Evidence for the Existence of Two Cu(I)4-Thiolate ClustersPountney, Dean L.; Schauwecker, Ivo; Zarn, Juerg; Vasak, MilanBiochemistry (1994), 33 (32), 9699-705CODEN: BICHAW; ISSN:0006-2960.Cu accumulates in metallothionein (Cu-MT) in Cu overload diseases, such as Wilson's disease and Bedlington Terriers disease. The in vitro formation of the Cu12-MT form comprising 2 Cu(I)6(CysS)9,11 cores is well documented. However, lysosomal Cu-MT isolated from canine liver was previously shown to contain 8 Cu(I) ions in 2 proposed adamantane-like Cu4-thiolate clusters. The present studies were carried out in an effort to learn more about the Cu(I)-thiolate cluster species formed upon the sequential incorporation of Cu(I) ions into metal-free MT from rabbit liver. On the basis of changes in the electronic absorption, CD, MCD, and luminescence spectra, besides the formation of a mol. species with 12 Cu(I) equiv., evidence for the existence of a distinct MT complex with 8 Cu(I) equiv. (Cu8-MT) was obtained. Anal. of the metal-dependent absorption envelope of Cu(I)-MT between 240 and 360 nm permitted the discrimination between predominantly CysS-Cu(I) charge-transfer (LMCT) (240-260 nm) and cluster-localized Cu(I) (d-s) transitions (260-360 nm). Accordingly, the decrease in the ratio of intensities of LMCT to d-s bands from 2.6 to 2.4 on going from 8 to 12 Cu(I) equiv. was attributed to the formation of Cu-MT species with different cysteine ligand to metal stoichiometries. The results suggested that while in Cu12-MT all 20 thiolate ligands participate in metal binding, in the Cu8-MT species between 12 and 14 cysteines take part in Cu(I) coordination. The low-temp. luminescence spectrum (77 K) of Cu8-MT was characterized by low- and high-energy emission bands at 610 nm (τ = ∼130 μs) and 425 nm (τ = ∼50 μs), resp. In contrast, the corresponding spectrum of the 2 Cu6 clusters in Cu12-MT exhibited only the low-energy band at 610 nm (τ = ∼130 μs). The measured lifetimes (τ) were consistent with emissions from triplet excited states assigned to mixed d-s/LMCT in origin. Similar luminescence behavior was previously obsd. with crystallog. defined inorg. Cu(I)6 and Cu(I)4 cluster models where it was attributed to a shorter Cu···Cu sepn. in the latter case. Accordingly, the occurrence of the high-energy band in Cu8-MT provided evidence for the existence of Cu(I)4 clusters in this species. On the basis of these data, it was concluded that the Cu8-MT species contains 2 Cu(I)4(CysS)6-7 clusters and is thus analogous to that isolated from canine liver (Bedlington Terriers disease). It was suggested that the formation of distinct Cu8- and Cu12-MT species reflects the relative concns. of Cu and protein present in soln.
- 227Roschitzki, B.; Vašák, M. A distinct Cu4-thiolate cluster of human metallothionein-3 is located in the N-terminal domain. JBIC, J. Biol. Inorg. Chem. 2002, 7, 611– 616, DOI: 10.1007/s00775-002-0339-1227A distinct Cu4-thiolate cluster of human metallothionein-3 is located in the N-terminal domainRoschitzki, Bernd; Vasak, MilanJBIC, Journal of Biological Inorganic Chemistry (2002), 7 (6), 611-616CODEN: JJBCFA; ISSN:0949-8257. (Springer-Verlag)Metallothionein-3 (MT-3), also known as neuronal growth inhibitory factor, is a metalloprotein expressed almost exclusively in the brain. Isolated MT-3 contains four Cu(I) and three Zn(II) ions organized in homometallic metal-thiolate clusters located in two independent protein domains. In this work a Cu(I) binding to metal-free MT-3 has been studied, aiming at the better understanding of the domain specificity for this metal ion. The cluster formation was followed by electronic absorption, CD, and by luminescence spectroscopy at room temp. and 77 K. The stepwise incorporation of Cu(I) into recombinant human apo-MT-3 revealed the cooperative formation of two Cu4S9 clusters in succession, formed in both protein domains, i.e. Cu4- and Cu8-MT-3. Further binding of four Cu(I) caused an expansion of these Cu(I) cores, leading to fully metal-loaded Cu12-MT-3 contg. Cu6S9 and Cu6S11 clusters in the β- and α-domains of the protein, resp. The location of the preferentially formed Cu4 cluster in the protein was established by immunochem. Using domain-specific antibodies, in combination with limited tryptic digestion of a partially metal-occupied Cu4-MT-3, we could demonstrate that the Cu4S9 cluster is located in the N-terminal β-domain of the protein that contains a total of nine cysteine ligands. Electronic supplementary material to this paper, comprising Table S1 (amino acid sequences of peptides used in immunization) and Fig. S1 (luminescence spectra of Cu(I) titrated apo-MT-3).
- 228Hofbeck, T.; Monkowius, U.; Yersin, H. Highly efficient luminescence of Cu(I) compounds – TADF combined with short-lived phosphorescence. J. Am. Chem. Soc. 2015, 137, 399– 404, DOI: 10.1021/ja5109672228Highly Efficient Luminescence of Cu(I) Compounds: Thermally Activated Delayed Fluorescence Combined with Short-Lived PhosphorescenceHofbeck, Thomas; Monkowius, Uwe; Yersin, HartmutJournal of the American Chemical Society (2015), 137 (1), 399-404CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Luminescent materials showing thermally-activated delayed fluorescence (TADF) have gained high attractiveness as emitters in org. light emitting diodes (OLEDs) and other photonic applications. Even use of TADF can be further improved, introducing a novel concept. This is demonstrated by a new class of brightly luminescent low-cost Cu(I) compds., for which the emission stems from both the lowest excited triplet T1 and singlet S1 state. At T = 300 K, these materials exhibit quantum yields of more than ΦPL = 90% at short emission decay times. About 80% of the emission intensity stems from the singlet due to TADF, but importantly, an addnl. 20% is contributed by the lower lying triplet state according to effective spin-orbit coupling (SOC). SOC induces also a relatively large zero-field splitting of the triplet being unusual for Cu(I) complexes. Thus, the overall emission decay time is distinctly reduced. Combined use of both decay paths opens novel photonic applications, in particular, for OLEDs. Crystallog. data are given.
- 229Scheller, J. S.; Irvine, G. W.; Wong, D. L.; Hartwig, A.; Stillman, M. J. Stepwise copper(I) binding to metallothionein: A mixed cooperative and non-cooperative mechanism for all 20 copper ions. Metallomics 2017, 9, 447– 462, DOI: 10.1039/C7MT00041C229Stepwise copper(I) binding to metallothionein: a mixed cooperative and non-cooperative mechanism for all 20 copper ionsScheller, Judith S.; Irvine, Gordon W.; Wong, Daisy L.; Hartwig, Andrea; Stillman, Martin J.Metallomics (2017), 9 (5), 447-462CODEN: METAJS; ISSN:1756-591X. (Royal Society of Chemistry)Copper is a ubiquitous trace metal of vital importance in that it serves as a cofactor in many metalloenzymes. Excess copper becomes harmful if not sequestered appropriately in the cell. As a metal ion chaperone, metallothionein (MT) has been proposed as a key player in zinc and copper homeostasis within the cell. The underlying mechanisms by which MT sequesters and transfers copper ions, and subsequently achieves its proposed biol. function remain unknown. Using a combination of electrospray ionization mass spectrometry (ESI-MS), CD (CD), and emission spectroscopy, we report that the Cu(I) to human apo-MT1a binding mechanism is highly pH-dependent. The 20 relative Kf-values for the binding of 1 to 20 Cu(I) to the 20 cysteines of MT were obtained from computational simulation of the exptl. mass spectral results. These data identified the pH-dependent formation of three sequential but completely different Cu-SCYS clusters, as a function of Cu(I) loading. These data provide the first overall sequence for Cu(I) binding in terms of domain specificity and transient binding site structures. Under cooperative binding at pH 7.4, a series of four clusters form: Cu4SCYS-6, followed by Cu6SCYS-9 (β), then a second Cu4SCYS-6 (α), and finally Cu7SCYS-x (α) (x = up to 11). Upon further addn. of Cu(I), a mixt. of species is formed in a non-cooperative mechanism, satg. the 20 cysteines of MT1a. Using benzoquinone, a cysteine modifier, we were able to confirm that Cu6SCYS-9 formed solely in the N-terminal β-domain, as well as confirming the existence of the presumed Cu4SCYS-6 cluster in the α-domain. Based on the results of ESI-MS and computational simulation we were able to identify Cu:MT speciation that resulted in specific emission and CD spectral properties.
- 230Nielson, K. B.; Winge, D. R. Preferential binding of copper to the β-domain metallothionein. J. Biol. Chem. 1984, 259, 4941– 4946, DOI: 10.1016/S0021-9258(17)42937-1230Preferential binding of copper to the β domain of metallothioneinNielson, Kirk B.; Winge, Dennis R.Journal of Biological Chemistry (1984), 259 (8), 4941-6CODEN: JBCHA3; ISSN:0021-9258.Proteolytic studies of rat liver metallothionein reconstituted in vitro with Cu salts revealed that the 2 metal centers fill in an ordered fashion. The B cluster in the N-terminal β domain fills prior to Cu binding in cluster A. This is contrary to cluster formation induced by the binding of Cd2+ or Zn2+ in which cluster A is the center of initial binding. The formation of metal cluster B by Cu occurs in a cooperative fashion yielding a satd. cluster with ∼6 Cu+ bound. The B cluster is satd. with Cd or Zn after binding of only 3 metal ions. The preferential binding of Cd and Cu to the α and β domains, resp., and the tolerance toward proteolysis of these 2 different half-satd. mols. permit the isolation of each domain. The metal cluster in each isolated domain can be reversibly formed with predicted stoichiometries of Cd and Cu. The folding of the polypeptide therefore appears to create each cluster independently. The metal binding data suggest that Cu-metallothionein contains 11-12 Cu ions, 6 bound in the β domain and 5-6 in the α domain. In contrast, Cd-metallothionein contains 7 Cd ions, 3 bound to β and 4 to α.
- 231Young, T. R.; Xiao, Z. Principles and practice of determining metal-protein affinities. Biochem. J. 2021, 478, 1085– 1116, DOI: 10.1042/BCJ20200838231Principles and practice of determining metal-protein affinitiesYoung, Tessa R.; Xiao, ZhiguangBiochemical Journal (2021), 478 (5), 1085-1116CODEN: BIJOAK; ISSN:0264-6021. (Portland Press Ltd.)A review. Metal ions play many crit. roles in biol., as structural and catalytic cofactors, and as cell regulatory and signalling elements. The metal-protein affinity, expressed conveniently by the metal dissocn. const., KD, describes the thermodn. strength of a metal-protein interaction and is a key parameter that can be used, for example, to understand how proteins may acquire metals in a cell and to identify dynamic elements (e.g. cofactor binding, changing metal availabilities) which regulate protein metalation in vivo. Here, we outline the fundamental principles and practical considerations that are key to the reliable quantification of metal-protein affinities. We review a selection of spectroscopic probes which can be used to det. protein affinities for essential biol. transition metals (including Mn(II), Fe(II), Co(II), Ni(II), Cu(I), Cu(II) and Zn(II)) and, using selected examples, demonstrate how rational probe selection combined with prudent exptl. design can be applied to det. accurate KD values.
- 232Morgan, M. T.; Yang, B.; Harankhedkar, S.; Nabatilan, A.; Bourassa, D.; McCallum, A. M.; Sun, F.; Wu, R.; Forest, C. R.; Fahrni, C. J. Stabilization of aliphatic phosphines by auxiliary phosphine sulfides offers zeptomolar affinity and unprecedented selectivity for probing biological CuI. Angew. Chem., Int. Ed. 2018, 57, 9711– 9715, DOI: 10.1002/anie.201804072232Stabilization of Aliphatic Phosphines by Auxiliary Phosphine Sulfides Offers Zeptomolar Affinity and Unprecedented Selectivity for Probing Biological CuIMorgan, M. Thomas; Yang, Bo; Harankhedkar, Shefali; Nabatilan, Arielle; Bourassa, Daisy; McCallum, Adam M.; Sun, Fangxu; Wu, Ronghu; Forest, Craig R.; Fahrni, Christoph J.Angewandte Chemie, International Edition (2018), 57 (31), 9711-9715CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)Full elucidation of the functions and homeostatic pathways of biol. copper requires tools that can selectively recognize and manipulate this trace nutrient within living cells and tissues, where it exists primarily as CuI. Buffered at attomolar concns., intracellular CuI is, however, not readily accessible to commonly employed amine and thioether-based chelators. Herein, the authors reveal a chelator design strategy in which phosphine sulfides aid in CuI coordination while simultaneously stabilizing aliph. phosphine donors, producing a charge-neutral ligand with low-zeptomolar dissocn. const. and 1017-fold selectivity for CuI over ZnII, FeII, and MnII. As illustrated by reversing ATP7A trafficking in cells and blocking long-term potentiation of neurons in mouse hippocampal brain tissue, the ligand is capable of intercepting copper-dependent processes. The phosphine sulfide-stabilized phosphine (PSP) design approach, which confers resistance towards protonation, dioxygen, and disulfides, could be readily expanded towards ligands and probes with tailored properties for exploring CuI in a broad range of biol. systems.
- 233Bagchi, P.; Morgan, M. T.; Bacsa, J.; Fahrni, C. J. Robust affinity standards for Cu(I) biochemistry. J. Am. Chem. Soc. 2013, 135, 18549– 18559, DOI: 10.1021/ja408827d233Robust Affinity Standards for Cu(I) BiochemistryBagchi, Pritha; Morgan, M. Thomas; Bacsa, John; Fahrni, Christoph J.Journal of the American Chemical Society (2013), 135 (49), 18549-18559CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)The measurement of reliable Cu-(I) protein binding affinities requires competing ref. ligands with similar binding strengths; however, the literature on such ref. ligands is not only sparse but often conflicting. To address this deficiency, we have created and characterized a series of water-sol. monovalent copper ligands, MCL-1,/ MCL-2, and MCL-3, that form well-defined, air-stable, and colorless complexes with Cu-(I) in aq. soln. X-ray structural data, electrochem. measurements, and an extensive network of equil. titrns. showed that all three ligands form discrete Cu-(I) complexes with 1:1 stoichiometry and are capable of buffering Cu-(I) concns. between 10-10 and 10-17 M. As most Cu-(I) protein affinities have been obtained from competition expts. with bathocuproine disulfonate or 2,2'-bicinchoninic acid, we further calibrated their Cu-(I) stability consts. against the MCL series. To demonstrate the application of these reagents, we detd. the Cu-(I) binding affinity of CusF (log K = 14.3 ± 0.1), a periplasmic metalloprotein required for the detoxification of elevated copper levels in Escherichia coli. Altogether, this interconnected set of affinity stds. establishes a reliable foundation that will facilitate the precise detn. of Cu-(I) binding affinities of proteins and small-mol. ligands.
- 234Hemmerich, P.; Sigwart, C. Cu(CH3CN)2+, ein Mittel zum Studium homogener Reaktionen des einwertigen Kupfers in wässriger Lösung. Experientia 1963, 19, 488– 489, DOI: 10.1007/BF02150666234Cu(CH3CN)2+, a method for the study of the homogeneous reactions of univalent copper in aqueous solutionHemmerich, P.; Sigwart, C.Experientia (1963), 19 (9), 488-9CODEN: EXPEAM; ISSN:0014-4754.In the presence of excess MeCN, the formation consts. of Cu(I) complexes may be detd. by pH titration. Log β2 for Cu(CH3CN)2 is 4.35.
- 235Xiao, Z.; Loughlin, F.; George, G. N.; Howlett, G. J.; Wedd, A. G. C-terminal domain of the membrane copper transporter Ctr1 from Saccharomyces cerevisiae binds four Cu(I) ions as a cuprous-thiolate polynuclear cluster: sub-femtomolar Cu(I) affinity of three proteins involved in copper trafficking. J. Am. Chem. Soc. 2004, 126, 3081– 3090, DOI: 10.1021/ja0390350235C-Terminal Domain of the Membrane Copper Transporter Ctr1 from Saccharomyces cerevisiae Binds Four Cu(I) Ions as a Cuprous-Thiolate Polynuclear Cluster: Sub-femtomolar Cu(I) Affinity of Three Proteins Involved in Copper TraffickingXiao, Zhiguang; Loughlin, Fionna; George, Graham N.; Howlett, Geoffrey J.; Wedd, Anthony G.Journal of the American Chemical Society (2004), 126 (10), 3081-3090CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)The cytosolic C-terminal domain of the membrane copper transporter Ctr1 from the yeast Saccharomyces cerevisiae, Ctr1c, was expressed in E. coli as an oxygen-sensitive sol. protein with no significant secondary structure. Visible-UV spectroscopy demonstrated that Ctr1c bound four Cu(I) ions, structurally identified as a CuI4(μ-S-Cys)6 cluster by X-ray absorption spectroscopy. This was the only metalated form detected by electrospray ionization mass spectrometry. An av. dissocn. const. Ka = (K1K2K3K4)1/4 = 10-19 for binding of Cu(I) to Ctr1c was estd. via competition with the ligand bathocuproine disulfonate bcs (β2 = 1019.8). Equivalent expts. for the yeast chaperone Atx1 and an N-terminal domain of the yeast Golgi pump Ccc2, which both bind a single Cu(I) ion, provided similar KD values. The ests. of KD were supported by independent ests. of the equil. consts. Kex for exchange of Cu(I) between pairs of these three proteins. It is apparent that, in vitro, the three proteins buffer "free" Cu(I) concns. in a narrow range around 10-19 M. The results provide quant. support for the proposals that, in yeast, (a) "free" copper concns. are very low in the cytosol and (b) the Cu(I) trafficking gradient is shallow along the putative Ctrlc → Atx1 → Ccc2n metabolic pathway. In addn., both Ctr1c and its copper-responsive transcription factor Mac1 contain similar clusters which may be important in signaling copper status in yeast.
- 236Banci, L.; Bertini, I.; Ciofi-Baffoni, S.; Kozyreva, T.; Zovo, K.; Palumaa, P. Affinity gradients drive copper to cellular destinations. Nature 2010, 465, 645– 648, DOI: 10.1038/nature09018236Affinity gradients drive copper to cellular destinationsBanci, Lucia; Bertini, Ivano; Ciofi-Baffoni, Simone; Kozyreva, Tatiana; Zovo, Kairit; Palumaa, PeepNature (London, United Kingdom) (2010), 465 (7298), 645-648CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)Copper is an essential trace element for eukaryotes and most prokaryotes. However, intracellular free copper must be strictly limited because of its toxic side effects. Complex systems for copper trafficking evolved to satisfy cellular requirements while minimizing toxicity. The factors driving the copper transfer between protein partners along cellular copper routes are, however, not fully rationalized. Until now, inconsistent, scattered and incomparable data on the copper-binding affinities of copper proteins have been reported. Here we det., through a unified electrospray ionization mass spectrometry (ESI-MS)-based strategy, in an environment that mimics the cellular redox milieu, the apparent Cu(I)-binding affinities for a representative set of intracellular copper proteins involved in enzymic redox catalysis, in copper trafficking to and within various cellular compartments, and in copper storage. The resulting thermodn. data show that copper is drawn to the enzymes that require it by passing from one copper protein site to another, exploiting gradients of increasing copper-binding affinity. This result complements the finding that fast copper-transfer pathways require metal-mediated protein-protein interactions and therefore protein-protein specific recognition. Together with Cu,Zn-SOD1, metallothioneins have the highest affinity for copper(I), and may play special roles in the regulation of cellular copper distribution; however, for kinetic reasons they cannot demetallate copper enzymes. Our study provides the thermodn. basis for the kinetic processes that lead to the distribution of cellular copper.
- 237Krężel, A.; Leśniak, W.; Jeżowska-Bojczuk, M.; Młynarz, P.; Brasuń, J.; Kozłowski, H.; Bal, W. Coordination of heavy metals by dithiothreitol, a commonly used thiol group protectant. J. Inorg. Biochem. 2001, 84, 77– 88, DOI: 10.1016/S0162-0134(00)00212-9237Coordination of heavy metals by dithiothreitol, a commonly used thiol group protectantKrezel, A.; Lesniak, W.; Jezowska-Bojczuk, M.; Mlynarz, P.; Brasun, J.; Kozlowski, H.; Bal, W.Journal of Inorganic Biochemistry (2001), 84 (1-2), 77-88CODEN: JIBIDJ; ISSN:0162-0134. (Elsevier Science Inc.)D,l-Dithiothreitol (DTT), known also as Cleland reagent, is a thiol group protectant, used commonly in peptide and protein chem. Therefore, it is often added at high concns. in prepns. of proteins relevant to heavy metal biochem. The coordination of five of these metal ions, Zn(II), Cd(II), Pb(II), Ni(II) and Cu(I) to DTT was studied by potentiometric titrns., and UV-Vis and NMR spectroscopies. It was found that DTT forms specific and very stable polymeric and monomeric complexes with all of these metal ions, using both of its sulfur donors. The quant. description of these complexes in soln. and the solid state provides the basis for predictions of interference from DTT in studies of metal ion binding of thiol-contg. biomols.
- 238Smirnova, J.; Kabin, E.; Järving, I.; Bragina, O.; Tõugu, V.; Plitz, T.; Palumaa, P. Copper(I)-binding properties of de-coppering drugs for the treatment of Wilson disease. α-Lipoic acid as a potential anti-copper agent. Sci. Rep. 2018, 8, 1463, DOI: 10.1038/s41598-018-19873-2238Copper(I)-binding properties of de-coppering drugs for the treatment of Wilson disease. α-Lipoic acid as a potential anti-copper agentSmirnova Julia; Kabin Ekaterina; Jarving Ivar; Bragina Olga; Tougu Vello; Palumaa Peep; Plitz ThomasScientific reports (2018), 8 (1), 1463 ISSN:.Wilson disease is an autosomal recessive genetic disorder caused by loss-of-function mutations in the P-type copper ATPase, ATP7B, which leads to toxic accumulation of copper mainly in the liver and brain. Wilson disease is treatable, primarily by copper-chelation therapy, which promotes copper excretion. Although several de-coppering drugs are currently available, their Cu(I)-binding affinities have not been quantitatively characterized. Here we determined the Cu(I)-binding affinities of five major de-coppering drugs - D-penicillamine, trientine, 2,3-dimercapto-1-propanol, meso-2,3-dimercaptosuccinate and tetrathiomolybdate - by exploring their ability to extract Cu(I) ions from two Cu(I)-binding proteins, the copper chaperone for cytochrome c oxidase, Cox17, and metallothionein. We report that the Cu(I)-binding affinity of these drugs varies by four orders of magnitude and correlates positively with the number of sulfur atoms in the drug molecule and negatively with the number of atoms separating two SH groups. Based on the analysis of structure-activity relationship and determined Cu(I)-binding affinity, we hypothesize that the endogenous biologically active substance, α-lipoic acid, may be suitable for the treatment of Wilson disease. Our hypothesis is supported by cell culture experiments where α-lipoic acid protected hepatic cells from copper toxicity. These results provide a basis for elaboration of new generation drugs that may provide better therapeutic outcomes.
- 239Meloni, G.; Faller, P.; Vašák, M. Redox silencing of copper in metal-linked neurodegenerative disorders. J. Biol. Chem. 2007, 282, 16068– 16078, DOI: 10.1074/jbc.M701357200239Redox Silencing of Copper in Metal-linked Neurodegenerative Disorders: Reaction of Zn7metallothionein-3 with Cu2+ ionsMeloni, Gabriele; Faller, Peter; Vasak, MilanJournal of Biological Chemistry (2007), 282 (22), 16068-16078CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)Dysregulation of copper and zinc homeostasis in the brain plays a crit. role in Alzheimer disease (AD). Copper binding to amyloid-β peptide (Aβ) is linked with the neurotoxicity of Aβ and free radical damage. Metallothionein-3 (MT-3) is a small cysteine- and metal-rich protein expressed in the brain and found down-regulated in AD. This protein occurs intra- and extracellularly, and it plays an important role in the metab. of zinc and copper. In cell cultures Zn7MT-3, by an unknown mechanism, protects neurons from the toxicity of Aβ. We have, therefore, used a range of complementary spectroscopic and biochem. methods to characterize the interaction of Zn7MT-3 with free Cu2+ ions. We show that Zn7MT-3 scavenges free Cu2+ ions through their redn. to Cu+ and binding to the protein. In this reaction thiolate ligands are oxidized to disulfides concomitant with Zn2+ release. The binding of the first four Cu2+ is cooperative forming a Cu(I)4-thiolate cluster in the N-terminal domain of Cu4,Zn4MT-3 together with two disulfides bonds. The Cu4-thiolate cluster exhibits an unusual stability toward air oxygen. The results of UV-visible, CD, and Cu(I) phosphorescence at 77 K suggest the existence of metal-metal interactions in this cluster. We have demonstrated that Zn7MT-3 in the presence of ascorbate completely quenches the copper-catalyzed hydroxyl radical (OH•) prodn. Thus, zinc-thiolate clusters in Zn7MT-3 can efficiently silence the redox-active free Cu2+ ions. The biol. implication of our studies as to the protective role of Zn7MT-3 from the Cu2+ toxicity in AD and other neurodegenerative disorders is discussed.
- 240Ohta, T.; Tachiyama, T.; Yoshizawa, K.; Yamabe, T.; Uchida, T.; Kitagawa, T. Synthesis, structure, and H2O2-dependent catalytic functions of disulfide-bridged dicopper(I) and related thioether-copper(I) and thioether-copper(II) complexes. Inorg. Chem. 2000, 39, 4358– 4369, DOI: 10.1021/ic000018a240Synthesis, Structure, and H2O2-Dependent Catalytic Functions of Disulfide-Bridged Dicopper(I) and Related Thioether-Copper(I) and Thioether-Copper(II) ComplexesOhta, Takehiro; Tachiyama, Takashi; Yoshizawa, Kazunari; Yamabe, Tokio; Uchida, Takeshi; Kitagawa, TeizoInorganic Chemistry (2000), 39 (19), 4358-4369CODEN: INOCAJ; ISSN:0020-1669. (American Chemical Society)A disulfide-bridged dicopper(I) complex, [Cu2(Py2SSPy2)](ClO4)2 (1) (Py2SSPy2 = bis{2-[N,N-bis(2-pyridylethyl)amino]-1,1-dimethylethyl}disulfide), a thioether-Cu(I) complex, [Cu(iPrSPy2)](ClO4) (2) (iPrSPy2 = N-(2-isopropylthio-2-methyl)propyl-N,N-bis-2-(2-pyridyl)ethylamine), and a thioether-Cu(II) complex, [Cu(PheSPy2)(H2O)](ClO4)2 (3) (PheSPy2 = N-(2-methyl-2-phenethylthio)propyl-N,N-bis-2-(2-pyridyl)ethylamine), were newly synthesized by the reactions of Cu(ClO4)2·6H2O with a thiol ligand of Py2SH (N,N-bis[2-(2-pyridyl)ethyl]-1,1-dimethyl-2-mercaptoethylamine) and thioether ligands of iPrSPy2 and PheSPy2, resp. For complexes 1 and 2, x-ray analyses were performed. Complex 1 crystallizes in the triclinic space group P‾1, and complex 2 crystallizes in the orthorhombic space group Pbca with the following unit cell parameters: for 1, a 15.165(3) Å, b 22.185(4) Å, c 14.989(3) Å, α 105.76(1), β 90.82(2), γ 75.23(1)°, and Z = 2; for 2, a 17.78(2) Å, b 17.70(1) Å, c 15.75(1) Å, and Z = 8. Complex 1 is the 1st structurally characterized example obtained by the redox reaction Cu(II) + RSH → Cu(I) + RSSR and has two independent structures (1a, 1b) which mainly differ in S-S bond distances, Cu(I)···Cu(I) sepns., and C-S-S-C dihedral angles of the disulfide units. The S-S bond distances of 2.088(7) Å in 1a and 2.070(7) Å in 1b are indicative of significant activation of the S-S bonds by the dicopper centers. Fragment MO (FMO) analyses and MO overlap population (MOOP) analyses based on the extended Huckel method clarify the preferable formation of the disulfide S-S bond in 1 rather than the formation of a thiolate-Cu(II) complex within the Py2S- ligand framework. Catalytic functions of complexes 1-3 were studied with peroxides (H2O2 and tBuOOH) as oxidants. Complex 1 catalyzed the selective oxidn. of cyclohexane to cyclohexanol and mediated the cyclohexene epoxidn. in the presence of H2O2. A transient dark green intermediate obsd. in the reaction of 1 with H2O2 was characterized by UV-visible, EPR, and resonance Raman spectroscopies, identifying it as a Cu(II)-OOH species, 1(OOH). The resonance Raman features of the ν(O-O) bands at 822 and 836 cm-1, which are red shifted to 781 and 791 cm-1, resp., upon introduction of H218O2, are indicative of formation of two kinds of Cu-OOH species rather than the Fermi doublet and the significant weakening of the O-O bonds. These mechanistic studies demonstrate that by virtue of the electron-donating ability of the disulfide unit the Cu-OOH species can be actually activated for 1-electron oxidn., which is reported so far unfavorable for other vibrationally characterized Cu-OOH species.
- 241Belle, C.; Rammal, W.; Pierre, J. L. Sulfur ligation in copper enzymes and models. J. Inorg. Biochem. 2005, 99, 1929– 1936, DOI: 10.1016/j.jinorgbio.2005.06.013241Sulfur ligation in copper enzymes and modelsBelle, Catherine; Rammal, Wassim; Pierre, Jean-LouisJournal of Inorganic Biochemistry (2005), 99 (10), 1929-1936CODEN: JIBIDJ; ISSN:0162-0134. (Elsevier B.V.)A review. Biol. Cu-S entities display versatile and unusual coordination chem. The role of S ligation is briefly reviewed through examples from selected Cu-contg. enzymes and relevant biomimetic models. Copper thiolate complexes are of particular interest because of their key roles in a no. of ubiquitous metalloenzymes such as type I (blue Cu proteins) or in the binuclear CuA electron transfer site found in both cytochrome c oxidase (CcO) and nitrous oxide reductase (N2OR). The possible roles of the S(Met) ligand in monoxygenases are described in relation to recently proposed pathways. Some perspectives regarding the biol. relevance of disulfide copper ligation and possible radical Cu bonds in catalytic cycle are also discussed.
- 242Weser, U. Redox reactions of sulphur-containing amino-acid residues in proteins and metalloproteins, an XPS study. In Cation Ordering and Electron Transfer. Structure and Bonding; Springer, Berlin, Germany, 1985; Vol. 61, pp 145– 160.There is no corresponding record for this reference.
- 243Sievers, C.; Deters, D.; Hartman, H. J.; Weser, U. Stable thiyl radicals in dried yeast Cu(I)6-thionein. J. Inorg. Biochem. 1996, 62, 199– 205, DOI: 10.1016/0162-0134(95)00153-0243Stable thiyl radicals in dried yeast Cu(I)6-thioneinSievers, Christian; Deters, Dirk; Hartmann, Hans-Juergen; Weser, UlrichJournal of Inorganic Biochemistry (1996), 62 (3), 199-205CODEN: JIBIDJ; ISSN:0162-0134. (Elsevier)It was of interest to obtain long-lived thiyl radicals embedded in org. matrixes. Solid thiol compds. including penicillamine, glutathione, and cysteine were UV irradiated under anaerobic conditions at 293 K for 60 min. The formed radicals were identified by ESR (EPR) (g = 2.0265 ± 0.0015) at 293 K as thiyl radicals. The blue-colored radical species were subjected to reflection spectrometry (λmax = 601 ± 3 nm). The color and the EPR signal remained unchanged for six months. At the same time, UV irradn. of lyophilized yeast Cu(I)6-thionein generated stable EPR detectable thiyl radicals at a g-value of 2.2026 ± 0.001. Unlike irradiated cysteine, a five times higher concn. of thiyl radicals was seen when the Cu(I)-thiolate protein was used. No EPR detectable thiyl radicals were measured in the Cu(I)-thiolates of penicillamine, glutathione, and thiophenole, indicating that the hexanuclear copper arrangement in Cu(I)-thionein is most suitable for both the formation and stabilization of this sulfur radical species.
- 244Roschitzki, B.; Vašák, M. Redox labile site in a Zn4 cluster of Cu4,Zn4-metallothionein-3. Biochemistry 2003, 42, 9822– 9828, DOI: 10.1021/bi034816z244Redox Labile Site in a Zn4 Cluster of Cu4,Zn4-Metallothionein-3Roschitzki, Bernd; Vasak, MilanBiochemistry (2003), 42 (32), 9822-9828CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)Human metallothionein-3 (MT-3) is a neuronal inhibitory factor mainly expressed in brain and downregulated in Alzheimer's disease. The neuroinhibitory activity has been established for native Cu4,Zn3-MT-3 and recombinant Zn7-MT-3. However, there is only limited knowledge about the structure and properties of the former metalloform. The authors have now generated native-like MT-3 through direct Cu(I) and Zn(II) incorporation into the recombinant apoprotein. Its characterization revealed monomeric Cu4,Zn4-MT-3 contg. metal-thiolate clusters located in two mutually interacting protein domains, a Cu4 cluster in the β-domain and a Zn4 cluster in the α-domain. Using the PC12 cell line, the nontoxic nature of the protein was demonstrated. The results of electronic absorption and Cu(I) luminescence at 77 K showed that the Cu4 cluster possesses an unprecedented stability in air. In contrast, the Zn4 cluster is air sensitive. Its oxidn. in the release of one Zn(II) and the formation of a Zn3 cluster, i.e., Cu4,-Zn3-MT-3. This process can be prevented or reversed under reducing conditions. The detd. apparent stability const. for the Zn4 cluster of 2.4 × 1011 M-1 is similar to that obtained for other zinc-contg. MTs. This suggests that a substantially increased nucleophilic reactivity of specific thiolate ligands is responsible for this effect. Thus, the Zn4 cluster in MT-3 may play a redox-dependent regulatory role.
- 245Bogumil, R.; Faller, P.; Binz, P. A.; Vašák, M.; Charnock, J. M.; Garner, C. D. Structural characterization of Cu(I) and Zn(II) sites in neuronal-growth-inhibitory factor by extended X-ray absorption fine structure (EXAFS). Eur. J. Biochem. 1998, 255, 172– 177, DOI: 10.1046/j.1432-1327.1998.2550172.x245Structural characterization of Cu(I) and Zn (II) sites in neuronal-growth-inhibitory factor by extended x-ray absorption fine structure (EXAFS)Bogumil, Ralf; Faller, Peter; Binz, Pierre-Alain; Vasak, Milan; Charnock, John M.; Garner, C. DavidEuropean Journal of Biochemistry (1998), 255 (1), 172-177CODEN: EJBCAI; ISSN:0014-2956. (Springer-Verlag)Neuronal-growth-inhibitory factor (GIF) is a metalloprotein specific to the central nervous system which has been linked to Alzheimer's disease. The high metal content, approx. seven metal atoms/protein mol., and 70% sequence identity to mammalian metallothioneins (MT), including a preserved array of 20 cysteinyl residues, place GIF in the family of MT. In contrast to MT, native GIF isolated from human or bovine brain contains an unusual metal compn., viz. four Cu(I) and three Zn(II) per polypeptide chain. Cu and/or Zn K-edge x-ray absorption spectra have been recorded for native Cu, Zn-GIF, Zn-substituted GIF, and these metals bound to the 32-residue N-terminal domain, Cu4-, Cu6- or Zn3- GIF-(1-32) at 77 K. The results are consistent with the metals being bound to the protein by cysteinyl residues in every case. The Cu-S distance is approx. 2.25 Å and the EXAFS is considered to be consistent with primarily trigonal coordination of the Cu(I); Cu···Cu backscattering is obsd. at approx. 2.67 Å, indicative of the formation of Cux(Scys)y clusters. Thus, the Cu(I) environment is similar to that obsd. in MT. This is also the case for Zn(II), with 4 S at approx. 2.34 Å. However, in contrast to Zn-MT for Zn-substituted GIF and Zn3-GIF-(1-32), Zn···Zn backscattering is obsd. at approx. 3.28 Å. The significance of these results are discussed with respect to the specific biol. activity of GIF.
- 246Meloni, G.; Sonois, V.; Delaine, T.; Guilloreau, L.; Gillet, A.; Teissié, J.; Faller, P.; Vašák, M. Metal swap between Zn7-metallothionein-3 and amyloid-β-Cu protects against amyloid-β toxicity. Nat. Chem. Biol. 2008, 4, 366– 372, DOI: 10.1038/nchembio.89246Metal swap between Zn7-metallothionein-3 and amyloid-β-Cu protects against amyloid-β toxicityMeloni, Gabriele; Sonois, Vanessa; Delaine, Tamara; Guilloreau, Luc; Gillet, Audrey; Teissie, Justin; Faller, Peter; Vasak, MilanNature Chemical Biology (2008), 4 (6), 366-372CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)Aberrant interactions of copper and zinc ions with the amyloid-β peptide (Aβ) potentiate Alzheimer's disease (AD) by participating in the aggregation process of Aβ and in the generation of reactive oxygen species (ROS). The ROS prodn. and the neurotoxicity of Aβ are assocd. with copper binding. Metallothionein-3 (Zn7MT-3), an intra- and extracellularly occurring metalloprotein, is highly expressed in the brain and downregulated in AD. This protein protects, by an unknown mechanism, cultured neurons from the toxicity of Aβ. Here, the authors show that a metal swap between Zn7MT-3 and sol. and aggregated Aβ1-40-Cu(II) abolishes the ROS prodn. and the related cellular toxicity. In this process, copper is reduced by the protein thiolates forming Cu(I)4Zn4MT-3, in which an air-stable Cu(I)4-thiolate cluster and two disulfide bonds are present. The discovered protective effect of Zn7MT-3 from the copper-mediated Aβ1-40 toxicity may lead to new therapeutic strategies for treating AD.
- 247Wezynfeld, N. E.; Stefaniak, E.; Stachucy, K.; Drozd, A.; Płonka, D.; Drew, S. C.; Krężel, A.; Bal, W. Resistance of Cu(Aβ4–16) to copper capture by metallothionein-3 supports a function for the Aβ4–42 peptide as a synaptic Cu(II) scavenger. Angew. Chem., Int. Ed. 2016, 55, 8235– 8238, DOI: 10.1002/anie.201511968247Resistance of Cu(Aβ4-16) to copper capture by metallothionein-3 supports a function for the Aβ4-42 peptide as a synaptic CuII scavengerWezynfeld, Nina E.; Stefaniak, Ewelina; Stachucy, Kinga; Drozd, Agnieszka; Plonka, Dawid; Drew, Simon C.; Krezel, Artur; Bal, WojciechAngewandte Chemie, International Edition (2016), 55 (29), 8235-8238CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)Aβ4-42 is a major species of amyloid-β (Aβ) peptide in the brains of both healthy individuals and those affected by Alzheimer's disease. It has recently been demonstrated to bind Cu2+ with an affinity ∼3000-fold higher than the commonly studied Aβ1-42 and Aβ1-40 peptides, which are implicated in the pathogenesis of Alzheimer's disease. Metallothionein-3, a protein considered to orchestrate Cu2+ and Zn2+ metab. in the brain and provide antioxidant protection, was shown to ext. Cu2+ from Aβ1-40 when acting in its native Zn7MT-3 form. This reaction is assumed to underlie the neuroprotective effect of Zn7MT-3 against Aβ toxicity. Here, the authors used truncated model peptides Aβ1-16 and Aβ4-16 to demonstrate that the high-affinity Cu2+ complex of Aβ4-16 is resistant to Zn7MT-3 reactivity. This indicated that the analogous complex of the full-length peptide, Cu(Aβ4-42), will not yield Cu2+ to MT-3 in the brain, thus supporting the concept of a physiol. role for Aβ4-42 as a Cu2+ scavenger in the synaptic cleft.
- 248Santoro, A.; Wezynfeld, N. E.; Stefaniak, E.; Pomorski, A.; Płonka, D.; Krężel, A.; Bal, W.; Faller, P. Cu transfer from amyloid-β4–16 to metallothionein-3: the role of the neurotransmitter glutamate and metallothionein-3 Zn(II)-load states. Chem. Commun. (Cambridge, U. K.) 2018, 54, 12634– 12637, DOI: 10.1039/C8CC06221H248Cu transfer from amyloid-β4-16 to metallothionein-3: the role of the neurotransmitter glutamate and metallothionein-3 Zn(II)-load statesSantoro, Alice; Wezynfeld, Nina Ewa; Stefaniak, Ewelina; Pomorski, Adam; Plonka, Dawid; Krezel, Artur; Bal, Wojciech; Faller, PeterChemical Communications (Cambridge, United Kingdom) (2018), 54 (89), 12634-12637CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)Copper transfer from Cu(II)amyloid-β4-16 to human Zn7-metallothionein-3 can be accelerated by glutamate and by lowering the Zn-load of metallothionein-3 with EDTA. Glutamate facilitates the Cu(II) release, and Zn4-6-metallothionein-3 react more rapidly. These mechanisms are additive, proving the intricate and interconnected network of zinc and copper trafficking between biomols.
- 249Santoro, A.; Calvo, J. S.; Peris-Díaz, M. D.; Krężel, A.; Meloni, G.; Faller, P. The glutathione/metallothionein system challenges the design of efficient O2-activating copper complexes. Angew. Chem., Int. Ed. 2020, 59, 7830– 7835, DOI: 10.1002/anie.201916316249The Glutathione/Metallothionein System Challenges the Design of Efficient O2-Activating Copper ComplexesSantoro, Alice; Calvo, Jenifer S.; Peris-Diaz, Manuel David; Krezel, Artur; Meloni, Gabriele; Faller, PeterAngewandte Chemie, International Edition (2020), 59 (20), 7830-7835CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)Copper complexes are of medicinal and biol. interest, including as anticancer drugs designed to cleave intracellular biomols. by O2 activation. To exhibit such activity, the copper complex must be redox active and resistant to dissocn. Metallothioneins (MTs) and glutathione (GSH) are abundant in the cytosol and nucleus. Because they are thiol-rich reducing mols. with high CuI affinity, they are potential competitors for a copper ion bound in a copper drug. Herein, we report the investigation of a panel of CuI/CuII complexes often used as drugs, with diverse coordination chemistries and redox potentials. We evaluated their catalytic activity in ascorbate oxidn. based on redox cycling between CuI and CuII, as well as their resistance to dissocn. or inactivation under cytosolically relevant concns. of GSH and MT. O2-activating CuI/CuII complexes for cytosolic/nuclear targets are generally not stable against the GSH/MT system, which creates a challenge for their future design.
- 250Palumaa, P.; Mackay, E. A.; Vašák, M. Nonoxidative cadmium-dependent dimerization of Cd7-metallothionein from rabbit liver. Biochemistry 1992, 31, 2181– 2186, DOI: 10.1021/bi00122a040250Nonoxidative cadmium-dependent dimerization of cadmium-metallothionein from rabbit liverPalumaa, Peep; Mackay, Elaine A.; Vasak, MilanBiochemistry (1992), 31 (7), 2181-6CODEN: BICHAW; ISSN:0006-2960.The effect of free Cd(II) ions on monomeric Cd7-metallothionein-2 (MT) from rabbit liver has been studied. Slow, concn.-dependent dimerization of this protein was obsd. by gel filtration chromatog. studies. The dimeric MT form, isolated by gel filtration, contains approx. two addnl. and more weakly bound Cd(II) ions per monomer. The incubation of MT dimers with complexing agents EDTA and 2-mercaptoethanol leads to the dissocn. of dimers to monomers. The results of CD and electronic absorption studies indicate that the slow dimerization process is preceded by an initial rapid Cd-induced rearrangement of the monomeric Cd7-MT structure. The 113Cd NMR spectrum of the MT dimer revealed only four 113Cd resonances at chem. shift positions similar to those obsd. for the Cd4 cluster of the well-characterized monomeric 113Cd7-MT. This result suggests that on dimer formation major structural changes occur in the original three-metal cluster domain of Cd7-MT.
- 251Palumaa, P.; Vašák, M. Binding of inorganic phosphate to the cadmium-induced dimeric form of metallothionein from rabbit liver. Eur. J. Biochem. 1992, 205, 1131– 1135, DOI: 10.1111/j.1432-1033.1992.tb16882.x251Binding of inorganic phosphate to the cadmium-induced dimeric form of metallothionein from rabbit liverPalumaa, Peep; Vasak, MilanEuropean Journal of Biochemistry (1992), 205 (3), 1131-5CODEN: EJBCAI; ISSN:0014-2956.Recently it was demonstrated that the exposure of monomeric Cd7-metallothionein (MT) to Cd(II) ions in potassium phosphate buffer results in the nonoxidative formation of MT dimers contg. ∼2 addnl. Cd(II) ions/monomer subunit. The present data shows that inorg. phosphate participates in the Cd-induced dimerization of MT. In the absence of phosphate, Cd-induced oligomerization of MT still takes place, but a substantially lower apparent yield of the dimeric form and an addnl. peak of MT tetramers were detected in gel-filtration expts. Arsenate exhibits a similar effect to that of phosphate, whereas a no. of other anions, i.e. F-, NO3-, SO42-, ClO4-, BO3-, SCN-, HCOO-, and CH3COO- had no effect on Cd-induced oligomerization of MT. Studies on the pH dependence of MT dimerization indicate that the dianionic form of phosphate is involved in this process. Equil.-dialysis expts. using potassium [32P]phosphate established binding of 2 mols. of phosphate to the dimeric MT form with a dissocn. const., Kd, of 23 ± 3 μM (20 mM Tris-HCl and 0.1 M KCl, pH 8.0 at 25°), whereas binding of phosphate was not obsd. with the monomeric Cd7-MT. The noncovalent nature of phosphate binding to the Cd-induced MT dimers has been demonstrated. This is the first evidence for the binding of a nonmetallic cellular component to MT.
- 252Schmid, R. F. Kinetische Untersuchungen des Metallaustauschs in Metallothionein. Diploma Thesis, University of Zürich: Switzerland, 1991.There is no corresponding record for this reference.
- 253Zangger, K.; Öz, G.; Otvos, J. D.; Armitage, I. M. Three-dimensional solution structure of mouse Cd7-metallothionein-1 by homonuclear and heteronuclear NMR spectroscopy. Protein Sci. 1999, 8, 2630– 2638, DOI: 10.1110/ps.8.12.2630253Three-dimensional solution structure of mouse [Cd7]-metallothionein-1 by homonuclear and heteronuclear NMR spectroscopyZangger, Klaus; Oz, Gulin; Otvos, James D.; Armitage, Ian M.Protein Science (1999), 8 (12), 2630-2638CODEN: PRCIEI; ISSN:0961-8368. (Cambridge University Press)Sequential 1H-NMR assignments of mouse [Cd7]-metallothionein-1 (MT1) have been carried out by std. homonuclear NMR methods and the use of an accordion-heteronuclear multiple quantum correlation (HMQC) expt. for establishing the metal, 113Cd2+, to cysteine connectivities. The three-dimensional structure was then calcd. using the distance constraints from two-dimensional nuclear Overhauser effect (NOE) spectroscopy spectra and the Cys-Cd connectivities as input for a distance geometry-dynamical simulated annealing protocol in X-PLOR 3.851. Similar to the mammalian MT2 isoforms, the homologous primary structure of MT1 suggested two sep. domains, each contg. one metal cluster. Because there were no interdomain constraints, the structure calcn. for the N-terminal β- and the C-terminal α-domain were carried out sep. The structures are based on 409 NMR constraints, consisting of 381 NOEs and 28 cysteine-metal connectivities. The only elements of regular secondary structure found were two short stretches of 310 helixes along with some half-turns in the α-domain. Structural comparison with rat liver MT2 showed high similarity, with the β-domain structure in mouse MT1 showing evidence of increased flexibility compared to the same domain in MT2. The latter was reflected by the presence of fewer interresidue NOEs, no slowly exchanging backbone amide protons, and enhanced cadmium-cadmium exchange rates found in the β-domain of MT1.
- 254Vašák, M.; Bauer, R. Evidence for two types of binding sites in cadmium metallothionein determined by perturbed angular correlation of γ-rays. J. Am. Chem. Soc. 1982, 104, 3236– 3238, DOI: 10.1021/ja00375a059254Evidence for two types of binding sites in cadmium metallothionein determined by perturbed angular correlation of γ-raysVasak, Milan; Bauer, RogertJournal of the American Chemical Society (1982), 104 (11), 3236-8CODEN: JACSAT; ISSN:0002-7863.The metal-binding sites in rabbit metallothionein-1 were probed by perturbed angular correlation of γ-ray spectroscopy (PAC) with excited 114Cd. The presence of 2 distinct frequencies, ω1 ∼120 and ω2 ∼580 MHz, is consistent with the existence of 2 different coordination geometries for Cd binding in this protein. The 120 MHz frequency is comparable to the 65 MHz frequency displayed by excited 111Cd when substituted for Zn in the slightly distorted tetrahedral structural metal site of horse liver alc. dehydrogenase. Judged from the greater amplitude of the 120 MHz signal, it would appear that this symmetry type applies to ∼80% of all metal-binding sites in metallothionein. The 580 MHz frequency is close to the 880 MHz frequency calcd. for the square planar Cd (Cys-S)42- complex and, hence, it could arise from an extremely distorted tetrahedron. Less likely, it could originate also from an octahedral-type complex having 2 axial carboxylate ligands and yielding a frequency of 640 MHz. The 2 frequencies are obsd. both in partially and in fully complexed metallothionein and are, thus, independent of the degree of metal-binding site occupation. Hence, the clustering of the metals in metallothionein, as proposed from homonuclear 113Cd-decoupling expts. and from magnetic studies of the Co(II)-deriv., does not measurably alter the basic coordination feature of the metal environment.
- 255Vašák, M.; Meloni, G. Metallothionein structure and reactivity. In Metallothionein in Biochemistry and Pathology; Zatta, P., Ed.; World Scientific: Singapore, 2008; pp 3– 26.There is no corresponding record for this reference.
- 256Jones, W. B.; Elgren, T. E.; Morelock, M. M.; Elder, R. C.; Wilcox, D. E. Technetium metallothionein: Spectroscopic and EXAFS study of 99TcO3+ binding to Zn7-metallothionein. Inorg. Chem. 1994, 33, 5571– 5578, DOI: 10.1021/ic00102a034256Technetium Metallothionein: Spectroscopic and EXAFS Study of 99TcO3+ Binding to Zn7-MetallothioneinJones, William B.; Elgren, Timothy E.; Morelock, Maurice M.; Elder, R. C.; Wilcox, Dean E.Inorganic Chemistry (1994), 33 (24), 5571-8CODEN: INOCAJ; ISSN:0020-1669.Vibrational, absorption, CD, and Tc K-edge extended x-ray absorption fine structure (EXAFS) measurements have been made on three samples of metallothionein (MT) contg. 1.6, 3.9, and 6.2 mol equiv of 99Tc, prepd. by the metal displacement (transchelation) reaction of TcO(glucoheptonate)2- with Zn7-MT. For all three samples the TcO3+ ion is bound to the protein in a square pyramidal coordination with equatorial ligands consisting of ∼2 cysteine thiolates and, surprisingly, ∼2 O/N donor ligands. As the Tc content increases, there is very little change in the Tc coordination, except for a decrease in the no. of cysteine ligands per TcO3+ for Tc6.2-MT. The unusual ligand set for this metal ion when it is bound to MT, the absence of bridging cysteine ligands, and the significant no. of cysteines not involved in binding TcO3+, indicate that transchelation does not involve a simple substitution of TcO3+ for Zn(II).
- 257Good, M.; Vašák, M. Iron(II)-substituted metallothionein: evidence for the existence of iron-thiolate clusters. Biochemistry 1986, 25, 8353– 8356, DOI: 10.1021/bi00374a003257Iron(II)-substituted metallothionein: evidence for the existence of iron-thiolate clustersGood, Meinrad; Vasak, MilanBiochemistry (1986), 25 (26), 8353-6CODEN: BICHAW; ISSN:0006-2960.Metallothioneins (MTs) are unique low-mol.-wt. (Mr 6000-7000) metal- and cysteine-rich proteins characterized by 2 tetrahedral tetrathiolate clusters contg. 3 and 4 metal ions. Naturally occurring proteins usually contain the diamagnetic metal ions Zn(II) and(or) Cd(II). These ions were substituted with paramagnetic Fe(II) and the resulting Fe-MT was characterized. Rabbit liver MT-1 in which all 7 metal binding sites were occupied by Fe(II) ions displays absorption features typical of tetrahedral tetrathiolate Fe(II) coordination. This is documented by the presence of a ligand field 5E→2T2 transition in the near-IR region centered at ∼1850 nm absorption coeff of Fe, (εFe, ≈100 M-1 cm-1) and a broad charge-transfer absorption in the UV region with a shoulder at 314 nm. A metal-thiolate cluster structure is inferred from the 7/20 ratio of metal ions to cysteine residues and from spectral studies in which successive increments of Fe(II) were incorporated into the metal-free protein. Thus, to ∼4 equiv, the charge-transfer absorption and magnetic CD (MCD) features of the complexes formed resemble closely those of reduced rubredoxin from Desulfovibro gigas, in which tetrahedral tetrathiolate Fe(II) coordination is documented. However, upon further addn. of Fe(II), the charge-transfer absorption bands undergo a progressive red-shift until the full metal occupancy of 7 Fe(II) ions/mol. is reached. The bathochromic shift which is also manifested in the MCD spectra can be ascribed to the transformation of some of the terminal thiolate ligands to bridging when the fall complement of Fe(II) is bound. The concomitant loss in amplitude of the MCD bands at >4 equiv is thought to arise from exchange coupling of vicinal Fe(II) via the thiolate bridges.
- 258Werth, M. T.; Johnson, M. K. Magnetic circular dichroism and electron paramagnetic resonance studies of iron(II)-metallothionein. Biochemistry 1989, 28, 3982– 3988, DOI: 10.1021/bi00435a053258Magnetic circular dichroism and electron paramagnetic resonance studies of iron(II)-metallothioneinWerth, Mark T.; Johnson, Michael K.Biochemistry (1989), 28 (9), 3982-8CODEN: BICHAW; ISSN:0006-2960.The electronic and magnetic properties of the Fe(II)-thiolate centers in Fe(II)-metallothionein were investigated by low-temp. MCD and EPR spectroscopies at various levels of Fe(II) incorporation. In agreement with previous results, rabbit liver metallothionein was found to bind a max. of 7 Fe(II) ions, with cluster formation occurring when >4 Fe(II) ions are bound at pH 8.5. The results indicate that all the Fe in fully loaded Fe(II)-metallothionein is accommodated in Fe(II)-thiolate clusters that have either S = 0 or S = 2 ground states as a result of antiferromagnetic coupling between high-spin Fe(II) ions. By analogy with the cluster compn. and mechanism of assembly that have been established for other divalent metal ions, the clusters with S = 0 and S = 2 ground states are attributed to tetranuclear and trinuclear centers, resp. EPR signals indicative of S = 2 species were obsd. for samples contg. monomeric tetrathiolate-Fe(II) centers and trinuclear Fe(II)-thiolate clusters. However, the nature of the zero field splitting of the S = 2 ground states that is indicated by the EPR signals is not consistent with that deduced from Moessbauer and MCD studies, suggesting heterogeneity in both types of center.
- 259Ding, X. Q.; Butzlaff, C.; Bill, E.; Pountney, D. L.; Henkel, G.; Winkler, H.; Vašák, M.; Trautwein, A. X. Mössbauer and magnetic susceptibility studies on iron(II) metallothionein from rabbit liver. Evidence for the existence of an unusual type of [M3(CysS)9]3- cluster. Eur. J. Biochem. 1994, 220, 827– 837, DOI: 10.1111/j.1432-1033.1994.tb18685.x259Moessbauer and magnetic susceptibility studies on iron(II) metallothionein from rabbit liver. Evidence for the existence of an unusual type of [M3(CysS)9]3- clusterDing, Xiao-Qi; Butzlaff, Christian; Bill, Eckhard; Pountney, Dean L.; Henkel, Gerald; Winkler, Heiner; Vasak, Milan; Trautwein, Alfred XaverEuropean Journal of Biochemistry (1994), 220 (3), 827-37CODEN: EJBCAI; ISSN:0014-2956.The magnetic properties of the Fe(II)-binding-sites in Fe(II)7-metallothionein (MT) have been studied using Moessbauer spectroscopy and magnetic-susceptibility measurements. In agreement with the authors' previous results, simulation of the Moessbauer spectra showed the presence of paramagnetic and diamagnetic subspectra in the ratio 3:4. By comparison with Moessbauer spectra of the inorg. adamantane-like (Et4N)2[Fe4(SEt)10] model compd., the diamagnetic component in Fe(II)7-MT has been assigned to a four-metal cluster in which there is antiferromagnetic coupling between the high-spin Fe(II) ions. It is suggested that the organization of this cluster is similar to that detd. in the three-dimensional structure of the protein, contg. diamagnetic Zn(II) and/or Cd(II) ions. From magnetic-susceptibility studies, an av. magnetic moment of approx. 8.5 μn was obtained for the three remaining bound Fe(II) ions, responsible for the paramagnetic component obsd. in the Moessbauer studies. This value is slightly lower than that for three completely uncoupled Fe(II) ions, suggesting the existence of a three-metal cluster within which there is weak exchange coupling between adjacent Fe(II) ions. The spin-Hamiltonian formalism including, besides zero-field and Zeeman interaction, also exchange interaction among the three Fe(II) ions in the three-metal cluster, H = -J12(S1·S2)-J23(S2·S3)-J13 (S1·S3), was applied to simulate both magnetic-Moessbauer and magnetic-susceptibility date. Reasonable fits were achieved only with values |J12| = |J23| = |J13| = |J| < 1 cm-1. Such a situation could not be reconciled with the chair-like geometry of the [M3(CysS)9]3- cluster detd. with paramagnetic metal ions, where significantly stronger coupling would be anticipated (|J| = 50-70 cm-1). However, modest exchange-coupling properties have been reported for a no. of crystallog. characterized trinuclear [Fe3(SR)3X6]3- clusters (X = Cl, Br; R = Phe, p-tolyl, 2,6-Me2C6H3) distinguished by the preferential formation of a planar Fe3(μ2-SR)3 ring. It is therefore more likely that a pseudo-planar geometry rather than a chair-like geometry is present in the Fe3 cluster of Fe(II)7-MT. This would represent the first example of structural differences on binding divalent metal ions to this protein.
- 260Orihuela, R.; Fernández, B.; Palacios, O.; Valero, E.; Atrian, S.; Watt, R. K.; Domínguez-Vera, J. M.; Capdevila, M. Ferritin and metallothionein: dangerous liaisons. Chem. Commun. (Cambridge, U. K.) 2011, 47, 12155– 12157, DOI: 10.1039/c1cc14819b260Ferritin and metallothionein: dangerous liaisonsOrihuela, Ruben; Fernandez, Belen; Palacios, Oscar; Valero, Elsa; Atrian, Silvia; Watt, Richard K.; Dominguez-Vera, Jose M.; Capdevila, MerceChemical Communications (Cambridge, United Kingdom) (2011), 47 (44), 12155-12157CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)Ferritin (Ft) interaction with the Zn-complexes of mammalian MT1, MT2 and MT3 metallothioneins (MT) leads to simultaneous FeII and ZnII release.
- 261Baird, S. K.; Kurz, T.; Brunk, U. T. Metallothionein protects against oxidative stress-induced lysosomal destabilization. Biochem. J. 2006, 394, 275– 283, DOI: 10.1042/BJ20051143261Metallothionein protects against oxidative stress-induced lysosomal destabilizationBaird, Sarah K.; Kurz, Tino; Brunk, Ulf T.Biochemical Journal (2006), 394 (1), 275-283CODEN: BIJOAK; ISSN:0264-6021. (Portland Press Ltd.)The introduction of apo-ferritin or the iron chelator DFO (desferrioxamine) conjugated to starch into the lysosomal compartment protects cells against oxidative stress, lysosomal rupture and ensuing apoptosis/necrosis by binding intralysosomal redox-active iron, thus preventing Fenton-type reactions and ensuing peroxidn. of lysosomal membranes. Because up-regulation of MTs (metallothioneins) also generates enhanced cellular resistance to oxidative stress, including X-irradn., and MTs were found to be capable of iron binding in an acidic and reducing lysosomal-like environment, we propose that these proteins might similarly stabilize lysosomes following autophagocytotic delivery to the lysosomal compartment. Here, we report that Zn-mediated MT up-regulation, assayed by Western blotting and immunocytochem., results in lysosomal stabilization and decreased apoptosis following oxidative stress, similar to the protection afforded by fluid-phase endocytosis of apo-ferritin or DFO. In contrast, the endocytotic uptake of an iron phosphate complex destabilized lysosomes against oxidative stress, but this was suppressed in cells with up-regulated MT. It is suggested that the resistance against oxidative stress, known to occur in MT-rich cells, may be a consequence of autophagic turnover of MT, resulting in reduced iron-catalyzed intralysosomal peroxidative reactions.
- 262Kennedy, M. C.; Gan, T.; Antholine, W. E.; Petering, D. H. Metallothionein reacts with Fe2+ and NO to form products with a g = 2.039 ESR signal. Biochem. Biophys. Res. Commun. 1993, 196, 632– 635, DOI: 10.1006/bbrc.1993.2296262Metallothionein reacts with iron(2+) and nitric oxide to form products with a g = 2.039 ESR signalKennedy, Mary Claire; Gan, Tong; Antholine, William E.; Petering, David H.Biochemical and Biophysical Research Communications (1993), 196 (2), 632-5CODEN: BBRCA9; ISSN:0006-291X.The interaction of metallothionein (MT), Fe2+, and nitric oxide has been examd. by ESR spectroscopy. When either apoMT 2 or Zn-MT 2 from rabbit liver was mixed with Fe2+ and NO, paramagnetic products with g values of 2.013 and 2.039 were found. These are characteristic of iron nitrosyl thiolates with stoichiometry, Fe(NO)2(SR)2. This product was not generated when beef heart mitochondrial aconitase was used as the source of iron.
- 263Sano, Y.; Onoda, A.; Sakurai, R.; Kitagishi, H.; Hayashi, T. Preparation and reactivity of a tetranuclear Fe(II) core in the metallothionein α-domain. J. Inorg. Biochem. 2011, 105, 702– 708, DOI: 10.1016/j.jinorgbio.2011.01.011263Preparation and reactivity of a tetranuclear Fe(II) core in the metallothionein α-domainSano, Yohei; Onoda, Akira; Sakurai, Rie; Kitagishi, Hiroaki; Hayashi, TakashiJournal of Inorganic Biochemistry (2011), 105 (5), 702-708CODEN: JIBIDJ; ISSN:0162-0134. (Elsevier)Metallothioneins (MTs) are small cysteine-rich proteins which exhibit high affinities for various metal ions and play roles in storage of essential metals and detoxification of toxic metals. Studies on the redox properties of MTs have been quite limited. Recently, the authors focused on the α-domain of MT (MTα) as a protein matrix and incorporated a tetranuclear metal cluster as a reductant. UV-visible, CD and MS data indicate the formation of the stable tetranuclear metal-cysteine cluster in the MTα matrix with FeII4-MTα and CoII4-MTα species existing in water. Furthermore, the FeII4-MTα species was found to promote the redn. of met-myoglobin and azobenzene derivs. under mild conditions. Particularly, the stoichiometric redn. of methyl red with FeII4-MTα (1:1) proceeds with a conversion of 98% over a period of 6 h at 25°. All four Fe(II) cores contribute to the redn. The authors describe the prepn. and reactivity of the tetranuclear iron cluster in the protein matrix in this paper.
- 264Irving, H.; Williams, R. J. Order of stability of metal complexes. Nature 1948, 162, 746– 747, DOI: 10.1038/162746a0264Order of stability of metal complexesIrving, H.; Williams, R. J. P.Nature (London, United Kingdom) (1948), 162 (), 746-7CODEN: NATUAS; ISSN:0028-0836.cf. Mellor and Maley, C.A. 41, 4398e; 42, 4809d. In the first transition series (Mn, Fe, Co, Ni, Cu, Zn) the stability of complexes increases steadily to a max. at Cu; ligands may be NH3, (CH2NH2)2, CH2(CH2NH2)2, or salicyl-aldehyde. Zn complexes are less stable than those of CuII (M-shell is completely filled); however, attachment of 6 groups through d2sp3 or dsp2 orbitals is possible from Ca to Cu(II). A similar trend is noted in the 2nd transition series with Pd(II) > Cd(II). In the 3rd transition series, the order is Pt(II) > Hg(II) and Pb(II). The stability of complexes increases with the electronegativity of the metal involved.
- 265Irving, H. M. N. H.; Williams, R. J. P. The stability of transition-metal complexes. J. Chem. Soc. 1953, 3192– 3210, DOI: 10.1039/jr9530003192265Stability of transition metal complexesIrving, H. M.; Williams, R. J. P.Journal of the Chemical Society (1953), (), 3192-3210CODEN: JCSOA9; ISSN:0368-1769.The published values of the stability consts. for the bivalent ions of Mn, Fe, Co, Ni, Cu, and Zn with ligands exhibiting NN, NO, and OO co.ovrddot.ordinating patterns are tabulated. In almost all cases the order of stability is Mn < Fe < Co < Ni < Cu > Zn irrespective of the nature of the ligand. This order is a consequence of the monotonic change in the 2nd ionization potentials and the reciprocal radii, the parameters serving as a guide to the magnitude of the covalent and electrostatic interactions, resp., of the ions involved. When other ions are inserted in the order of stability, the new order varies with the ligand because there is not generally the same relation for both factors throughout the series. Characteristic co.ovrddot.ordination no., stereochem. considerations, and entropy factors may, however, affect the series given here. Examples of the former two effects are the low value for k3 in the Cu++-(NH2CH2)2 system, the high values for the consts. for the Fe++-ο-phenanthroline system, and the low values for k2 in systems with (MeNHCH2)2. The entropy effect is such that the predominant factor, the entropy of hydration of M++, follows the order of stabilization and hence will not be a factor in exceptions.
- 266Williams, R. J. P.; Fraústo da Silva, J. J. R. The distribution of elements in cells. Coord. Chem. Rev. 2000, 200–202, 247– 348, DOI: 10.1016/S0010-8545(00)00324-6266The distribution of elements in cellsWilliams, R. J. P.; Frausto da Silva, J. J. R.Coordination Chemistry Reviews (2000), 200-202 (), 247-348CODEN: CCHRAM; ISSN:0010-8545. (Elsevier Science S.A.)A review, with 82 refs. The selective uptake of elements by proteins can be based on several routes: (1) the equil. binding of different elements by different protein ligands using charge, size, electron affinity, and stereochem. preferences; (2) kinetic insertion of an element into such a coordination site of a protein; (3) removal of the element to a special compartment by pumping followed by (1) or (2). A cellular system also limits the amt. of each type of metal-binding apoprotein by genetic regulation of its symbiosis with element uptake. Such a limitation generates much greater selectivity. Finally we consider how the obsd. selection of elements by proteins has changed in evolution through changes of availability of elements and their combinations in the environment1.
- 267Fraústo da Silva, J. J. R.; Williams, R. J. P. The natural selection of the chemical elements; Clarendon Press: Oxford, UK, 1997.There is no corresponding record for this reference.
- 268Foster, A. W.; Osman, D.; Robinson, N. J. Metal preferences and metallation. J. Biol. Chem. 2014, 289, 28095– 28103, DOI: 10.1074/jbc.R114.588145268Metal preferences and metalationFoster, Andrew W.; Osman, Deenah; Robinson, Nigel J.Journal of Biological Chemistry (2014), 289 (41), 28095-28103CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)A review. The metal binding preferences of most metalloproteins do not match their metal requirements. Thus, metalation of an estd. 30% of metalloenzymes is aided by metal delivery systems, with ∼25% acquiring preassembled metal cofactors. The remaining ∼70% are presumed to compete for metals from buffered metal pools. Metalation is further aided by maintaining the relative concns. of these pools as an inverse function of the stabilities of the resp. metal complexes. For example, Mg2+-contg. enzymes always prefer to bind Zn2+, and these metals dominate the metalloenzymes without metal delivery systems. Therefore, the buffered concn. of Zn2+ is held at least a million-fold below Mg2+ inside most cells.
- 269Robinson, N. J.; Glasfeld, A. Metalation: Nature’s challenge in bioinorganic chemistry. JBIC, J. Biol. Inorg. Chem. 2020, 25, 543– 545, DOI: 10.1007/s00775-020-01790-3269Metalation: nature's challenge in bioinorganic chemistryRobinson, Nigel J.; Glasfeld, ArthurJBIC, Journal of Biological Inorganic Chemistry (2020), 25 (4), 543-545CODEN: JJBCFA; ISSN:0949-8257. (Springer)Abstr.: The assocn. of proteins with metals, metalation, is challenging because the tightest binding metals are rarely the correct ones. Inside cells, correct metalation is enabled by controlled bioavailability plus extra mechanisms for tricky combinations such as iron and manganese.
- 270Kepp, K. P. A quantitative scale of oxophilicity and thiophilicity. Inorg. Chem. 2016, 55, 9461– 9470, DOI: 10.1021/acs.inorgchem.6b01702270A Quantitative Scale of Oxophilicity and ThiophilicityKepp, Kasper P.Inorganic Chemistry (2016), 55 (18), 9461-9470CODEN: INOCAJ; ISSN:0020-1669. (American Chemical Society)Oxophilicity and thiophilicity are widely used concepts with no quant. definition. In this paper, a simple, generic scale is developed that solves issues with ref. states and system dependencies and captures empirically known tendencies toward oxygen. This enables a detailed anal. of the fundamental causes of oxophilicity. Notably, the notion that oxophilicity relates to Lewis acid hardness is invalid. Rather, oxophilicity correlates only modestly and inversely with abs. hardness and more strongly with electronegativity and effective nuclear charge. Since oxygen is highly electroneg., ionic bonding is stronger to metals of low electronegativity. Left-side d-block elements with low effective nuclear charges and electronegativities are thus highly oxophilic, and the f-block elements, not because of their hardness, which is normal, but as a result of the small ionization energies of their outermost valence electrons, can easily transfer electrons to fulfill the electron demands of oxygen. Consistent with empirical experience, the most oxophilic elements are found in the left part of the d block, the lanthanides, and the actinides. The d-block elements differ substantially in oxophilicity, quantifying their different uses in a wide range of chem. reactions; thus, the use of mixed oxo- and thiophilic (i.e., "mesophilic") surfaces and catalysts as a design principle can explain the success of many recent applications. The proposed scale may therefore help to rationalize and improve chem. reactions more effectively than current qual. considerations of oxophilicity.
- 271Martin, R. B. A stability ruler for metal ion complexes. J. Chem. Educ. 1987, 64, 402, DOI: 10.1021/ed064p402271A stability ruler for metal ion complexesMartin, R. BruceJournal of Chemical Education (1987), 64 (5), 402CODEN: JCEDA8; ISSN:0021-9584.A stability ruler is described that makes use of the Irving-Williams series, the regular increase in stability of the dipos. metal ions of the 1st transition row, from Mg2+ through Cu2+, against which metal ions with more variable stabilities such as Zn2+ may be compared. The stabilities of Pb2+, Cd2+, and Hg2+ are characterized using the stability ruler.
- 272Romans, A. Y.; Graichen, M. E.; Lochmüller, C. H.; Henkens, R. W. Kinetics and mechanism of dissociation of zinc ion from carbonic anhydrase. Bioinorg. Chem. 1978, 9, 217– 229, DOI: 10.1016/S0006-3061(78)80007-6272Kinetics and mechanism of dissociation of zinc ion from carbonic anhydraseRomans, Alice Y.; Graichen, Mary E.; Lochmueller, C. H.; Henkens, Robert W.Bioinorganic Chemistry (1978), 9 (3), 217-29CODEN: BICHBX; ISSN:0006-3061.The kinetics of dissocn. of Zn2+ from carbonic anhydrase was measured over a range of pH, temp., and acetate concn. The rate of dissocn. was extremely slow at neutral pH, but increased in almost direct proportion to the H+ concn. and was enhanced in the presence of 1,10-phenanthroline or acetate. The thermodn. stability of the Zn-apoenzyme complex was detd. over a range of pH from rate data on binding and dissocn. (stability consts. 109 to 1011 M-1, 25°. The great stability of the complex and slow exchange of the apoenzyme ligand was attributed, at least in part, to the rigidity of the multidentate protein ligand.
- 273Heinz, U.; Kiefer, M.; Tholey, A.; Adolph, H. W. On the competition for available zinc. J. Biol. Chem. 2005, 280, 3197– 3207, DOI: 10.1074/jbc.M409425200273On the Competition for Available ZincHeinz, Uwe; Kiefer, Martin; Tholey, Andreas; Adolph, Hans-WernerJournal of Biological Chemistry (2005), 280 (5), 3197-3207CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)Extended x-ray absorption fine structure (EXAFS) spectroscopy was combined with thermodn. and kinetic approaches to investigate zinc binding to a zinc finger (C2H2) and a tetrathiolate (C4) peptide. Both peptides represent structural zinc sites of proteins and rapidly bind a single zinc ion with picomolar dissocn. consts. In competition with EDTA the transfer of peptide-bound zinc ions proved to be 6 orders of magnitude faster than predicted for a dissocn.-assocn. mechanism thus requiring ligand exchange mechanisms via peptide-zinc-EDTA complexes. EXAFS spectra of C2H2 showed the expected Cys2His2-ligand geometry when fully loaded with zinc. For a 2-fold excess of peptide, however, the existence of zinc-bridged peptide-peptide complexes with dominating sulfur coordination could be clearly shown. Whereas zinc binding kinetics of C2H2 appeared as a simple second order process, the suggested mechanism for C4 comprises a zinc-bridged Zn-(C4)2 species as well as a Zn-C4 species with less than 4 metal-bound thiolates, which is supported by EXAFS results. A rapid equil. of bound and unbound states of individual ligands might explain the kinetic instability of zinc-peptide complexes, which enables fast ligand exchange during the encounter of occupied and unoccupied acceptor sites. Depending on relative concns. and stabilities, this results in a rapid transfer of zinc ions in the virtual absence of free zinc ions, as seen for the zinc transfer to EDTA, or in the formation of zinc-bridged complexes, as seen for both peptides with excess of peptides over available zinc.
- 274Colvin, R. A.; Holmes, W. R.; Fontaine, C. P.; Maret, W. Cytosolic zinc buffering and muffling: their role in intracellular zinc homeostasis. Metallomics 2010, 2, 306– 317, DOI: 10.1039/b926662c274Cytosolic zinc buffering and muffling: their role in intracellular zinc homeostasisColvin, Robert A.; Holmes, William R.; Fontaine, Charles P.; Maret, WolfgangMetallomics (2010), 2 (5), 306-317CODEN: METAJS; ISSN:1756-591X. (Royal Society of Chemistry)A review. Our knowledge of the mol. mechanisms of intracellular homeostatic control of zinc ions is now firmly grounded on exptl. findings gleaned from the study of zinc proteomes and metallomes, zinc transporters, and insights from the use of computational approaches. A cell's repertoire of zinc homeostatic mols. includes cytosolic zinc-binding proteins, transporters localized to cytoplasmic and organellar membranes, and sensors of cytoplasmic free zinc ions. Under steady state conditions, a primary function of cytosolic zinc-binding proteins is to buffer the relatively large zinc content found in most cells to a cytosolic zinc(II) ion concn. in the picomolar range. Under non-steady state conditions, zinc-binding proteins and transporters act in concert to modulate transient changes in cytosolic zinc ion concn. in a process that is called zinc muffling. For example, if a cell is challenged by an influx of zinc ions, muffling reactions will dampen the resulting rise in cytosolic zinc ion concn. and eventually restore the cytosolic zinc ion concn. to its original value by shuttling zinc ions into subcellular stores or by removing zinc ions from the cell. In addn., muffling reactions provide a potential means to control changes in cytosolic zinc ion concns. for purposes of cell signalling in what would otherwise be considered a buffered environment not conducive for signalling. Such intracellular zinc ion signals are known to derive from redox modifications of zinc-thiolate coordination environments, release from subcellular zinc stores, and zinc ion influx via channels. Recently, it has been discovered that metallothionein binds its seven zinc ions with different affinities. This property makes metallothionein particularly well positioned to participate in zinc buffering and muffling reactions. In addn., it is well established that metallothionein is a source of zinc ions under conditions of redox signalling. We suggest that the biol. functions of transient changes in cytosolic zinc ion concns. (presumptive zinc signals) complement those of calcium ions in both spatial and temporal dimensions.
- 275Foster, A. W.; Robinson, N. J. Promiscuity and preferencs of metallothioneins: the cell rules. BMC Biol. 2011, 9, 25, DOI: 10.1186/1741-7007-9-25275Promiscuity and preferences of metallothioneins: the cell rulesFoster, Andrew W.; Robinson, Nigel J.BMC Biology (2011), 9 (), 25CODEN: BBMIF7; ISSN:1741-7007. (BioMed Central Ltd.)A review. Metalloproteins are essential for many cellular functions, but it has not been clear how they distinguish between the different metals to bind the correct ones. A report in BMC Biol. finds that preferences of two metallothionein isoforms for two different cations are due to inherent properties of these usually less discriminating proteins. Here these observations are discussed in the context of the cellular mechanisms that regulate metal binding to proteins.
- 276Osman, D.; Martini, M. A.; Foster, A. W.; Chen, J.; Scott, A. J. P.; Morton, R. J.; Steed, J. W.; Lurie-Luke, E.; Huggins, T. G.; Lawrence, A. D.; Deery, E.; Warren, M. J.; Chivers, P. T.; Robinson, N. J. Bacterial sensors define intracellular free energies for correct enzyme metalation. Nat. Chem. Biol. 2019, 15, 241– 249, DOI: 10.1038/s41589-018-0211-4276Bacterial sensors define intracellular free energies for correct enzyme metalationOsman, Deenah; Martini, Maria Alessandra; Foster, Andrew W.; Chen, Junjun; Scott, Andrew J. P.; Morton, Richard J.; Steed, Jonathan W.; Lurie-Luke, Elena; Huggins, Thomas G.; Lawrence, Andrew D.; Deery, Evelyne; Warren, Martin J.; Chivers, Peter T.; Robinson, Nigel J.Nature Chemical Biology (2019), 15 (3), 241-249CODEN: NCBABT; ISSN:1552-4450. (Nature Research)There is a challenge for metalloenzymes to acquire their correct metals because some inorg. elements form more stable complexes with proteins than do others. These preferences can be overcome provided some metals are more available than others. However, while the total amt. of cellular metal can be readily measured, the available levels of each metal have been more difficult to define. Metal-sensing transcriptional regulators are tuned to the intracellular availabilities of their cognate ions. Here the authors detd. the std. free energy for metal complex formation to which each sensor, in a set of bacterial metal sensors, is attuned: the less competitive the metal, the less favorable the free energy and hence the greater availability to which the cognate allosteric mechanism is tuned. Comparing these free energies with values derived from the metal affinities of a metalloprotein reveals the mechanism of correct metalation exemplified here by a cobalt chelatase for vitamin B12.
- 277Nar, H.; Huber, R.; Messerschmidt, A.; Filippou, A. C.; Barth, M.; Jaquinod, M.; Kamp, M.; Canters, G. W. Characterization and crystal structure of zinc azurin, a by-product of heterologous expression in Escherichia coli of Pseudomonas aeruginosa copper azurin. Eur. J. Biochem. 1992, 205, 1123– 1129, DOI: 10.1111/j.1432-1033.1992.tb16881.x277Characterization and crystal structure of zinc azurin, a by-product of heterologous expression in Escherichia coli of Pseudomonas aeruginosa copper azurinNar, Herbert; Huber, Robert; Messerschmidt, Albrecht; Filippou, Alexander C.; Barth, Manfred; Jaquinod, Michel; Van de Kamp, Mart; Canters, Gerard W.European Journal of Biochemistry (1992), 205 (3), 1123-9CODEN: EJBCAI; ISSN:0014-2956.Azurin*, a byproduct of heterologous expression of the gene encoding the blue copper protein azurin from P. aeruginosa in E. coli, was characterized by chem. anal. and electrospray ionization mass spectrometry, and its structure detd. by x-ray crystallog. It was shown that azurin* is native azurin with its copper atom replaced by zinc in the metal binding site. Zinc is probably incorporated in the apo-protein after its expression and transport into the periplasm. Holo-azurin can be reconstituted from azurin* by prolonged exposure of the protein to high copper ion concns. or unfolding of the protein and refolding in the presence of copper ions. An x-ray crystallog. anal. of azurin* at 0.21-nm resoln. revealed that the overall structure of azurin is not perturbed by the metal exchange. However, the geometry of the coordination sphere changes from trigonal bipyramidal in the case of copper azurin to distorted tetrahedral for the zinc protein. The copper ligand Met121 is no longer coordinated to zinc which adopts a position close to the carbonyl oxygen atom from residue Gly45. The polypeptide structure surrounding the metal site undergoes moderate reorganization upon zinc binding. The largest displacement obsd. is for the carbonyl oxygen from residue Gly45, which is involved in copper and zinc binding. It moves by 0.03 nm towards the zinc, thereby reducing its distance to the metal from 0.29 nm in the copper protein to 0.23 nm in the deriv.
- 278Maret, W.; Shiemke, A. K.; Wheeler, W. D.; Loehr, T. M.; Sanders-Loehr, J. Resonance Raman spectroscopy of blue copper proteins: Ligand and coenzyme effects in Cu(II)-substituted liver alcohol dehydrogenase. J. Am. Chem. Soc. 1986, 108, 6351– 6359, DOI: 10.1021/ja00280a036278Resonance Raman spectroscopy of blue copper proteins: ligand and coenzyme effects in copper(II)-substituted liver alcohol dehydrogenaseMaret, Wolfgang; Shiemke, Andrew K.; Wheeler, William D.; Loehr, Thomas M.; Sanders-Loehr, JoannJournal of the American Chemical Society (1986), 108 (20), 6351-9CODEN: JACSAT; ISSN:0002-7863.Liver alc. dehydrogenase (LADH) with Cu(II) substituted for the native Zn(II) at the catalytic metal site exhibits a resonance Raman spectrum characteristic of type 1 Cu of blue Cu proteins when excited within its cysteinate → Cu(II) charge-transfer band (620 nm at 298 K, 570 nm at 125 K). The 2 most intense Raman peaks at 350 and 415 cm-1 and the 2 weaker peaks at 330 and 342 cm-1 are assigned to Cu-cysteinate vibrations of 1 or both cysteine ligands. The multiplicity of peaks indicates coupling of ν(Cu-S) with ligand modes. The peak at 254 cm-1 which is shifted 2 cm-1 to lower energy in D2O is assigned to a Cu-imidazole vibration of the histidine ligand. All of the resonance-enhanced modes display narrow excitation profiles are max. at ∼600 nm (at 15 and 90 K) and appear to coincide with 1 component of the 570-nm electronic absorption envelope. The peaks in the resonance Raman spectra of Ni(II)- and Fe(III)-substituted LADH are very close in frequency and intensity to those of the Cu(II) protein, supporting the view that the protein imposes a tetrahedral structure at the catalytic metal site with ≥1 short metal-S(cysteine) bond. Binding of ligands such as pyrazole, imidazole, β-mercaptoethanol, and cyanide to Cu(II)-LADH induces 3-20-cm-1 shifts in the 4 main Cu-cysteinate vibrations and causes a substantial decrease in the intensities of the 2 high-energy Cu-cysteinate peaks. Low-temp. electronic spectra of these binary complexes show absorption max. close to 500 nm which are significantly blue-shifted from the 570-nm max. in the low-temp. spectrum of Cu(II)-LADH. These spectral alterations can be interpreted in terms of a distortion toward a tetragonal geometry at the metal site. In contrast, the binary NADH complex and the ternary NAD/pyrazole complex both exhibit intensity increases in the 2 high-frequency Cu-cysteinate modes are well as a rich set of overtone and combination bands. These resonance Raman features are new signatures of tetrahedral coordination which correlate well with optical and ESR indicators of type 1 Cu sites. The Cu(II)-LADH/NADH complex further resembles the blue Cu proteins in that the Cu-cysteinate peaks are strongly affected by deuteration but differs in that there are D-induced alterations in intensities as well as frequencies. It is proposed that these D isotope effects are due to H bonding of the cysteinate ligand S atoms to suitable protein donors, as is typical of metal-S centers in other metalloproteins.
- 279Goch, W.; Bal, W. Stochastic or not? Method to predict and quantify the stochastic effects on the association equilibria in nanoscopic systems. J. Phys. Chem. A 2020, 124, 1421– 1428, DOI: 10.1021/acs.jpca.9b09441279Stochastic or Not? Method To Predict and Quantify the Stochastic Effects on the Association Reaction Equilibria in Nanoscopic SystemsGoch, Wojciech; Bal, WojciechJournal of Physical Chemistry A (2020), 124 (7), 1421-1428CODEN: JPCAFH; ISSN:1089-5639. (American Chemical Society)The stochastic nature of chem. reaction and impact of the stochasticity on their evolution is soundly documented. Both theor. predictions and emerging exptl. evidence indicate the influence of stochastic effects on the equil. state of assocn. reaction. In this work simple math. formulas are introduced to est. these effects. First, the dependence of the ratio of obsd. reactants (apparent assocn. const., equiv. of macroscopic assocn. const. in stochastic anal.) on the vol. and the no. of mols. of reagents is discussed and the limiting factors of this effect are shown. Next, the apparent assocn. const. is approximated for nanoscale systems by closed-form formulas derived for this purpose. Finally, an estn. for the macroscopic const. value from the apparent one is provided and validated on the published exptl. data. This work was inspired by chem. reactions occurring in biol. compartments, but the results can be used for all systems belonging to the stochastic regime of chem. reactions.
- 280Andreini, C.; Banci, L.; Bertini, I.; Rosato, A. Counting the zinc-proteins encoded in the human genome. J. Proteome Res. 2006, 5, 196– 201, DOI: 10.1021/pr050361j280Counting the zinc-proteins encoded in the human genomeAndreini, Claudia; Banci, Lucia; Bertini, Ivano; Rosato, AntonioJournal of Proteome Research (2006), 5 (1), 196-201CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)Metalloproteins are proteins capable of binding one or more metal ions, which may be required for their biol. function, or for regulation of their activities or for structural purposes. Genome sequencing projects have provided a huge no. of protein primary sequences, but, even though several different elaborate analyses and annotations have been enabled by a rich and ever-increasing portfolio of bioinformatic tools, metal-binding properties remain difficult to predict as well as to investigate exptl. Consequently, the present knowledge about metalloproteins is only partial. The present bioinformatic research proposes a strategy to answer the question of how many and which proteins encoded in the human genome may require zinc for their physiol. function. This is achieved by a combination of approaches, which include: (i) searching in the proteome for the zinc-binding patterns that, on their turn, are obtained from all available X-ray data; (ii) using libraries of metal-binding protein domains based on multiple sequence alignments of known metalloproteins obtained from the Pfam database; and (iii) mining the annotations of human gene sequences, which are based on any type of information available. It is found that 1684 proteins in the human proteome are independently identified by all three approaches as zinc-proteins, 746 are identified by two, and 777 are identified by only one method. By assuming that all proteins identified by at least two approaches are truly zinc-binding and inspecting the proteins identified by a single method, it can be proposed that ca. 2800 Human proteins are potentially zinc-binding in vivo, corresponding to 10% of the human proteome, with an uncertainty of 400 sequences. Available functional information suggests that the large majority of human zinc-binding proteins are involved in the regulation of gene expression. The most abundant class of zinc-binding proteins in humans is that of zinc-fingers, with Cys4 and Cys2His2 being the most common types of coordination environment.
- 281Andreini, C.; Banci, L.; Bertini, I.; Rosato, A. Zinc through the three domains of life. J. Proteome Res. 2006, 5, 3173– 3178, DOI: 10.1021/pr0603699281Zinc through the Three Domains of LifeAndreini, Claudia; Banci, Lucia; Bertini, Ivano; Rosato, AntonioJournal of Proteome Research (2006), 5 (11), 3173-3178CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)Zinc is one of the metal ions essential for life, as it is required for the proper functioning of a large no. of proteins. Despite its importance, the annotation of zinc-binding proteins in gene banks or protein domain databases still has significant room for improvement. In the present work, we compiled a list of known zinc-binding protein domains and of known zinc-binding sequence motifs (zinc-binding patterns), and then used them jointly to analyze the proteome of 57 different organisms to obtain an overview of zinc usage by archaeal, bacterial, and eukaryotic organisms. Zinc-binding proteins are an abundant fraction of these proteomes, ranging between 4% and 10%. The no. of zinc-binding proteins correlates linearly with the total no. of proteins encoded by the genome of an organism, but the proportionality const. of Eukaryota (8.8%) is significantly higher than that obsd. in Bacteria and Archaea (from 5% to 6%). Most of this enrichment is due to the larger portfolio of regulatory proteins in Eukaryota.
- 282Maret, W. New perspectives of zinc coordination environments in proteins. J. Inorg. Biochem. 2012, 111, 110– 116, DOI: 10.1016/j.jinorgbio.2011.11.018282New perspectives of zinc coordination environments in proteinsMaret, WolfgangJournal of Inorganic Biochemistry (2012), 111 (), 110-116CODEN: JIBIDJ; ISSN:0162-0134. (Elsevier)A review. Zn(II) is more widely used as a cofactor in proteins than any other transition metal ion. In addn. to catalytic and structural functions, zinc(II) ions play a role in information transfer and cellular control. They bind transiently when proteins regulate Zn(II) concns. and re-distribute Zn(II) and when proteins are regulated by Zn(II). Transient Zn-binding sites employ the same donors of amino acid side-chains as catalytic and structural sites, but differ in their coordination chem. that can modulate Zn(II) affinities over at least 10 orders of magnitude. Redox activity of Cys ligands, multiple binding modes of O, S, and N donors, and protein conformational changes induce coordination dynamics in Zn sites and Zn(II) ion mobility. Functional annotations of the marked variation of coordination environments in Zn proteomes need to consider how the primary coordination spheres interact with protein structure and dynamics, and the adaptation of coordination properties to the biol. context in extracellular, cellular, or subcellular locations.
- 283Kambe, T.; Taylor, K. M.; Fu, D. Zinc transporters and their functional integration into mammalian cells. J. Biol. Chem. 2021, 296, 100320, DOI: 10.1016/j.jbc.2021.100320283Zinc transporters and their functional integration in mammalian cellsKambe, Taiho; Taylor, Kathryn M.; Fu, DaxJournal of Biological Chemistry (2021), 296 (), 100320CODEN: JBCHA3; ISSN:1083-351X. (Elsevier Inc.)A review. Zinc is a ubiquitous biol. metal in all living organisms. The spatiotemporal zinc dynamics in cells provide crucial cellular signaling opportunities, but also challenges for intracellular zinc homeostasis with broad disease implications. Zinc transporters play a central role in regulating cellular zinc balance and subcellular zinc distributions. The discoveries of two complementary families of mammalian zinc transporters (ZnTs and ZIPs) in the mid-1990s spurred much speculation on their metal selectivity and cellular functions. After two decades of research, we have arrived at a biochem. description of zinc transport. However, in vitro functions are fundamentally different from those in living cells, where mammalian zinc transporters are directed to specific subcellular locations, engaged in dedicated macromol. machineries, and connected with diverse cellular processes. Hence, the mol. functions of individual zinc transporters are reshaped and deeply integrated in cells to promote the utilization of zinc chem. to perform enzymic reactions, tune cellular responsiveness to pathophysiol. signals, and safeguard cellular homeostasis. At present, the underlying mechanisms driving the functional integration of mammalian zinc transporters are largely unknown. This knowledge gap has motivated a shift of the research focus from in vitro studies of purified zinc transporters to in cell studies of mammalian zinc transporters in the context of their subcellular locations and protein interactions. In this review, we will outline how knowledge of zinc transporters has been accumulated from in-test-tube to in-cell studies, highlighting new insights and paradigm shifts in our understanding of the mol. and cellular basis of mammalian zinc transporter functions.
- 284Hirose, J.; Ohsaki, T.; Nishimoto, N.; Matuoka, S.; Hiromoto, T.; Yoshida, T.; Minoura, T.; Iwamoto, H.; Fukasawa, K. M. Characterization of the metal-binding site in aminopeptidase B. Biol. Pharm. Bull. 2006, 29, 2378– 2382, DOI: 10.1248/bpb.29.2378284Characterization of the metal-binding site in aminopeptidase BHirose, Junzo; Ohsaki, Takamichi; Nishimoto, Naoyo; Matuoka, Shouji; Hiromoto, Takashi; Yoshida, Takahide; Minoura, Takatosi; Iwamoto, Hiroyuki; Fukasawa, Kayoko M.Biological & Pharmaceutical Bulletin (2006), 29 (12), 2378-2382CODEN: BPBLEO; ISSN:0918-6158. (Pharmaceutical Society of Japan)A recombinant rat aminopeptidase-B (Ap-B) was expressed as a glutathione S-transferase (GST) fusion protein in Escherichia coli BL21 harboring a plasmid pGEX-Ap-B and was purified by glutathione-Sepharose 4B and Q-Sepharose columns. The metal-substituted derivs. of Ap-B, Co(II)- and Cu(II)-Ap-B contain almost 1 mol of cobalt(II) and copper(II) ions per enzyme mol., resp. The specific activity of Co(II)-Ap-B is very similar to that of recombinant Ap-B but that of Cu(II)-Ap-B is very low. The dissocn. consts. of the zinc ions of recombinant Ap-B and of the cobalt ions of Co(II)-Ap-B calcd. from the relationships between the free metal ions and the residual enzyme activities are 3.7(±1.0) × 10-13 and 4.7(±1.0) × 10-12 M, resp. The EPR parameters (g.perp., g‖ and A‖) of Cu(II)-Ap-B were 2.06, 2.27, and 156 × 10-4 cm-1. The A‖ value and the g‖ of Cu(II)-Ap-B are very similar to those of Cu(II)-thermolysin or Cu(II)-dipeptidyl peptidase III, in which the coordination geometry is a distorted tetrahedral.
- 285Kleemann, S. G.; Keung, W. M.; Riordan, J. F. Metal binding to angiotensin converting enzyme: implications for the metal binding site. J. Inorg. Biochem. 1986, 26, 93– 106, DOI: 10.1016/0162-0134(86)80002-2285Metal binding to angiotensin converting enzyme: implications for the metal binding siteKleemann, Stephan G.; Keung, Wing Ming; Riordan, James F.Journal of Inorganic Biochemistry (1986), 26 (2), 93-106CODEN: JIBIDJ; ISSN:0162-0134.Angiotensin-converting enzyme (ACE) interacts with the chelator 1,10-phenanthroline (OP) to form a OP-Zn-ACE ternary complex, which subsequently dissocs. to OP-Zn and apoenzyme. The assocn. and dissocn. rate consts. for the reaction OP + Zn-ACE ↹ OP-Zn-ACE have been detd. and compared with those of known OP-metal complexes. Such consts. were also used to calc. the rate const. for formation of the OP-Zn complex from OP-Zn-ACE. The rate of dissocn. of Zn from ACE has been measured in the presence of EDTA (which acts only as a metal scavenger) as a function of chelator concn., at different pH values, and with different buffers. The stability const. for the binding of Zn to apo-ACE, log Kc = 8.2, detd. by equil. dialysis using at. absorption spectroscopy to assess metal concn., is much smaller than that for Zn-carboxypeptidase A, Zn-thermolysin, or Zn-carbonic anhydrase. This weak binding is attributable to the Zn dissocn. rate const. of ACE, 7.5 × 10-3 sec-1 at pH 7.0, which is much greater than that of the other Zn metalloenzymes. These results lead to inferences regarding the metal binding site of ACE.
- 286Ippolito, J. A.; Baird, T. T., Jr.; McGee, S. A.; Christianson, D. W.; Fierke, C. A. Structure-assisted redesign of aprotein-zinc binding site with femtomolar affinity. Proc. Natl. Acad. Sci. U. S. A. 1995, 92, 5017– 5021, DOI: 10.1073/pnas.92.11.5017286Structure-assisted redesign of a protein-zinc-binding site with femtomolar affinityIppolito, Joseph A.; Baird, Teaster T., Jr.; McGee, Sharon A.; Christianson, David W.; Fierke, Carol A.Proceedings of the National Academy of Sciences of the United States of America (1995), 92 (11), 5017-21CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)The authors have inserted a fourth protein ligand into the zinc coordination polyhedron of carbonic anhydrase II (CAII) that increases metal affinity 200-fold (Kd = 20 fM). The three-dimensional structures of threonine-199 → aspartate (T199D) and threonine-199 → glutamate (T199E) CAIIs, detd. by x-ray crystallog. methods to resolns. of 2.35 Å and 2.2 Å, resp., reveal a tetrahedral metal-binding site consisting of H94, H96, H119, and the engineered carboxylate side chain, which displaces zinc-bound hydroxide. Although the stereochem. of neither engineered carboxylate-zinc interaction is comparable to that found in naturally occurring protein zinc-binding sites, protein-zinc affinity is enhanced in T199E CAII demonstrating that ligand-metal sepn. is a significant determinant of carboxylate-zinc affinity. In contrast, the three-dimensional structure of threonine-199 → histidine (T199H) CAII, detd. to 2.25-Å resoln., indicates that the engineered imidazole side chain rotates away from the metal and does not coordinate to zinc; this results in a weaker zinc-binding site. All three of these substitutions nearly obliterate CO2 hydrase activity, consistent with the role of zinc-bound hydroxide as catalytic nucleophile. The engineering of an addnl. protein ligand represents a general approach for increasing protein-metal affinity if the side chain can adopt a reasonable conformation and achieve inner-sphere zinc coordination. Moreover, this structure-assisted design approach may be effective in the development of high-sensitivity metal ion biosensors.
- 287McCall, K. A.; Fierke, C. A. Probing determinants of the metal ion selectivity in carbonic anhydrase using mutagenesis. Biochemistry 2004, 43, 3979– 3986, DOI: 10.1021/bi0498914287Probing Determinants of the Metal Ion Selectivity in Carbonic Anhydrase Using MutagenesisMcCall, Keith A.; Fierke, Carol A.Biochemistry (2004), 43 (13), 3979-3986CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)Few studies measuring thermodn. metal ion selectivity of metalloproteins have been performed, and the major determinants of metal ion selectivity in proteins are not yet well understood. Several features of metal ion binding sites and metal coordination have been hypothesized to alter the transition metal selectivity of chelators, including (1) the polarizability of the coordinating atom, (2) the relative sizes of the binding site and the metal ion, and (3) the metal ion binding site geometry. To test these hypotheses, we have measured the metal ion affinity and selectivity of a prototypical zinc enzyme, human carbonic anhydrase II (CAII), and a no. of active site variants where one of the coordinating ligands is substituted by another side chain capable of coordinating metal. CAII and almost all of the variants follow the inherent metal ion affinity trend suggested by the Irving-Williams series, demonstrating that this trend operates within proteins as well as within small mol. chelators and may be a dominant factor in metal ion selectivity in biol. Neither the polarizability of the liganding side chains nor the size of the metal ion binding site correlates strongly with metal ion specificity; instead, changes in metal ion specificity in the variants correlate with the preferred coordination no. and geometry of the metal ion. This correlation suggests that a primary feature driving deviations from the inherent ligand affinity trend is the positioning of active site groups such that a given metal ion can adopt a preferred coordination no./geometry.
- 288Coleman, J. E.; Vallee, B. L. Metallocarboxypeptidases: Stability constants and enzymatic characteristics. J. Biol. Chem. 1961, 236, 2244– 2249, DOI: 10.1016/S0021-9258(18)64065-7288Metallocarboxypeptidases: stability constants and enzymic characteristicsColeman, Joseph E.; Vallee, Bert L.Journal of Biological Chemistry (1961), 236 (), 2244-9CODEN: JBCHA3; ISSN:0021-9258.cf. CA 54, 13212c; 55, 4619d. Apocarboxypeptidase forms enzymically active complexes with a series of metal ions. The ranking order of stability consts. for the complexes of a series of metal ions with simple ligands is characteristic of its constituent donor atoms. Thus, when N and N or N and O function in this capacity, the ranking order for the stability consts. of metal complexes follows the sequence Hg(II) > Cu(II) ≥ Ni(II) > Co(II) ≥ Zn(II) ≥ Cd(II) > Fe(II) > Mn(II). The substitution of S for one of the donor atoms, to yield a S.sbd.N ligand markedly changes this sequence to Hg(II) » Cd(II) > Zn(II) > Ni(II) > Co(II) > Fe(II) > Mn. Cu is indeterminant because of the oxidn. of the mercapto group. The complexes of Cd and Zn are more distinctly stabilized over those of Co and Ni, the characteristic feature of the S-ligand series. The close correlation of both the order and magnitudes of the consts. imply that in carboxypeptidase a N.sbd.S site binds metal ions to yield enzymic activity. This interpretation is supported by titrations showing 2 metal-binding groups with pK values of 7.7 and 9.1, resp., compatible with published values for α-amino and SH groups. Mn, Co, Ni, and Zn carboxypeptidase hydrolyze both peptides and an ester substrate, hippuryl-DL,-β-phenyllactate. The ranking order of peptidase activities for these metal atoms varies as a function of the primary structure of the synthetic peptide substrate. Hg, Cd, and Pd carboxypeptidases exhibit marked esterase activity but do not hydrolyze the synthetic peptides tested. Thus, the metal atom known to function in substrate binding also plays a role in the detn. of enzymic specificity. The role of metal ions in the action of carboxypeptidase is apparent in yet another manner. Under standard conditions of assay, the relative order of the catalytic efficiencies of different metallocarboxypeptidases varies as a function of the primary structure of the synthetic peptide substrate. Thus the ranking order Co > Ni > Zn > Mn observed for carbobenzyloxyglycyl-L-phenylalanine is confirmed and is preserved over a wide range of substrate concns., ionic strengths, and other conditions of assay, but is inverted to Zn > Co > Ni > Mn for both carbobenzyloxyglycyl-L-tryptophan and benzoylglycyl-L-phenylalanine, and also the ester substrate, hippuryl-DL-β-phenylacetate. The spectral changes accompanying the formation of Co carboxypeptidase show the formation of a mercaptide linkage with apocarboxypeptidase. Co carboxypeptidase exhibits a distinctive red color with an absorption max. at 530 mμ and an extinction coeff. of 150. The shift in the absorption max. from 512 in the hydrated Co(II) to 530 mμ in Co carboxypeptidase, together with the increase in the extinction coeff. from 10 to 150, suggest binding to S. Similar binding of Ni, Mn, Hg, and Cd to a S atom is indicated by the relative order of the stability consts. for the resp. metallocarboxypeptidases which follow that expected for a S-contg. ligand. The S.sbd.N nature of the bidentate binding site is implied by the magnitude of the stability consts. and by the release of 2 H ions on combination of the apoenzyme with Zn ions. Equil. dialysis expts. in which the Zn atom of carboxypeptidase is exchanged for Hg or Cd demonstrate that the same site of the enzyme is involved in binding all of these, as shown previously for Co. The evidence suggests that the active center of native carboxypeptidase A includes 1 S, 1 N, and 1 Zn atom.
- 289Hirose, J.; Iwamoto, H.; Nagao, I.; Enmyo, K.; Sugao, H.; Kanemitu, N.; Ikeda, K.; Takeda, M.; Inoue, M.; Ikeda, T.; Matsuura, F.; Fukasawa, K. M.; Fukasawa, K. Characterization of the metal-substituted dipeptidyl peptidase III (rat liver). Biochemistry 2001, 40, 11860– 11865, DOI: 10.1021/bi0110903289Characterization of the Metal-Substituted Dipeptidyl Peptidase III (Rat Liver)Hirose, Junzo; Iwamoto, Hiroyuki; Nagao, Ikuko; Enmyo, Kanako; Sugao, Hidenori; Kanemitu, Nobuharu; Ikeda, Keiichi; Takeda, Mitsunori; Inoue, Masaki; Ikeda, Tomoyuki; Matsuura, Fumito; Fukasawa, Kayoko M.; Fukasawa, KatsuhikoBiochemistry (2001), 40 (39), 11860-11865CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)Dipeptidyl peptidase III (DPP III) (EC 3.4.14.4), which has a HELLGH-E (residues 450-455, 508) motif as the zinc binding site, is classified as a zinc metallopeptidase. The zinc dissocn. consts. of the wild type, Leu453-deleted, and E508D mutant of DPP III at pH 7.4 were 4.5 (±0.7) × 10-13, 5.8 (±0.7) × 10-12, and 3.2 (±0.9) × 10-10 M, resp. The recoveries of the enzyme activities by the addn. of various metal ions to apo-DPP III were also measured, and Co2+, Ni2+, and Cu2+ ions completely recovered the enzyme activities as did Zn2+. The dissocn. consts. of Co2+, Ni2+, and Cu2+ ions for apo-DPP III at pH 7.4 were 8.2 (±0.9) × 10-13, 2.7 (±0.3) × 10-12, and 1.1 (±0.1) × 10-14 M, resp. The shape of the absorption spectrum of Co2+-DPP III was very similar to that of Co2+-carboxypeptidase A or Co2+-thermolysin, in which the Co2+ is bound to two histidyl nitrogens, a water mol., and a glutamate residue. The absorption spectrum of Cu2+-DPP III is also very similar to that of Cu2+-thermolysin. The EPR spectrum and the EPR parameters of Cu2+-DPP III were very similar to those of Cu2+-thermolysin but slightly different from those of Cu2+-carboxypeptidase A. The five lines of the superfine structure in the perpendicular region of the EPR spectrum in Cu2+-DPP III suggest that nitrogen atoms should coordinate to the cupric ion in Cu2+-DPP III. All of these data suggest that the donor set and the coordination geometry of the metal ions in DPP III, which has the HExxxH motif as the metal binding site, are very similar to those of the metal ions in thermolysin, which has the HExxH motif.
- 290Sellin, S.; Mannervik, B. (1984) Metal dissociation constants for glyoxalase I reconstituted with Zn2+, Co2+, Mn2+, and Mg2+. J. Biol. Chem. 1984, 259, 11426– 11429, DOI: 10.1016/S0021-9258(18)90878-1290Metal dissociation constants for glyoxalase I reconstituted with zinc(2+), cobalt(2+), manganese(2+), and magnesium(2+)Sellin, Siv; Mannervik, BengtJournal of Biological Chemistry (1984), 259 (18), 11426-9CODEN: JBCHA3; ISSN:0021-9258.Metal dissocn. consts. for glyoxalase I from human erythrocytes were detd. by use of nitrilotriacetic acid as a metal buffer. The consts. for Zn2+, Co2+, Mn2+, and Mg2+ were 2.7 × 10-11, 3.0 × 10-10, 4.9 × 10-9, and 1.0 × 10-6M, resp., demonstrating that the natural cofactor, Zn2+, has the highest affinity for the apoprotein. The results were consistent with the proposal of N and O atoms as ligands to the metal in the active site of glyoxalase I. Both 1:1 and 1:2 complexes of the metal ions and nitrilotriacetic acid must be considered in the application of the metal buffer technique.
- 291Lasch, J. Kinetic properties of bovine lens leucine aminopeptidase. Ophthalmic Res. 1979, 11, 372– 376, DOI: 10.1159/000265036291Kinetic properties of bovine lens leucine aminopeptidaseLasch, JuergenOphthalmic Research (1979), 11 (5-6), 372-6CODEN: OPRSAQ; ISSN:0030-3747.Kinetic studies with bovine eye lens leucine aminopeptidase are reported. Particular attention is paid to the substrate specificity, the transferase activity, the correlation between binding and catalytic parameters, and the catalytic properties of the different metal ion hybrids of the enzyme.
- 292Simons, T. J. B. The affinity of human erythrocyte porphobilinogen synthase for Zn2+ and Pb2+. Eur. J. Biochem. 1995, 234, 178– 183, DOI: 10.1111/j.1432-1033.1995.178_c.x292The affinity of human erythrocyte porphobilinogen synthase for Zn2+ and Pb2+Simons, Timothy J. B.European Journal of Biochemistry (1995), 234 (1), 178-83CODEN: EJBCAI; ISSN:0014-2956. (Springer)Porphobilinogen synthase activity was measured in human erythrocyte lysates supplemented with metal-ion buffers to control free Zn2+ and Pb2+ concns. The enzyme was activated by Zn2+ with a Km of 1.6 pM and inhibited by Pb2+ with a Ki of 0.07 pM. Pb2+ and Zn2+ appear to compete for a single metal-binding site. The half-time for loss of Zn2+ from the active site, or replacement of Pb2+ by Zn2+, were in the 10-20 min range at 37°. Zn2+ did not affect the affinity for the substrate 5-aminolevulinate, but Pb2+ reduced it non-competitively. All the expts. were conducted with a blood sample of the common 1-1 phenotype.
- 293Day, E. S.; Wen, D.; Garber, E. A.; Hong, J.; Avedissian, L. S.; Rayhorn, P.; Shen, W.; Zeng, C.; Bailey, V. R.; Reilly, J. O.; Roden, J. A.; Moore, C. B.; Williams, K. P.; Galdes, A.; Whitty, A.; Baker, D. P. Zinc-dependent structural stability of human sonic hedgehog. Biochemistry 1999, 38, 14868– 14880, DOI: 10.1021/bi9910068293Zinc-Dependent Structural Stability of Human Sonic HedgehogDay, Eric S.; Wen, Dingyi; Garber, Ellen A.; Hong, Jin; Avedissian, Lena S.; Rayhorn, Paul; Shen, Weihong; Zeng, Chenhui; Bailey, Voilaine R.; Reilly, Jennifer O.; Roden, Julie A.; Moore, Claire B.; Williams, Kevin P.; Galdes, Alphonse; Whitty, Adrian; Baker, Darren P.Biochemistry (1999), 38 (45), 14868-14880CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)The role of the zinc site in the N-terminal fragment of human Sonic hedgehog (ShhN) was explored by comparing the biophys. and functional properties of wild-type ShhN with those of mutants in which the zinc-coordinating residues H140, D147, and H182, or E176 which interacts with the metal ion via a bridging water mol., were mutated to alanine. The wild-type and E176A mutant proteins retained 1 mol of zinc/mol of protein after extensive dialysis, whereas the H140A and D147A mutants retained only 0.03 and 0.05 mol of zinc/mol of protein, resp. Assay of the wild-type and mutant proteins in two activity assays indicated that the wild-type and E176A mutant proteins had similar activity, whereas the H140A and D147A mutants were significantly less active. These assays also indicated that the H140A and D147A mutants were susceptible to proteolysis. CD, fluorescence, and 1H NMR spectra of the H140A, D147A, and E176A mutants measured at 20 or 25° were very similar to those obsd. for wild-type ShhN. However, CD measurements at 37° showed evidence of some structural differences in the H140A and D147A mutants. Guanidine hydrochloride (GuHCl) denaturation studies revealed that the loss of zinc from the H140A and D147A mutants destabilized the folded proteins by ∼3.5 kcal/mol, comparable to the effect of removing zinc from wild-type ShhN by treatment with EDTA. Thermal melting curves of wild-type ShhN gave a single unfolding transition with a midpoint Tm of ∼59°, whereas both the H140A and D147A mutants displayed two distinct transitions with Tm values of 37-38 and 52-54°, similar to that obsd. for EDTA-treated wild-type ShhN. Addn. of zinc to the H140A and D147A mutants resulted in a partial restoration of stability against thermal and GuHCl denaturation. The ability of these mutants to bind zinc was confirmed using a fluorescence-based binding assay that indicated that they bound zinc with Kd values of ∼1.6 and ∼15 nM, resp., as compared to a value of ≤100 pM for wild-type ShhN. The properties of the E176A mutant were indistinguishable from those of wild-type ShhN in all biophys. and functional assays, indicating that this residue does not contribute significantly to stabilization of the zinc-binding site and that ShhN does not require hydrolase activity for in vitro biol. function.
- 294Masuoka, J.; Hegenauer, J.; Van Dyke, B. R.; Saltman, P. Intrinsic stoichiometric equilibrium constants for the binding of zinc(II) and copper(II) to the high affinity site of serum albumin. J. Biol. Chem. 1993, 268, 21533– 21537, DOI: 10.1016/S0021-9258(20)80574-2294Intrinsic stoichiometric equilibrium constants for the binding of zinc(II) and copper(II) to the high affinity site of serum albuminMasuoka, James; Hegenauer, Jack; Van Dyke, Bruce R.; Saltman, PaulJournal of Biological Chemistry (1993), 268 (29), 21533-7CODEN: JBCHA3; ISSN:0021-9258.Intrinsic stoichiometric equil. consts. were detd. for zinc(II) and copper(II) binding to bovine and human serum albumin. Data were obtained from equil. dialysis expts. Metals were presented to apoprotein as metal chelates in order to avoid metal hydrolysis and to minimize nonspecific metal-protein interactions. Scatchard anal. of the binding data indicated that the high affinity class for both zinc and copper was comprised of one site. Results of binding expts. done at several pH values suggested that while both histidyl and carboxyl groups appear to be involved in copper binding, histidyl residues alone were sufficient for zinc binding. These amino acid residues were used in combination to model several binding sites used in the formulation of equil. expressions from which stoichiometric consts. were calcd. The log10K for bovine serum albumin was calcd. to be 7.28 for Zn(II) and 11.12 for Cu(II). That for human serum albumin was detd. to be 7.53 and 11.18 for Zn(II) and Cu(II), resp. These consts. were used in equil. to simulate speciation of metal-albumin and metal-chelator and to illustrate relative binding affinities. This comparison of binding strengths was possible only through the calcn. of an intrinsic stoichiometric binding const.
- 295Krężel, A.; Maret, W. Thionein/metallothionein control Zn(II) availability and the activity of enzymes. JBIC, J. Biol. Inorg. Chem. 2008, 13, 401– 409, DOI: 10.1007/s00775-007-0330-y295Thionein/metallothionein control Zn(II) availability and the activity of enzymesKrezel, Artur; Maret, WolfgangJBIC, Journal of Biological Inorganic Chemistry (2008), 13 (3), 401-409CODEN: JJBCFA; ISSN:0949-8257. (Springer GmbH)Fundamental issues in zinc biol. are how proteins control the concns. of free Zn(II) ions and how tightly they interact with them. Since, basically, the Zn(II) stability consts. of only two cytosolic zinc enzymes, carbonic anhydrase and superoxide dismutase, have been reported, the affinity for Zn(II) of another zinc enzyme, sorbitol dehydrogenase (SDH), was detd. Its log K is 11.2±0.1, which is similar to the log K values of carbonic anhydrase and superoxide dismutase despite considerable differences in the coordination environments of Zn(II) in these enzymes. Protein tyrosine phosphatase 1B (PTP 1B), on the other hand, is not classified as a zinc enzyme but is strongly inhibited by Zn(II), with log K = 7.8±0.1. In order to test whether or not metallothionein (MT) can serve as a source for Zn(II) ions, it was used to control free Zn(II) ion concns. MT makes Zn(II) available for both PTP 1B and the apoform of SDH. However, whether or not Zn(II) ions are indeed available for interaction with these enzymes depends on the thionein (T) to MT ratio and the redox poise. At ratios [T/(MT + T) = 0.08-0.31] prevailing in tissues and cells, picomolar concns. of free Zn(II) are available from MT for reconstituting apoenzymes with Zn(II). Under conditions of decreased ratios, nanomolar concns. of free Zn(II) become available and affect enzymes that are not zinc metalloenzymes. The match between the Zn(II) buffering capacity of MT and the Zn(II) affinity of proteins suggests a function of MT in controlling cellular Zn(II) availability.
- 296Payne, J. C.; Rous, B. W.; Tenderholt, A. L.; Godwin, H. A. Spectroscopic determination of the binding affinity of zinc to the DNA-binding domains of nuclear hormone receptors. Biochemistry 2003, 42, 14214– 14224, DOI: 10.1021/bi035002l296Spectroscopic Determination of the Binding Affinity of Zinc to the DNA-Binding Domains of Nuclear Hormone ReceptorsPayne, John C.; Rous, Brian W.; Tenderholt, Adam L.; Godwin, Hilary ArnoldBiochemistry (2003), 42 (48), 14214-14224CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)Zinc binding to the two Cys4 sites present in the DNA-binding domain (DBD) of nuclear hormone receptor proteins is required for proper folding of the domain and for protein activity. By utilizing Co2+ as a spectroscopic probe, we have characterized the metal-binding properties of the two Cys4 structural zinc-binding sites found in the DBD of human estrogen receptor α (hERα-DBD) and rat glucocorticoid receptor (GR-DBD). The binding affinity of Co2+ to the two proteins was detd. relative to the binding affinity of Co2+ to the zinc finger consensus peptide, CP-1. Using the known dissocn. const. of Co2+ from CP-1, the dissocn. consts. of cobalt from hERα-DBD were calcd.: KCod1 = 2.2 (± 1.0) × 10-7 M and KCod2 = 6.1 (± 1.5) × 10-7 M. Similarly, the dissocn. consts. of Co2+ from GR-DBD were calcd.:KCod1 = 4.1 (± 0.6) × 10-7 M and KCod2 = 1.7 (± 0.3) × 10-7 M. Metal-binding studies conducted in which Zn2+ displaces Co2+ from the metal-binding sites of hERα-DBD and GR-DBD indicate that Zn2+ binds to each of the Cys4 metal-binding sites approx. 3 orders of magnitude more tightly than Co2+ does: the stoichiometric dissocn. consts. are KZnd1 = 1 (± 1) × 10-10 M and KZnd2 = 5 (± 1) × 10-10 M for hERα-DBD and KZnd1 = 2 (± 1) × 10-10 M and KZnd2 = 3 (± 1) × 10-10 M for GR-DBD. These affinities are comparable to those obsd. for most other naturally occurring structural zinc-binding sites. In contrast to the recent prediction by Low et. al. that zinc binding in these systems should be cooperative [Low, L. Y., Hernandez, H., Robinson, C. V., O'Brien, R., Grossmann, J. G., Ladbury, J. E., and Luisi, B. (2002) J. Mol. Biol. 319, 87-106], these data suggest that the zincs that bind to the two sites in the DBDs of hERα-DBD and GR-DBD do not interact.
- 297Dinkova-Kostova, A. T.; Holtzclaw, W. D.; Wakabayashi, N. Keap1, the sensor for electrophiles and oxidants that regulates the phase 2 response, is a zinc metalloprotein. Biochemistry 2005, 44, 6889– 6899, DOI: 10.1021/bi047434h297Keap1, the sensor for electrophiles and oxidants that regulates the phase 2 response, is a zinc metalloproteinDinkova-Kostova, Albena T.; Holtzclaw, W. David; Wakabayashi, NobunaoBiochemistry (2005), 44 (18), 6889-6899CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)Induction of the phase 2 response, a major cellular reaction to oxidative/electrophile stress depends on a protein triad: actin-tethered Keap1 that binds to Nrf2. Inducers react with Keap1 releasing Nrf2 for nuclear translocation and activation of the antioxidant response element (ARE), which regulates phase 2 genes. The primary sensors for inducers are certain uniquely reactive Cys SH groups of Keap1. Recombinant murine Keap1 contains 0.9 Zn atoms per monomer as detd. by inductively coupled plasma-optical emission spectrometry: its Zn content depends on the metal compn. of the overexpression medium. Simultaneous direct measurement of bound Zn using a pyridazoresorcinol chelator and protein SH groups using 4,4'-dipyridyl disulfide established that (1) Zn is bound to reactive Cys SH groups of Keap1 and is displaced stoichiometrically by inducers; (2) with these Cys residues mutated to Ala, the affinity for Zn was reduced by nearly 2 orders of magnitude; and (3) the assocn. const. of Keap1 for Zn2+ was 1.02 × 1011 M-1, consistent with a Zn-metalloprotein. Co substitution for Zn yielded an optical spectrum consistent with tetrahedral metal coordination. Coincident binding of inducers and release of Zn altered the conformation of Keap1, as shown by a profound decline of its Trp fluorescence and depression of fluorescence of a hydrophobicity probe. Therefore, regulation of the phase 2 response involves chem. modification of crit. Cys residues of Keap1, whose reactivity is modulated by Zn2+ binding. Thus, Keap1 is a Zn-thiol protein endowed with a delicate switch controlled by both metal-binding and thiol reactivity.
- 298Muller, H. N.; Skerra, A. Grafting of a high-affinity Zn(II)-binding site on the beta-barrel of retinol-binding protein results in enhanced folding stability and enables simplified purification. Biochemistry 1994, 33, 14126– 14135, DOI: 10.1021/bi00251a023298Grafting of a high-affinity Zn(II)-binding site on the beta-barrel of retinol-binding protein results in enhanced folding stability and enables simplified purificationMuller H N; Skerra ABiochemistry (1994), 33 (47), 14126-35 ISSN:0006-2960.In a rational protein design approach, the His3 Zn(II)-binding site from the active center of human carbonic anhydrase II was transplanted on the beta-barrel of mammalian serum retinol-binding protein (RBP) in a solvent-accessible location on the protein's outer surface. Several mutants of RBP were generated and produced in Escherichia coli, and their Zn(II)-binding properties were investigated in equilibrium dialysis experiments. One mutant, RBP/H3(A), with His residues introduced at the positions 46, 54, and 56 in the polypeptide sequence was shown to bind Zn(II) specifically with a stoichiometry of 1 and a corresponding dissociation constant equal to 36 +/- 10 nM. Binding of Zn(II) had no influence on the binding of retinoic acid, a natural ligand of RBP. In guanidinium chloride-induced unfolding experiments the mutant was found to be significantly stabilized in the presence of small concentrations of ZnSO4. This effect could be quantitatively explained using thermodynamic theory. Furthermore, it was demonstrated that the protein-bound Zn(II) is accessible to iminodiacetic acid as an additional chelating ligand without competition for the metal ion. Thus it was possible to use the grafted metal-binding site for the efficient purification of the engineered, bifunctional RBP via immobilized metal affinity chromatography from the bacterial protein extract.
- 299Guerrerio, A. L.; Berg, J. M. Metal ion affinities of the zinc finger domains of the metal responsive element-binding transcription factor-1 (MTF1). Biochemistry 2004, 43, 5437– 5444, DOI: 10.1021/bi0358418299Metal Ion Affinities of the Zinc Finger Domains of the Metal Responsive Element-Binding Transcription Factor-1 (MTF1)Guerrerio, Anthony L.; Berg, Jeremy M.Biochemistry (2004), 43 (18), 5437-5444CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)Metal response element (MRE) binding transcription factor-1 (MTF1) is a six Cys2His2 zinc finger-contg. transcription factor required for basal and zinc-induced transcription of metallothionein genes. The cobalt(II) and zinc(II) affinities of a protein fragment comprising the six zinc finger domains have been examd. to reveal apparent dissocn. consts. (for the six domains collectively) of 0.5±0.2 μM for cobalt(II) and 31±14 pM for zinc(II). Two approaches have been used to det. the metal ion affinities of the individual domains. First, the six domains have been examd. as single domain peptides revealing dissocn. consts. ranging from 0.3 to 1.7 μM for cobalt(II). The domains fall into two sets with peptides corresponding to domains 2, 3, and 4 showing relatively high affinity (Kd(Co(II)) 0.3-0.5 μM) and peptides corresponding to domains 1, 5, and 6 showing lower affinity (Kd(Co(II)) 1.6-1.7 μM). Second, we examd. the affinity of each domain in the context of the six zinc finger domain protein by individually mutating one metal-binding His residue to Cys to allow independent monitoring of the cobalt(II) occupancy of each site. The affinity of each domain was higher in this context than as a single domain peptide with affinities (cor. for the effect of the mutation) ranging from 0.02 to 0.5 μM. The increase in affinity for the individual domains ranged from factors of 1.1 to 20. The order of affinities (from higher to lowest) was obsd. to be 4>2 ≈ 5>6 ≈ 3≈1. These results reveal that none of the Cys2His2 zinc finger domains of MTF1 have dramatically low metal ion affinities, certainly none low enough to respond to changes in free zinc ion concns. in the micromolar range. Nonetheless, the metal ion affinities of some domains do differ by a factor of 25 with domains at both the amino- and carboxyl-termini showing lower intrinsic affinities for metal ions than the central domains.
- 300Miłoch, A.; Krężel, A. Metal binding properties of the zinc finger metallome – Insights into variations of stability. Metallomics 2014, 6, 2015– 2024, DOI: 10.1039/C4MT00149D300Metal binding properties of the zinc finger metallome - insights into variations in stabilityMiloch, Anna; Krezel, ArturMetallomics (2014), 6 (11), 2015-2024CODEN: METAJS; ISSN:1756-591X. (Royal Society of Chemistry)Zinc is one of the most widespread metal ions found in biol. systems. Of the expected 3000 zinc proteins in the human proteome, most contain zinc in structural sites. Among these structures, the most important are zinc fingers, which are well suited to facilitate interactions with DNA, RNA, proteins and lipid mols. Knowledge regarding their stability is a crit. issue in understanding the function of zinc fingers and their reactivity under fluxing cellular Zn(II) availability and different redox states. Zinc stability consts. that have been detd. using a variety of methods demonstrate wide diversity. Recent studies on the stability of consensus zinc fingers have demonstrated that the known metal-ion affinities for zinc fingers may have been underestimated by as much as three or more orders of magnitude. Here, using four natural ββα zinc fingers, we compare in detail several different methods that have been used for the detn. of zinc finger stability consts., such as common reverse-titrn., potentiometry, competition with metal chelators, and a new approach based on a three-step spectrophotometric titrn. We discuss why the stabilities of zinc fingers that are detd. spectrophotometrically are frequently underestimated due to the lack of effective equil. competition, which leads to large errors during the processing of the titrn. data. The literature stability consts. of many natural zinc fingers have been underestimated, and they are significantly lower when compared with the consensus peptides. Our data show that in the cell, some naturally occurring zinc fingers may potentially be unoccupied and are instead loaded transiently with Zn(II). Large variations in stability within the same class of zinc fingers have demonstrated that the thermodn. effects hidden in the sequence and structure are the key elements responsible for the differentiation of the stability of the zinc finger metallome.
- 301Sikorska, M.; Krężel, A.; Otlewski, J. Femtomolar Zn2+ affinity of LIM domain of PDLIM1 protein uncovers crucial contribution of protein-protein interactions to protein stability. J. Inorg. Biochem. 2012, 115, 28– 35, DOI: 10.1016/j.jinorgbio.2012.05.009301Femtomolar Zn2+ affinity of LIM domain of PDLIM1 protein uncovers crucial contribution of protein-protein interactions to protein stabilitySikorska, Malgorzata; Krezel, Artur; Otlewski, JacekJournal of Inorganic Biochemistry (2012), 115 (), 28-35CODEN: JIBIDJ; ISSN:0162-0134. (Elsevier)An individual LIM domain has approx. 55 amino acids with 8 highly conserved residues responsible for binding of two Zn2+ into two distinct zinc finger motifs. We examd. LIM domain stability of PDLIM1 protein (known also as Elfin protein), its C-terminally extended constructs as well as sep. zinc fingers, and several full domain mutants in terms of Zn2+ affinity and domain stability. Thermal denaturation, mass spectrometry, limited proteolysis, protein oxidn. and CD techniques were used to det. a set of thermodn. stability parameters. The results demonstrate unambiguously very high (femtomolar) affinity of both Zn2+ to the conserved LIM domain (Kdav = 2.5 × 10-14 M) and its addnl. elevation in the C-terminally extended domain construct (Kdav = 3.1 × 10-15 M). We demonstrate in the example of PDLIM1 using a set of LIM protein constructs and its zinc finger peptides that stability of the entire zinc-contg. domain is not only defined by the Zn2+ coordination environment but significantly depends on the set of protein-protein interactions with the C-terminus of the protein. We discuss structural similarities of LIM domains and suggest the prolongation of the conserved LIM sequence to its C-terminal helix that has a significant impact on domain stability. We also discuss the functionality of LIM domains in terms of different physiol. zinc and redox buffering capacity.
- 302Park, Y. B.; Hohl, M.; Padjasek, M.; Jeong, E.; Jin, K. S.; Krężel, A.; Petrini, J. H.; Cho, Y. Eukaryotic Rad50 functions as a rod-shaped dimer. Nat. Struct. Mol. Biol. 2017, 24, 248– 257, DOI: 10.1038/nsmb.3369302Eukaryotic Rad50 functions as a rod-shaped dimerPark, Young Bong; Hohl, Marcel; Padjasek, Michal; Jeong, Eunyoung; Jin, Kyeong Sik; Krezel, Artur; Petrini, John H. J.; Cho, YunjeNature Structural & Molecular Biology (2017), 24 (3), 248-257CODEN: NSMBCU; ISSN:1545-9993. (Nature Publishing Group)The Rad50 hook interface is crucial for assembly and various functions of the Mre11 complex. Previous analyses suggested that Rad50 mols. interact within (intracomplex) or between (intercomplex) dimeric complexes. In this study, we detd. the structure of the human Rad50 hook and coiled-coil domains. The data suggest that the predominant structure is the intracomplex, in which the two parallel coiled coils proximal to the hook form a rod shape, and that a novel interface within the coiled-coil domains of Rad50 stabilizes the interaction of Rad50 protomers in the dimeric assembly. In yeast, removal of the coiled-coil interface compromised Tel1 activation without affecting DNA repair, while simultaneous disruption of that interface and the hook phenocopied a null mutation. The results demonstrate that the hook and coiled-coil interfaces coordinately promote intracomplex assembly and define the intracomplex as the functional form of the Mre11 complex.
- 303Crow, J. P.; Sampson, J. B.; Zhuang, Y.; Thompson, J. A.; Beckman, J. S. Decreased zinc affinity of amyotrophic lateral sclerosis-associated superoxide dismutase mutants leads to enhanced catalysis of tyrosine nitration by peroxynitrite. J. Neurochem. 1997, 69, 1936– 1944, DOI: 10.1046/j.1471-4159.1997.69051936.x303Decreased zinc affinity of amyotrophic lateral sclerosis-associated superoxide dismutase mutants leads to enhanced catalysis of tyrosine nitration by peroxynitriteCrow, John P.; Sampson, Jacinda B.; Zhuang, Yingxin; Thompson, John A.; Beckman, Joseph S.Journal of Neurochemistry (1997), 69 (5), 1936-1944CODEN: JONRA9; ISSN:0022-3042. (Lippincott-Raven)Mutations to Cu/Zn superoxide dismutase (SOD) linked to familial amyotrophic lateral sclerosis (ALS) enhance an unknown toxic reaction that leads to the selective degeneration of motor neurons. However, the question of how >50 different missense mutations produce a common toxic phenotype remains perplexing. The authors found that the zinc affinity of four ALS-assocd. SOD mutants was decreased up to 30-fold compared to wild-type SOD but that both mutants and wild-type SOD retained copper with similar affinity. Neurofilament-L (NF-L), one of the most abundant proteins in motor neurons, bound multiple zinc atoms with sufficient affinity to potentially remove zinc from both wild-type and mutant SOD while having a lower affinity for copper. The loss of zinc from wild-type SOD approx. doubled its efficiency for catalyzing peroxynitrite-mediated tyrosine nitration, suggesting that one gained function by SOD in ALS may be an indirect consequence of zinc loss. Nitration of protein-bound tyrosines is a permanent modification that can adversely affect protein function. Thus, the toxicity of ALS-assocd. SOD mutants may be related to enhanced catalysis of protein nitration subsequent to zinc loss. By acting as a high-capacity zinc sink, NF-L could foster the formation of zinc-deficient SOD within motor neurons.
- 304Posewitz, M. C.; Wilcox, D. E. Properties of the Sp1 zinc finger 3 peptide: coordination chemistry, redox reactions, and metal binding competition with metallothionein. Chem. Res. Toxicol. 1995, 8, 1020– 1028, DOI: 10.1021/tx00050a005304Properties of the Sp1 Zinc Finger 3 Peptide: Coordination Chemistry, Redox Reactions, and Metal Binding Competition with MetallothioneinPosewitz, Matthew C.; Wilcox, Dean E.Chemical Research in Toxicology (1995), 8 (8), 1020-8CODEN: CRTOEC; ISSN:0893-228X. (American Chemical Society)Toxic and/or carcinogenic consequences may result from metal ion substitution for the Zn(II) in transcription factors contg. zinc fingers, and the small Cys-rich metal-binding protein metallothionein (MT) may play a role in this metal substitution. To begin to evaluate this hypothesis, with regard to the carcinogenic metal ion Ni(II), a peptide corresponding to the third finger of the transcription factor Sp1 (Sp1-3) has been synthesized and its metal binding and redox reactions have been studied. The peptide binds Zn(II), Co(II), and Ni(II), with spectroscopic data indicating a tetrahedral coordination for the latter two; metal ion affinities have been quantified (Kd = 6 × 10-10, 3 × 10-7, and 4 × 10-6, resp.) and less than those of an optimized zinc finger peptide (B. A. Krizek et al. (1993)) but greater than those of the second finger of transcription factor IIIA (J. M. Berg and D. L. Merkle (1989)). Reactions of the peptide and its metal-bound forms with dioxygen or hydrogen peroxide did not produce oxygen radical species; however, oxidn. of the two Sp1-3 cysteines was modulated by metal ions (Zn < Co = apo < Ni), suggesting a protective role for Zn(II) but an enhancing role for Ni(II). Metal binding competition between Sp1-3 and the α domain of human liver MT-2 (α-hMT2) indicates a similar affinity for Zn(II). However, α-hMT2 has a higher affinity for Ni(II), suggesting that MT may play a protective role by ensuring Zn(II), rather than Ni(II), coordination to zinc finger sequences of transcription factors.
- 305diTargiani, R. C.; Lee, S. J.; Wassink, S.; Michel, S. L. Functional characterization of iron-substituted tristetraprolin-2D (TTP-2D, NUP475–2D): RNA binding affinity and selectivity. Biochemistry 2006, 45, 13641– 13649, DOI: 10.1021/bi060747n305Functional characterization of iron-substituted tristetraprolin-2D (TTP-2D, NUP475-2D): RNA binding affinity and selectivitydiTargiani Robert C; Lee Seung Jae; Wassink Sarah; Michel Sarah L JBiochemistry (2006), 45 (45), 13641-9 ISSN:0006-2960.The protein tristetraprolin (TTP, also known as NUP475 and TIS11) is a nonclassical zinc finger protein that is involved in regulating the inflammatory response. Specifically, TTP binds to AU-rich sequence elements located at the 3'-untranslated region of cytokine mRNAs forming a complex that is degraded by the exosome. The nucleic acid binding region of TTP is comprised of two CysX(8)CysX(5)CysX(3)His domains that are activated in the presence of zinc. A two-domain construct of TTP (TTP-2D) has been cloned and overexpressed in E. coli. TTP-2D picks up visible red coloration from the expression media, unless it is expressed under iron-restricted conditions. The iron-binding properties of TTP-2D and the effect of iron substitution on RNA recognition have been investigated. Both Fe(II) and Fe(III) bind to TTP-2D and a full titration of Fe(III) with TTP-2D revealed that this metal ion binds with micromolar affinity. Upon reconstitution of TTP-2D with either Fe(II) or Fe(III), the protein recognizes a canonical RNA-binding sequence, UUUAUUUAUUU, with nanomolar affinity. Substitution of a single adenine or both adenines results in a decreased affinity of TTP-2D for the RNA molecule, demonstrating that both Fe(II)-TTP-2D and Fe(III)-TTP-2D selectively recognize a physiologically relevant RNA sequence. The relative affinities of Fe(II)-TTP-2D and Fe(III)-TTP-2D for the series of RNA sequences mirror those observed for Zn(II)-TTP-2D and suggest that iron is a viable substitute for zinc in this protein.
- 306Bal, W.; Schwerdtle, T.; Hartwig, A. Mechanism of nickel assault on the zinc finger of DNA repair protein XPA. Chem. Res. Toxicol. 2003, 16, 242– 248, DOI: 10.1021/tx025639q306Mechanism of Nickel Assault on the Zinc Finger of DNA Repair Protein XPABal, Wojciech; Schwerdtle, Tanja; Hartwig, AndreaChemical Research in Toxicology (2003), 16 (2), 242-248CODEN: CRTOEC; ISSN:0893-228X. (American Chemical Society)Xeroderma pigmentosum group A complementing protein (XPA) is a member of the protein complex of the nucleotide excision repair (NER) pathway of DNA repair, participating in the assembly of the incision complex. The 4S zinc finger domain of XPA is involved the interactions with other NER proteins. As demonstrated previously, the activity of XPA is compromised by several metal ions implicated in DNA repair inhibition, including Ni(II), Cd(II), and Co(II) (M. Asmuss et al., 2000). To study the possible mol. mechanisms of XPA inhibition, we investigated Zn(II) and Ni(II) interactions with the synthetic 37 peptide (XPAzf), representing the XPA zinc finger sequence AcDYVICEECGKEFMDSYLMNHFDLPTCDNCRDADDKHKam. The binding consts. were detd. using fluorescence and UV-vis spectroscopies, structural insights were provided by CD, and oxidative damage to XPAzf was studied with HPLC. The binding consts. for Zn(II) and Ni(II) are (8.5±1.5) × 108 (log value 8.93(7)) and (1.05±0.07) × 106 M-1 (6.02(3)), resp., in 10 mM phosphate buffer, pH 7.4, and (6±4) × 109 (9.8(2)) and (2.9±0.5) × 106 M-1 (6.46(8)) in 50 mM phosphate buffer, pH 7.4, yielding binding const. ratios Zn(II)/Ni(II) of 800±100 and 2300±500, resp. The Ni(II) ion forms a square planar complex with the sulfurs of XPAzf, opposed to the tetrahedral structure of the native Zn(II) complex. Consequently, the overall zinc finger structure is lost in the Ni(II)-substituted peptide. Zn(II)-satd. XPAzf is remarkably resistant to air oxidn. and is only slowly oxidized by 0.01 mM, 0.1 mM, and 1 mM H2O2 in a concn.-dependent fashion. However, the presence of just 10-fold molar excess of Ni(II) is sufficient to accelerate this process for all three H2O2 concns. tested. Overall, our results indicate that XPAzf can undergo Ni(II) assault in specific conditions.
- 307Kocyła, A.; Krężel, A. Zinc clasp-based reversible toolset for selective metal-mediated protein heterodimerization. Chem. Commun. (Cambridge, U. K.) 2018, 54, 13539– 13542, DOI: 10.1039/C8CC06301J307Zinc clasp-based reversible toolset for selective metal-mediated protein heterodimerizationKocyla, Anna; Krezel, ArturChemical Communications (Cambridge, United Kingdom) (2018), 54 (96), 13539-13542CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)Considering the complex biol. quandaries of the tightly woven networks of biol. macromols., we present an optimized zinc clasp-based toolset from the CD4 co-receptor and Lck protein tyrosine kinase complex for selective, tight and fully reversible protein heterodimerization (log K12 = 18.6). We demonstrated its utility on CD4-tagged proteins with capture from bacterial lysate and constructed mol. baits using a new small-mol. tether.
- 308Kocyła, A.; Adamczyk, J.; Krężel, A. Interdependence of free zinc changes and protein complex assembly - insights into zinc signal regulation. Metallomics 2018, 10, 120– 131, DOI: 10.1039/C7MT00301C308Interdependence of free zinc changes and protein complex assembly - insights into zinc signal regulationKocyla, Anna; Adamczyk, Justyna; Krezel, ArturMetallomics (2018), 10 (1), 120-131CODEN: METAJS; ISSN:1756-591X. (Royal Society of Chemistry)Cellular zinc (Zn(ii)) is bound with proteins that are part of the proteomes of all domains of life. It is mostly utilized as a catalytic or structural protein cofactor, which results in a vast no. of binding architectures. The Zn(ii) ion is also important for the formation of transient protein complexes with a Zn(ii)-dependent quaternary structure that is formed upon cellular zinc signals. The mechanisms by which proteins assoc. with and dissoc. from Zn(ii) and the connection with cellular Zn(ii) changes remain incompletely understood. In this study, we aimed to examine how zinc protein domains with various Zn(ii)-binding architectures are formed under free Zn(ii) concn. changes and how formation of the Zn(ii)-dependent assemblies is related to the protein concn. and reactivity. To accomplish these goals we chose four zinc domains with different Zn(ii)-to-protein binding stoichiometries: classical zinc finger (ZnP), LIM domain (Zn2P), zinc hook (ZnP2) and zinc clasp (ZnP1P2) folds. Our research demonstrated a lack of changes in the satn. level of intraprotein zinc binding sites, despite various peptide concns., while homo- and heterodimers indicated a concn.-dependent tendency. In other words, at a certain free Zn(ii) concn., the fraction of a formed dimeric complex increases or decreases with subunit concn. changes. Secondly, even small or local changes in free Zn(ii) may significantly affect protein satn. depending on its architecture, function and subcellular concn. In our paper, we indicate the importance of interdependence of free Zn(ii) availability and protein subunit concns. for cellular zinc signal regulation.
- 309Hogstrand, C.; Verbost, P. M.; Wendelaar Bonga, S. E. Inhibition of human erythrocyte Ca2+-ATPase by Zn2+. Toxicology 1999, 133, 139– 145, DOI: 10.1016/S0300-483X(99)00020-7309Inhibition of human erythrocyte Ca2+-ATPase by Zn2+Hogstrand, Christer; Verbost, Pieter M.; Wendelaar Bonga, Sjoerd E.Toxicology (1999), 133 (2,3), 139-145CODEN: TXCYAC; ISSN:0300-483X. (Elsevier Science Ireland Ltd.)Recent investigations suggest that Ca2+-ATPase from fish gills is very sensitive to Zn2+ (1996). The effect of free Zn2+ ion on the human erythrocyte plasma membrane Ca2+-ATPase was investigated to explore the possible extension of this finding to humans. Membrane vesicles were prepd. and the Ca2+-ATPase activity was measured as Ca2+-stimulated ATP hydrolysis and as ATP-dependent Ca2+ transport. The Zn2+ ion inhibited the erythrocyte Ca2+-ATPase by reducing Vmax and increasing the K0.5. While in the Ca2+ transport assay only the Vmax was affected at lower Zn2+ concns. (50-100 pM), redn. of Vmax was always accompanied by an affinity decrease in the ATP hydrolysis assay. The Ca2+-ATPase was found to be inhibited by Zn2+ at extremely low concns. The IC10 and IC50 for Zn2+, at a Ca2+ concn. of 1.0 μM, were estd. at 4 and 80 pM, resp. Although the Ca2+-ATPase might be more sensitive in vitro than in vivo conditions, the results suggest that physiol. concns. of Zn2+ may reduce the activity of the erythrocyte Ca2+-ATPase. Furthermore, disturbance of Ca homeostasis may be a mechanism causing Zn toxicity during exposure.
- 310Eron, S. J.; MacPherson, D. J.; Dagbay, K. B.; Hardy, J. A. Multiple mechanisms of zinc-mediated inhibition for the apoptotic caspases-3, −6, −7, and −8. ACS Chem. Biol. 2018, 13, 1279– 1290, DOI: 10.1021/acschembio.8b00064310Multiple Mechanisms of Zinc-Mediated Inhibition for the Apoptotic Caspases-3, -6, -7, and -8Eron, Scott J.; MacPherson, Derek J.; Dagbay, Kevin B.; Hardy, Jeanne A.ACS Chemical Biology (2018), 13 (5), 1279-1290CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)Zinc is emerging as a widely used and important biol. regulatory signal. Cellular zinc levels are tightly regulated by a complex array of zinc importer and exporters to control processes such as apoptotic cell death. While caspase inhibition by zinc has been reported previously, the reported inhibition consts. were too weak to suggest a crit. biol. role for zinc-mediated inhibition. In this work we have adopted a method of assessing available zinc. This allowed assessment of the accurate inhibition consts. for apoptotic caspases, caspase-3, -6, -7 and -8. Each of these caspases are inhibited by zinc at intracellular levels, however, with widely differing inhibition consts. and different zinc binding stoichiometries. Caspase -3, -6 and -8 appear to be constitutively inhibited by typical zinc levels and this inhibition must be lifted to allow activation. The inhibition const. for caspase-7 (76 nM) is much weaker than for the other apoptotic caspases (2.6-6.9 nM) suggesting that caspase-7 is not inactivated by normal zinc concns. but can be inhibited under conditions of zinc stress. Caspase-3, -7, and -8 were found to bind three, one, and two zincs resp. In each of these caspases, zinc was present in the active site, in contrast to caspase-6, which binds one zinc allosterically. The most notable new mechanism to emerge from this work is for zinc-mediated inhibition of caspase-8. Zinc binds caspase-8 directly at the active site and at a second site. Zinc binding inhibits formation of the caspase-8 dimer, the activated form of the enzyme. Together these findings suggest that zinc plays a crit. role in regulation of apoptosis by direct inactivation of caspases, in a manner that is unique for each caspase.
- 311Hao, Q.; Hong, S.-H.; Maret, W. Lipid-raft dependent endocytosis of metallothionein in HepG2 cells. J. Cell. Physiol. 2007, 210, 428– 435, DOI: 10.1002/jcp.20874311Lipid raft-dependent endocytosis of metallothionein in HepG2 cellsHao, Qiang; Hong, Sung-Hye; Maret, WolfgangJournal of Cellular Physiology (2007), 210 (2), 428-435CODEN: JCLLAX; ISSN:0021-9541. (Wiley-Liss, Inc.)Human hepatocellular carcinoma (HepG2) cells take up metallothionein (MT) by endocytosis. MT co-localizes with albumin but not with transferrin, indicating uptake via a non-classical pathway rather than via clathrin-mediated endocytosis. A lipid raft-dependent uptake is indicated by pravastatin inhibition of cholesterol synthesis and methyl-β-cyclodextrin inhibition of cholesterol translocation to the plasma membrane, reducing MT uptake by 29% and 69%, resp. Subcellular fractionation after MT uptake reveals significant amts. of MT in vesicular fractions including lysosomes but virtually no MT in the cytosol. Metals bound to MT are released into the cytosol, however. The findings define a pathway for cellular metal acquisition. Together with results from other studies demonstrating secretion of MT from different cells and the presence of MT in extracellular fluids, the results suggest a function of MT in intercellular communication.
- 312Knipp, M.; Charnock, J. M.; Garner, C. D.; Vašák, M. Structural and functional characterization of the Zn(II) site on dimethylarginase-1 (DDAH-1) from bovine brain. J. Biol. Chem. 2001, 276, 40449– 40456, DOI: 10.1074/jbc.M104056200312Structural and functional characterization of the Zn(II) site in dimethylargininase-1 (DDAH-1) from bovine brain: Zn(II) release activates DDAH-1Knipp, Markus; Charnock, John M.; Garner, C. David; Vasak, MilanJournal of Biological Chemistry (2001), 276 (44), 40449-40456CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)L-Nω,Nω-Dimethylarginine dimethylaminohydrolase-1 (DDAH-1) is a Zn(II)-contg. enzyme that, through hydrolysis of side-chain methylated L-arginines, regulates the activity of nitric-oxide synthase. Herein we report the structural and functional properties of the Zn(II)-binding site in DDAH-1 from bovine brain. Activity measurements of the native and metal-free enzyme have revealed that the endogenously bound Zn(II) inhibits the enzyme. Native DDAH-1 could be fully or partially activated using various concns. of phosphate, imidazole, histidine, and histamine, a process that is paralleled by the release of Zn(II). The slow activation of the enzyme by the bulky complexing agents EDTA and 1,10-phenanthroline suggests that the Zn(II)-binding site is partially buried in the protein structure. The apparent Zn(II)-dissocn. const. of 4.2 nM, detd. by 19F NMR using the chelator 5F-BAPTA (1,2-bis(2-amino-5-fluorophenoxy)ethane-N,N,N',N'-tetraacetic acid), lies in the range of intracellular free Zn(II) concns. These results suggest a regulatory role for the Zn(II)-binding site. The coordination environment of the Zn(II) in DDAH-1 has been examd. by Zn K-edge x-ray absorption spectroscopy. The extended x-ray absorption fine structure obsd. is consistent with Zn(II) being coordinated by 2 S and 2 N (or O) atoms. The biol. implications of these findings are discussed.
- 313Maret, W. Inhibitory zinc sites in enzymes. BioMetals 2013, 26, 197– 204, DOI: 10.1007/s10534-013-9613-7313Inhibitory zinc sites in enzymesMaret, WolfgangBioMetals (2013), 26 (2), 197-204CODEN: BOMEEH; ISSN:0966-0844. (Springer)A review. Several pathways increase the concns. of cellular free zinc(II) ions. Such fluctuations suggest that zinc(II) ions are signalling ions used for the regulation of proteins. One function is the inhibition of enzymes. It is quite common that enzymes bind zinc(II) ions with micro- or nanomolar affinities in their active sites that contain catalytic dyads or triads with a combination of glutamate (aspartate), histidine and cysteine residues, which are all typical zinc-binding ligands. However, for such binding to be physiol. significant, the binding consts. must be compatible with the cellular availability of zinc(II) ions. The affinity of inhibitory zinc(II) ions for receptor protein tyrosine phosphatase β is particularly high (K i = 21 pM, pH 7.4), indicating that some enzymes bind zinc almost as strongly as zinc metalloenzymes. The competitive pattern of zinc inhibition for this phosphatase implicates its active site cysteine and nearby residues in the coordination of zinc. Quant. biophys. data on both affinities of proteins for zinc and cellular zinc(II) ion concns. provide the basis for examg. the physiol. significance of inhibitory zinc-binding sites in proteins and the role of zinc(II) ions in cellular signalling. Regulatory functions of zinc(II) ions add a significant level of complexity to biol. control of metab. and signal transduction and embody a new paradigm for the role of transition metal ions in cell biol.
- 314Romero-Hernandez, A.; Simorowski, N.; Karakas, E.; Furukawa, H. Molecular Basis for subtype specificity and high-affinity zinc inhibition in the GluN1-GluN2A NMDA receptor amino-terminal domain. Neuron 2016, 92, 1324– 1336, DOI: 10.1016/j.neuron.2016.11.006314Molecular Basis for Subtype Specificity and High-Affinity Zinc Inhibition in the GluN1-GluN2A NMDA Receptor Amino-Terminal DomainRomero-Hernandez, Annabel; Simorowski, Noriko; Karakas, Erkan; Furukawa, HiroNeuron (2016), 92 (6), 1324-1336CODEN: NERNET; ISSN:0896-6273. (Cell Press)Zinc is vastly present in the mammalian brain and controls functions of various cell surface receptors to regulate neurotransmission. A distinctive characteristic of N-methyl-D-aspartate (NMDA) receptors contg. a GluN2A subunit is that their ion channel activity is allosterically inhibited by a nano-molar concn. of zinc that binds to an extracellular domain called an amino-terminal domain (ATD). Despite physiol. importance, the mol. mechanism underlying the high-affinity zinc inhibition has been incomplete because of the lack of a GluN2A ATD structure. Here we show the first crystal structures of the heterodimeric GluN1-GluN2A ATD, which provide the complete map of the high-affinity zinc-binding site and reveal distinctive features from the ATD of the GluN1-GluN2B subtype. Perturbation of hydrogen bond networks at the hinge of the GluN2A bi-lobe structure affects both zinc inhibition and open probability, supporting the general model in which the bi-lobe motion in ATD regulates the channel activity in NMDA receptors.
- 315Milstein, C. Inhibition of phosphoglucomutase by trace metals. Biochem. J. 1961, 79, 591– 596, DOI: 10.1042/bj0790591315Inhibition of phosphoglucomutase by trace metalsMilstein, C.Biochemical Journal (1961), 79 (), 591-6CODEN: BIJOAK; ISSN:0264-6021.A quant. study of the inhibition of phosphoglucomutase provides addnl. evidence that the necessity for a chelating agent is the result of the presence of minute traces of inhibitory metals. Zn is an inhibitor of phosphoglucomutase, competing with Mg ions, with dissocn. const. KzD 3.9 × 10-14M. Cu also competes with Mg ions, but the inhibition is not purely competitive. The inhibition by Cu++ ions can be explained by competition with Mg ions (KCu 2.3 × 10-17M), together with noncompetitive combination at a 2nd site (K'Cu 2.5 × 10-16M). Three to 4 SH groups are present in phosphoglucomutase. Although some inhibition is observed with high concns. of several SH reagents, phosphoglucomutase does not appear to be an enzyme requiring these groups for activity. The effects are discussed in the light of the amino acid sequence in the active center previously detd.
- 316Wilson, M.; Hogstrand, C.; Maret, W. Picomolar concentrations of free zinc(II) ions regulate receptor protein-tyrosine phosphatase β activity. J. Biol. Chem. 2012, 287, 9322– 9326, DOI: 10.1074/jbc.C111.320796316Picomolar concentrations of free zinc(II) ions regulate receptor protein-tyrosine phosphatase β activityWilson, Matthew; Hogstrand, Christer; Maret, WolfgangJournal of Biological Chemistry (2012), 287 (12), 9322-9326CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)As key enzymes in the regulation of biol. phosphorylations, protein tyrosine phosphatases are central to the control of cellular signaling and metab. Zn(II) ions are known to inhibit these enzymes, but the physiol. significance of this inhibition has remained elusive. Here, employing metal buffering for strict metal control and performing a kinetic anal., the authors demonstrate that Zn(II) ions are reversible inhibitors of the cytoplasmic catalytic domain of receptor protein tyrosine phosphatase β (also known as vascular endothelial protein tyrosine phosphatase). The Ki(Zn) value was 21 pM, 6 orders of magnitude lower than Zn(II) inhibition reported previously for this enzyme. It exceeded the affinity of the most potent synthetic small mol. inhibitors targeting these enzymes. The inhibition was in the range of cellular Zn(II) ion concns., suggesting that Zn(II) regulates this enzyme, which is involved in vascular physiol. and angiogenesis. Thus, for some enzymes that are not recognized as Zn-metalloenzymes, Zn(II) binding inhibits rather than activates as in classical Zn-enzymes. Activation then requires removal of the inhibitory Zn(II).
- 317Hardyman, J. E. J.; Tyson, J.; Jackson, K. A.; Aldridge, C.; Cockell, S. J.; Wakeling, L. A.; Valentine, R. A.; Ford, D. Zinc sensing by metal-responsive transcription factor 1 (MTF1) controls metallothionein and ZnT1 expression to buffer the sensitivity of the transcriptome response to zinc. Metallomics 2016, 8, 337– 343, DOI: 10.1039/C5MT00305A317Zinc sensing by metal-responsive transcription factor 1 (MTF1) controls metallothionein and ZnT1 expression to buffer the sensitivity of the transcriptome response to zincHardyman, J. E. J.; Tyson, J.; Jackson, K. A.; Aldridge, C.; Cockell, S. J.; Wakeling, L. A.; Valentine, R. A.; Ford, D.Metallomics (2016), 8 (3), 337-343CODEN: METAJS; ISSN:1756-591X. (Royal Society of Chemistry)Only a small no. of genes are known direct targets of the zinc-responsive transcription factor MTF1; therefore, the aim of this study was to gain a more complete understanding of the MTF-1 regulated zinc-responsive component of the transcriptome. A targeted siRNA was used to deplete MTF1 expression in the human intestinal cell line Caco-2. We predicted that the response to zinc of direct MTF1 target genes would be abrogated by MTF1 knockdown. Surprisingly, a greater no. of genes were regulated by zinc following MFT1 knockdown, and most genes that responded to zinc under both control and MTF1-depleted conditions had an augmented response in the latter condition. Exceptions were the zinc effluxer ZnT1 and a suite of metallothionein genes, suggesting that responses of other genes to zinc are usually buffered by increases in these proteins. We propose that MTF1 heads a hierarchy of zinc sensors, and through controlling the expression of a raft of metallothioneins and other key proteins involved in controlling intracellular zinc levels (e.g. ZnT1) alters zinc buffering capacity and total cellular zinc content. We tested and validated this model by overexpressing metallothionein and observing the predicted curtailment in response of the zinc-repressed SLC30A5 (ZnT5) promoter. The model provides the framework for an integrated understanding of cellular zinc homeostasis. Because MTs can bind metals other than zinc, this framework links with overall cellular metal homeostasis.
- 318Krężel, A.; Hao, Q.; Maret, W. The zinc/thiolate redox biochemistry of metallothionein and the control of zinc ion fluctuations in cell signalling. Arch. Biochem. Biophys. 2007, 463, 188– 200, DOI: 10.1016/j.abb.2007.02.017318The zinc/thiolate redox biochemistry of metallothionein and the control of zinc ion fluctuations in cell signalingKrezel, Artur; Hao, Qiang; Maret, WolfgangArchives of Biochemistry and Biophysics (2007), 463 (2), 188-200CODEN: ABBIA4; ISSN:0003-9861. (Elsevier)A review. Free Zn2+ ions are potent effectors of proteins. Their tightly controlled fluctuations ("Zn2+ signals") in the picomolar range of concns. modulate cellular signaling pathways. S (cysteine) donors generate redox-active coordination environments in proteins for the redox-inert Zn2+ ion and make it possible for redox signals to induce Zn2+ signals. The amplitudes of Zn2+ signals are detd. by the cellular Zn2+ buffering capacity, which itself is redox-sensitive. In part by interfering with Zn2+ and redox buffering, reactive species, drugs, toxins, and metal ions can elicit Zn2+ signals that initiate physiol. and pathobiochem. changes or lead to cellular injury when free Zn2+ ions are sustained at higher concns. These interactions establish redox-inert Zn2+ as an important factor in redox signaling. At the center of Zn2+/redox signaling are the Zn/thiolate clusters of metallothionein. They can transduce Zn2+ and redox signals and thereby attenuate or amplify these signals.
- 319Han, Y.; Sanford, L.; Simpson, D. M.; Dowell, R. D.; Palmer, A. E. Remodeling of Zn2+ homeostasis upon differentiation of mammary epithelial cells. Metallomics 2020, 12, 346– 362, DOI: 10.1039/C9MT00301K319Remodeling of Zn2+ homeostasis upon differentiation of mammary epithelial cellsHan, Yu; Sanford, Lynn; Simpson, David M.; Dowell, Robin D.; Palmer, Amy E.Metallomics (2020), 12 (3), 346-362CODEN: METAJS; ISSN:1756-591X. (Royal Society of Chemistry)Using mouse mammary epithelial cells as a model system, we investigated the remodeling of zinc homeostasis during differentiation induced by treatment with the lactogenic hormones cortisol and prolactin. RNA-Seq at different stages of differentiation revealed changes in global gene expression, including genes encoding zinc-dependent proteins and regulators of zinc homeostasis. Increases in mRNA levels of three zinc homeostasis genes, Slc39a14 (ZIP14) and metallothioneins (MTs) I and II were induced by cortisol but not by prolactin. The cortisol-induced increase was partially mediated by the nuclear glucocorticoid receptor signaling pathway. An increase in the cytosolic labile Zn2+ pool was also detected in lactating mammary cells, consistent with upregulation of MTs. We found that the zinc transporter ZIP14 was important for the expression of a major milk protein, whey acid protein (WAP), as knockdown of ZIP14 dramatically decreased WAP mRNA levels. In summary, our study demonstrated remodeling of zinc homeostasis upon differentiation of mammary epithelial cells resulting in changes in cytosolic Zn2+ and differential expression of zinc homeostasis genes, and these changes are important for establishing the lactation phenotype.
- 320Sanford, L.; Carpenter, M. C.; Palmer, A. E. Intracellular Zn2+ transients modulate global gene expression in dissociated rat hippocampal neurons. Sci. Rep. 2019, 9, 9411, DOI: 10.1038/s41598-019-45844-2320Intracellular Zn(2+) transients modulate global gene expression in dissociated rat hippocampal neuronsSanford Lynn; Carpenter Margaret C; Palmer Amy EScientific reports (2019), 9 (1), 9411 ISSN:.Zinc (Zn(2+)) is an integral component of many proteins and has been shown to act in a regulatory capacity in different mammalian systems, including as a neurotransmitter in neurons throughout the brain. While Zn(2+) plays an important role in modulating neuronal potentiation and synaptic plasticity, little is known about the signaling mechanisms of this regulation. In dissociated rat hippocampal neuron cultures, we used fluorescent Zn(2+) sensors to rigorously define resting Zn(2+) levels and stimulation-dependent intracellular Zn(2+) dynamics, and we performed RNA-Seq to characterize Zn(2+)-dependent transcriptional effects upon stimulation. We found that relatively small changes in cytosolic Zn(2+) during stimulation altered expression levels of 931 genes, and these Zn(2+) dynamics induced transcription of many genes implicated in neurite expansion and synaptic growth. Additionally, while we were unable to verify the presence of synaptic Zn(2+) in these cultures, we did detect the synaptic vesicle Zn(2+) transporter ZnT3 and found it to be substantially upregulated by cytosolic Zn(2+) increases. These results provide the first global sequencing-based examination of Zn(2+)-dependent changes in transcription and identify genes that may mediate Zn(2+)-dependent processes and functions.
- 321Zhang, C.; Maslar, D.; Minckley, T. F.; LeJeune, K. D.; Qin, Y. Spontaneous, synchronous zinc spikes oscillate with neural excitabiliity and calcium spikes in primary hippocampal neuron culture. J. Neurochem. 2021, 157, 1838– 1849, DOI: 10.1111/jnc.15334321Spontaneous, synchronous zinc spikes oscillate with neural excitability and calcium spikes in primary hippocampal neuron cultureZhang, Chen; Maslar, Drew; Minckley, Taylor F.; LeJeune, Kate D.; Qin, YanJournal of Neurochemistry (2021), 157 (6), 1838-1849CODEN: JONRA9; ISSN:0022-3042. (Wiley-Blackwell)Zinc has been suggested to act as an intracellular signaling mol. due to its regulatory effects on numerous protein targets including enzymes, transcription factors, ion channels, neurotrophic factors, and postsynaptic scaffolding proteins. However, intracellular zinc concn. is tightly maintained at steady levels under natural physiol. conditions. In this work, we made the novel observation that the developing neurons generated spontaneous and synchronous zinc spikes in primary hippocampal cultures using a fluorescent zinc sensor, FluoZin-3. Blocking of glutamate receptor-dependent calcium influx depleted the zinc spikes, suggesting that these zinc spikes were driven by the glutamate-mediated spontaneous neural excitability and calcium spikes that have been characterized in early developing neurons. Simultaneous imaging of calcium or pH together with zinc, we uncovered that a downward pH spike was evoked with each zinc spike and this transient cellular acidification occurred downstream of calcium spikes but upstream of zinc spikes. Our results suggest that spontaneous, synchronous zinc spikes were generated through calcium influx-induced cellular acidification, which liberates zinc from intracellular zinc binding ligands. Given that changes in zinc concn. can modulate activities of proteins essential for synapse maturation and neuronal differentiation, these zinc spikes might act as important signaling roles in neuronal development.
- 322Blockhuys, S.; Celauro, E.; Hildesjö, C.; Feizi, A.; Stål, O.; Fierro-Gonzalez, J. C.; Wittung-Stafshede, P. Defining the human copper proteome and analysis of its expression variation in cancers. Metallomics 2017, 9, 112– 123, DOI: 10.1039/C6MT00202A322Defining the human copper proteome and analysis of its expression variation in cancersBlockhuys, S.; Celauro, E.; Hildesjoe, C.; Feizi, A.; Staal, O.; Fierro-Gonzalez, J. C.; Wittung-Stafshede, P.Metallomics (2017), 9 (2), 112-123CODEN: METAJS; ISSN:1756-591X. (Royal Society of Chemistry)Copper (Cu) is essential for living organisms, and acts as a cofactor in many metabolic enzymes. To avoid the toxicity of free Cu, organisms have specific transport systems that 'chaperone' the metal to targets. Cancer progression is assocd. with increased cellular Cu concns., whereby proliferative immortality, angiogenesis and metastasis are cancer hallmarks with defined requirements for Cu. The aim of this study is to gather all known Cu-binding proteins and reveal their putative involvement in cancers using the available database resources of RNA transcript levels. Using the Uniprot.org database along with manual curation, we identified a total of 54 Cu-binding proteins (named the human Cu proteome). Next, we retrieved RNA expression levels in cancer vs. normal tissues from the TCGA database for the human Cu proteome in 18 cancer types, and noted an intricate pattern of up- and downregulation of the genes in different cancers. Hierarchical clustering in combination with bioinformatics and functional genomics analyses allowed for the prediction of cancer-related Cu-binding proteins; these were specifically inspected for the breast cancer data. Finally, for the Cu chaperone ATOX1, which is the only Cu-binding protein proposed to have transcription factor activities, we validated its predicted over-expression in patient breast cancer tissue at the protein level. This collection of Cu-binding proteins, with RNA expression patterns in different cancers, will serve as an excellent resource for mechanistic-mol. studies of Cu-dependent processes in cancer.
- 323Hartmann, H. J.; Weser, U. Copper-thionein from fetal bovine liver. Biochim. Biophys. Acta, Protein Struct. 1977, 491, 211– 222, DOI: 10.1016/0005-2795(77)90057-5323Copper-thionein from fetal bovine liverHartmann, Hans Juergen; Weser, UlrichBiochimica et Biophysica Acta, Protein Structure (1977), 491 (1), 211-22CODEN: BBPTBH; ISSN:0005-2795.An 8 Cu-2 Zn thionein, having a mol. wt. of 11,500, was isolated from bovine fetal liver. Sixteen percent of the total Cu present in the whole liver was recovered in this protein. This Cu-thionein was similar to the polymeric neonatal type mitochondrocuprein. A comparison of different Cu-thioneins contg. variable amts. of Cu was possible when εCu from 280 nm to longer wavelengths was detd. With respect to the UV properties, there were no detectable differences between Cu-thioneins prepd. either in vivo or in vitro and the fetal Cu-thionein. Furthermore, the positions of the Cotton effects were rather similar. X-ray photoelectron spectrometric studies revealed a Cu(2p3/2) binding energy value of 932.9 eV. Unlike the S(2p1/2,3/2) value near 162 eV using Cu-thioneins from chicken liver or yeast, the higher S(2p1/2,3/2) binding energy of 163.0 eV employing fetal Cu-thionein was attributed to partial oxidn. of the protein moiety and (or) a particular chem. environment. The 2nd S(2p1/2,3/2) peak was assigned to the Cu-catalyzed oxidn. of S via OH radicals to yield RSO3-. In the x-ray photoelectron spectrum of the apoprotein, 1 homogeneous S(2p1/2,3/2) band at 163.7 eV was attributable to RSSR.
- 324Luchinat, E.; Barbieri, L.; Banci, L. A molecular chaperone activity of CCS restores the maturation of SOD1 fALS mutants. Sci. Rep. 2017, 7, 17433, DOI: 10.1038/s41598-017-17815-y324A molecular chaperone activity of CCS restores the maturation of SOD1 fALS mutantsLuchinat Enrico; Barbieri Letizia; Banci Lucia; Luchinat Enrico; Barbieri Letizia; Banci LuciaScientific reports (2017), 7 (1), 17433 ISSN:.Superoxide dismutase 1 (SOD1) is an important metalloprotein for cellular oxidative stress defence, that is mutated in familiar variants of Amyotrophic Lateral Sclerosis (fALS). Some mutations destabilize the apo protein, leading to the formation of misfolded, toxic species. The Copper Chaperone for SOD1 (CCS) transiently interacts with SOD1 and promotes its correct maturation by transferring copper and catalyzing disulfide bond formation. By in vitro and in-cell NMR, we investigated the role of the SOD-like domain of CCS (CCS-D2). We showed that CCS-D2 forms a stable complex with zinc-bound SOD1 in human cells, that has a twofold stabilizing effect: it both prevents the accumulation of unstructured mutant SOD1 and promotes zinc binding. We further showed that CCS-D2 interacts with apo-SOD1 in vitro, suggesting that in cells CCS stabilizes mutant apo-SOD1 prior to zinc binding. Such molecular chaperone function of CCS-D2 is novel and its implications in SOD-linked fALS deserve further investigation.
- 325Skopp, A.; Boyd, S. D.; Ullrich, M. S.; Liu, L.; Winkler, D. D. Copper-zinc superoxide dismutase (Sod1) activation terminates interaction between its copper chaperone (Ccs) and the cytosolic metal-binding domain of the copper importer Ctr1. BioMetals 2019, 32, 695– 705, DOI: 10.1007/s10534-019-00206-3325Copper-zinc superoxide dismutase (Sod1) activation terminates interaction between its copper chaperone (Ccs) and the cytosolic metal-binding domain of the copper importer Ctr1Skopp, Amelie; Boyd, Stefanie D.; Ullrich, Morgan S.; Liu, Li; Winkler, Duane D.BioMetals (2019), 32 (4), 695-705CODEN: BOMEEH; ISSN:0966-0844. (Springer)Copper-zinc superoxide dismutase (Sod1) is a crit. antioxidant enzyme that rids the cell of reactive oxygen through the redox cycling of a catalytic copper ion provided by its copper chaperone (Ccs). Ccs must first acquire this copper ion, directly or indirectly, from the influx copper transporter, Ctr1. The three proteins of this transport pathway ensure careful trafficking of copper ions from cell entry to target delivery, but the intricacies remain undefined. Biochem. examn. of each step in the pathway detd. that the activation of the target (Sod1) regulates the Ccs·Ctr1 interaction. Ccs stably interacts with the cytosolic C-terminal tail of Ctr1 (Ctr1c) in a copper-dependent manner. This interaction becomes tripartite upon the addn. of an engineered immature form of Sod1 creating a stable Cu(I)-Ctr1c·Ccs·Sod1 heterotrimer in soln. This heterotrimer can also be made by the addn. of a preformed Sod1·Ccs heterodimer to Cu(I)-Ctr1c, suggestive of multiple routes to the same destination. Only complete Sod1 activation (i.e. active site copper delivery and intra-subunit disulfide bond formation) breaks the Sod1·Ccs·Ctr1c complex. The results provide a new and extended view of the Sod1 activation pathway(s) originating at cellular copper import.
- 326Maryon, E. B.; Molloy, S. A.; Kaplan, J. H. Cellular glutathione plays a key role in copper uptake mediated by human copper transporter 1. Am. J. Phyiol. Cell Physiol. 2013, 304, C768– 779, DOI: 10.1152/ajpcell.00417.2012There is no corresponding record for this reference.
- 327Morgan, M. T.; Nguyen, L. A. H.; Hancock, H. L.; Fahrni, C. J. Glutathione limits aquacopper(I) to sub-femtomolar concentrations through cooperative assembly of a tetranuclear cluster. J. Biol. Chem. 2017, 292, 21558– 21567, DOI: 10.1074/jbc.M117.817452327Glutathione limits aquacopper(I) to sub-femtomolar concentrations through cooperative assembly of a tetranuclear clusterMorgan, M. Thomas; Nguyen, Lily Anh H.; Hancock, Haylie L.; Fahrni, Christoph J.Journal of Biological Chemistry (2017), 292 (52), 21558-21567CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)The tripeptide glutathione (GSH) is a crucial intracellular reductant and radical scavenger, but it may also coordinate the soft Cu(I) cation and thereby yield pro-oxidant species. The GSH-Cu(I) interaction is thus a key consideration for both redox and copper homeostasis in cells. However, even after nearly four decades of investigation, the nature and stability of the GSH-Cu(I) complexes formed under biol. relevant conditions remain controversial. Here, we revealed the unexpected predominance of a tetranuclear [Cu4(GS)6] cluster that is sufficiently stable to limit the effective free aquacopper(I) concn. to the sub-femtomolar regime. Combined spectrophotometric-potentiometric titrns. at biol. realistic GSH/Cu(I) ratios, enabled by our recently developed Cu(I) affinity stds. and corroborated by low-temp. phosphorescence studies, established cooperative assembly of [Cu4(GS)6] as the dominant species over a wide pH range, from 5.5 to 7.5. Our robust model for the glutathione-Cu(I) equil. system sets a firm upper limit on the thermodn. availability of intracellular copper that is 3 orders of magnitude lower than previously estd. Taking into account their ability to catalyze the prodn. of deleterious superoxide, the formation of Cu(I)-glutathione complexes might be avoided under normal physiol. conditions. The actual intracellular Cu(I) availability may thus be regulated a further 3 orders of magnitude below the GSH/Cu(I) affinity limit, consistent with the most recent affinity detns. of Cu(I) chaperones.
- 328Chung, C. Y.-S.; Posimo, J. M.; Lee, S.; Tsang, T.; Davis, J. M.; Brady, D. C.; Chang, C. J. Activity-based ratiometric FRET probe reveals oncogene-driven changes in labile copper pools induced by altered glutathione metabolism. Proc. Natl. Acad. Sci. U. S. A. 2019, 116, 18285– 18294, DOI: 10.1073/pnas.1904610116328Activity-based ratiometric FRET probe reveals oncogene-driven changes in labile copper pools induced by altered glutathione metabolismChung Clive Yik-Sham; Lee Sumin; Chang Christopher J; Posimo Jessica M; Brady Donita C; Tsang Tiffany; Davis Julianne M; Brady Donita C; Chang Christopher J; Chang Christopher JProceedings of the National Academy of Sciences of the United States of America (2019), 116 (37), 18285-18294 ISSN:.Copper is essential for life, and beyond its well-established ability to serve as a tightly bound, redox-active active site cofactor for enzyme function, emerging data suggest that cellular copper also exists in labile pools, defined as loosely bound to low-molecular-weight ligands, which can regulate diverse transition metal signaling processes spanning neural communication and olfaction, lipolysis, rest-activity cycles, and kinase pathways critical for oncogenic signaling. To help decipher this growing biology, we report a first-generation ratiometric fluorescence resonance energy transfer (FRET) copper probe, FCP-1, for activity-based sensing of labile Cu(I) pools in live cells. FCP-1 links fluorescein and rhodamine dyes through a Tris[(2-pyridyl)methyl]amine bridge. Bioinspired Cu(I)-induced oxidative cleavage decreases FRET between fluorescein donor and rhodamine acceptor. FCP-1 responds to Cu(I) with high metal selectivity and oxidation-state specificity and facilitates ratiometric measurements that minimize potential interferences arising from variations in sample thickness, dye concentration, and light intensity. FCP-1 enables imaging of dynamic changes in labile Cu(I) pools in live cells in response to copper supplementation/depletion, differential expression of the copper importer CTR1, and redox stress induced by manipulating intracellular glutathione levels and reduced/oxidized glutathione (GSH/GSSG) ratios. FCP-1 imaging reveals a labile Cu(I) deficiency induced by oncogene-driven cellular transformation that promotes fluctuations in glutathione metabolism, where lower GSH/GSSG ratios decrease labile Cu(I) availability without affecting total copper levels. By connecting copper dysregulation and glutathione stress in cancer, this work provides a valuable starting point to study broader cross-talk between metal and redox pathways in health and disease with activity-based probes.
- 329Rae, T. D.; Schmidt, P. J.; Pufahl, R. A.; Culotta, V. C.; O’Halloran, T. V. Undetectable free copper: The requirement of copper chaperone for superoxide dismutase. Science 1999, 284, 805– 808, DOI: 10.1126/science.284.5415.805329Undetectable intracellular free copper: the requirement of a copper chaperone for superoxide dismutaseRae, T. D.; Schmidt, P. J.; Pufahl, R. A.; Culotta, V. C.; O'Halloran, T. V.Science (Washington, D. C.) (1999), 284 (5415), 805-808CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)The copper chaperone for the superoxide dismutase (CCS) gene is necessary for expression of an active, copper-bound form of superoxide dismutase (SOD1) in vivo in spite of the high affinity of SOD1 for copper (dissocn. const. = 6 fM) and the high intracellular concns. of both SOD1 (10 μM in yeast) and copper (70 μM in yeast). In vitro studies demonstrated that purified Cu(I)-yCCS protein is sufficient for direct copper activation of apo-ySOD1 but is necessary only when the concn. of free copper ions ([Cu]free) is strictly limited. Moreover, the physiol. requirement for yCCS in vivo was readily bypassed by elevated copper concns. and abrogation of intracellular copper-scavenging systems such as the metallothioneins. This metallochaperone protein activates the target enzyme through direct insertion of the copper cofactor and apparently functions to protect the metal ion from binding to intracellular copper scavengers. These results indicate that intracellular [Cu]free is limited to less than one free copper ion per cell and suggest that a pool of free copper ions is not used in physiol. activation of metalloenzymes.
- 330Bethin, K. E.; Cimato, T. R.; Ettinger, M. J. Copper binding to mouse liver S-adenosylhomocysteine hydrolase and the effects of copper on its levels. J. Biol. Chem. 1995, 270, 20703– 20711, DOI: 10.1074/jbc.270.35.20703330Copper binding to mouse liver S-adenosylhomocysteine hydrolase and the effects of copper on its levelsBethin, Kathleen E.; Cimato, Thomas R.; Ettinger, Murray J.Journal of Biological Chemistry (1995), 270 (35), 20703-11CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)The dissocn. const. and stoichiometry of copper binding to mouse liver S-adenosylhomocysteine hydrolase (SAHH) was detd. as part of characterizing the possible roles of SAHH in copper metab. Copper (64Cu(II)) binding was measured by an ultrafiltration method in the presence of EDTA as a competing ligand. The KD was 3.9×10-16 M, and the stoichiometry was one g atom of copper per 48-kDa subunit. Western blots indicated that the liver contains ≈12 times more SAHH than the kidney, which in turn contains ≃5 times more SAHH than the brain. The high concn. and copper affinity of SAHH in the liver may contribute to the liver's ability to preferentially accumulate copper, and the low levels of SAHH in the brain may contribute to the sensitivity of the brain to copper deficiency. The effects of genetic defects of copper metab. and copper deficiency on SAHH were also detd. Normal SAHH levels were detected in brindled mouse liver, kidney, and brain. However, SAHH from brindled mouse liver eluted abnormally from Ph Superose columns implying an effect of the brindled mouse defect on SAHH protein structure. Hepatic cytosols from the toxic milk mouse contained ≈42% the amt. of SAHH detected in controls, and hepatic levels of SAHH were also decreased by 45% in copper-deficient mice. The binding properties of SAHH and the effects of abnormal states of copper metab. on its levels are consistent with significant roles for SAHH in normal and abnormal copper metab. SAHH may have roles in regulating tissue copper levels and the distribution of intracellular copper.
- 331Chen, P.; Onana, P.; Shaw, C. F., III; Petering, D. H. Characterization of calf liver Cu,Zn-metallothionein: naturally variable Cu and Zn stoichiometries. Biochem. J. 1996, 317, 389– 394, DOI: 10.1042/bj3170389331Characterization of calf liver Cu,Zn-metallothionein: naturally variable Cu and Zn stoichiometriesChen, Pu; Onana, Patrick; Shaw, C. Frank, III; Petering, David H.Biochemical Journal (1996), 317 (2), 389-394CODEN: BIJOAK; ISSN:0264-6021. (Portland Press)Cu,Zn-metallothioneins were purified from bovine calf liver in order to examine the stoichiometry of metal binding to the protein. Cu and Zn analyses were carried out by at. absorption spectrophotometry. Consistent quant. thiolate analyses were obtained spectrophotometrically with Ellman's reagent and amperometrically with phenylmercuric acetate. These were used to define protein concn. A complementary method to assess the sum of the thiol and Cu(I) content of metallothionein involved titrn. of the reducing equiv. of the protein with ferricyanide. The reaction stoichiometry was consistent with the oxidn. of all of the SH groups to disulfides and all of the bound Cu from the Cu(I) to the Cu(II) oxidn. state. According to these methods, the total no. of Zn plus Cu ions bound to metallothionein isolated from a no. of calf livers centered on approx. 7, 10-12, or 15 g-atoms of metal per mol of protein. The reactivity of ferricyanide and 4,7-phenylsulfonyl-2,9-dimethyl-1,10-phenanthroline (BCS) with Cu,Zn-metallothioneins of various metal ratios was assessed. Zn-metallothionein reacted almost entirely in 2 slow steps with ferricyanide. As the Cu content of the protein increased, the fraction of reaction occurring in the time of mixing increased in parallel. BCS was able to remove 70-80% of metallothionein-bound Cu as Cu(I). The rest was resistant to reaction.
- 332Chang, C. J. Searching for harmony in transition-metal signaling. Nat. Chem. Biol. 2015, 11, 744– 747, DOI: 10.1038/nchembio.1913332Searching for harmony in transition-metal signalingChang, Christopher J.Nature Chemical Biology (2015), 11 (10), 744-747CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)A review. The recent emergence of signaling roles for transition metals presages a broader contribution of these elements beyond their traditional functions as metabolic cofactors. New chem. approaches to identify the sources, targets and physiologies of transition-metal signaling can help expand understanding of the periodic table in a biol. context.
- 333Leary, S. C.; Ralle, M. Advances in visualization of copper in mammalian systems using X-ray fluorescence microscopy. Curr. Opin. Chem. Biol. 2020, 55, 19– 25, DOI: 10.1016/j.cbpa.2019.12.002333Advances in visualization of copper in mammalian systems using X-ray fluorescence microscopyLeary, Scot C.; Ralle, MartinaCurrent Opinion in Chemical Biology (2020), 55 (), 19-25CODEN: COCBF4; ISSN:1367-5931. (Elsevier B.V.)Synchrotron-based X-ray fluorescence microscopy (XFM) has become an important imaging technique to investigate elemental concns. and distributions in biol. specimens. Advances in technol. now permit imaging at resolns. rivaling that of electron microscopy, and researchers can now visualize elemental concns. in subcellular organelles when using appropriate correlative methods. XFM is an esp. valuable tool to det. the distribution of endogenous trace metals that are involved in neurodegenerative diseases. Here, we discuss the latest research on the unusual copper (Cu) storage vesicles that were originally identified in mouse brains and the involvement of Cu in Alzheimer's disease. Finally, we provide an outlook of how future improvements to XFM will drive current trace element research forward.
- 334Grubman, A.; White, A. R. Copper as a key regulator of cell signalling pathways. Expert Rev. Mol. Med. 2014, 16, e11, DOI: 10.1017/erm.2014.11334Copper as a key regulator of cell signalling pathwaysGrubman, Alexandra; White, Anthony R.Expert Reviews in Molecular Medicine (2014), 16 (), e11/1-e11/27CODEN: ERMMFS; ISSN:1462-3994. (Cambridge University Press)A review. Copper is an essential element in many biol. processes. The crit. functions assocd. with copper have resulted from evolutionary harnessing of its potent redox activity. This same property also places copper in a unique role as a key modulator of cell signal transduction pathways. These pathways are the complex sequence of mol. interactions that drive all cellular mechanisms and are often assocd. with the interplay of key enzymes including kinases and phosphatases but also including intracellular changes in pools of smaller mols. A growing body of evidence is beginning to delineate the how, when and where of copper-mediated control over cell signal transduction. This has been driven by research demonstrating crit. changes to copper homeostasis in many disorders including cancer and neurodegeneration and therapeutic potential through control of disease assocd. cell signalling changes by modulation of copper-protein interactions. This timely review brings together for the first time the diverse actions of copper as a key regulator of cell signalling pathways and discusses the potential strategies for controlling disease-assocd. signalling processes using copper modulators. It is hoped that this review will provide a valuable insight into copper as a key signal regulator and stimulate further research to promote our understanding of copper in disease and therapy.
- 335Kardos, J.; Heja, L.; Simon, A.; Jablonkai, I.; Kovacs, R.; Jemnitz, K. Copper signalling: causes and consequences. Cell Commun. Signaling 2018, 16, 71, DOI: 10.1186/s12964-018-0292-4335Copper signalling: causes and consequencesKardos, Julianna; Heja, Laszlo; Simon, Agnes; Jablonkai, Istvan; Kovacs, Richard; Jemnitz, KatalinCell Communication and Signaling (2018), 16 (), 71CODEN: CCSEC6; ISSN:1478-811X. (BioMed Central Ltd.)Copper-contg. enzymes perform fundamental functions by activating dioxygen (O2) and therefore allowing chem. energy-transfer for aerobic metab. The copper-dependence of O2 transport, metab. and prodn. of signalling mols. are supported by mol. systems that regulate and preserve tightly-bound static and weakly-bound dynamic cellular copper pools. Disruption of the reducing intracellular environment, characterized by glutathione shortage and ambient Cu(II) abundance drives oxidative stress and interferes with the bidirectional, copper-dependent communication between neurons and astrocytes, eventually leading to various brain disease forms. A deeper understanding of of the regulatory effects of copper on neuro-glia coupling via polyamine metab. may reveal novel copper signalling functions and new directions for therapeutic intervention in brain disorders assocd. with aberrant copper metab.
- 336Ackerman, C. M.; Chang, C. J. Copper signaling in the brain and beyond. J. Biol. Chem. 2018, 293, 4628– 4635, DOI: 10.1074/jbc.R117.000176336Copper signaling in the brain and beyondAckerman, Cheri M.; Chang, Christopher J.Journal of Biological Chemistry (2018), 293 (13), 4628-4635CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)A review. Transition metals have been recognized and studied primarily in the context of their essential roles as structural and metabolic cofactors for biomols. that compose living systems. More recently, an emerging paradigm of transition-metal signaling, where dynamic changes in transitional metal pools can modulate protein function, cell fate, and organism health and disease, has broadened our view of the potential contributions of these essential nutrients in biol. Using Cu as a canonical example of transition-metal signaling, we highlight key expts. where direct measurement and/or visualization of dynamic Cu pools, in combination with biochem., physiol., and behavioral studies, have deciphered sources, targets, and physiol. effects of Cu signals.
- 337Krishnamoorthy, L.; Cotruvo, J. A., Jr; Chan, J.; Kaluarachchi, H.; Muchenditsi, A.; Pendyala, V. S.; Jia, S.; Aron, A. T.; Ackerman, C. M.; Vander Wal, M. N.; Guan, T.; Smaga, L. P.; Farhi, S. L.; New, E. J.; Lutsenko, S.; Chang, C. J. Copper regulates cyclic AMP-dependent lipolysis. Nat. Chem. Biol. 2016, 12, 586– 592, DOI: 10.1038/nchembio.2098337Copper regulates cyclic-AMP-dependent lipolysisKrishnamoorthy, Lakshmi; Cotruvo, Joseph A., Jr.; Chan, Jefferson; Kaluarachchi, Harini; Muchenditsi, Abigael; Pendyala, Venkata S.; Jia, Shang; Aron, Allegra T.; Ackerman, Cheri M.; Vander Wal, Mark N.; Guan, Timothy; Smaga, Lukas P.; Farhi, Samouil L.; New, Elizabeth J.; Lutsenko, Svetlana; Chang, Christopher J.Nature Chemical Biology (2016), 12 (8), 586-592CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)Cell signaling relies extensively on dynamic pools of redox-inactive metal ions such as sodium, potassium, calcium and zinc, but their redox-active transition metal counterparts such as copper and iron have been studied primarily as static enzyme cofactors. Here we report that copper is an endogenous regulator of lipolysis, the breakdown of fat, which is an essential process in maintaining body wt. and energy stores. Using a mouse model of genetic copper misregulation, in combination with pharmacol. alterations in copper status and imaging studies in a 3T3-L1 white adipocyte model, we found that copper regulates lipolysis at the level of the second messenger, cAMP, by altering the activity of the cAMP-degrading phosphodiesterase PDE3B. Biochem. studies of the copper-PDE3B interaction establish copper-dependent inhibition of enzyme activity and identify a key conserved cysteine residue in a PDE3-specific loop that is essential for the obsd. copper-dependent lipolytic phenotype.
- 338Angeletti, B.; Waldron, K. J.; Freeman, K. B.; Bawagan, H.; Hussain, I.; Miller, C. C.; Lau, K. F.; Tennant, M. E.; Dennison, C.; Robinson, N. J.; Dingwall, C. BACE1 cytoplasmic domain interacts with the copper chaperone for superoxide dismutase-1 and binds copper. J. Biol. Chem. 2005, 280, 17930– 17937, DOI: 10.1074/jbc.M412034200338BACE1 Cytoplasmic Domain Interacts with the Copper Chaperone for Superoxide Dismutase-1 and Binds CopperAngeletti, Barbara; Waldron, Kevin J.; Freeman, Katie B.; Bawagan, Hinayana; Hussain, Ishrut; Miller, Christopher C. J.; Lau, Kwok-Fai; Tennant, Maria E.; Dennison, Christopher; Robinson, Nigel J.; Dingwall, ColinJournal of Biological Chemistry (2005), 280 (18), 17930-17937CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)The amyloidogenic pathway leading to the prodn. and deposition of Aβ peptides, major constituents of Alzheimer's disease senile plaques, is linked to neuronal metal homeostasis. The amyloid precursor protein binds copper and zinc in its extracellular domain, and the Aβ peptides also bind copper, zinc, and iron. The first step in the generation of Aβ is cleavage of amyloid precursor protein by the aspartic protease BACE1. Here we show that BACE1 interacts with CCS (the copper chaperone for superoxide dismutase-1 (SOD1)) through domain I and the proteins co-immunoppt. from rat brain exts. We have also been able to visualize the co-transport of membranous BACE1 and sol. CCS through axons. BACE1 expression reduces the activity of SOD1 in cells consistent with direct competition for available CCS as overexpression of CCS restores SOD1 activity. Finally, we demonstrate that the twenty-four residue C-terminal domain of BACE1 binds a single Cu(I) atom with high affinity through cysteine residues.
- 339Udom, A. O.; Brady, F. O. Reactivation in vitro of zinc-requiring apo-enzymes by rat liver zinc-thionein. Biochem. J. 1980, 187, 329– 335, DOI: 10.1042/bj1870329339Reactivation in vitro of zinc-requiring apo-enzymes by rat liver zinc-thioneinUdom, Albert O.; Brady, Frank O.Biochemical Journal (1980), 187 (2), 329-35CODEN: BIJOAK; ISSN:0264-6021.Liver Zn-thionein was as good as, or better than ZnSO4, Zn(OAc)2, or Zn(NO3)2 in donating Zn to apoenzymes of alc. dehydrogenase (I), aldolase, thermolysin, alk. phosphatase (II), and carbonic anhydrase (III). Apo-I could not be reactivated by Zn salts or Zn-thionein, whereas incubation of the other apoenzymes with near satg. amts. of Zn as ZnSO4, Zn(OAc)2, Zn(NO3)2, or Zn-thionein caused reactivation. Apo-aldolase was 100% reactivated by Zn-thionein in 30 min, whereas reactivation by ZnSO4 and Zn(OAc)2 was complete and instantaneous. Zn-thionein was better than Zn(NO3)2 in completely reactivating apo-thermolysin. Apo-II was 43 and 18% reactivated by Zn(OAc)2 and Zn-thionein, resp. Zn-thionein was better than ZnSO4, Zn(OAc)2, or Zn(NO3)2 in reactivating apo-III, giving max. reactivation of 54%. Zn was apparently being transferred from Zn-thionein to apo-III, as indicated by the effects of 2,6-pyridinedicarboxylic acid and 1,10-phenanthroline on the reactivation of apo-III. Thus, Zn-thionein can apparently function in Zn homeostasis as a Zn reservoir releasing Zn to Zn-requiring metalloenzymes according to need.
- 340Li, T. Y.; Kraker, A. J.; Shaw, C. F., 3rd; Petering, D. H. Ligand substitution reactions of metallothioneins with EDTA and apo-carbonic anhydrase. Proc. Natl. Acad. Sci. U. S. A. 1980, 77, 6334– 6338, DOI: 10.1073/pnas.77.11.6334340Ligand substitution reactions of metallothioneins with EDTA and apo-carbonic anhydraseLi, Ta-Yuen; Kraker, Alan J.; Shaw, C. Frank, III; Petering, David H.Proceedings of the National Academy of Sciences of the United States of America (1980), 77 (11), 6334-8CODEN: PNASA6; ISSN:0027-8424.The reactions of Zn-, Cd-, and Zn,Cd-thioneins with EDTA and apo-carbonic anhydrase were studied. The ligand substitution reaction of Zn with EDTA is multiphasic having both associative and dissociative components in the rate expression. The Cd sites are ∼2 orders of magnitude less reactive. In contrast, apo-carbonic anhydrase abstrs. Zn from Zn-thionein and Zn,Cd-thionein in 2nd-order processes that are 2-3 orders of magnitude more rapid than those involving EDTA and approach the rate for unligated Zn2+ with the apo-protein. In comparison with other Zn proteins, Zn-thionein contains unusually reactive metal sites, suggesting that this protein may be a physiol. Zn transfer protein, able to donate Zn to Zn-requiring apo macromols.
- 341Brady, F. O. The physiological function of metallothionein. Trends Biochem. Sci. 1982, 7, 143– 145, DOI: 10.1016/0968-0004(82)90206-7341The physiological function of metallothioneinBrady, Frank O.Trends in Biochemical Sciences (1982), 7 (4), 143-5CODEN: TBSCDB; ISSN:0968-0004.A review with 29 refs. on the physiol. function of metallothionein, the nature of its Zn and Cu binding, and the regulation of its gene.
- 342Hathout, Y.; Reynolds, K. J.; Szilagyi, Z.; Fenselau, C. Metallothionein dimers studied by nano-spray mass spectrometry. J. Inorg. Biochem. 2002, 88, 119– 122, DOI: 10.1016/S0162-0134(01)00375-0342Metallothionein dimers studied by nano-spray mass spectrometryHathout, Yetrib; Reynolds, Kristy J.; Szilagyi, Zoltan; Fenselau, CatherineJournal of Inorganic Biochemistry (2002), 88 (2), 119-122CODEN: JIBIDJ; ISSN:0162-0134. (Elsevier Science Inc.)Both transient and stable dimers of metallothionein have been characterized, based on earlier studies using NMR, CD and size-exclusion chromatog. Here addnl. characterization is provided by nanospray mass spectrometry. Rapid redistribution of metal ions between monomeric Cd7- and Zn7-metallothionein 2a is monitored by nanospray. An expt. in which theses two forms of the monomeric protein are sepd. by a dialysis membrane, which will pass metal ions but not proteins, confirms that a transient dimer must form for metal ions to be redistributed. On the other hand, size-exclusion chromatog. of reconstituted Zn7- or Cd7-metallothionein revealed the presence of monomeric and dimeric species. These dimers do not equilibrate readily to form monomers and they are shown to be covalent.
- 343Andersson, I.; Maret, W.; Zeppezauer, M.; Brown, R. D., III; Koenig, S. H. Metal ion substitution at the catalytic site of horse liver alcohol dehydrogenase: Results from solvent magnetic relaxation studies. 2. Binding of manganese(II) and competition with zinc(II) and cadmium(II) ions. Biochemistry 1981, 20, 3433– 3438, DOI: 10.1021/bi00515a020343Metal ion substitution at the catalytic site of horse-liver alcohol dehydrogenase: results from solvent magnetic relaxation studies. 2. Binding of manganese(2+) and competition with zinc(2+) and cadmium(2+) ionsAndersson, Inger; Maret, Wolfgang; Zeppezauer, Michael; Brown, Rodney D., III; Koenig, Seymour H.Biochemistry (1981), 20 (12), 3433-8CODEN: BICHAW; ISSN:0006-2960.The interaction of Mn2+ with native horse liver alc. dehydrogenase demetalized specifically at the catalytic sites has been investigated by studying the magnetic field dependence and time dependence of the magnetic spin-lattice relaxation rate of solvent water protons. No detectable binding of Mn2+ to the catalytic sites was found in times on the order of hs; however, it was found that these ions bound to the enzyme at 2 previously unreported types of sites: one characterized by a low dissocn. const. (0.01 mM at pH 7.7, 5°), low relaxivity, and a stoichiometry of 1/2 catalytic sites, and a 2nd, with a high dissocn. const. (1.5 mM at pH 7.7, 5°) and high relaxivity. The stoichiometry of the 2nd type of site could not be detd. because of the relatively weak binding of Mn2+ to these sites. Both Zn2+ and Cd2+ bound to the newly found tight-binding sites, displacing Mn2+ and thereby altering the relaxation rates of solvent protons. By monitoring the return to equil. of these altered rates, it was found that Zn2+ entered the catalytic sites from the new tight-binding sites with an on-rate of ∼0.1 M-1s-1. It was not clear whether binding to these new sites was an obligatory intermediate for reintroduction of Zn2+ into the catalytic sites, but a small excess of Zn2+ beyond 1/monomer caused the protein to ppt. Cd2+ by contrast, entered the catalytic sites at least 1 order of magnitude more rapidly than did Zn2+, a rate too rapid to observe by the techniques employed. However, once the catalytic sites were filled, Cd2+ displaced Mn2+ at the new sites as did Zn2+.
- 344Mason, A. Z.; Perico, N.; Moeller, R.; Thrippleton, K.; Potter, T.; Lloyd, D. Metal donation and apo-metalloenzyme activation by stable isotopically labelled metallothionein. Mar. Environ. Res. 2004, 58, 371– 375, DOI: 10.1016/j.marenvres.2004.03.082344Metal donation and apo-metalloenzyme activation by stable isotopically labeled metallothioneinMason, Andrew. Z.; Perico, Natalie; Moeller, Rhonda; Thrippleton, Kelly; Potter, Tiffany; Lloyd, DouglasMarine Environmental Research (2004), 58 (2-5), 371-375CODEN: MERSDW; ISSN:0141-1136. (Elsevier Science B.V.)Coupled HPLC-ICP-MS has been used to quant. study the effects of GSSG and GSH on the ability of metallothionein (MTII) to donate essential and non-essential metals to apo-carbonic anhydrase. Stable isotopically labeled 67Zn3Cd4 in gills and liver MTII was used to enable Zn donated from MTII to be differentiated from extraneous sources of Zn. Transfer of both 67Zn and Cd from MTII to apo-carbonic anhydrase was noted in the absence of either GSSG or GSH. GSSG increased the initial transfer of both Zn and Cd. Thereafter, a gradual increase in the 67Zn content at the expense of Cd was noted over 24-h indicating continued interaction and exchange between MTII and the enzyme commensurate with the relative preferences shown by the proteins for these two metals. Although GSH also increased transfer of 67Zn from MT it reduced the simultaneous transfer of Cd to the enzyme thereby conferring protection against Cd induced activation.
- 345Roesijadi, G. Metal transfer as a mechanism for metallothionein-mediated metal detoxification. Cell. Mol. Biol. 2000, 46, 393– 405345Metal transfer as a mechanism for metallothionein-mediated metal detoxificationRoesijadi, G.Cellular and Molecular Biology (Paris) (2000), 46 (2), 393-405CODEN: CMOBEF; ISSN:0145-5680. (C.M.B. Association)A review with 101 refs. High kinetic reactivity and high metal affinity of the metal-binding sites of metallothionein are characteristics that would facilitate involvement of the thionein-zinc metallothionein couple in metal transfer or exchange reactions. Studies demonstrating thionein-metallothionein-mediated activation or inhibition of various zinc metalloenzymes and transcription factors provide support for a potential role for metallothionein in metal transfer reactions with receptor mols. Although a role in basal zinc regulation is currently a topic of debate, less controversial is a role for metallothionein in the detoxification of metals such as cadmium. The toxicity of metals can, in part, be due to adventitious binding to charged sites of target proteins or the displacement of zinc bound to zinc metalloproteins. Zinc metallothionein has the capability of repairing such structures through abstraction of a toxic metal in the former case or through a reciprocal metal transfer reaction that involves abstraction of the toxic metal and donation of the essential metal zinc in the latter. This would confer on metallothionein an active role in the protective response to metal toxicity, rather than a passive one that is solely dependent on the high metal affinity for binding free metal ions. The efficacy of such a mechanism for metal detoxification has been demonstrated with enzymes, actin and zinc finger proteins. With zinc finger proteins, zinc metallothionein can restore both altered secondary structure and inhibited DNA-binding function to functional states through a zinc for cadmium exchange.
- 346Costello, L. C.; Liu, Y.; Franklin, R. B.; Kennedy, M. C. Zinc inhibition of mitochondrial aconitase and its importance in citrate metabolism of prostate epithelial cells. J. Biol. Chem. 1997, 272, 28875– 28881, DOI: 10.1074/jbc.272.46.28875346Zinc inhibition of mitochondrial aconitase and its importance in citrate metabolism of prostate epithelial cellsCostello L C; Liu Y; Franklin R B; Kennedy M CThe Journal of biological chemistry (1997), 272 (46), 28875-81 ISSN:0021-9258.Prostate epithelial cells possess a uniquely limiting mitochondrial (m-) aconitase activity that minimizes their ability to oxidize citrate. These cells also possess uniquely high cellular and mitochondrial zinc levels. Correlations among zinc, citrate, and m-aconitase in prostate indicated that zinc might be an inhibitor of prostate m-aconitase activity and citrate oxidation. The present studies reveal that zinc at near physiological levels inhibited m-aconitase activity of mitochondrial sonicate preparations obtained from rat ventral prostate epithelial cells. Corresponding studies conducted with mitochondrial sonicates of rat kidney cells revealed that zinc also inhibited the kidney m-aconitase activity. However the inhibitory effect of zinc was more sensitive with the prostate m-aconitase activity. Zinc inhibition fit the competitive inhibitor model. The inhibitory effect of zinc occurred only with citrate as substrate and was specific for the citrate --> cis-aconitate reaction. Other cations (Ca2+, Mn2+, Cd2+) did not result in the inhibitory effects obtained with zinc. The presence of endogenous zinc inhibited the m-aconitase activity of the prostate mitochondrial preparations. Kidney preparations that contain lower endogenous zinc levels exhibited no endogenous inhibition of m-aconitase activity. Studies with pig prostate and seminal vesicle mitochondrial preparations also revealed that zinc was a competitive inhibitor against citrate of m-aconitase activity. The effects of zinc on purified beef heart m-aconitase verified the competitive inhibitor action of zinc. In contrast, zinc had no inhibitory effect on purified cytosolic aconitase. These studies reveal for the first time that zinc is a specific inhibitor of m-aconitase of mammalian cells. In prostate epithelial cells, in situ mitochondrial zinc levels inhibit m-aconitase activity, which provides a mechanism by which citrate oxidation is limited.
- 347Feng, W.; Cai, J.; Pierce, W. M.; Franklin, R. B.; Maret, W.; Benz, F. M.; Kang, Y. J. Metallothionein transfers zinc to mitochondrial-aconitase through a direct interaction in mouse hearts. Biochem. Biophys. Res. Commun. 2005, 332, 853– 858, DOI: 10.1016/j.bbrc.2005.04.170347Metallothionein transfers zinc to mitochondrial aconitase through a direct interaction in mouse heartsFeng, Wenke; Cai, Jian; Pierce, William M.; Franklin, Renty B.; Maret, Wolfgang; Benz, Frederick W.; Kang, Y. JamesBiochemical and Biophysical Research Communications (2005), 332 (3), 853-858CODEN: BBRCA9; ISSN:0006-291X. (Elsevier)Previous studies have shown that in a cell-free system, metallothionein (MT) releases zinc when the environment becomes oxidized and the released zinc is transferred to a zinc-binding protein if such a protein is present. However, it is unknown whether and how zinc transfers from MT to other proteins in vivo. The present study was undertaken to test the hypothesis that if zinc transfer from MT to other proteins occurs in vivo, the transfer would proceed through a direct interaction between MT and a specific group of proteins. The heart ext. obtained from MT-null mice was incubated with 65Zn-MT or 65ZnCl2 and the proteins receiving 65Zn were sepd. by blue-native PAGE (BN-PAGE) or SDS-PAGE, and detected by autoradiog. A unique 65Zn-binding band was obsd. from the 65Zn-MT-incubated, but not the 65ZnCl2-incubated prepn. The anal. using matrix assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometry revealed that mitochondrial aconitase (m-aconitase) was among the proteins accepting Zn directly from Zn-MT. The m-aconitase, not the cytosolic aconitase (c-aconitase), was co-immunopptd. with MT. This study demonstrates that MT transfers zinc to m-aconitase through a direct interaction.
- 348Maret, W. Zinc biochemistry: from a single zinc enzyme to a key element of life. Adv. Nutr. 2013, 4, 82– 91, DOI: 10.3945/an.112.003038348Zinc biochemistry: from a single zinc enzyme to a key element of lifeMaret, WolfgangAdvances in Nutrition (2013), 4 (1), 82-91CODEN: ANDUAW; ISSN:2156-5376. (American Society for Nutrition)A review. The nutritional essentiality of zinc for the growth of living organisms had been recognized long before zinc biochem. began with the discovery of zinc in carbonic anhydrase in 1939. Painstaking anal. work then demonstrated the presence of zinc as a catalytic and structural cofactor in a few hundred enzymes. In the 1980s, the field again gained momentum with the new principle of "zinc finger" proteins, in which zinc has structural functions in domains that interact with other biomols. Advances in structural biol. and a rapid increase in the availability of gene/protein databases now made it possible to predict zinc-binding sites from metal-binding motifs detected in sequences. This procedure resulted in the definition of zinc proteomes and the remarkable est. that the human genome encodes ∼3000 zinc proteins. More recent developments focus on the regulatory functions of zinc(II) ions in intra- and intercellular information transfer and have tantalizing implications for yet addnl. functions of zinc in signal transduction and cellular control. At least three dozen proteins homeostatically control the vesicular storage and subcellular distribution of zinc and the concns. of zinc(II) ions. Novel principles emerge from quant. investigations on how strongly zinc interacts with proteins and how it is buffered to control the remarkably low cellular and subcellular concns. of free zinc (II) ions. It is fair to conclude that the impact of zinc for health and disease will be at least as far-reaching as that of iron.
- 349Maret, W.; Jacob, C.; Vallee, B. L.; Fischer, E. H. Inhibitory sites in enzymes: Zinc removal and reactivation by thionein. Proc. Natl. Acad. Sci. U. S. A. 1999, 96, 1936– 1940, DOI: 10.1073/pnas.96.5.1936349Inhibitory sites in enzymes: zinc removal and reactivation by thioneinMaret, Wolfgang; Jacob, Claus; Vallee, Bert L.; Fischer, Edmond H.Proceedings of the National Academy of Sciences of the United States of America (1999), 96 (5), 1936-1940CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Thionein (T) has not been isolated previously from biol. material. However, it is generated transiently in situ by removal of zinc from metallothionein under oxidoreductive conditions, particularly in the presence of selenium compds. T very rapidly activates a group of enzymes in which zinc is bound at an inhibitory site. The reaction is selective, as is apparent from the fact that T does not remove zinc from the catalytic sites of zinc metalloenzymes. T instantaneously reverses the zinc inhibition with a stoichiometry commensurate with its known capacity to bind seven zinc atoms in the form of clusters in metallothionein. The zinc inhibition is much more pronounced than was previously reported, with dissocn. consts. in the low nanomolar range. Thus, T is an effective, endogenous chelating agent, suggesting the existence of a hitherto unknown and unrecognized biol. regulatory system. T removes the metal from an inhibitory zinc-specific enzymic site with a resultant marked increase of activity. The potential significance of this system is supported by the demonstration of its operations in enzymes involved in glycolysis and signal transduction.
- 350Bellomo, E.; Birla Singh, K.; Massarotti, A.; Hogstrand, C.; Maret, W. The metal face of protein tyrosine phosphatase 1B. Coord. Chem. Rev. 2016, 327–328, 70– 83, DOI: 10.1016/j.ccr.2016.07.002350The metal face of protein tyrosine phosphatase 1BBellomo, Elisa; Birla Singh, Kshetrimayum; Massarotti, Alberto; Hogstrand, Christer; Maret, WolfgangCoordination Chemistry Reviews (2016), 327-328 (), 70-83CODEN: CCHRAM; ISSN:0010-8545. (Elsevier B.V.)A review. A new paradigm in metallobiochem. describes the activation of inactive metalloenzymes by metal ion removal. Protein tyrosine phosphatases (PTPs) do not seem to require a metal ion for enzymic activity. However, both metal cations and metal anions modulate their enzymic activity. One binding site is the phosphate binding site at the catalytic cysteine residue. Oxyanions with structural similarity to phosphate, such as vanadate, inhibit the enzyme with nanomolar to micromolar affinities. In addn., zinc ions (Zn2+) inhibit with picomolar to nanomolar affinities. We mapped the cation binding site close to the anion binding site and established a specific mechanism of inhibition occurring only in the closed conformation of the enzyme when the catalytic cysteine is phosphorylated and the catalytic aspartate moves into the active site. We discuss this dual inhibition by anions and cations here for PTP1B, the most thoroughly investigated protein tyrosine phosphatase. The significance of the inhibition in phosphorylation signaling is becoming apparent only from the functions of PTP1B in the biol. context of metal cations as cellular signaling ions. Zinc ion signals complement redox signals but provide a different type of control and longer lasting inhibition on a biol. time scale owing to the specificity and affinity of zinc ions for coordination environments. Inhibitor design for PTP1B and other PTPs is a major area of research activity and interest owing to their prominent roles in metabolic regulation in health and disease, in particular cancer and diabetes. Our results explain the apparent dichotomy of both cations (Zn2+) and oxyanions such as vanadate inhibiting PTP1B and having insulin-enhancing ("anti-diabetic") effects and suggest different approaches, namely targeting PTPs in the cell by affecting their physiol. modulators and considering a metallodrug approach that builds on the knowledge of the insulin-enhancing effects of both zinc and vanadium compds.
- 351Chong, C. R.; Auld, D. S. Inhibition of carboxypeptidase A by D-penicillamine: mechanism and implications for drug design. Biochemistry 2000, 39, 7580– 7588, DOI: 10.1021/bi000101+351Inhibition of carboxypeptidase A by D-penicillamine: mechanism and implications for drug designChong, Curtis R.; Auld, David S.Biochemistry (2000), 39 (25), 7580-7588CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)Zinc metalloprotease inhibitors are usually designed to inactivate the enzyme by forming a stable ternary complex with the enzyme and active-site zinc. D-Cysteine inhibits carboxypeptidase, ZnCPD, by forming such a complex, with a Ki of 2.3 μM. In contrast, the antiarthritis drug D-penicillamine, D-PEN, which differs from D-Cys only by the presence of two Me groups on the β-carbon, inhibits ZnCPD by promoting the release of the active-site zinc. We have given the name catalytic chelator to such inhibitors. Inhibition is a two-step process characterized by formation of a complex with the enzyme (Ki(initial) = 1.2 mM) followed by release of the active-site zinc at rates up to 420-fold faster than the spontaneous release. The initial rate of substrate hydrolysis at completion of the second step also depends on D-PEN concn., reflecting formation of a thermodn. equil. governed by the stability consts. of chelator and apocarboxypeptidase for zinc (Ki(final) = 0.25 mM). The interaction of D-PEN and D-Cys with the active-site metal has been examd. by replacing the active-site zinc by a chromophoric cobalt atom. Both inhibitors perturb the d-d transitions of CoCPD in the 500-600 nm region within milliseconds of mixing but only the CoCPD·D-Cys complex displays a strong S → Co(II) charge-transfer band at 340 nm indicative of a metal-sulfur bond. While the D-Cys complex is stable, the CoCPD·D-PEN complex breaks down to apoenzyme and Co(D-PEN)2 with a half-life of 0.5 s. D-PEN is the first drug found to inhibit a metalloprotease by increasing the dissocn. rate const. of the active-site metal. The ability of D-PEN to catalyze metal removal from carboxypeptidase A and other zinc proteases suggests a possible mechanism of action in arthritis and Wilson's disease and may also underlie complications assocd. with its clin. use.
- 352Chong, C. R.; Auld, D. S. Catalysis of zinc transfer by D-penicillamine to secondary chelators. J. Med. Chem. 2007, 50, 5524– 5527, DOI: 10.1021/jm070803y352Catalysis of Zinc Transfer by D-Penicillamine to Secondary ChelatorsChong, Curtis R.; Auld, David S.Journal of Medicinal Chemistry (2007), 50 (22), 5524-5527CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)The antiarthritis drug D-penicillamine (D-PEN) catalyzes zinc(II) transfer from carboxypeptidase A to chelators such as thionein and EDTA at a rate const. up to 400-fold faster than the uncatalyzed release. Once D-PEN releases zinc(II) from enzyme stronger chelators can tightly bind zinc(II) leading to complete and essentially irreversible inhibition. D-PEN is the first drug to inhibit a zinc protease by catalyzing metal removal, and the name "catalytic chelation" is proposed for this mechanism.
- 353Zeng, J.; Heuchel, R.; Schaffner, W.; Kägi, J. H. R. Thionein (apometallothionein) can modulate DNA binding and transcription activation by zinc finger containing Sp1. FEBS Lett. 1991, 279, 310– 312, DOI: 10.1016/0014-5793(91)80175-3353Thionein (apometallothionein) can modulate DNA binding and transcription activation by zinc finger containing factor Sp1Zeng, Jin; Heuchel, Rainer; Schaffner, Walter; Kaegi, Jeremias H. R.FEBS Letters (1991), 279 (2), 310-12CODEN: FEBLAL; ISSN:0014-5793.A no. of transcription factors contain so-called Zn finger domains for the interaction with their cognate DNA sequence. Removal of the Zn ions complexed in these Zn fingers abrogates DNA binding and transcription activation. The hypothesis that the activity of transcription factors could be regulated by physiol. chelators of Zn was tested. A prominent candidate for such a chelator is the cysteine-rich protein thionein (apometallothionein) that is inducible by heavy metal loads, and by other environmental stimuli. DNA binding and in vitro transcription assays show that thionein indeed can inactivate the Zn finger-contg. Sp1 in a reversible manner. By contrast, transcription factor Oct-1, which binds DNA via a homeo-domain, i.e. a helix-turn-helix motif not involving Zn ions, is refractory to thionein action. It is proposed that modulation of intracellular thionein concn. is used for the coordinated regulation of a large subset of genes whose transcription depends on Zn finger proteins.
- 354Zeng, J.; Vallee, B. L.; Kägi, J. H. R. Zinc transfer from transcription factor IIIA fingers to thionein clusters. Proc. Natl. Acad. Sci. U. S. A. 1991, 88, 9984– 9988, DOI: 10.1073/pnas.88.22.9984354Zinc transfer from transcription factor IIIA fingers to thionein clustersZeng, Jin; Vallee, Bert L.; Kaegi, Jeremias H. R.Proceedings of the National Academy of Sciences of the United States of America (1991), 88 (22), 9984-8CODEN: PNASA6; ISSN:0027-8424.The rapid induction of thionein (apometallothionein) by many endogenous stimuli such as steroid hormones, cytokines, and second messengers suggests that this cysteine-rich, metal-binding protein participates in an as yet undefined role in cellular regulatory processes. This study demonstrates with DNA and RNA binding assays and in vitro transcription measurements that thionein suppresses the binding of the Xenopus laevis zinc finger transcription factor IIIA (TFIIIA) to 5 S RNA and to the 5 S RNA gene and abrogates the capacity of synthesis of 5 S RNA. The effect is reversed by the addn. of zinc and is not obsd. in the TFIIIA-independent transcription of a tRNA gene by the same RNA polymerase. In view of the strong tendency of thionein to complex posttransition metals such as zinc, one effect of its enhanced synthesis in vivo could be to reduce the intracellular disposability of zinc and thus modulate the actions of zinc-dependent enzymes and proteins, most notably those of the zinc finger transcription factors.
- 355Cano-Gauci, D. F.; Sarkar, B. Reversible zinc exchange between metallothionein and the estrogen receptor zinc finger. FEBS Lett. 1996, 386, 1– 4, DOI: 10.1016/0014-5793(96)00356-0355Reversible zinc exchange between metallothionein and the estrogen receptor zinc fingerCano-Gauci D F; Sarkar BFEBS letters (1996), 386 (1), 1-4 ISSN:0014-5793.We report here the first demonstration that reversible metal exchange occurs between metallothionein (MT) and full-length estrogen receptor (ER). Specific binding of ER to estrogen response element is inhibited in the presence of 40 microM thionein and restored by 120 microM zinc. Moreover, ER in metal-depleted nuclear extracts exhibits reduced DNA binding which can be restored by 140 microM native MT. Hence, thionein inhibits DNA binding by abstracting zinc from functional ER while native MT is capable of restoring binding to metal-depleted extracts by donating metal to ER. This indicates MT may be an important physiological regulator of intracellular zinc and/or other metals.
- 356Petering, D. H.; Zhu, J.; Krezoski, S.; Meeusen, J.; Kiekenbush, C.; Krull, S.; Specher, T.; Dughish, M. Apo-metallothionein emerging as a major player in the cellular activities of metallothionein. Exp. Biol. Med. (London, U. K.) 2006, 231, 1528– 1534, DOI: 10.1177/153537020623100912356Apo-metallothionein emerging as a major player in the cellular activities of metallothioneinPetering, David H.; Zhu, Jianyu; Krezoski, Susan; Meeusen, Jeffrey; Kiekenbush, Christy; Krull, Sara; Specher, Todd; Dughish, MuniraExperimental Biology and Medicine (Maywood, NJ, United States) (2006), 231 (9), 1528-1534CODEN: EBMMBE; ISSN:1535-3702. (Society for Experimental Biology and Medicine)A review. Observations of apo-metallothionein (apo-MT) have been made under a variety of physiol. circumstances, including Zn deficiency in cell culture and in rodents, cellular induction of MT by dexamethasone with concurrent Zn deficiency, a variety of tumors under normal Zn conditions, MT induction by Zn and Bi citrate, induction of hepatic MT after tumor cell injection into nude mice, and overexpression of cardiac MT in MT transgenic mice. Expts. demonstrating both the lability of Zn and Cu bound to MT and the cellular stability of apo-MT are described to help rationalize the widespread presence of this metal-depleted species. Finally, comparative in vitro and cellular expts. have examd. the relative reactivity of Zn- and apo-MT with NO species, showing that apo-MT is much more reactive chem. and that in cells it may be a principal reactive species within the MT pool.
- 357Kluska, K.; Adamczyk, J.; Krężel, A. Metal binding properties of zinc fingers with a naturally altered metal binding site. Metallomics 2018, 10, 248– 263, DOI: 10.1039/C7MT00256D357Metal binding properties of zinc fingers with a naturally altered metal binding siteKluska, Katarzyna; Adamczyk, Justyna; Krezel, ArturMetallomics (2018), 10 (2), 248-263CODEN: METAJS; ISSN:1756-591X. (Royal Society of Chemistry)A review. Zinc fingers (ZFs) are among the most abundant motifs found in proteins, and are commonly known for their structural role. Classical ZFs (CCHH) are part of the transcription factors that participate in DNA binding. Although biochem. studies of classical ZFs have a long history, there is limited knowledge about the sequential and structural diversity of ZFs. We have found that classical ZFs, with metal binding sites consisting of amino acids other than conserved Cys or His residues, are frequently encoded in the human genome, and we refer to these peptides as ZFs with a naturally altered metal binding site. The biol. role of the altered ZFs remains undiscovered. In this study, we characterized nine natural XCHH, CXHH, CCXH and CCHX ZFs in terms of their Zn(II) and Co(II) binding properties, such as complex stoichiometry, spectroscopic properties and metal-to-peptide affinity. We revealed that XCHH and CXHH ZFs form ML complexes that are 4-5 orders of magnitude weaker in comparison to CCHH ZFs. Nevertheless, spectroscopic studies demonstrate that, depending on the altered position, they may adopt an open coordination geometry with one or two water mols. bound to a central metal ion, which has not been demonstrated in natural ZFs before. Stability data show that both CCXH and CCHX peptides have high Zn(II) affinity (with a Kd of 10-9 to 10-11 M), suggesting their potential biol. function. This study is a comprehensive overview of the relationship between the sequence, structure, and stability of ZFs.
- 358Apuy, J. L.; Chen, X.; Russell, D. H.; Baldwin, T. O.; Giedroc, D. P. Ratiometric pulsed alkylation/mass spectrometry of the cysteine pairs in individual zinc fingers of MRE-binding transcription factor-1 (MTF-1) as a probe of zinc chelate stability. Biochemistry 2001, 40, 15164– 15175, DOI: 10.1021/bi0112208358Ratiometric Pulsed Alkylation/Mass Spectrometry of the Cysteine Pairs in Individual Zinc Fingers of MRE-Binding Transcription Factor-1 (MTF-1) as a Probe of Zinc Chelate StabilityApuy, Julius L.; Chen, Xiaohua; Russell, David H.; Baldwin, Thomas O.; Giedroc, David P.Biochemistry (2001), 40 (50), 15164-15175CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)Metal-response element (MRE)-binding transcription factor-1 (MTF-1) is a zinc-regulated transcriptional activator of metallothionein (MT) genes in mammalian cells. The MRE-binding domain of MTF-1 (MTF-zf) has six canonical Cys2-His2 zinc finger domains that are distinguished on the basis of their apparent affinities for zinc and their specific roles in MRE-binding. In this paper, pulsed alkylation of the zinc-liganding cysteine thiolate pairs with the sulfhydryl-specific alkylating reagent d5-N-ethylmaleimide (d5-NEM) is used as a residue-specific probe of the relative stabilities of the individual zinc finger coordination complexes in Zn6 MTF-zf. A chase with excess H5-N-ethylmaleimide (H5-NEM) to fully derivatize MTF-zf concomitant with complete proteolysis, followed by MALDI-TOF mass spectrometry allows quantitation of the mole fraction of d5,d5-, d5,H5-, and H5,H5-NEM derivatized peptides corresponding to each individual zinc finger domain as a function of d5-NEM pulse time. This expt. establishes the hierarchy of cysteine thiolate reactivity in MTF-zf as F5 > F6 » F1 > F2 ≈ F3 ≈ F4. The apparent second-order rate of reaction of F1 thiolates is comparable to that detd. for the DNA binding domain of Sp1, Zn3 Sp1-zf, under identical soln. conditions. The reactivities of all Cys residues in MTF-zf are significantly reduced when bound to an MREd-contg. oligonucleotide. An identical expt. carried out with Zn5 MTF-zf26, an MTF-zf domain lacking the N-terminal F1 zinc finger, reveals that MTF-zf26 binds to the MREd very weakly, and is characterized by strongly increased reactivity of nonadjacent F4 thiolates. These findings are discussed in the context of existing models for metalloregulation by MTF-1.
- 359Potter, B. M.; Feng, L. S.; Parasuram, P.; Matskevich, V. A.; Wilson, J. A.; Andrews, G. K.; Laity, J. H. The six zinc fingers of metal-responsive element binding transcription factor-1 form stable and quasi-ordered structures with relatively small differences in zinc affinities. J. Biol. Chem. 2005, 280, 28529– 28540, DOI: 10.1074/jbc.M505217200359The Six Zinc Fingers of Metal-responsive Element Binding Transcription Factor-1 Form Stable and Quasi-ordered Structures with Relatively Small Differences in Zinc AffinitiesPotter, Belinda M.; Feng, Linda S.; Parasuram, Priya; Matskevich, Viktor A.; Wilson, Jed A.; Andrews, Glen K.; Laity, John H.Journal of Biological Chemistry (2005), 280 (31), 28529-28540CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)Six Cys2His2 zinc fingers (F1-6) comprise the DNA binding domain of metal-responsive element binding transcription factor-1 (MTF-1). F1-6 is necessary for basal and zinc-induced expression of metallothionein genes. Anal. of NMR structural and dynamic data for an F1-6 protein construct demonstrates that each zinc finger adopts a stable ββα fold in the presence of stoichiometric Zn(II), provided that all cysteine ligands are in a reduced state. Parallel studies of protein constructs spanning the four N-terminal core DNA binding fingers (F1-4) and two C-terminal low DNA affinity fingers (F5-6) reveal similar stable zinc finger structures. In both the F1-6 and F5-6 proteins, the finger 5 cysteines were found to readily oxidize at neutral pH. Detailed spectral d. and hydrodynamic anal. of 15N relaxation data revealed quasi-ordered anisotropic rotational diffusion properties of the six F1-6 zinc fingers that could influence MTF-1 DNA binding function. A more general effect on the rotational diffusion properties of Cys2His2 zinc fingers was also uncovered that is dependent upon the position of each finger within multifinger domains. Anal. of NMR 1H-15N-heteronuclear single quantum coherence spectral peak intensities measured as a function of added Zn(II) in conjunction with Zn(II) binding modeling studies indicated that the Zn(II) affinities of all MTF-1 zinc fingers are within ∼10-50-fold. These analyses further suggested that metal sensing by MTF-1 in eukaryotic cells involves multiple zinc fingers and occurs over a 100-fold or less range of accessible Zn(II) concn.
- 360Vallee, B. L. Historical review and perspectives. EXS 1979, 34, 19– 40, DOI: 10.1007/978-3-0348-6493-0_1There is no corresponding record for this reference.
- 361Jiang, L.-J.; Maret, W.; Vallee, B. L. The ATP-metallothionein complex. Proc. Natl. Acad. Sci. U. S. A. 1998, 95, 9146– 9149, DOI: 10.1073/pnas.95.16.9146361The ATP-metallothionein complexJiang, Li-Juan; Maret, Wolfgang; Vallee, Bert L.Proceedings of the National Academy of Sciences of the United States of America (1998), 95 (16), 9146-9149CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)We have previously shown that glutathione (GSH) and glutathione disulfide interact with metallothionein (MT) and modulate its capacity to donate and transfer zinc. In this paper, we show that ATP also forms a 1:1 complex with MT (Kd = 176±33 μM, pH 7.4) that enhances the transfer of zinc to zinc-depleted sorbitol dehydrogenase, increases the rate of thiol-disulfide interchange with Ellman's reagent [5,5'-dithiobis (Z-nitrobenzoic acid)], and changes the apparent shape of the protein. GTP produces almost identical effects. The corresponding di- or monophosphates and pyrimidine nucleotides, however, neither bind as strongly as ATP nor enhance zinc transfer. Carbamoylation of MT lysines abolishes ATP binding, indicating that these highly conserved residues are part of the binding site. GSH decreases, whereas glutathione disulfide increases, ATP binding. The interaction of MT with two crit. cellular ligands, i.e., GSH and ATP, and ensuing effects on zinc transfer and reactivity suggest that MT is not merely a cellular zinc buffer but, rather, actively participates in zinc distribution. Apparently, when isolated, MT lacks two important effectors that affect its redox behavior and function. The magnitude of the binding const. and the cellular concn. of ATP indicate that in the cell MT could be essentially satd. with ATP at low concns. of GSH. Both the redox and energy states of the cell seem to control zinc distribution from MT, but their relative contributions require further studies.
- 362Zangger, K.; Öz, G.; Armitage, I. M. Re-evaluation of the binding of ATP to metallothioneins. J. Biol. Chem. 2000, 275, 7534– 7538, DOI: 10.1074/jbc.275.11.7534362Re-evaluation of the binding of ATP to metallothioneinZangger, Klaus; Oz, Gulin; Armitage, Ian M.Journal of Biological Chemistry (2000), 275 (11), 7534-7538CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)In a recent paper Jiang et al. reported that metallothionein interacts with ATP to form a 1:1 complex with a dissocn. const. of Kd = 176 ± 33 μM at pH 7.4. In an effort to characterize further this interaction using NMR spectroscopy, titrn. calorimetry, gel-filtration chromatog., affinity chromatog., and ultrafiltration, we were unable to find any evidence for the binding of ATP to metallothionein.
- 363Kangur, L.; Palumaa, P. The effects of physiologically important non-metallic ligands in the reactivity of metallothionein towards 5,5′-dithiobis(2-nitrobenzoic acid). Eur. J. Biochem. 2001, 268, 4979– 4984, DOI: 10.1046/j.0014-2956.2001.02430.x363The effects of physiologically important nonmetallic ligands in the reactivity of metallothionein towards 5,5'-dithiobis(2-nitrobenzoic acid). A new method for the determination of ligand interactions with metallothioneinKangur, Liina; Palumaa, PeepEuropean Journal of Biochemistry (2001), 268 (18), 4979-4984CODEN: EJBCAI; ISSN:0014-2956. (Blackwell Science Ltd.)The reaction of Cd5Zn2-metallothionein (MT) with 5,5'-dithiobis(2-nitrobenzoic acid) (Nbs2) has been studied at different reagent stoichiometries, pH and temp. conditions and in the presence of several ligands. At stoichiometries of Nbs2 to MT from 0.5 to 5, the reaction followed first order kinetics. The first order rate consts. obtained were independent from the concn. of Nbs2 but were linearly dependent on the concn. of MT. At higher Nbs2/MT stoichiometries, the reaction deviates from first order kinetics and the obsd. rate const. increases. The reactivity of MT towards Nbs2 has been probed at 4 μM concn. of both reagents where the reaction is monophasic and is characterized by a linear Arrhenius plot (Ea = 45.8±2.7 kJ·mol-1). It has been demonstrated that metal release at low pH or subtraction from MT by EDTA substantially increases the reactivity of MT towards Nbs2. At the same time, a no. of nonmetallic ligands moderately accelerate the reaction of MT with Nbs2 and hyperbolic dose-response curves were obtained. The data have been interpreted with the binding of ligands to MT and following MT. Ligand binding consts. were calcd. as follows: ATP, K = 0.31±0.06 mM; ADP, K = 0.26±0.07 mM. Several compds. such as AMP, S-methylglutathione, and phosphate had no effect on the reaction, but Zn2+ ions showed an inhibitory effect at micromolar concns.
- 364Kwok, F.; Churchich, J. E. Brain pyridoxal kinase. Purification, substrate specificities, and sensitized photodestruction of an essential histidine. J. Biol. Chem. 1979, 254, 6489– 6495, DOI: 10.1016/S0021-9258(18)50394-X364Brain pyridoxal kinase. Purification, substrate specificities, and sensitized photodestruction of an essential histidineKwok, Francis; Churchich, Jorge E.Journal of Biological Chemistry (1979), 254 (14), 6489-95CODEN: JBCHA3; ISSN:0021-9258.Pyridoxal kinase (I) was purified 2000-fold from pig brain. The enzyme prepn. migrates as a single protein and activity band on anal. gel electrophoresis. I, 60,000 mol. wt., catalyzes the phosphorylation of pyridoxal (Km = 2.5 × 10-5 M) and pyridoxine (Km = 1.7 × 10-5 M). Pyridoxamine is not a substrate of the purified kinase. Irradn. of I in the presence of riboflavin leads to irreversible loss of catalytic activity. Riboflavin binds to I with a KD = 5 μM as shown by fluorometric titrns. Singlet excited O, generated by energy transfer from the lowest triplet of riboflavin to O, acts as the oxidizing agent of approx. 1 histidine residue/mol of I. The amino acid residues tyrosine, tryptophan, and cysteine are not photooxidized by the sensitizer bound to the enzyme. Probably, histidine is involved in the binding of the substrate ATP to the catalytic site of I.
- 365Nakano, H.; McCormick, D. B. Stereospecificity of the metal.ATP complex in flavokinase from rat small intestine. J. Biol. Chem. 1991, 266, 22125– 22128, DOI: 10.1016/S0021-9258(18)54542-7365Stereospecificity of the metal·ATP complex in flavokinase from rat small intestineNakano, Hideko; McCormick, Donald B.Journal of Biological Chemistry (1991), 266 (33), 22125-8CODEN: JBCHA3; ISSN:0021-9258.Transfer of the γ-phosphoryl group of ATP to riboflavin is catalyzed by flavokinase, which prefers Zn(II), and is essential in the biosynthesis of the flavocoenzyme, FMN. To study the mechanism and steric disposition of ATP binding, adenosine 5'-O-(2-thiotriphosphate) (ATPβS) and adenosine 5'-O-(3-thiotriphosphate) (ATPγS) were tested in comparison to ATP with representative divalent metal ions. Activities with 0.1 mM Zn(II) for 1 mM ATP thio analogs compared to ATP with flavokinase were 60% for the S-isomer of ATPβS, 312% for the R-isomer of ATPβS, and 14% for ATPγS. With Mg(II), flavokinase showed stereospecificity for the R-isomer of ATPβS with a Vmax ratio for the stereoisomers of S/R = 0.125. With Cd(II), the enzyme showed preference for the S-isomer of ATPβS with a Vmax ratio, S/R = 1.261. The Vmax ratio progressively increases from Mg(II) to Cd(II) in the order of affinity for sulfur. The ratios, (Vmax/Km)Mg/(Vmax/Km)Cd, for the diastereomers of ATPβS were expectedly >1 for one diastereomer (R = 6.597) and <1 for the other (S = 0.292). Activities with ATPγS were much slower than with ATP or ATPβS. With Mg(II), the γ analog was a good substrate; however, with Cd(II), it was hardly active. Altogether these results indicate that flavokinase uses the Λ,β,γ-bidentate Zn·ATP as a substrate.
- 366Lerner, F.; Niere, M.; Ludwig, A.; Ziegler, M. Structural and functional characterization of human NAD kinase. Biochem. Biophys. Res. Commun. 2001, 288, 69– 74, DOI: 10.1006/bbrc.2001.5735366Structural and Functional Characterization of Human NAD KinaseLerner, Felicitas; Niere, Marc; Ludwig, Antje; Ziegler, MathiasBiochemical and Biophysical Research Communications (2001), 288 (1), 69-74CODEN: BBRCA9; ISSN:0006-291X. (Academic Press)NADP is essential for biosynthetic pathways, energy, and signal transduction. Its synthesis is catalyzed by NAD kinase. Very little is known about the structure, function, and regulation of this enzyme from multicellular organisms. We identified a human NAD kinase cDNA and the corresponding gene using available database information. A cDNA was amplified from a human fibroblast cDNA library and functionally overexpressed in Escherichia coli. The obtained cDNA, slightly different from that deposited in the database, encodes a protein of 49 kDa. The gene is expressed in most human tissues, but not in skeletal muscle. Human NAD kinase differs considerably from that of prokaryotes by subunit mol. mass (49 kDa vs. 30-35 kDa). The catalytically active homotetramer is highly selective for its substrates, NAD and ATP. It did not phosphorylate the nicotinic acid deriv. of NAD (NAAD) suggesting that the potent calcium-mobilizing pyridine nucleotide NAADP is synthesized by an alternative route. (c) 2001 Academic Press.
- 367McCormick, D. Micronutrient cofactor research with extensions to applications. Nutr. Res. Rev. 2002, 15, 245– 262, DOI: 10.1079/NRR200241367Micronutrient cofactor research with extensions to applicationsMcCormick, Donald B.Nutrition Research Reviews (2002), 15 (2), 245-262CODEN: NREREX; ISSN:0954-4224. (CABI Publishing)A review. Following identification of essential micronutrients, there has been a continuum of research aimed at revealing their absorption, transport, utilization as cofactors, and excretion and secretion. Among those cases that have received our attention are vitamin B6, riboflavin, biotin, lipoate, ascorbate, and certain metal ions. Circulatory transport and cellular uptake of the water-sol. vitamins exhibit relative specificity and facilitated mechanisms at physiol. concns. Isolation of enzymes and metabolites from micro-organisms and mammals has provided information on pathways involved in cofactor formation and metab. Kinases catalyzing phosphorylation of B6 and riboflavin have a preference for Zn2+ in stereospecific chelates with ATP. The synthetase for FAD prefers Mg2+. The FMN-dependent oxidase that converts the 5'-phosphates of pyridoxine and of pyridoxamine to pyridoxal phosphate is a connection between B6 and riboflavin and is a primary control point for conversion of B6 to its coenzyme. Sequencing and cloning of a side-chain oxidase for riboflavin was achieved. Details on binding and function have been delineated for some cofactor systems, esp. in several flavoproteins. There is both photochem. oxidn. and oxidative catabolism of B6 and riboflavin. Both biotin and lipoate undergo oxidn. of their acid side chains with redox cleavage of the rings. Applications from our findings include the development of affinity absorbents, enhanced drug delivery, delineation of residues in biopolymer modification, pathogen photoinactivation in blood components, and input into human dietary recommendations. Ongoing and future research in the cofactor arena can be expected to add to this panoply. At the mol. level, the way in which the same cofactor can participate in diverse catalytic reactions resides in interactions with surrounding enzyme strictures that must be detd. case by case. At the level of human intake, more knowledge is desirable for making micronutrient recommendations based on biochem. indicators, esp. for the span between infancy and adulthood.
- 368Li, M. H.; Kwok, F.; Chang, W. R.; Lau, C. K.; Zhang, J. P.; Lo, S. C.; Jiang, T.; Liang, D. C. Crystal structure of brain pyridoxal kinase, a novel member of the ribokinase superfamily. J. Biol. Chem. 2002, 277, 46385– 46390, DOI: 10.1074/jbc.M208600200368Crystal structure of brain pyridoxal kinase, a novel member of the ribokinase superfamilyLi, Ming-Hui; Kwok, Francis; Chang, Wen-Rui; Lau, Chi-Kong; Zhang, Ji-Ping; Lo, Samuel C. L.; Jiang, Tao; Liang, Dong-CaiJournal of Biological Chemistry (2002), 277 (48), 46385-46390CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)The 3-dimensional structures of sheep brain pyridoxal kinase (I) and its complex with ATP were elucidated in the dimeric form at 2.1 and 2.6 Å, resp. The results showed that I, as an enzyme obeying random sequential kinetics in catalysis, did not possess a lid shape structure common to all kinases in the ribokinase superfamily. This finding was shown to be in line with the condition that I binds substrates with variable sizes of chem. groups at position 4 of vitamin B6 and its derivs. In addn., I contained a 12-residue peptide loop in the active site for the prevention of premature hydrolysis of ATP. Conserved amino acid residues Asp-118 and Tyr-127 in the peptide loop could be moved to a position covering the nucleotide after its binding so that its chance to hydrolyze in the aq. environment of the active site was reduced. With respect to the evolutionary trend of kinases, the existence of this loop in I could be classified as an independent category in the ribokinase superfamily according to the structural feature found and mechanism followed in catalysis.
- 369Bauer, S.; Kemter, K.; Bacher, A.; Huber, R.; Fischer, M.; Steinbacher, S. Crystal structure of Schizosaccharomyces pombe riboflavin kinase reveals a novel ATP and riboflavin-binding fold. J. Mol. Biol. 2003, 326, 1463– 1473, DOI: 10.1016/S0022-2836(03)00059-7369Crystal Structure of Schizosaccharomyces pombe Riboflavin Kinase Reveals a Novel ATP and Riboflavin-Binding FoldBauer, Stefanie; Kemter, Kristina; Bacher, Adelbert; Huber, Robert; Fischer, Markus; Steinbacher, StefanJournal of Molecular Biology (2003), 326 (5), 1463-1473CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Science Ltd.)The essential redox cofactors riboflavin monophosphate (FMN) and FAD are synthesized from their precursor, riboflavin, in sequential reactions by the metal-dependent riboflavin kinase and FAD synthetase. Here, we describe the 1.6 Å crystal structure of the Schizosaccharomyces pombe riboflavin kinase. The enzyme represents a novel family of phosphoryl transferring enzymes. It is a monomer comprising a central β-barrel clasped on one side by two C-terminal helixes that display an L-like shape. The opposite side of the β-barrel serves as a platform for substrate binding as demonstrated by complexes with ADP and FMN. Formation of the ATP-binding site requires significant rearrangements in a short α-helix as compared to the substrate free form. The diphosphate moiety of ADP is covered by the glycine-rich flap I formed from parts of this α-helix. In contrast, no significant changes are obsd. upon binding of riboflavin. The ribityl side-chain might be covered by a rather flexible flap II. The unusual metal-binding site involves, in addn. to the ADP phosphates, only the strictly conserved Thr45. This may explain the preference for zinc obsd. in vitro.
- 370Churchich, J. E.; Scholz, G. F.; Kwok, F. Activation of pyridoxal kinase by metallothionein. Biochim. Biophys. Acta, Protein Struct. Mol. Enzymol. 1989, 996, 181– 186, DOI: 10.1016/0167-4838(89)90245-8370Activation of pyridoxal kinase by metallothioneinChurchich, Jorge E.; Scholz, Glen; Kwok, FrancisBiochimica et Biophysica Acta, Protein Structure and Molecular Enzymology (1989), 996 (3), 181-6CODEN: BBAEDZ; ISSN:0167-4838.Brain pyridoxal kinase, which uses ATP complexed to either Zn(II) or Co(II) as substrates, displays high catalytic activity in the presence of Zn-thionein and Co-thionein. Several steps intervene in the process of pyridoxal kinase activation, i.e., binding of Zn2+ to ATP and interaction between Zn-ATP and the enzyme. Equil. binding studies show that ATP mediates the release of Zn2+ from the metal-thiolate clusters of the thioneins, whereas spectroscopic measurements conducted on Co-thionein reveal that the absorption transitions corresponding to the metal-thiolate of the protein are perturbed by ATP. The binding Zn-ATP to the kinase proceeds with a ΔG = -6.3 kcal/mol as demonstrated by fluorometric titrns. Direct interaction between the kinase and derivatized-metallothionein could not be detected by emission anisotropy measurements, indicating that juxtaposition of the proteins does not influence the exchange of metal ions. Since the concn. of free Zn in several mammalian tissues is <1 nM, it is postulated that under in vivo conditions the concn. of metallothionein regulates the catalytic activity of pyridoxal kinase.
- 371Sigel, H.; Griesser, R. Nucleoside 5′triphosphates: self-association, acid-base, and metal ion-binding properties in solution. Chem. Soc. Rev. 2005, 34, 875– 900, DOI: 10.1039/b505986k371Nucleoside 5'-triphosphates: self-association, acid-base, and metal ion-binding properties in solutionSigel, Helmut; Griesser, RolfChemical Society Reviews (2005), 34 (10), 875-900CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)A review. ATP (ATP4-) and related nucleoside 5'-triphosphates (NTP4-) serve as substrates in the form of metal ion complexes in enzymic reactions taking part thus in central metabolic processes. With this in mind, the coordination chem. of NTPs is critically reviewed and the conditions are defined for studies aiming to describe the properties of monomeric complexes because at higher concns. (>1 mM) self-stacking may take place. The metal ion (M2+) complexes of purine-NTPs are more stable than those of pyrimidine-NTPs; this stability enhancement is attributed, in accord with NMR studies, to macrochelate formation of the phosphate-coordinated M2+ with N7 of the purine residue and the formation degrees of the resulting isomeric complexes are listed. Furthermore, the formation of mixed-ligand complexes (including also those with buffer mols.), the effect of a reduced solvent polarity on complex stability and structure (giving rise to selectivity), the use of nucleotide analogs as antiviral agents, and the effect of metal ions on group transfer reactions are summarized.
- 372Si, M.; Lang, J. The roles of metallothioneins in carcinogenesis. J. Hematol. Oncol. 2018, 11, 107, DOI: 10.1186/s13045-018-0645-x372The roles of metallothioneins in carcinogenesisSi, Manfei; Lang, JingheJournal of Hematology & Oncology (2018), 11 (), 107/1-107/20CODEN: JHOOAO; ISSN:1756-8722. (BioMed Central Ltd.)A review. Metallothioneins (MTs) are small cysteine-rich proteins that play important roles in metal homeostasis and protection against heavy metal toxicity, DNA damage, and oxidative stress. In humans, MTs have four main isoforms (MT1, MT2, MT3, and MT4) that are encoded by genes located on chromosome 16q13. MT1 comprises eight known functional (sub)isoforms (MT1A, MT1B, MT1E, MT1F, MT1G, MT1H, MT1M, and MT1X). Emerging evidence shows that MTs play a pivotal role in tumor formation, progression, and drug resistance. However, the expression of MTs is not universal in all human tumors and may depend on the type and differentiation status of tumors, as well as other environmental stimuli or gene mutations. More importantly, the differential expression of particular MT isoforms can be utilized for tumor diagnosis and therapy. This review summarizes the recent knowledge on the functions and mechanisms of MTs in carcinogenesis and describes the differential expression and regulation of MT isoforms in various malignant tumors. The roles of MTs in tumor growth, differentiation, angiogenesis, metastasis, microenvironment remodeling, immune escape, and drug resistance are also discussed. Finally, this review highlights the potential of MTs as biomarkers for cancer diagnosis and prognosis and introduces some current applications of targeting MT isoforms in cancer therapy. The knowledge on the MTs may provide new insights for treating cancer and bring hope for the elimination of cancer.
- 373Vasconcelos, M. H.; Tam, S. C.; Hesketh, J. E.; Reid, M.; Beattie, J. H. Metal and tissue-dependent relationship between metallothionein mRNA and protein. Toxicol. Appl. Pharmacol. 2002, 182, 91– 97, DOI: 10.1006/taap.2002.9428373Metal- and Tissue-Dependent Relationship between Metallothionein mRNA and ProteinVasconcelos, M. Helena; Tam, Shuk-Ching; Hesketh, John E.; Reid, Martin; Beattie, John H.Toxicology and Applied Pharmacology (2002), 182 (2), 91-97CODEN: TXAPA9; ISSN:0041-008X. (Elsevier Science)Metallothionein (MT) expression is transcriptionally regulated but recent evidence suggests that translation of MT mRNA may be regulated under some circumstances (1995). A systematic investigation of MT mRNA, protein, and metal levels in liver and kidney of cadmium- or copper-treated rats was made to further understand the relationship between mRNA and protein in particular. Adult rats were injected once with either Cd (8.9 μmol/kg) or Cu2+ (8.7 μmol/kg) as the chloride salts, and the liver and kidney concns. of MT-1 and MT-2 mRNA, total sol. MT protein, and tissue Cd, Cu, and Zn were monitored over 48-72 h. The metal compn. in the sol. MT protein fraction was also analyzed by online size-exclusion chromatog.-ICP/MS. Discrepancies between mRNA and protein levels were found in both tissues, but particularly in kidney. Cd treatment significantly increased renal MT-1 and MT-2 mRNA levels but protein was unaffected. In contrast, Cu actually decreased renal MT-1 and MT-2 mRNA but significantly increased MT protein. Cd induced considerably more MT-1 than MT-2 mRNA in liver, but induction of both isoforms was similar in kidney and in liver of Cu-treated rats. Changes in tissue metal levels tended to reflect MT protein levels and Cd appeared to bind to existing MT in the kidney. The results support the contention that MT protein levels often bear no clear relationship with mRNA levels and emphasizes the importance of measuring both in studies of MT expression.
- 374Raudenska, M.; Gumulec, J.; Podlaha, O.; Sztalmachova, M.; Babula, P.; Eckschlager, T.; Adam, V.; Kizek, R.; Masarik, M. Metallothionein polymorphisms in pathological processes. Metallomics 2014, 6, 55– 68, DOI: 10.1039/C3MT00132F374Metallothionein polymorphisms in pathological processesRaudenska, Martina; Gumulec, Jaromir; Podlaha, Ondrej; Sztalmachova, Marketa; Babula, Petr; Eckschlager, Tomas; Adam, Vojtech; Kizek, Rene; Masarik, MichalMetallomics (2014), 6 (1), 55-68CODEN: METAJS; ISSN:1756-591X. (Royal Society of Chemistry)A review. Metallothioneins (MTs) are a class of metal-binding proteins characterized by a high cysteine content and low mol. wt. MTs play an important role in metal metab. and protect cells against the toxic effects of radiation, alkylating agents and oxygen free radicals. The evidence that individual genetic characteristics of MTs play an important role in physiol. and pathol. processes assocd. with antioxidant defense and detoxification inspired targeted studies of genetic polymorphisms in a clin. context. In recent years, common MT polymorphisms were identified and assocd. with, particularly, western lifestyle diseases such as cancer, complications of atherosclerosis, and type 2 diabetes mellitus along with related complications. This review summarizes all evidence regarding MT polymorphisms of major human MTs (MT1, MT2, MT3 and MT4), their relation to pathol. processes, and outlines specific applications of MTs as a set of genetic markers for certain pathologies.
- 375West, A. K.; Stallings, R.; Hildebrand, C. E.; Chiu, R.; Karin, M.; Richards, R. I. Human metallothionein genes: structure of the functional locus at 16q13. Genomics 1990, 8, 513– 518, DOI: 10.1016/0888-7543(90)90038-V375Human metallothionein genes: structure of the functional locus at 16q13West, A. K.; Stallings, R.; Hildebrand, C. E.; Chiu, R.; Karin, M.; Richards, R. I.Genomics (1990), 8 (3), 513-18CODEN: GNMCEP; ISSN:0888-7543.The functional human metallothionein (MT) genes are located on chromosome 16q13. The functional human MT locus was mapped by isolation and restriction digest mapping of cloned DNA. The mapped region contains all sequences on chromosome 16 that hybridize to metallothionein gene probes and comprises 14 tightly linked MT genes, 6 of which have not been previously described. This anal. defines the genetic limits of metallothionein functional diversity in the human genome.
- 376Stennard, F. A.; Holloway, A. F.; Hamilton, J.; West, A. K. Characterisation of six additional human metallothionein genes. Biochim. Biophys. Acta, Gene Struct. Expression 1994, 1218, 357– 365, DOI: 10.1016/0167-4781(94)90189-9376Characterization of six additional human metallothionein genesStennard, Fiona A.; Holloway, Adele F.; Hamilton, Jenny; West, Adrian K.Biochimica et Biophysica Acta, Gene Structure and Expression (1994), 1218 (3), 357-65CODEN: BBGSD5; ISSN:0167-4781. (Elsevier B.V.)Human metallothionein (MT) genes are clustered in a locus on chromosome 16, and this report presents the characterization of the remaining six uninvestigated members of the family. Nucleotide sequencing in whole or part suggested that four of these genes, MT1I, MT1J, MT1K and MT1L do not encode expressed MT proteins, based on the presence of structural faults or atypical amino acid assignments. On the other hand, the structures of MT1H and MT1X are consistent with these genes being functional and encoding unique type 1 isoforms. The promoters of both genes conferred activity to CAT expression constructs when transfected into HeLa cells, and showed differential responses to inducers of MT synthesis. Endogenous MT1H and MT1X genes were expressed at the mRNA level in HeLa cells following cadmium treatment. This work brings the no. of functional class 1 and 2 MT genes in the human to eight, and confirms that each encodes structurally unique proteins.
- 377Rao, P. S.; Jaggi, M.; Smith, D. J.; Hemstreet, G. P.; Balaji, K. C. Metallothionein 2A interacts with the kinase domain of PKCmu in prostate cancer. Biochem. Biophys. Res. Commun. 2003, 310, 1032– 1038, DOI: 10.1016/j.bbrc.2003.09.118377Metallothionein 2A interacts with the kinase domain of PKCμ in prostate cancerRao, Prema S.; Jaggi, Meena; Smith, David J.; Hemstreet, George P.; Balaji, K. C.Biochemical and Biophysical Research Communications (2003), 310 (3), 1032-1038CODEN: BBRCA9; ISSN:0006-291X. (Elsevier Science)Prostate cancer (PC) patients die from progression to androgen independence (AI) and chemoresistance (CR). Protein kinase Cμ (PKCμ) a novel member of the PKC family of signal transduction proteins is downregulated in AI PC. Studying PKCμ interactors in the yeast two-hybrid system identified metallothionein 2A (MT 2A) as an interactor of PKCμ kinase domain (KD) in PC, which was quantified by β-gal assay, confirmed in PC cells by immunopptn., and PKCμ-MT 2A co-localization in vivo by immunofluorescence studies. PKCμ domain interaction studies revealed that MT 2A interacted strongly with KD, relatively weakly with C1, and failed to interact with C2, PH or kinase mutant domains. Peptide library and in silico anal. strongly suggest that MT 2A is a novel substrate of PKCμ and our data indicate that the PKCμ-MT 2A interaction depends on PKCμ kinase activity. Because metallothioneins are assocd. with cell proliferation and CR, the PKCμ-MT 2A interaction may contribute to CR and/or AI in PC.
- 378Aras, M. A.; Hara, H.; Hartnett, K. A.; Kandler, K.; Aizenman, E. Protein kinase C regulation of neuronal zinc signaling mediates survival during preconditioning. J. Neurochem. 2009, 110, 106– 117, DOI: 10.1111/j.1471-4159.2009.06106.x378Protein kinase C regulation of neuronal zinc signaling mediates survival during preconditioningAras, Mandar A.; Hara, Hirokazu; Hartnett, Karen A.; Kandler, Karl; Aizenman, EliasJournal of Neurochemistry (2009), 110 (1), 106-117CODEN: JONRA9; ISSN:0022-3042. (Wiley-Blackwell)Sublethal activation of cell death processes initiate pro-survival signaling cascades. As intracellular Zn2+ liberation mediates neuronal death pathways, we tested whether a sublethal increase in free Zn2+ could also trigger neuroprotection. Neuronal free Zn2+ transiently increased following preconditioning, and was both necessary and sufficient for conferring excitotoxic tolerance. Lethal exposure to NMDA led to a delayed increase in Zn2+ that contributed significantly to excitotoxicity in non-preconditioned neurons, but not in tolerant neurons, unless preconditioning-induced free Zn2+ was chelated. Thus, preconditioning may trigger the expression of Zn2+-regulating processes, which, in turn, prevent subsequent Zn2+-mediated toxicity. Indeed, preconditioning increased Zn2+-regulated gene expression in neurons. Examn. of the mol. signaling mechanism leading to this early Zn2+ signal revealed a crit. role for protein kinase C (PKC) activity, suggesting that PKC may act directly on the intracellular source of Zn2+. We identified a conserved PKC phosphorylation site at serine-32 (S32) of metallothionein (MT) that was important in modulating Zn2+-regulated gene expression and conferring excitotoxic tolerance. Importantly, we obsd. increased PKC-induced serine phosphorylation in immunopurified MT1, but not in mutant MT1(S32A). These results indicate that neuronal Zn2+ serves as an important, highly regulated signaling component responsible for the initiation of a neuroprotective pathway.
- 379Merlos-Rodrigo, M. A.; Jimenez Jimemez, A. M.; Haddad, Y.; Bodoor, K.; Adam, P.; Krizkova, S.; Heger, Z.; Adam, V. Metallothionein isoforms as double agents – Their roles in carcinogenesis, cancer progression and chemoresistance. Drug Resist. Updates 2020, 52, 100691, DOI: 10.1016/j.drup.2020.100691379Metallothionein isoforms as double agents - Their roles in carcinogenesis, cancer progression and chemoresistanceMerlos Rodrigo Miguel Angel; Adam Vojtech; Jimenez Jimemez Ana Maria; Haddad Yazan; Krizkova Sona; Heger Zbynek; Bodoor Khaldon; Adam PavlinaDrug resistance updates : reviews and commentaries in antimicrobial and anticancer chemotherapy (2020), 52 (), 100691 ISSN:.Metallothioneins (MTs) are small cysteine-rich intracellular proteins with four major isoforms identified in mammals, designated MT-1 through MT-4. The best known biological functions of MTs are their ability to bind and sequester metal ions as well as their active role in redox homeostasis. Despite these protective roles, numerous studies have demonstrated that changes in MT expression could be associated with the process of carcinogenesis and participation in cell differentiation, proliferation, migration, and angiogenesis. Hence, MTs have the role of double agents, i.e., working with and against cancer. In view of their rich biochemical properties, it is not surprising that MTs participate in the emergence of chemoresistance in tumor cells. Many studies have demonstrated that MT overexpression is involved in the acquisition of resistance to anticancer drugs including cisplatin, anthracyclines, tyrosine kinase inhibitors and mitomycin. The evidence is gradually increasing for a cellular switch in MT functions, showing that they indeed have two faces: protector and saboteur. Initially, MTs display anti-oncogenic and protective roles; however, once the oncogenic process was launched, MTs are utilized by cancer cells for progression, survival, and contribution to chemoresistance. The duality of MTs can serve as a potential prognostic/diagnostic biomarker and can therefore pave the way towards the development of new cancer treatment strategies. Herein, we review and discuss MTs as tumor disease markers and describe their role in chemoresistance to distinct anticancer drugs.
- 380Maret, W. Zinc in pancreatic islet biology, insulin sensitivity, and diabetes. Prev. Nutr. Food Sci. 2017, 22, 1– 8, DOI: 10.3746/pnf.2017.22.1.1380Zinc in pancreatic islet biology, insulin sensitivity, and diabetesMaret, WolfgangPreventive Nutrition and Food Science (2017), 22 (1), 1-8CODEN: PNFSBW; ISSN:2287-1098. (Korean Society of Food Science and Nutrition)About 20 chem. elements are nutritionally essential for humans with defined mol. functions. Several essential and nonessential biometals are either functional nutrients with antidiabetic actions or can be diabetogenic. A key question remains whether changes in the metab. of biometals and biominerals are a consequence of diabetes or are involved in its etiol. Exploration of the roles of zinc (Zn) in this regard is most revealing because 80 years of scientific discoveries link zinc and diabetes. In pancreatic β- and α-cells, zinc has specific functions in the biochem. of insulin and glucagon. When zinc ions are secreted during vesicular exocytosis, they have autocrine, paracrine, and endocrine roles. The membrane protein ZnT8 transports zinc ions into the insulin and glucagon granules. ZnT8 has a risk allele that predisposes the majority of humans to developing diabetes. In target tissues, increased availability of zinc enhances the insulin response by inhibiting protein tyrosine phosphatase 1B, which controls the phosphorylation state of the insulin receptor and hence downstream signalling. Inherited diseases of zinc metab., environmental exposures that interfere with the control of cellular zinc homeostasis, and nutritional or conditioned zinc deficiency influence the pathobiochem. of diabetes. Accepting the view that zinc is one of the many factors in multiple gene-environment interactions that cause the functional demise of β-cells generates an immense potential for treating and perhaps preventing diabetes. Personalized nutrition, bioactive food, and pharmaceuticals targeting the control of cellular zinc in precision medicine are among the possible interventions.
- 381Li, X.; Cai, L.; Feng, W. Diabetes and metallothionein. Mini-Rev. Med. Chem. 2007, 7, 761– 768, DOI: 10.2174/138955707781024490381Diabetes and metallothioneinLi, Xiaokun; Cai, Lu; Feng, WenkeMini-Reviews in Medicinal Chemistry (2007), 7 (7), 761-768CODEN: MMCIAE; ISSN:1389-5575. (Bentham Science Publishers Ltd.)A review. Diabetes is a widespread disease, and its development and toxic effects on various organs were attributed to increased oxidative stress. Metallothionein (MT) is a group of intracellular metal-binding and cysteine-rich proteins, being highly inducible in many tissues. Although it mainly acts as a regulator of metal homeostasis such as zinc and copper in tissues, MT was found to be a potent antioxidant and adaptive (or stress) protein to protect cells and tissues from oxidative stress. Studies showed that zinc-induced or genetically enhanced MT synthesis in the pancreas prevented the development of spontaneous or chem.-induced diabetes. Genetically or pharmacol. enhanced MT expression in various organs including heart and kidney provided significant protection from diabetes-induced organ dysfunction such as cardiomyopathy and nephropathy. These studies suggest that MT as an adaptive protein can prevent both diabetes development and diabetic complications. This mini-review will thus briefly describe MT's biochem. features and then summarize the data on the protective effect of MT against diabetes and diabetic complications. In addn., the coordinative role of MT with zinc in the prevention of diabetes and its complications will also be discussed.
- 382Bensellam, M.; Laybutt, D. R.; Jonas, H.-C. Emerging roles of metallothioneins in beta cell pathophysiology: Beyond and above metal homeostasis and antioxidant response. Biology 2021, 10, 176, DOI: 10.3390/biology10030176382Emerging roles of metallothioneins in beta cell pathophysiology: beyond and above metal homeostasis and antioxidant responseBensellam, Mohammed; Laybutt, D. Ross; Jonas, Jean-ChristopheBiology (Basel, Switzerland) (2021), 10 (3), 176CODEN: BBSIBX; ISSN:2079-7737. (MDPI AG)Metallothioneins (MTs) are low mol. wt., cysteine-rich, metal-binding proteins whose precise biol. roles have not been fully characterized. Existing evidence implicated MTs in heavy metal detoxification, metal ion homeostasis and antioxidant defense. MTs were thus categorized as protective effectors that contribute to cellular homeostasis and survival. This view has, however, been challenged by emerging evidence in different medical fields revealing novel pathophysiol. roles of MTs, including inflammatory bowel disease, neurodegenerative disorders, carcinogenesis and diabetes. In the present focused review, we discuss the evidence for the role of MTs in pancreatic beta-cell biol. and insulin secretion. We highlight the pattern of specific isoforms of MT gene expression in rodents and human beta-cells. We then discuss the mechanisms involved in the regulation of MTs in islets under physiol. and pathol. conditions, particularly type 2 diabetes, and analyze the evidence revealing adaptive and neg. roles of MTs in beta-cells and the potential mechanisms involved. Finally, we underscore the unsettled questions in the field and propose some future research directions. Keywords: metallothionein; pancreatic beta-cel.
- 383Koh, J.-Y.; Lee, S.-J. Metallothionein-3 as a multifunctional player in the control of cellular processes and diseases. Mol. Brain 2020, 13, 116, DOI: 10.1186/s13041-020-00654-w383Metallothionein-3 as a multifunctional player in the control of cellular processes and diseasesKoh, Jae-Young; Lee, Sook-JeongMolecular Brain (2020), 13 (1), 116CODEN: MBORAO; ISSN:1756-6606. (BioMed Central Ltd.)A review. Transition metals, such as iron, copper, and zinc, play a very important role in life as the regulators of various physiochem. reactions in cells. Abnormal distribution and concn. of these metals in the body are closely assocd. with various diseases including ischemic seizure, Alzheimer's disease, diabetes, and cancer. Iron and copper are known to be mainly involved in in vivo redox reaction. Zinc controls a variety of intracellular metab. via binding to lots of proteins in cells and altering their structure and function. Metallothionein-3 (MT3) is a representative zinc binding protein predominant in the brain. Although the role of MT3 in other organs still needs to be elucidated, many reports have suggested crit. roles for the protein in the control of a variety of cellular homeostasis. Here, we review various biol. functions of MT3, focusing on different cellular mols. and diseases involving MT3 in the body.
- 384Fung, S. J.; Chung, R. S.; West, A. The role of metallothionein and astrocyte-neuron interactions in injury to the CNS. In Metallothioneins in Biochemistry and Pathology; Zatta, P., Ed.; World Scientific Publishing Co.: Singapore, 2008; pp 117– 138.There is no corresponding record for this reference.
- 385Wang, B.; Wood, I. S.; Trayhurn, P. PCR arrays identify metallthionein-3 as a highly hypoxia-inducible gene in human adipocytes. Biochem. Biophys. Res. Commun. 2008, 368, 88– 93, DOI: 10.1016/j.bbrc.2008.01.036385PCR arrays identify metallothionein-3 as a highly hypoxia-inducible gene in human adipocytesWang, Bohan; Stuart Wood, I.; Trayhurn, PaulBiochemical and Biophysical Research Communications (2008), 368 (1), 88-93CODEN: BBRCA9; ISSN:0006-291X. (Elsevier)Hypoxia-signalling pathway PCR arrays were used to examine the integrated response of human adipocytes to low O2 tension. Incubation of adipocytes in 1% O2 for 24 h resulted in no change in the expression of 63 of the 84 genes on the arrays, a redn. in expression of 9 genes (including uncoupling protein 2) and increased expression of 12 genes. Substantial increases (>10-fold) in leptin, angiopoietin-like protein 4, VEGF and GLUT-1 mRNA levels were obsd. The expression of one gene, metallothionein-3 (MT-3), was dramatically (>600-fold) and rapidly (by 60 min) increased by hypoxia. MT-3 gene expression was also substantially induced by hypoxia mimetics (CoCl2, desferrioxamine, dimethyloxalylglycine), indicating transcriptional regulation through HIF-1. Hypoxia addnl. induced MT-3 expression in preadipocytes, and MT-3 mRNA was detected in human (obese) s.c. and omental adipose tissue. MT-3 is a highly hypoxia-inducible gene in human adipocytes; the protein may protect adipocytes from hypoxic damage.
- 386Bousleiman, J.; Pinsky, A.; Ki, S.; Su, A.; Morozova, I.; Kalachikov, S.; Wiqas, A.; Silver, R.; Sever, M.; Austin, R. N. Function of metallothionein-3 in neuronal cells: Do metal ions alter expression levels of MT3?. Int. J. Mol. Sci. 2017, 18, 1133, DOI: 10.3390/ijms18061133386Function of metallothionein-3 in neuronal cells: do metal ions alter expression levels of MT3?Bousleiman, Jamie; Pinsky, Alexa; Ki, Sohee; Su, Angela; Morozova, Irina; Kalachikov, Sergey; Wiqas, Amen; Silver, Rae; Sever, Mary; Austin, Rachel NarehoodInternational Journal of Molecular Sciences (2017), 18 (6), 1133/1-1133/17CODEN: IJMCFK; ISSN:1422-0067. (MDPI AG)A study of factors proposed to affect metallothionein-3 (MT3) function was carried out to elucidate the opaque role MT3 plays in human metalloneurochem. Gene expression of Mt2 and Mt3 was examd. in tissues extd. from the dentate gyrus of mouse brains and in human neuronal cell cultures. The whole-genome gene expression anal. identified significant variations in the mRNA levels of genes assocd. with zinc homeostasis, including Mt2 and Mt3. Mt3 was found to be the most differentially expressed gene in the identified groups, pointing to the existence of a factor, not yet identified, that differentially controls Mt3 expression. To examine the expression of the human metallothioneins in neurons, mRNA levels of MT3 and MT2 were compared in BE(2)C and SH-SY5Y cell cultures treated with lead, zinc, cobalt, and lithium. MT2 was highly upregulated by Zn2+ in both cell cultures, while MT3 was not affected, and no other metal had an effect on either MT2 or MT3.
- 387Subramanian Vignesh, K.; Landero Figueroa, J. A.; Porollo, A.; Divanovic, S.; Caruso, J. A.; Deepe, G. S., Jr. IL-4 induces metallothionein3- and SLC30A4-dependent increase in intracellular Zn2+ that promotes pathogen persistence in macrophages. Cell Rep. 2016, 16, 3232– 3246, DOI: 10.1016/j.celrep.2016.08.057387IL-4 Induces Metallothionein 3- and SLC30A4-Dependent Increase in Intracellular Zn(2+) that Promotes Pathogen Persistence in MacrophagesSubramanian Vignesh Kavitha; Landero Figueroa Julio A; Caruso Joseph A; Porollo Aleksey; Divanovic Senad; Deepe George S JrCell reports (2016), 16 (12), 3232-3246 ISSN:.Alternative activation of macrophages promotes wound healing but weakens antimicrobial defenses against intracellular pathogens. The mechanisms that suppress macrophage function to create a favorable environment for pathogen growth remain elusive. We show that interleukin (IL)-4 triggers a metallothionein 3 (MT3)- and Zn exporter SLC30A4-dependent increase in the labile Zn(2+) stores in macrophages and that intracellular pathogens can exploit this increase in Zn to survive. IL-4 regulates this pathway by shuttling extracellular Zn into macrophages and by activating cathepsins that act on MT3 to release bound Zn. We show that IL-4 can modulate Zn homeostasis in both human monocytes and mice. In vivo, MT3 can repress macrophage function in an M2-polarizing environment to promote pathogen persistence. Thus, MT3 and SLC30A4 dictate the size of the labile Zn(2+) pool and promote the survival of a prototypical intracellular pathogen in M2 macrophages.
- 388Laukens, D.; Waeytens, A.; De Bleser, P.; Cuvelier, C.; De Vos, M. Human metallothionein expression under normal and pathological conditions: Mechanism of gene regulation based on in silico promoter analysis. Crit. Rev. Eukaryotic Gene Expression 2009, 19, 301– 317, DOI: 10.1615/CritRevEukarGeneExpr.v19.i4.40388Human metallothionein expression under normal and pathological conditions: mechanisms of gene regulation based on in silico promoter analysisLaukens, Debby; Waeytens, Anouk; De Bleser, Pieter; Cuvelier, Claude; De Vos, MartineCritical Reviews in Eukaryotic Gene Expression (2009), 19 (4), 301-317CODEN: CRGEEJ; ISSN:1045-4403. (Begell House, Inc.)A review. Metallothioneins (MTs) are ubiquitous metal-binding proteins that have been highly conserved throughout evolution. Although their physiol. function is not completely understood, they are involved in diverse processes including metal homeostasis and detoxification, the oxidative stress response, inflammation, and cell proliferation. The human MT gene family consists of at least 18 isoforms, contg. pseudogenes as well as genes encoding functional proteins. Most of the MT isoforms can be induced by a wide variety of substances, such as metals, cytokines, and hormones. Different cell types express discrete MT isoforms, which reflects the specifically adapted functions of MTs and a divergence in their regulation. The aberrant expression of MTs has been described in a no. of diseases, including Crohn's disease, cancer, Alzheimer's disease, amyotrophic lateral sclerosis, Menkes disease, and Wilson's disease. Therefore, a thorough understanding of MT gene regulation is imperative. To date, the transcriptional regulation of MTs has primarily been studied in mice. While only four murine MT isoforms exist, the homol. between murine and human MTs allows for the evaluation of the regulatory regions in their resp. promoters. Here, we review the aberrant expression of MTs in human diseases and the mechanisms that regulate MT1 expression based on an in silico evaluation of transcription factor binding sites.
- 389Laity, J. H.; Andrews, G. K. Understanding the mechanisms of zinc-sensing by metal-response element binding transcription factor-1 (MTF-1). Arch. Biochem. Biophys. 2007, 463, 201– 210, DOI: 10.1016/j.abb.2007.03.019389Understanding the mechanisms of zinc-sensing by metal-response element binding transcription factor-1 (MTF-1)Laity, John H.; Andrews, Glen K.Archives of Biochemistry and Biophysics (2007), 463 (2), 201-210CODEN: ABBIA4; ISSN:0003-9861. (Elsevier)A review. The regulation of Zn2+ has been obsd. in a wide range of organisms. Since this metal is an essential nutrient, but also toxic in excess, Zn2+ homeostasis is crucial for normal cellular functioning. Metal-responsive-element-binding transcription factor-1 (MTF-1) is a key regulator of Zn2+ in higher eukaryotes ranging from insects to mammals. MTF-1 controls the expression of metallothioneins (MTs) and a no. of other genes directly involved in the intracellular sequestration and transport of Zn2+. Although the diverse functions of MTF-1 extend well beyond Zn2+ homeostasis to include stress-responses to heavy metal toxicity, oxidative stress, and selected chem. agents, in this review the authors focus on recent advances in understanding the mechanisms whereby MTF-1 regulates MT gene expression to protect the cell from fluctuations in environmental Zn2+. Particular emphasis is devoted to recent studies involving the Cys2His2 zinc finger DNA-binding domain of MTF-1, which is an important contributor to the zinc-sensing and metal-dependent transcriptional activation functions of this protein.
- 390Günther, V.; Lindert, U.; Schaffner, W. The taste of heavy metals: gene regulation by MTF-1. Biochim. Biophys. Acta, Mol. Cell Res. 2012, 1823, 1416– 1425, DOI: 10.1016/j.bbamcr.2012.01.005There is no corresponding record for this reference.
- 391Zhang, B.; Georgiev, O.; Hagmann, M.; Günes, C.; Cramer, M.; Faller, P.; Vašák, M.; Schaffner, W. Activity of metal-responsive transcription factor 1 by toxic heavy metals and H2O2 in vitro is modulated by metallothionein. Mol. Cell. Biol. 2003, 23, 8471– 8485, DOI: 10.1128/MCB.23.23.8471-8485.2003391Activity of metal-responsive transcription factor 1 by toxic heavy metals and H2O2 in vitro is modulated by metallothioneinZhang, Bo; Georgiev, Oleg; Hagmann, Michael; Guenes, Cagatay; Cramer, Mirjam; Faller, Peter; Vasak, Milan; Schaffner, WalterMolecular and Cellular Biology (2003), 23 (23), 8471-8485CODEN: MCEBD4; ISSN:0270-7306. (American Society for Microbiology)Metallothioneins are small, cysteine-rich proteins that avidly bind heavy metals such as zinc, copper, and cadmium to reduce their concn. to a physiol. or nontoxic level. Metallothionein gene transcription is induced by several stimuli, notably heavy metal load and oxidative stress. Transcriptional induction of metallothionein genes is mediated by the metal-responsive transcription factor 1 (MTF-1), an essential zinc finger protein that binds to specific DNA motifs termed metal-response elements. In cell-free DNA binding reactions with nuclear exts., MTF-1 requires elevated zinc concns. for efficient DNA binding but paradoxically is inactivated by other in vivo inducers such as cadmium, copper, and hydrogen peroxide. Here the authors have developed a cell-free, MTF-1-dependent transcription system which accurately reproduces the activation of metallothionein gene promoters not only by zinc but also by these other inducers. Whereas transcriptional induction by zinc can be achieved by elevated zinc concn. alone, induction by cadmium, copper, or H2O2 addnl. requires the presence of zinc-satd. metallothionein. This is explained by the preferential binding of cadmium or copper to metallothionein or its oxidn. by H2O2; the concomitant release of zinc in turn leads to the activation of transcription factor MTF-1. Conversely, thionein, the metal-free form of metallothionein, inhibits activation of MTF-1. The release of zinc from cellular components, including metallothioneins, and the sequestration of zinc by newly produced apometallothionein might be a basic mechanism to regulate MTF-1 activity upon cellular stress.
- 392Lichten, L. A.; Ryu, M.-S.; Guo, L.; Embury, J.; Cousins, R. J. MTF-1-mediated repression of the zinc transporter Zip10 is alleviated by zinc restriction. PLoS One 2011, 6, e21526 DOI: 10.1371/journal.pone.0021526392MTF-1-mediated repression of the zinc transporter Zip10 is alleviated by zinc restrictionLichten, Louis A.; Ryu, Moon-Suhn; Guo, Liang; Embury, Jennifer; Cousins, Robert J.PLoS One (2011), 6 (6), e21526CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)The regulation of cellular zinc uptake is a key process in the overall mechanism governing mammalian zinc homeostasis and how zinc participates in cellular functions. We analyzed the zinc transporters of the Zip family in both the brain and liver of zinc-deficient animals and found a large, significant increase in Zip10 expression. Addnl., Zip10 expression decreased in response to zinc repletion. Moreover, isolated mouse hepatocytes, AML12 hepatocytes, and Neuro 2A cells also respond differentially to zinc availability in vitro. Measurement of Zip10 hnRNA and actinomycin D inhibition studies indicate that Zip10 was transcriptionally regulated by zinc deficiency. Through luciferase promoter constructs and ChIP anal., binding of MTF-1 to a metal response element located 17 bp downstream of the transcription start site was shown to be necessary for zinc-induced repression of Zip10. Furthermore, zinc-activated MTF-1 causes down-regulation of Zip10 transcription by phys. blocking Pol II movement through the gene. Lastly, ZIP10 is localized to the plasma membrane of hepatocytes and neuro 2A cells. Collectively, these results reveal a novel repressive role for MTF-1 in the regulation of the Zip10 zinc transporter expression by pausing Pol II transcription. ZIP10 may have roles in control of zinc homeostasis in specific sites particularly those of the brain and liver. Within that context ZIP10 may act as an important survival mechanism during periods of zinc inadequacy.
- 393Suhy, D. A.; Simon, K. D.; Linzer, D. I.; O’Halloran, T. V. Metallothionein is part of a zinc-scavenging mechanism for cell survival under conditions of extreme zinc deprivation. J. Biol. Chem. 1999, 274, 9183– 9192, DOI: 10.1074/jbc.274.14.9183393Metallothionein is part of a zinc-scavenging mechanism for cell survival under conditions of extreme zinc deprivationSuhy, David A.; Simon, Kathryn D.; Linzer, Daniel I. H.; O'Halloran, Thomas V.Journal of Biological Chemistry (1999), 274 (14), 9183-9192CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)Metallothionein (MT) is a small cysteine-rich protein thought to play a crit. role in cellular detoxification of inorg. species by sequestering metal ions that are present in elevated concns. We demonstrate here that metallothionein can play an important role at the other end of the homeostatic spectrum by scavenging an essential metal in a mouse fibroblast cell line that has been cultured under conditions of extreme zinc deprivation (LZA-LTK-). These cells unexpectedly produce constitutively high levels of metallothionein mRNA; however, the MT protein accumulates only when high concns. of zinc are provided in the media. Until this MT pool is satd., no measurable zinc remains in the external media. In this case, zinc deprivation leads to amplification of the MT gene locus in the LZA-LTK- cell line. Furthermore, the intracellular zinc levels in the fully adapted cells remain at the normal level of 0.4 fmol zinc/cell, even when extracellular zinc concn. is decreased by 2 orders of magnitude relative to normal media.
- 394Karin, M.; Andersen, R. D.; Slater, E.; Smith, K.; Herschman, H. R. Metallothionein mRNA induction in HeLa cells in response to zinc or dexamethasone is a primary induction response. Nature 1980, 286, 295– 297, DOI: 10.1038/286295a0394Metallothionein mRNA induction in HeLa cells in response to zinc or dexamethasone is a primary induction responseKarin, Michael; Andersen, Robert D.; Slater, Emily; Smith, Karen; Herschman, Harvey R.Nature (London, United Kingdom) (1980), 286 (5770), 295-7CODEN: NATUAS; ISSN:0028-0836.Induction of translatable metallothionein (MT) mRNA in HeLa cells by either Zn2+ or dexamethasone [50-02-2] was independent of concomitant protein synthesis but not RNA synthesis. Elevation of MT mRNA by 10-7M dexamethasone or 3.7 × 10-5M Zn2+, 5- or 6-fold, resp., was prevented by actinomycin D (1 μg/mL), which also lowered MT mRNA levels relative to control cultures by 50-60%. Variable cycloheximide enhancement of MT synthesis (40-100%) was obsd. in Zn-treated and control cultures but not in dexamethasone-induced cultures. In contrast to dexamethasone induction of MT mRNA, dexamethasone stimulation of Zn2+ transport was blocked by cycloheximide. MT induction by both dexamethasone and Zn2+ was, therefore, a primary induction response.
- 395Murphy, B. J.; Laderoute, K. R.; Chin, R. J.; Sutherland, R. M. Metallothionein IIA is up-regulated by hypoxia in human A431 squamous carcinoma cells. Cancer Res. 1994, 54, 5808– 5810395Metallothionein IIA is up-regulated by hypoxia in human A431 squamous carcinoma cellsMurphy, Brian J.; Laderoute, Keith R.; Chin, Roxanne J.; Sutherland, Robert M.Cancer Research (1994), 54 (22), 5808-10CODEN: CNREA8; ISSN:0008-5472. (American Association for Cancer Research)The expression of metallothionein IIA (MT-IIA) was investigated in A431 human squamous carcinoma cells exposed to hypoxia (pO2 ≤ 0.01% of atm. pO2) and subsequent reoxygenation. Northern anal. showed that MT-IIA mRNA levels were significantly increased during 14 h of hypoxia and during reoxygenation. Western blotting confirmed that total MT protein levels were also increased in response to these stresses. Evidence of the transcriptional control of MT-IIA expression in hypoxic and in reoxygenated A431 cells was found using a 0.2-kilobase sequence of the proximal 5'-regulatory region of the MT-IIA gene in a chloramphenicol acetyltransferase reporter gene construct. Thus the proximal promoter of the human MT-IIA gene appears to contain a hypoxic response elements(s). These observations indicate that MT-IIA may have an important role in the stress responses of cells in solid tumors.
- 396Murphy, B. J.; Kimura, T.; Sato, B. G.; Shi, Y.; Andrews, G. K. Metallothionein induction by hypoxia involves cooperative interactions between metal-responsive transcription factor-1 and hypoxia-inducible transcription factor-1α. Mol. Cancer Res. 2008, 6, 483– 490, DOI: 10.1158/1541-7786.MCR-07-0341396Metallothionein Induction by Hypoxia Involves Cooperative Interactions between Metal-Responsive Transcription Factor-1 and Hypoxia-Inducible Transcription Factor-1αMurphy, Brian J.; Kimura, Tomoki; Sato, Barbara G.; Shi, Yihui; Andrews, Glen K.Molecular Cancer Research (2008), 6 (3), 483-490CODEN: MCROC5; ISSN:1541-7786. (American Association for Cancer Research)Mammalian metallothionein (MT) genes are transcriptionally activated by the essential metal zinc as well as by environmental stresses, including toxic metal overload and redox fluctuations. In addn. to playing a key role in zinc homeostasis, MT proteins can protect against metal- and oxidant-induced cellular damage, and may participate in other fundamental physiol. and pathol. processes such as cell survival, proliferation, and neoplasia. Previously, our group reported a requirement for metal-responsive transcription factor-1 (MTF-1) in hypoxia-induced transcription of mouse MT-I and human MT-IIA genes. Here, we provide evidence that the protumorigenic hypoxia-inducible transcription factor-1α (HIF-1α) is essential for induction of MT-1 by hypoxia, but not zinc. Chromatin immunopptn. assays revealed that MTF-1 and HIF-1α are both recruited to the mouse MT-I promoter in response to hypoxia, but not zinc. In the absence of HIF-1α, MTF-1 is recruited to the MT-I promoter but fails to activate MT-I gene expression in response to hypoxia. Thus, HIF-1α seems to function as a coactivator of MT-I gene transcription by interacting with MTF-1 during hypoxia. Coimmunopptn. studies suggest interaction between MTF-1 and HIF-1α, either directly or as mediated by other factors. It is proposed that assocn. of these important transcription factors in a multi-protein complex represents a common strategy to control unique sets of hypoxia-inducible genes in both normal and diseased tissue.
- 397Kojima, I.; Tanaka, T.; Inagi, R.; Nishi, H.; Aburatani, H.; Kato, H.; Miyata, T.; Fujita, T.; Nangaku, M. Metallothionein is upregulated by hypoxia and stabilizes hypoxia-inducible factor in the kidney. Kidney Int. 2009, 75, 268– 277, DOI: 10.1038/ki.2008.488397Metallothionein is upregulated by hypoxia and stabilizes hypoxia-inducible factor in the kidneyKojima, Ichiro; Tanaka, Tetsuhiro; Inagi, Reiko; Nishi, Hiroshi; Aburatani, Hiroyuki; Kato, Hideki; Miyata, Toshio; Fujita, Toshiro; Nangaku, MasaomiKidney International (2009), 75 (3), 268-277CODEN: KDYIA5; ISSN:0085-2538. (Nature Publishing Group)Recent studies underscore that chronic hypoxia in the tubulointerstitium is a final common pathway to progression to end-stage renal failure regardless of etiol. We used microarray anal. of rat kidneys made hypoxic by unilateral renal artery stenosis to measure transcriptomic events and clarify pathophysiol. mechanisms of renal injury induced by chronic hypoxia. Many genes were upregulated in the kidney by chronic hypoxia, but we focused on metallothionein due to its antioxidative properties. Using tubular epithelial cells transfected with a reporter construct of luciferase, driven by the hypoxia-responsive elements (HRE), we found that addn. of metallothionein to the culture media increased luciferase activity. This was assocd. with upregulation of the target genes of hypoxia-inducible factor (HIF), such as vascular endothelial growth factor and glucose transporter-1. Stimulation of the HIF-HRE pathway by metallothionein was confirmed by metallothionein overexpression. Hypoxia and exogenous metallothionein increased HIF-1α protein without changes in its mRNA levels, suggesting protein stabilization. Upregulation of the HIF-HRE system by metallothionein was assocd. with phosphorylation of ERK but not Akt. MEK inhibition and rapamycin decreased metallothionein-induced HIF activity. Our study shows that upregulation of metallothionein expression by hypoxia activates the HIF-HRE system through the ERK/mTOR pathway and may be a novel defense against hypoxia. Kidney International (2009) 75, 268-277. doi:10.1038/ki.2008.488.
- 398Sato, S.; Shirakawa, H.; Tomita, S.; Tohkin, M.; Gonzalez, F. J.; Komai, M. The aryl hydrocarbon receptor and glucocorticoid receptor interact to activate human metallothionein 2A. Toxicol. Appl. Pharmacol. 2013, 273, 90– 99, DOI: 10.1016/j.taap.2013.08.017398The aryl hydrocarbon receptor and glucocorticoid receptor interact to activate human metallothionein 2ASato, Shoko; Shirakawa, Hitoshi; Tomita, Shuhei; Tohkin, Masahiro; Gonzalez, Frank J.; Komai, MichioToxicology and Applied Pharmacology (2013), 273 (1), 90-99CODEN: TXAPA9; ISSN:0041-008X. (Elsevier Inc.)Although the aryl hydrocarbon receptor (AHR) and glucocorticoid receptor (GR) play essential roles in mammalian development, stress responses, and other physiol. events, crosstalk between these receptors has been the subject of much debate. Metallothioneins are classic glucocorticoid-inducible genes that were reported to increase upon treatment with AHR agonists in rodent tissues and cultured human cells. In this study, the mechanism of human metallothionein 2A (MT2A) gene transcription activation by AHR was investigated. Cotreatment with 3-methylcholanthrene and dexamethasone, agonists of AHR and GR resp., synergistically increased MT2A mRNA levels in HepG2 cells. MT2A induction was suppressed by RNA interference against AHR or GR. Coimmunopptn. expts. revealed a phys. interaction between AHR and GR proteins. Moreover, chromatin immunopptn. assays indicated that AHR was recruited to the glucocorticoid response element in the MT2A promoter. Thus, we provide a novel mechanism whereby AHR modulates expression of human MT2A via the glucocorticoid response element and protein-protein interactions with GR.
- 399Rasinger, J. D.; Carroll, T. S.; Lundebye, A. K.; Hogstrand, C. Cross-omics gene and protein expression profiling in juvenile female mice highlights disruption of calcium and zinc signalling in the brain following dietary exposure to CB-153, BDE-47, HBCD or TCDD. Toxicology 2014, 321, 1– 12, DOI: 10.1016/j.tox.2014.03.006399Cross-omics gene and protein expression profiling in juvenile female mice highlights disruption of calcium and zinc signalling in the brain following dietary exposure to CB-153, BDE-47, HBCD or TCDDRasinger, J. D.; Carroll, T. S.; Lundebye, A. K.; Hogstrand, C.Toxicology (2014), 321 (), 1-12CODEN: TXCYAC; ISSN:0300-483X. (Elsevier Ltd.)The present study assessed if eating a diet of fish, spiked with persistent org. pollutants (POPs), affects gene and protein expression in the maturing mouse brain. Juvenile female Balb/c mice (22 days of age) were exposed for 28 days to fish-based diets spiked with the dioxin 2,3,7,8-tetrachlorodibenzodioxin (TCDD) or the non dioxin-like (NDL) chems. hexabromocyclodocecane (HBCD), 2,2'4,4'-tetrabromodiphenylether (BDE-47) or 2,2'4,4',5,5'-hexachlorobiphenyl (CB-153) at doses approximating their resp. lowest obsd. adverse effect levels (LOAEL). It was found that all POPs elicited changes in neural gene and protein expression profiles. Bioinformatic anal. of gene expression data highlighted the importance of the aryl hydrocarbon receptor (AHR) in dioxin toxicity and revealed that zinc regulation in the brain is targeted by TCDD through the AHR. Calcium homeostasis was affected by both TCDD and the NDL chems. In contrast to the transcriptomic anal., the proteomics data did not allow for a clear distinction between DL and NDL responses in the juvenile brain but indicated that proteins assocd. with excitotoxicity were affected in all exposure groups. Integrated interpretation of data led to the conclusion that the dietary contaminants investigated in the present study breach the blood brain barrier (BBB) and accumulate in the juvenile brain where they may induce excitotoxic insults by dysregulation of the otherwise tightly controlled homeostasis of calcium and zinc. Overall, the findings of the present study highlight the need for further assessment of the risks assocd. with early life exposure to foodborne POPs.
- 400Dalton, T.; Palmiter, R. D.; Andrews, G. K. Transcriptional induction of the mouse metallothionein-I gene in hydrogen peroxide-treated Hepa cells involves a composite major late transcription factor/antioxidant response element and metal response promoter elements. Nucleic Acids Res. 1994, 22, 5016– 5023, DOI: 10.1093/nar/22.23.5016400Transcriptional induction of the mouse metallothionein-I gene in hydrogen peroxide-treated Hepa cells involves a composite major late transcription factor/antioxidant response element and metal response promoter elementsDalton, Tim; Palmiter, Richard D.; Andrews, Glen K.Nucleic Acids Research (1994), 22 (23), 5016-23CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)Synthesis of metallothionein-I (MT-I) and heme oxygenase mRNAs is rapidly and transiently induced by H2O2 in mouse hepatomna cells (Hepa) and this effect is blocked by catalase. Menadione, which generates free radicals, also induces these mRNAs. Deletion mutagenesis revealed that a region between -42 and -153 in the mouse MT-I promoter was essential for induction of a CAT reporter gene. A multimer of a 16 bp sequence (-101 to -86) that includes an antioxidant response element and overlapping adenovirus major late transcription factor binding site elevated basal expression and allowed induction by H2O2 when inserted upstream of a minimal promoter. However, deletion of this region (-100 to -89) from the intact MT-I promoter (-153) did not completely eliminate response. Multiple copies of a metal response element also permitted response to H2O2. These results suggest that induction of MT-I gene transcription by H2O2 is mediated by at least two different elements within the proximal MT-I gene promoter and suggest a previously undescribed function of the MRE. Induction of MT gene transcription by ROS and the subsequent scavenging of ROS by the MT peptide is reminiscent of the metal regulatory loop and is consistent with the hypothesized protective functions of MT.
- 401Abdel-Mageed, A. B.; Agrawal, K. C. Activation of nuclear factor kappaB: potential role in metallothioinein-mediated mitogenic response. Cancer Res. 1998, 58, 2335– 2338401Activation of nuclear factor κB: potential role in metallothionein-mediated mitogenic responseAbdel-Mageed, Asim B.; Agrawal, Krishna C.Cancer Research (1998), 58 (11), 2335-2338CODEN: CNREA8; ISSN:0008-5472. (American Association for Cancer Research)The antiapoptotic response and enhanced cellular proliferation obsd. in neoplastic cells on overexpression of metallothionein (MT) have been well documented. We have investigated the mechanisms assocd. with this phenomenon by using MT inducers that increased MT transcripts and stimulated growth in MCF-7 cells. A MT antisense phosphorothioate oligonucleotide inhibited growth induction by >50%, suggesting a potential role of MT in mediating the mitogenic effects of these agents. Mobility shift assays using oligonucleotides encompassing the consensus nuclear factor κB (NFκB) binding site and anti-MT antibody revealed activation and a specific interaction of NFκB with MT. Cotransfection expts. using expression and reporter constructs demonstrated that MT caused transactivation of NFκB. Gel shift assays using purified proteins showed a specific interaction between MT and the p50 subunit of NFκB. These data indicate that MT may be involved in the interaction of NFκB with the DNA-binding domain and further suggest a potential role for NFκB in mediating the antiapoptotic effects of MT.
- 402Cramer, M.; Nagy, I.; Murphy, B. J.; Gassmann, M.; Hottiger, M. O.; Georgiev, O.; Schaffner, W. NF-kappaB contributes to transcription of placenta growth factor and interacts with metal-response transcription factor-1 in hypoxic animal cells. Biol. Chem. 2005, 386, 865– 872, DOI: 10.1515/BC.2005.101402NF-κB contributes to transcription of placenta growth factor and interacts with metal responsive transcription factor-1 in hypoxic human cellsCramer, Mirjam; Nagy, Ivana; Murphy, Brian J.; Gassmann, Max; Hottiger, Michael O.; Georgiev, Oleg; Schaffner, WalterBiological Chemistry (2005), 386 (9), 865-872CODEN: BICHF3; ISSN:1431-6730. (Walter de Gruyter GmbH & Co. KG)Placenta growth factor (PIGF) is a member of the vascular endothelial growth factor family of cytokines that control vascular and lymphatic endothelium development. It has been implicated in promoting angiogenesis in pathol. conditions via signaling to vascular endothelial growth factor receptor-1. PIGF expression is induced by hypoxia and proinflammatory stimuli. Metal responsive transcription factor 1 (MTF-1) was shown to take part in the hypoxic induction of PIGF in Ras-transformed mouse embryonic fibroblasts. Here we report that PIGF expression is also controlled by NF-κB. We identified several putative binding sites for NF-κB in the PIGF promoter/enhancer region by sequence analyses, and show binding and transcriptional activity of NF-κB p65 at these sites. Expression of NF-κB p65 from a plasmid vector in HEK293 cells caused a substantial increase of PIGF transcript levels. Furthermore, we found that hypoxic conditions induce nuclear translocation and interaction of MTF-1 and NF-κB p65 proteins, suggesting a role for this complex in hypoxia-induced transcription of PIGF.
- 403Maret, W. Zinc and Human Disease. In Interrelations between essential metal ions and human diseases; Metal Ions in Life Sciences; Sigel, A., Sigel, H., Sigel, R. K. O., Eds.; Springer Science + Business Media B.V.: Dordrecht, The Netherlands, 2013; Vol. 13, pp 389– 414.There is no corresponding record for this reference.
- 404Butcher, H. L.; Kennette, W. A.; Collins, O.; Zalups, R. K.; Koropatnick, J. Metallothionein mediates the level and activity of nuclear factor kappa B in murine fibroblasts. J. Pharmacol. Exp. Ther. 2004, 310, 589– 598, DOI: 10.1124/jpet.104.066126404Metallothionein mediates the level and activity of nuclear factor κB in murine fibroblastsButcher, Heather L.; Kennette, Wendy A.; Collins, Olga; Zalups, Rudolfs K.; Koropatnick, JamesJournal of Pharmacology and Experimental Therapeutics (2004), 310 (2), 589-598CODEN: JPETAB; ISSN:0022-3565. (American Society for Pharmacology and Experimental Therapeutics)The zinc-binding protein metallothionein (MT) is assocd. with resistance to apoptosis. We examd. whether MT regulates the zinc-dependent antiapoptotic transcription factor nuclear factor κB (NF-κB), which is up-regulated under many conditions that lead to elevated MT expression. NF-κB protein levels and NF-κB-dependent reporter gene activity were examd. in clonal MT(+) (MT-WT) and MT(-) (MT-KO) fibroblastic cell lines. The amt. of cellular NF-κB p65 protein in MT-KO was less than 20% of the amt. in MT-WT cells, in accord with increased sensitivity of MT-KO cells to apoptosis. NF-κB p65 mRNA levels, and NF-κB p50 subunit and IκBα protein levels, were unchanged. NF-κB activity assessed by expression of a transfected NF-κB reporter construct was less than half that obsd. in MT-KO cells. Decreased nuclear localization of NF-κB p65 in MT-KO clones was not responsible for differences in activity. In fact, MT-KO cells had higher nuclear levels of NF-κB p65 than did MT-WT cells, despite a lower cellular NF-κB level and function, suggesting that metallothionein mediated the specific activity of NF-κB. Reconstitution of MT by stable incorporation of an MT-1 expression vector in MT-KO cells resulted in increased NF-κB p65 (but not IκBα or NF-κB p50), increased NF-κB-dependent reporter activity, and increased resistance to apoptosis. These data support the hypothesis that metallothionein pos. regulates the cellular level and activity of NF-κB.
- 405Blanden, A. R.; Yu, X.; Blayney, A. J.; Demas, C.; Ha, J.-H.; Liu, Y.; Withers, T.; Carpizo, D. R.; Loh, S. N. Zinc shapes the folding landscape of p53 and established a pathway for reactivating structurally diverse cancer mutants. eLife 2020, 9, e61487 DOI: 10.7554/eLife.61487405Zinc shapes the folding landscape of p53 and establishes a pathway for reactivating structurally diverse cancer mutantsBlanden, Adam R.; Yu, Xin; Blayney, Alan J.; Demas, Christopher; Ha, Jeung-Hoi; Liu, Yue; Withers, Tracy; Carpizo, Darren R.; Loh, Stewart N.eLife (2020), 9 (), e61487CODEN: ELIFA8; ISSN:2050-084X. (eLife Sciences Publications Ltd.)Missense mutations in the p53 DNA-binding domain (DBD) contribute to half of new cancer cases annually. Here we present a thermodn. model that quantifies and links the major pathways by which mutations inactivate p53. We find that DBD possesses two unusual properties-one of the highest zinc affinities of any eukaryotic protein and extreme instability in the absence of zinc-which are predicted to poise p53 on the cusp of folding/unfolding in the cell, with a major determinant being available zinc concn. We analyze the 20 most common tumorigenic p53 mutations and find that 80% impair zinc affinity, thermodn. stability, or both. Biophys., cellbased, and murine xenograft expts. demonstrate that a synthetic zinc metallochaperone rescues not only mutations that decrease zinc affinity, but also mutations that destabilize DBD without impairing zinc binding. The results suggest that zinc metallochaperones have the capability to treat 120,500 patients annually in the U.S.
- 406Meplan, C.; Richard, M. J.; Hainaut, P. Metalloregulation of the tumor suppressor protein p53: zinc mediates the renaturation of p53 after exposure to metal chelators in vitro and in intact cells. Oncogene 2000, 19, 5227– 5236, DOI: 10.1038/sj.onc.1203907406Metalloregulation of the tumor suppressor protein p53: zinc mediates the renaturation of p53 after exposure to metal chelators in vitro and in intact cellsMeplan, Catherine; Richard, Marie-Jeanne; Hainaut, PierreOncogene (2000), 19 (46), 5227-5236CODEN: ONCNES; ISSN:0950-9232. (Nature Publishing Group)The tumor suppressor p53 is a transcription factor which binds DNA through a structurally complex domain stabilized by a zinc atom. Zinc chelation disrupts the architecture of this domain, inducing the protein to adopt an immunol. phenotype identical to that of many mutant forms of p53. In this report, we used 65Zn to show that incorporation of zinc within the protein was required for folding in the "wild-type" conformation capable of specific DNA-binding. Using a cellular assay, we show that addn. of extracellular zinc at concns. within the physiol. range (5 μM) was required for renaturation and reactivation of wild-type p53. Among other divalent metals tested (Cd2+, Cu2+, Co2+, Fe2+ and Ni2+), only Co2+ at 125 μM had a similar effect. Recombinant metallothionein (MT), a metal chelator protein, was found to modulate p53 conformation in vitro. In cultured cells, overexpression of MT by transfection could modulate p53 transcriptional activity. Taken together, these results suggest that zinc binding plays a regulatory role in the control of p53 folding and DNA-binding activity.
- 407Ostrakhovitch, E. A.; Olsson, P.-E.; Jiang, S.; Cherian, M. G. Interaction of metallothionein with tumor suppressor p53 protein. FEBS Lett. 2006, 580, 1235– 1238, DOI: 10.1016/j.febslet.2006.01.036407Interaction of metallothionein with tumor suppressor p53 proteinOstrakhovitch, Elena A.; Olsson, Per-Erik; Jiang, Sean; Cherian, M. GeorgeFEBS Letters (2006), 580 (5), 1235-1238CODEN: FEBLAL; ISSN:0014-5793. (Elsevier B.V.)Previous reports have shown that metallothionein (MT) may modulate p53 protein activity through Zn2+ exchange. However, little is known about a direct interaction between MT and p53 in cells. Here, the results demonstrated that an interaction between MT and p53 could occur in vitro. The complex between MT and p53 was obsd. in breast cancer epithelial cells with both wild-type (wt) and inactive type of p53. Furthermore, it was shown that wt-p53 was preferentially assocd. with apo-MT. The data suggested that co-expression of MT and p53 and their complex formation in tumor cells may be involved in regulation of apoptosis in these cells.
- 408Lee, B. M.; Buck-Koehntop, B. A.; Martinez-Yamout, M. A.; Dyson, H. J.; Wright, P. E. Embryonic neural inducing factor churchill is not a DNA-binding zinc finger protein: solution structure reveals a solvent-exposed beta-sheet and zinc binuclear cluster. J. Mol. Biol. 2007, 371, 1274– 1289, DOI: 10.1016/j.jmb.2007.06.021408Embryonic Neural Inducing Factor Churchill Is not a DNA-binding Zinc Finger Protein: Solution Structure Reveals a Solvent-exposed β-Sheet and Zinc Binuclear ClusterLee, Brian M.; Buck-Koehntop, Bethany A.; Martinez-Yamout, Maria A.; Dyson, H. Jane; Wright, Peter E.Journal of Molecular Biology (2007), 371 (5), 1274-1289CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Ltd.)Churchill is a zinc-contg. protein that is involved in neural induction during embryogenesis. At the time of its discovery, it was thought on the basis of sequence alignment to contain two zinc fingers of the C4 type. Further, binding of an N-terminal GST-Churchill fusion protein to a particular DNA sequence was demonstrated by immunopptn. selection assay, suggesting that Churchill may function as a transcriptional regulator by sequence-specific DNA binding. We show by NMR soln. structure detn. that, far from contg. canonical C4 zinc fingers, the protein contains three bound zinc ions in novel coordination sites, including an unusual binuclear zinc cluster. The secondary structure of Churchill is also unusual, consisting of a highly solvent-exposed single-layer β-sheet. Hydrogen-deuterium exchange and backbone relaxation measurements reveal that Churchill is unusually dynamic on a no. of time scales, with the exception of regions surrounding the zinc coordinating sites, which serve to stabilize the otherwise unstructured N terminus and the single-layer β-sheet. No binding of Churchill to the previously identified DNA sequence could be detected, and extensive searches using DNA sequence selection techniques could find no other DNA sequence that was bound by Churchill. Since the N-terminal amino acids of Churchill form part of the zinc-binding motif, the addn. of a fusion protein at the N terminus causes loss of zinc and unfolding of Churchill. This observation most likely explains the published DNA-binding results, which would arise due to non-specific interaction of the unfolded protein in the immunopptn. selection assay. Since Churchill does not appear to bind DNA, we suggest that it may function in embryogenesis as a protein-interaction factor.
- 409Bellon, S. F.; Rodgers, K. K.; Schatz, D. G.; Coleman, J. E.; Steitz, T. A. Crystal structure of the RAG1 dimerization domain reveals multiple zinc-binding motifs including a novel zinc binuclear cluster. Nat. Struct. Biol. 1997, 4, 586– 591, DOI: 10.1038/nsb0797-586409Crystal structure of the RAG1 dimerization domain reveals multiple zinc-binding motifs including a novel zinc binuclear clusterBellon, Steven F.; Rodgers, Karla K.; Schatz, David G.; Coleman, Joseph E.; Steitz, Thomas A.Nature Structural Biology (1997), 4 (7), 586-591CODEN: NSBIEW; ISSN:1072-8368. (Nature America)The crystal structure of the dimerization domain of the V(D)J recombination-activating protein, RAG1, was solved using zinc anomalous scattering. The structure reveals an unusual combination of multi-class zinc-binding motifs, including a zinc RING finger and a C2H2 zinc finger, that together form a single structural domain. The domain also contains a unique zinc binuclear cluster in place of normally mononuclear zinc site in the RING finger. Together, four zinc ions help organize the entire domain, including the two helixes that form the dimer interface.
- 410Wu, H.; Min, J.; Lunin, V. V.; Antoshenko, T.; Dombrovski, L.; Zeng, H.; Allali-Hassani, A.; Campagna-Slater, V.; Vedadi, M.; Arrowsmith, C. H.; Plotnikov, A. N.; Schapira, M. Structural biology of human H3K9 methyltransferases. PLoS One 2010, 5, e8570 DOI: 10.1371/journal.pone.0008570410Structural biology of human H3K9 methyltransferasesWu Hong; Min Jinrong; Lunin Vladimir V; Antoshenko Tatiana; Dombrovski Ludmila; Zeng Hong; Allali-Hassani Abdellah; Campagna-Slater Valerie; Vedadi Masoud; Arrowsmith Cheryl H; Plotnikov Alexander N; Schapira MatthieuPloS one (2010), 5 (1), e8570 ISSN:.SET domain methyltransferases deposit methyl marks on specific histone tail lysine residues and play a major role in epigenetic regulation of gene transcription. We solved the structures of the catalytic domains of GLP, G9a, Suv39H2 and PRDM2, four of the eight known human H3K9 methyltransferases in their apo conformation or in complex with the methyl donating cofactor, and peptide substrates. We analyzed the structural determinants for methylation state specificity, and designed a G9a mutant able to tri-methylate H3K9. We show that the I-SET domain acts as a rigid docking platform, while induced-fit of the Post-SET domain is necessary to achieve a catalytically competent conformation. We also propose a model where long-range electrostatics bring enzyme and histone substrate together, while the presence of an arginine upstream of the target lysine is critical for binding and specificity. ENHANCED VERSION: This article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the web plugin are available in Text S1.
- 411Takahashi, S. Positive and negative regulators of the metallothionein gene. Mol. Med. Rep. 2015, 12, 795– 799, DOI: 10.3892/mmr.2015.3459411Positive and negative regulators of the metallothionein gene (review)Takahashi, ShinichiroMolecular Medicine Reports (2015), 12 (1, Pt. B), 795-799CODEN: MMROA5; ISSN:1791-2997. (Spandidos Publications Ltd.)A review. Metallothioneins (MTs) are metal-binding proteins involved in diverse processes, including metal homeostasis and detoxification, the oxidative stress response and cell proliferation. Aberrant expression and silencing of these genes are important in a no. of diseases. Several pos. regulators of MT genes, including metal-responsive element-binding transcription factor (MTF)-1 and upstream stimulatory factor (USF)-1, have been identified and mechanisms of induction have been well described. However, the neg. regulators of MT genes remain to be elucidated. Previous studies from the group of the present review have revealed that the hematopoietic master transcription factor, PU.1, directly represses the expression levels of MT genes through its epigenetic activities, and upregulation of MT results in the potent inhibition of myeloid differentiation. The present review focuses on PU.1 and several other neg. regulators of this gene, including PZ120, DNA methyltransferase 3a with Mbd3 and Brg1 complex, CCAAT enhancer binding protein α and Ku protein, and describes the suppression of the MT genes through these transcription factors.
- 412Tang, C.-M.; Westling, J.; Seto, E. trans repression of the human metallothionein IIA gene promoter by PZ120, a novel 120-kilodalton zinc finger protein.. Mol. Cell. Biol. 1999, 19, 680– 689, DOI: 10.1128/MCB.19.1.680412Trans Repression of the human metallothionein IIA gene promoter by PZ120, a novel 120-kilodalton zinc finger proteinTang, Chih-Min; Westling, Jennifer; Seto, EdwardMolecular and Cellular Biology (1999), 19 (1), 680-689CODEN: MCEBD4; ISSN:0270-7306. (American Society for Microbiology)Metallothioneins are small, highly conserved, cysteine-rich proteins that bind a variety of metal ions. They are found in virtually all eukaryotic organisms and are regulated primarily at the transcriptional level. In humans, the predominant metallothionein gene is hMTIIA, which accounts for 50% of all metallothioneins expressed in cultured human cells. The hMTIIA promoter is quite complex. In addn. to cis-acting DNA sequences that serve as binding sites for trans-acting factors such as Sp1, AP1, AP2, AP4, and the glucocorticoid receptor, the hMTIIA promoter contains eight consensus metal response element sequences. We report here the cloning of a novel zinc finger protein with a mol. mass of 120 kDa (PZ120) that interacts specifically with the hMTIIA transcription initiation site. The PZ120 protein is ubiquitously expressed in most tissues and possesses a conserved poxvirus and zinc finger (POZ) motif previously found in several zinc finger transcription factors. Intriguingly, we found that a region of PZ120 outside of the zinc finger domain can bind specifically to the hMTIIA DNA. Using transient-transfection anal., we found that PZ120 repressed transcription of the hMTIIA promoter. These results suggest that the hMTIIA gene is regulated by an addnl. neg. regulator that has not been previously described.
- 413Sadhu, C.; Gedamu, L. Metal-specific posttranscriptional control of human metallothionein genes. Mol. Cell. Biol. 1989, 9, 5738– 5741, DOI: 10.1128/mcb.9.12.5738-5741.1989413Metal-specific posttranscriptional control of human metallothionein genesSadhu, Chanchal; Gedamu, LashitewMolecular and Cellular Biology (1989), 9 (12), 5738-41CODEN: MCEBD4; ISSN:0270-7306.During the initial 4 h of treatment, copper and zinc similarly activated the rates of transcription and mRNA accumulation from the 2 human metallothionein (MT) genes, viz., MTI-G and MTII-A, in the hepatoblastoma cell line HepG2. The levels of copper-induced MT mRNAs remained at a plateau for up to 15 h. In contrast, the levels of zinc-induced MT mRNAs gradually declined after about 4 h, despite substantial transcription. The decrease in the zinc-induced MT mRNA half-life is probably due to a posttranscriptional event(s).
- 414Vasconcelos, M. H.; Tam, S. C.; Beattie, J. H.; Hesketh, J. E. Evidence of differences in the post-transcritional regulation of rat metallothionein isoforms. Biochem. J. 1996, 315, 665– 671, DOI: 10.1042/bj3150665414Evidence for differences in the post-transcriptional regulation of rat metallothionein isoformsVasconcelos, M. Helena; Tam, Shuk-Ching; Beattie, John H.; Hesketh, John E.Biochemical Journal (1996), 315 (2), 665-71CODEN: BIJOAK; ISSN:0264-6021. (Portland Press)The expression of metallothionein (MT)-1 and -2 mRNAs in rat liver following administration of Cd or Cu was investigated using specific oligonucleotides. The specificity was confirmed using a competitive prehybridization assay. Cd injection caused a biphasic induction of both isoform mRNAs, whereas Cu induced a sustained, monophasic response. Anal. of polyribosomal RNA showed that, after both Cd and Cu treatments, the recruitment of MT-1 mRNA into polyribosomes paralleled the increase in transcription, but the increase of polyribosomal MT-2 mRNA was less than that of total MT-2 mRNA. This indicates that not all the MT-2 mRNA induced was translated, suggesting that there is translational control of MT-2 mRNA expression, but not of MT-1 mRNA. This hypothesis was supported by the observation that, after Cu treatment, the induction of MT-1 protein was induced to the same extent as MT-1 mRNA, whereas the total MT protein (MT-1 + MT-2) was increased far less (7-fold) than MT-2 mRNA (30-fold).
- 415Ehmke, V.; Winkler, E.; Banner, D. W.; Haap, W.; Schweizer, W. B.; Rottmann, M.; Kaiser, M.; Freymond, C.; Schirmeister, T.; Diederich, F. Optimization of triazine nitriles as rhodesain inhibitors: structure-activity relationships, bioisosteric imidazopyridine nitriles, and X-ray crystal structure analysis with human cathepsin L. ChemMedChem 2013, 8, 967– 975, DOI: 10.1002/cmdc.201300112415Optimization of Triazine Nitriles as Rhodesain Inhibitors: Structure-Activity Relationships, Bioisosteric Imidazopyridine Nitriles, and X-ray Crystal Structure Analysis with Human Cathepsin LEhmke, Veronika; Winkler, Edwin; Banner, David W.; Haap, Wolfgang; Schweizer, W. Bernd; Rottmann, Matthias; Kaiser, Marcel; Freymond, Celine; Schirmeister, Tanja; Diederich, FrancoisChemMedChem (2013), 8 (6), 967-975CODEN: CHEMGX; ISSN:1860-7179. (Wiley-VCH Verlag GmbH & Co. KGaA)The cysteine protease rhodesain of Trypanosoma brucei parasites causing African sleeping sickness has emerged as a target for the development of new drug candidates. Based on a triazine nitrile moiety as electrophilic headgroup, optimization studies on the substituents for the S1, S2, and S3 pockets of the enzyme were performed using structure-based design and resulted in inhibitors with inhibition consts. in the single-digit nanomolar range. Comprehensive structure-activity relationships clarified the binding preferences of the individual pockets of the active site. The S1 pocket tolerates various substituents with a preference for flexible and basic side chains. Variation of the S2 substituent led to high-affinity ligands with inhibition consts. down to 2 nM for compds. bearing cyclohexyl substituents. Systematic investigations on the S3 pocket revealed its potential to achieve high activities with arom. vectors that undergo stacking interactions with the planar peptide backbone forming part of the pocket. X-ray crystal structure anal. with the structurally related enzyme human cathepsin L confirmed the binding mode of the triazine ligand series as proposed by mol. modeling. Sub-micromolar inhibition of the proliferation of cultured parasites was achieved for ligands decorated with the best substituents identified through the optimization cycles. In cell-based assays, the introduction of a basic side chain on the inhibitors resulted in a 35-fold increase in antitrypanosomal activity. Finally, bioisosteric imidazopyridine nitriles were studied to prevent off-target effects with unselective nucleophiles by decreasing the inherent electrophilicity of the triazine nitrile headgroup. Using this ligand, the stabilization by intramol. hydrogen bonding of the thioimidate intermediate, formed upon attack of the catalytic cysteine residue, compensates for the lower reactivity of the headgroup. The imidazopyridine nitrile ligand showed excellent stability toward the thiol nucleophile glutathione in a quant. in vitro assay and fourfold lower cytotoxicity than the parent triazine nitrile.
- 416Ye, B.; Maret, W.; Vallee, B. L. Zinc metallothionein imported into liver mitochondria modulates respiration. Proc. Natl. Acad. Sci. U. S. A. 2001, 98, 2317– 2322, DOI: 10.1073/pnas.041619198416Zinc metallothionein imported into liver mitochondria modulates respirationYe, Bin; Maret, Wolfgang; Vallee, Bert L.Proceedings of the National Academy of Sciences of the United States of America (2001), 98 (5), 2317-2322CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Metallothionein (MT) localizes in the intermembrane space of liver mitochondria as well as in the cytosol and nucleus. Incubation of intact liver mitochondria with physiol., micromolar concns. of MT leads to the import of MT into the mitochondria where it inhibits respiration. This activity is caused by the N-terminal β-domain of MT; in this system, the isolated C-terminal α-domain is inactive. Free zinc inhibits respiration at concns. commensurate with the zinc content of either MT or the isolated β-domain, indicating that MT inhibition involves zinc delivery to mitochondria. Respiratory inhibition of uncoupled mitochondria identifies the electron transfer chain as the primary site of inhibition. The apo-form of MT, thionein, is an endogenous chelating agent and activates zinc-inhibited respiration with a 1:1 stoichiometry ([zinc binding sites]/[zinc]). Carbamoylation of the lysines of MT significantly attenuates the inhibitory effect, suggesting that these residues are crit. for the passage of MT through the outer mitochondrial membrane. Such an import pathway has been proposed for other proteins that also lack a mitochondrial targeting sequence, e.g., apo-cytochrome c, and possibly Cox17, a mitochondrial copper chaperone that is the only protein known so far to exhibit significant primary sequence homol. to MT. The presence and respiratory inhibition of MT in liver, but not heart, mitochondria suggest a hitherto unknown biol. modulating activity of MT in cellular respiration and energy metab. in a tissue-specific manner.
- 417Banerjee, D.; Onosaka, S.; George Cherian, M. Immunohistochemical localization and metallothionein in cell nucleus and cytoplasm of rat liver and kidney. Toxicology 1982, 24, 95– 105, DOI: 10.1016/0300-483X(82)90048-8417Immunohistochemical localization of metallothionein in cell nucleus and cytoplasm of rat liver and kidneyBanerjee, Diponkar; Onosaka, Satomi; Cherian, M. GeorgeToxicology (1982), 24 (2), 95-105CODEN: TXCYAC; ISSN:0300-483X.The intracellular distribution of metallothionein (Mt) in hepatic and renal cells from control and CdCl2-injected rats was investigated by immunohistochem. methods. Antiserum to purified rat liver Mt was prepd. in rabbits after partial polymn. of the protein. The unlabeled peroxidase-antiperoxidase method using the specific rabbit anti-rat liver Mt provided a sensitive technique to localize Mt in tissue sections. In control rats, Mt or thionein (metal-free protein) was mainly localized in the cytoplasm of hepatocytes, renal collecting duct epithelium, and distal convoluted tubular epithelium. In rats injected i.p. with CdCl2 (0.6 mg/kg) for 2 wk, Mt was present mainly in the nuclei which were largely neg. in control rats. Repeated injection with CdCl2 for 4-8 wk resulted in the appearance of Mt in the nucleus and cytoplasm. Intraluminal staining was also noted in proximal convoluted tubules along with marked vacuolation in the cytoplasm at 6 and 8 wk. High intensity staining was obsd. in proximal convoluted tubules and collecting duct epithelium of the kidneys in CdCl2-injected rats. The bile duct epithelium in liver samples, renal glomerular mesangial cells, glomerular visceral epithelial cells, and vascular smooth muscle cells showed weak to moderately intense staining. No staining was seen in vascular endothelial cells, fibroblasts, or leukocytes. The staining for Mt in this technique was abolished when the antibody was absorbed with rat liver Mt in vitro or by substitution of the antiserum with normal rabbit serum, demonstrating the specificity of the staining reaction for Mt. The results showed the presence of small amts. of Mt predominantly in the cytoplasm of control rat hepatocytes and renal tubular epithelium and its appearance in the nucleus and cytoplasm after its synthesis induced by CdCl2 injections.
- 418Tsujikawa, K.; Imai, T.; Kakutani, M.; Kayamori, Y.; Mimura, T.; Otaki, N.; Kimura, M.; Fukuyama, R.; Shimizu, N. Localization of metallothionein in nuclei of growing primary cultured adult rat hepatyocytes. FEBS Lett. 1991, 283, 239– 242, DOI: 10.1016/0014-5793(91)80597-V418Localization of metallothionein in nuclei of growing primary cultured adult rat hepatocytesTsujikawa, Kazutake; Imai, Takumi; Kakutani, Makoto; Kayamori, Yuzo; Mimura, Tsutomu; Otaki, Noriko; Kimura, Masami; Fukuyama, Ryuichi; Shimizu, NobuyoshiFEBS Letters (1991), 283 (2), 239-42CODEN: FEBLAL; ISSN:0014-5793.In primary cultured adult rat hepatocytes stimulated by EGF and insulin, dramatic changes in the subcellular distribution of metallothionein were clarified by indirect immunofluorescence using antisera specific for this protein. Metallothionein was detected only in the cytoplasm of cultured hepatocytes in the G0 and G1 phases, but was concd. in the cell nuclei in the early S phase. The strongest staining pattern in the nuclei was obsd. 12 h after stimulation. Subsequently, the intensity of metallothionein staining in the nuclei decreased. These results suggest that primary cultured hepatocytes are suitable for examg. the relation between subcellular localization of metallothionein and cell growth.
- 419Cherian, M. G.; Howell, S. B.; Imura, N.; Klaassen, C. D.; Koropatnick, J.; Lazo, J. S.; Waalkes, M. P. Role of metallothionein in carcinogenesis. Toxicol. Appl. Pharmacol. 1994, 126, 1– 5, DOI: 10.1006/taap.1994.1083419Role of metallothionein in carcinogenesisCherian, M. George; Howell, Stephen B.; Imura, Nobusama; Klaassen, Curtis D.; Koropatnick, James; Lazo, John S.; Waalkes, Michael P.Toxicology and Applied Pharmacology (1994), 126 (1), 1-5CODEN: TXAPA9; ISSN:0041-008X.A review, with 36 refs. Metallothionein (MT) is a low-mol.-wt. protein (6800 Da) and one-third of its amino acids are cysteine residues. The 20 cysteines coordinate 7 metal atoms (zinc, copper, and/or cadmium). This protein is extremely inducible by metals as well as a no. of org. compds. MT is thought to be an important intracellular storage site for zinc and possibly other essential trace elements. In addn., tolerance to cadmium toxicity is often due to the induction of MT, which sequesters cadmium and lowers its concn. at crit. intracellular sites. Recently it has been proposed that MT might play important roles in several aspects of the carcinogenic process. In this context a symposium was held recently on this topic at the 1993 Annual Society of Toxicol. Meeting. At this symposium Dr. Cherian discussed the expression of MT in various human tumors and its use as a potential marker of tumor differentiation or cell proliferation. Dr Imura provided data illustrating that induction of MT can be used as an adjunct in cancer chemotherapy, in preventing toxicity caused by γ-irradn. or cisplatin (CDDP) and other chemotherapeutics. Induction of MT has been suggested to be an important mechanism of resistance of tumor cells to chemotherapeutic agents, such as CDDP. This is controversial, and various views on this topic were presented by Drs. Howell, Lazo, and Koropatnick. Dr. Waalkes then discussed the role of MT in the carcinogenic and anticarcinogenic effects of metals.
- 420Nagel, W.; Vallee, B. L. Cell cycle regulation of metallothionein in human colonic cancer cells. Proc. Natl. Acad. Sci. U. S. A. 1995, 92, 579– 583, DOI: 10.1073/pnas.92.2.579420Cell cycle regulation of metallothionein in human colonic cancer cellsNagel, Wolfgang W.; Vallee, Bert L.Proceedings of the National Academy of Sciences of the United States of America (1995), 92 (2), 579-83CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Elevated levels of metallothionein (MT) found in rapidly growing tissues such as neonatal liver and various types of human tumors have suggested a role for MT in cell proliferation. To further explore this possibility we investigated the concn. of MT in human colonic cancer (HT-29) cells at different stages of proliferation by means of immunocytochem. and competitive binding. MT is increased in subconfluent proliferating cells relative to growth-inhibited confluent cells, much as it is in growing tissues. Cycling cells synchronized with compactin, an inhibitor of 3-hydroxy-3-methylglutaryl-CoA reductase, revealed an oscillation of cytoplasmic MT that reached a max. in successive late G1 phases and at the G1/S transition. Individual phases of the cell cycle were assessed by [3H]thymidine incorporation and by immunofluorescence employing an antibody that detects a nuclear antigen assocd. with proliferation. An ELISA was used to quantify the relative amts. of MT in homogenate supernatants of HT-29 cells. A 2- to 3-fold increase in MT in actively proliferating cells and the regulation of the protein during the mitotic cell cycle point to a physiol. role for MT in cellular proliferation and suggest that it may also serve as a proliferation marker.
- 421Apostolova, M. D.; Ivanova, I. A.; Cherian, M. G. Signal transduction pathways, and nuclear translocation of zinc and metallothionein during differentiation of myoblast. Biochem. Cell Biol. 2000, 78, 27– 37, DOI: 10.1139/o99-070421Signal transduction pathways, and nuclear translocation of zinc and metallothionein during differentiation of myoblastsApostolova, Margarita D.; Ivanova, Iordanka A.; Cherian, M. GeorgeBiochemistry and Cell Biology (2000), 78 (1), 27-37CODEN: BCBIEQ; ISSN:0829-8211. (National Research Council of Canada)The changes in subcellular localization of metallothionein during differentiation were studied in two myoblast cell lines, L6 and H9C2. Addn. of insulin like growth factor-I or lowering fetal bovine serum to 1% can induce differentiation of myoblasts to myotubes. Metallothionein and zinc were localized mainly in the cytoplasm in myoblasts but were translocated into the nucleus of newly formed myotubes during early differentiation. In fully differentiated myotubes, metallothionein content was decreased with a cytoplasmic localization. Addn. of an inhibitor of mitogen-activated protein kinase, PD 98059, did not affect differentiation but blocked nuclear translocation of metallothionein. LY 294092, an inhibitor of PI3 kinase, and rapamycin, an inhibitor of p70S6 serine/threonine kinase, abolished insulin-like growth factor-I induced differentiation of myoblasts, retained metallothionein in the cytoplasm, and decreased metallothionein content. These results demonstrate that the cytoplasmic-nuclear translocation of metallothionein occurs during the early stage of differentiation of myoblasts to myotubes and can be blocked by inhibition of certain signal transduction pathways. The transient nuclear localization of metallothionein and zinc may be related to a high requirement for zinc for metabolic activities during the early stage of differentiation.
- 422Levadoux, M.; Mahon, C.; Beattie, J. H.; Wallace, H. M.; Hesketh, J. E. Nuclear import of metallothionein requires its mRNA to be associated with the perinuclear cytoskeleton. J. Biol. Chem. 1999, 274, 34961– 34966, DOI: 10.1074/jbc.274.49.34961422Nuclear import of metallothionein requires its mRNA to be associated with the perinuclear cytoskeletonLevadoux, Marilyne; Mahon, Connor; Beattie, John H.; Wallace, Heather M.; Hesketh, John E.Journal of Biological Chemistry (1999), 274 (49), 34961-34966CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)The influence of mRNA localization on metallothionein-1 protein distribution was studied by immunocytochem. We used Chinese hamster ovary cells that had been transfected with either a native metallothionein-1 gene construct or metallothionein-1 5'-untranslated region and coding sequences linked to the 3'-untranslated region from glutathione peroxidase. The change in the 3'-untranslated region caused the delocalization of the mRNA with a loss of the perinuclear localization and assocn. with the cytoskeleton. Clones were selected which expressed similar levels of metallothionein-1 protein, as assessed by RIA. The results showed that loss of metallothionein-1 mRNA localization was assocd. with a loss of metallothionein-1 protein localization, most notably with a lack of metallothionein-1 protein in the nucleus of synchronized cells which were beginning to synthesize DNA. This indicates that the assocn. of metallothionein-1 mRNA with the cytoskeleton around the nucleus is essential for efficient shuttling of the protein into the nucleus during the G1 to S phase transition. This is the first demonstration of a physiol. role for perinuclear mRNA localization and we propose that such localization may be important for a wide range of nuclear proteins, including those that shuttle between nucleus and cytoplasm in a cell cycle dependent manner.
- 423Nagano, T.; Itoh, N.; Ebisutani, C.; Takatani, T.; Miyoshi, T.; Nakanishi, T.; Tanaka, K. The transport mechanism of metallothionein is different from that of classical NLS-bearing protein. J. Cell. Physiol. 2000, 185, 440– 446, DOI: 10.1002/1097-4652(200012)185:3<440::AID-JCP15>3.0.CO;2-N423The transport mechanism of metallothionein is different from that of classical NLS-bearing proteinNagano, Takayuki; Itoh, Norio; Ebisutani, Chikara; Takatani, Tomoka; Miyoshi, Tomoya; Nakanishi, Tsuyoshi; Tanaka, KeiichiJournal of Cellular Physiology (2000), 185 (3), 440-446CODEN: JCLLAX; ISSN:0021-9541. (Wiley-Liss, Inc.)A nuclear localization signal (NLS) has been detected in several nuclear proteins. Classical NLS-mediated nuclear pore targeting is performed by using the cytosolic factors, importin α and importin β, whereas nuclear translocation requires the small GTPase, Ran. In the present study, we demonstrated that nuclear localization of metallothionein (MT) differs from that of classical NLS-mediated substrates. In digitonin-permeabilized BALB/c3T3 cells, biotinylated MT was localized in the nucleus in the presence of ATP and erythrocyte cytosol in the same manner as for SV40 large T NLS-conjugated allophycocyanin (APC-NLS). Under ATP-free conditions, nuclear rim-binding was obsd. in both transport substrates. Rim-binding of labeled MT was competitively inhibited by the addn. of an excess amt. of unlabeled MT. Different elution profiles were obsd. for the localization-promotion activities of MT in the cytosol compared to those of APC-NLS. Furthermore, nuclear localization of MT was detd. to be a wheat germ agglutinin-insensitive, GTPγS-sensitive, and anti-Ran antibody-sensitive process. Green fluorescent protein-metallothionein (GFP-MT) fusion protein was also localized in the nucleus in the stable transformant of CHL-IU cells. These results strongly suggest that the targeting by MT of the nuclear pore is mediated by cytosolic factor(s) other than importins and that MT requires Ran for its nuclear localization.
- 424Woo, E. S.; Dellapiazza, D.; Wang, A. S.; Lazo, J. S. Energy-dependent nuclear binding dictates metallothionein localization. J. Cell. Physiol. 2000, 182, 69– 76, DOI: 10.1002/(SICI)1097-4652(200001)182:1<69::AID-JCP8>3.0.CO;2-9424Energy-dependent nuclear binding dictates metallothionein localizationWoo, Elizabeth S.; Dellapiazza, Dana; Wang, Angela S.; Lazo, John S.Journal of Cellular Physiology (2000), 182 (1), 69-76CODEN: JCLLAX; ISSN:0021-9541. (Wiley-Liss, Inc.)Metallothioneins (MTs) are low-mol.-wt., stress-activated proteins that protect cells against heavy metals, oxidants, and some electrophilic drugs. Both nuclear and cytoplasmic MT phenotypes have been obsd. in cells even though MTs (6 kDa) are well below the size exclusion limit for diffusion through the nuclear envelope. To study the factors controlling MT subcellular partitioning, the authors covalently linked MTII to a fluorescent label and examd. its subcellular distribution in response both to pharmacol. and phys. perturbations. Fluorescent MTII localized to the nucleus of digitonin-permeabilized human SCC25 carcinoma cells, consistent with its endogenous distribution in these cells. Nuclear sequestration of the fluorescent MTII was inhibited by a 100-fold molar excess of unlabeled MTII and by wheat germ agglutinin, indicating a saturable binding mechanism and the involvement of one or more glycoproteins, resp. Depletion of ATP (ATP) inhibited MTII nuclear localization, implying energy-dependent nuclear translocation or retention of MT. Neither chilling nor the absence of cytosolic exts. inhibited nuclear sequestration of MTII, supporting diffusion-based entry mechanism. In situ biochem. extns. of the nuclear MTII revealed at least two distinct binding activities. Collectively, these data indicate that MTII diffuses into the nucleus of SCC25 cells, where it is selectively and actively retained by nuclear binding factors, imparting its localization phenotype.
- 425Takahashi, Y.; Ogra, Y.; Suzuki, K. T. Nuclear trafficking of metallothionein requires oxidation of a cytosolic partner. J. Cell. Physiol. 2005, 202, 563– 569, DOI: 10.1002/jcp.20158425Nuclear trafficking of metallothionein requires oxidation of a cytosolic partnerTakahashi Yukihisa; Ogra Yasumitsu; Suzuki Kazuo TJournal of cellular physiology (2005), 202 (2), 563-9 ISSN:0021-9541.The present study revealed the mechanism underlying the nuclear trafficking of metallothionein (MT). Nuclear localization of MT in digitonin-permeabilized BALB 3T3 cells was enhanced in the presence of a cytosolic factor added as a rat red blood cell lysate by oxidation with H2O2 in a dose-dependent manner, but inhibited with excess glutathione. A cytosolic partner was assumed to bind MT and retain it in the cytoplasm, and its oxidation can mobilize MT to the nuclei on cellular oxidation. Pre-treatment of nuclei with H2O2 did not enhance the localization, and MT that had been localized in the nuclei was washed out, indicating that MT is in the nuclei as a result of a higher rate of uptake by the nuclei than the rate of diffusion from the nuclei. Nuclear localization of lysozyme and nuclear localization signal (NLS)-bearing allophycocyanin were not enhanced by the oxidation in the presence of cytosolic factor, suggesting that the nuclear traffic occurring on oxidation is specific to MT. Moreover, when cells were arrested the cell cycle at the S phase, MT was localized in the nuclei in response to coincidental generation of a feeble reactive oxygen species (ROS). These observations suggest that MT comes localized in the nuclei on the sensing of intracellular oxidation, whereby a cytosolic partner specific to MT comes oxidized as a cargo system, MT being localized as a result of enhanced uptake in the nuclei and re-localized in the cytoplasm diffusely. Nuclear MT was proposed to protect the nuclei from the oxidation occurring with progression of the cell cycle.
- 426Lee, S. J.; Park, M. H.; Kim, H. J.; Koh, J. Y. Metallothionein-3 regulates lysosomal function in cultured astrocytes under both normal and oxidative conditions. Glia 2010, 58, 1186– 1196, DOI: 10.1002/glia.20998426Metallothionein-3 regulates lysosomal function in cultured astrocytes under both normal and oxidative conditionsLee Sook-Jeong; Park Mi-Ha; Kim Hyun-Jae; Koh Jae-YoungGlia (2010), 58 (10), 1186-96 ISSN:.Cellular zinc plays a key role in lysosomal change and cell death in neurons and astrocytes under oxidative stress. Here, using astrocytes lacking metallothionein-3 (MT3), a potential source of labile zinc in the brain, we studied the role of MT3 in oxidative stress responses. H(2)O(2) induced a large increase in labile zinc in wild-type (WT) astrocytes, but stimulated only a modest rise in MT3-null astrocytes. In addition, H(2)O(2)-induced lysosomal membrane permeabilization (LMP) and cell death were comparably attenuated in MT3-null astrocytes. Expression and glycosylation of Lamp1 (lysosome-associated membrane protein 1) and Lamp2 were increased in MT3-null astrocytes, and the activities of several lysosomal enzymes were significantly reduced, indicating an effect of MT3 on lysosomal components. Consistent with lysosomal dysfunction in MT3-null cells, the level of LC3-II (microtubule-associated protein 1 light chain 3), a marker of early autophagy, was increased by oxidative stress in WT astrocytes, but not in MT3-null cells. Similar changes in Lamp1, LC3, and cathepsin-D were induced by the lysosomal inhibitors bafilomycin A1, chloroquine, and monensin, indicating that lysosomal dysfunction may lie upstream of changes observed in MT3-null astrocytes. Consistent with this idea, lysosomal accumulation of cholesterol and lipofuscin were augmented in MT3-null astrocytes. Similar to the results seen in MT3-null cells, MT3 knockdown by siRNA inhibited oxidative stress-induced increases in zinc and LMP. These results indicate that MT3 may play a key role in normal lysosomal function in cultured astrocytes.
- 427Nordberg, G. F.; Garvey, J. S.; Chang, C. C. Metallothionein in plasma and urine of cadmium workers. Environ. Res. 1982, 28, 179– 182, DOI: 10.1016/0013-9351(82)90167-0427Metallothionein in plasma and urine of cadmium workersNordberg, Gunnar F.; Garvey, Justine S.; Chang, Chin C.Environmental Research (1982), 28 (1), 179-82CODEN: ENVRAL; ISSN:0013-9351.Metallothionein (MT) and β-2-microglobulin (BM) concns. were detd. in samples of plasma and urine from workers in a Swedish factory manufg. Cd batteries. Concns. of metallothionein in plasma was 2-11 ng/g and in urine from 2-155 ng/g. In workers with urinary BM concns. >500 μg/L, the median value of MT in urine was statistically significantly higher than that in workers with urinary BM concns. <500 μg/L. In individual cases, however, a considerable increase in urinary BM may occur without any increase in urinary MT. The possible significance of increased MT concns. in plasma, as seen in some of the workers in the present study, the relation to transport of Cd-MT to the renal cortex is pointed out.
- 428Nordberg, M.; Nordberg, G. F. Metallothioneins: Historical developments and overview. Met. Ions Life Sci. 2009, 5, 1– 29, DOI: 10.1039/9781847559531-00001428Metallothioneins: historical development and overviewNordberg, Monica; Nordberg, Gunnar F.Metal Ions in Life Sciences (2009), 5 (Metallothioneins and Related Chelators), 1-29CODEN: MILSCT; ISSN:1559-0836. (Royal Society of Chemistry)A review. The history on research of metallothionein is reviewed. Various methods for isolation, characterization, and quantification are evaluated. The role of metallothionein in metal metab. and toxicity is explained. Gender differences and polymorphism as well as possible relationships with diseases are discussed. The review is based on data from the literature and on own original exptl. and epidemiol. data. Aspects on future research within the metallothionein field are indicated.
- 429Sabolić, I.; Breljak, D.; Skarica, M.; Herak-Kramberger, C. M. Role of metallothionein in cadmium traffic and toxicity in kidneys and other mammalian organs. BioMetals 2010, 23, 897– 926, DOI: 10.1007/s10534-010-9351-z429Role of metallothionein in cadmium traffic and toxicity in kidneys and other mammalian organsSabolic, Ivan; Breljak, Davorka; Skarica, Mario; Herak-Kramberger, Carol M.BioMetals (2010), 23 (5), 897-926CODEN: BOMEEH; ISSN:0966-0844. (Springer)A review. Metallothioneins are cysteine-rich, small metal-binding proteins present in various mammalian tissues. Of the four common metallothioneins, MT-1 and MT-2 (MTs) are expressed in most tissues, MT-3 is predominantly present in brain, whereas MT-4 is restricted to the squamous epithelia. The expression of MT-1 and MT-2 in some organs exhibits sex, age, and strain differences, and inducibility with a variety of stimuli. In adult mammals, MTs have been localized largely in the cell cytoplasm, but also in lysosomes, mitochondria and nuclei. The major physiol. functions of MTs include homeostasis of essential metals Zn and Cu, protection against cytotoxicity of Cd and other toxic metals, and scavenging free radicals generated in oxidative stress. The role of MTs in Cd-induced acute and chronic toxicity, particularly in liver and kidneys, is reviewed in more details. In acute toxicity, liver is the primary target, whereas in chronic toxicity, kidneys are major targets of Cd. The intracellular MTs bind Cd ions and form CdMT. In chronic intoxication, Cd stimulates de novo synthesis of MTs; it is assumed that toxicity in the cells starts when loading with Cd ions exceeds the buffering capacity of intracellular MTs. CdMT, released from the Cd-injured organs, or when applied parenterally for exptl. purposes, reaches the kidneys via circulation, where it is filtered, endocytosed in the proximal tubule cells, and degraded in lysosomes. Liberated Cd can immediately affect the cell structures and functions. The resulting proteinuria and CdMT in the urine can be used as biomarkers of tubular injury.
- 430Bremner, I.; Mehra, R. K. Assay of extracellular metallothionein. Methods Enzymol. 1991, 205, 60– 70, DOI: 10.1016/0076-6879(91)05086-B430Assay of extracellular metallothioneinBremner, Ian; Mehra, Rajesh K.Methods in Enzymology (1991), 205 (Metallobiochem. Pt. B), 60-70CODEN: MENZAU; ISSN:0076-6879.Purifn. of antigen, prepn. of antibodies, detection of antibodies, immunoassay procedure, and assay of metallothionein in extracellular fluids are discussed.
- 431Akintola, D. F.; Sampson, B.; Fleck, A. Development of an enzyme-linked immunosorbent assay for human metallothionein-1 in plasma and urine. J. Lab. Clin. Med. 1995, 126, 119– 127431Development of an enzyme-linked immunosorbent assay for human metallothionein-1 in plasma and urineAkintola, D. F.; Sampson, B.; Fleck, A.Journal of Laboratory and Clinical Medicine (1995), 126 (2), 119-27CODEN: JLCMAK; ISSN:0022-2143. (Mosby-Year Book)The development of a sensitive ELISA for human metallothionein-1 (MT-1) is reported. MT was purified from postmortem human liver and used to raise high-titer antibodies in rabbits. The assay was specific for human MT-1, and there was no significant cross-reaction with human MT-2. The detection limit (sensitivity) of the assay was 5 ng/mL, and the added MT-1 could be fully recovered from plasma and urine. The normal ref. range for MT-1 was 32 ng/mL in plasma and 10 ng MT-1 per μmol of creatinine in random samples of urine. No significant differences were found between the values for males and females. The concn. of MT-1 was greatly increased between 24 and 48 h after surgery, indicating that the protein behaves like an acute phase reactant in human subjects.
- 432Maret, W. Metallothionein and the acute phase response. J. Lab. Clin. Med. 1995, 126, 106– 107432Metallothionein and the acute phase responseMaret WThe Journal of laboratory and clinical medicine (1995), 126 (2), 106-7 ISSN:0022-2143.There is no expanded citation for this reference.
- 433Knipp, M.; Meloni, G.; Roschitzki, B.; Vašák, M. Zn7-metallothionein-3 and the synaptic vesicle cycle: interaction of metallothionein-3 with the small GTPase Rab3A. Biochemistry 2005, 44, 3159– 3165, DOI: 10.1021/bi047636d433Zn7Metallothionein-3 and the Synaptic Vesicle Cycle: Interaction of Metallothionein-3 with the Small GTPase Rab3AKnipp, Markus; Meloni, Gabriele; Roschitzki, Bernd; Vasak, MilanBiochemistry (2005), 44 (9), 3159-3165CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)In the central nervous system, a large amt. of chelatable Zn2+ is sequestered in presynaptic vesicles of certain glutamatergic nerve terminals. The exo-endocytic cycle of synaptic vesicles is strictly linked to the small GTPase Rab3A. Metallothionein-3 (Zn7MT-3) has been proposed to be involved in the intracellular trafficking of Zn2+ in zinc-contg. neurons, but its role in this process is not understood. By using affinity pptn. and surface plasmon resonance anal., we show that Zn7MT-3 binds reversibly to Rab3A·GDP (KD = 2.6 μM), but not to Rab3A·GTP. The binding of Zn7MT-3 to Rab3A·GDP is specific as no binding was obsd. with the metal-free form of MT-3. Mutational studies of Rab3A mapped the interaction site to the effector binding site of the protein. This location is further supported by the kinetics of GDP exchange, which was found to be unaffected by binding of Zn7MT-3 to Rab3A·GDP. The interaction of Zn7MT-3 with Rab3A indicates that Zn7MT-3 is not merely a cellular Zn2+ buffer, but actively participates in synaptic vesicle trafficking upstream of vesicle fusion.
- 434Chung, R. S.; Penkowa, M.; Dittmann, J.; King, C. E.; Bartlett, C.; Asmussen, J. W.; Hidalgo, J.; Carrasco, J.; Leung, Y. K.; Walker, A. K.; Fung, S. J.; Dunlop, S. A.; Fitzgerald, M.; Beazley, L. D.; Chuah, M. I.; Vickers, J. C.; West, A. K. Redefining the role of metallothionein within the injured brain: Extracellular metallothioneins play and important role in the astrocyte-neuron response to injury. J. Biol. Chem. 2008, 283, 15349– 15358, DOI: 10.1074/jbc.M708446200434Redefining the Role of Metallothionein within the Injured Brain: extracellular metallothioneins play an important role in the astrocyte-neuron response to injuryChung, Roger S.; Penkowa, Milena; Dittmann, Justin; King, Carolyn E.; Bartlett, Carole; Asmussen, Johanne W.; Hidalgo, Juan; Carrasco, Javier; Leung, Yee Kee J.; Walker, Adam K.; Fung, Samantha J.; Dunlop, Sarah A.; Fitzgerald, Melinda; Beazley, Lyn D.; Chuah, Meng I.; Vickers, James C.; West, Adrian K.Journal of Biological Chemistry (2008), 283 (22), 15349-15358CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)A no. of intracellular proteins that are protective after brain injury are classically thought to exert their effect within the expressing cell. The astrocytic metallothioneins (MT) are one example and are thought to act via intracellular free radical scavenging and heavy metal regulation, and in particular zinc. Indeed, we have previously established that astrocytic MTs are required for successful brain healing. Here we provide evidence for a fundamentally different mode of action relying upon intercellular transfer from astrocytes to neurons, which in turn leads to uptake-dependent axonal regeneration. First, we show that MT can be detected within the extracellular fluid of the injured brain, and that cultured astrocytes are capable of actively secreting MT in a regulatable manner. Second, we identify a receptor, megalin, that mediates MT transport into neurons. Third, we directly demonstrate for the first time the transfer of MT from astrocytes to neurons over a specific time course in vitro. Finally, we show that MT is rapidly internalized via the cell bodies of retinal ganglion cells in vivo and is a powerful promoter of axonal regeneration through the inhibitory environment of the completely severed mature optic nerve. Our work suggests that the protective functions of MT in the central nervous system should be widened from a purely astrocytic focus to include extracellular and intra-neuronal roles. This unsuspected action of MT represents a novel paradigm of astrocyte-neuronal interaction after injury and may have implications for the development of MT-based therapeutic agents.
- 435El Refaey, H.; Ebadi, M.; Kuszynski, C. A.; Sweeney, J.; Hamada, F. M.; Hamed, A. Identification of metallothionein receptors in human astrocytes. Neurosci. Lett. 1997, 231, 131– 134, DOI: 10.1016/S0304-3940(97)00548-X435Identification of metallothionein receptors in human astrocytesEl Refaey H; Ebadi M; Kuszynski C A; Sweeney J; Hamada F M; Hamed ANeuroscience letters (1997), 231 (3), 131-4 ISSN:0304-3940.Metallothionein (MT) isoforms are low molecular weight (6000-7000 Da) zinc binding proteins containing 60-68 amino acid residues, 25-30% cysteine, no aromatic amino acids, and binding between 5-7 g zinc/mol of protein. Since the synthesis of MT is induced by endotoxin, cytokines, and glucocorticoids, MT is now considered to be an acute phase protein protecting against oxygen radicals and oxidative damages caused by inflammation, tissue injury, and stress to the central nervous system. By postulating that a specific mechanism must exist to foster the induction of MTs I and II by numerous and diversified factors, we searched for and identified for the first time, MT receptors on U373MG cell membrane preparations, by using fluoresceinated MT I isoform probe; and by employing cysteine, glutathione, and four MT isoforms to determine high affinity and specific binding. MT receptors revealed a Kd value of 0.84 nM and a Bmax of 99.82 fmol/mg protein. Moreover, MT receptors were found in greater density on the surface of aggregated astrocytes. We postulate that conditions or agents generating reactive oxygen species may influence the expression of MT receptors.
- 436Wolff, N. A.; Abouhamed, M.; Verroust, P. J.; Thévenod, F. Megalin-dependent internalization of cadmium-metallothionein and cytotoxicity in cultured renal proximal tubule cells. J. Pharmacol. Exp. Ther. 2006, 318, 782– 787, DOI: 10.1124/jpet.106.102574436Megalin-dependent internalization of cadmium-metallothionein and cytotoxicity in cultured renal proximal tubule cellsWolff, Natascha A.; Abouhamed, Marouan; Verroust, Pierre J.; Thevenod, FrankJournal of Pharmacology and Experimental Therapeutics (2006), 318 (2), 782-791CODEN: JPETAB; ISSN:0022-3565. (American Society for Pharmacology and Experimental Therapeutics)Chronic cadmium (Cd2+) exposure results in renal proximal tubular cell damage. Delivery of Cd2+ to the kidney occurs mainly as complexes with metallothionein-1 (mol. mass ∼ 7 kDa), freely filtered at the glomerulus. For Cd2+ to gain access to the proximal tubule cells, these complexes are thought to be internalized via receptors for small protein ligands, such as megalin and cubilin, followed by release of Cd2+ from metallothionein-1 in endosomal/lysosomal compartments. To investigate the role of megalin in renal cadmium-metallothionein-1 resorption, megalin expression and dependence of cadmium-metallothionein-1 internalization and cytotoxicity on megalin were studied in a renal proximal tubular cell model (WKPT-0293 Cl.2 cells). Expression of megalin was detected by reverse transcriptase-polymerase chain reaction and visualized by immunofluorescence both at the cell surface (live staining) and intracellularly (permeabilized cells). Internalization of Alexa Fluor 488-coupled metallothionein-1 was concn.-dependent, satg. at approx. 15 μM. At 14.3 μM, metallothionein-1 uptake could be significantly attenuated by 30.9±6.6% (n = 4) by 1 μM of the receptor-assocd. protein (RAP) used as a competitive inhibitor of cadmium-metallothionein-1 binding to megalin and cubilin. Consistently, cytotoxicity of a 24-h treatment with 7.14 μM cadmium-metallothionein-1 was significantly reduced by 41.0±7.6%, 61.6±3.4%, and 26.2±1.8% (n = 4-5 each) by the presence of 1 μM RAP, 400 μg/mL anti-megalin antibody, or 5 μM of the cubilin-specific ligand, apo-transferrin, resp. Cubilin expression in proximal tubule cells was also confirmed at the mRNA and protein level. The data indicate that renal proximal tubular cadmium-metallothionein-1 uptake and cell death are mediated at least in part by megalin.
- 437Thévenod, F.; Fels, J.; Lee, W. K.; Zarbock, R. Channels, transporters and receptors for cadmium and cadmium complexes in eukaryotic cells: myths and facts. BioMetals 2019, 32, 469– 489, DOI: 10.1007/s10534-019-00176-6437Channels, transporters and receptors for cadmium and cadmium complexes in eukaryotic cells: myths and factsThevenod, Frank; Fels, Johannes; Lee, Wing-Kee; Zarbock, RalfBioMetals (2019), 32 (3), 469-489CODEN: BOMEEH; ISSN:0966-0844. (Springer)A review. Cadmium (Cd2+) is a toxic and non-essential divalent metal ion in eukaryotic cells. Cells can only be targeted by Cd2+ if it hijacks physiol. high-affinity entry pathways, which transport essential divalent metal ions in a process termed "ionic and mol. mimicry". Hence, "free" Cd2+ ions and Cd2+ complexed with small org. mols. are transported across cellular membranes via ion channels, carriers and ATP hydrolyzing pumps, whereas receptor-mediated endocytosis (RME) internalizes Cd2+-protein complexes. Only Cd2+ transport pathways validated by stringent methodol., namely electrophysiol., 109Cd2+ tracer studies, inductively coupled plasma mass spectrometry, at. absorption spectroscopy, Cd2+-sensitive fluorescent dyes, or specific ligand binding and internalization assays for RME are reviewed whereas indirect correlative studies are excluded. At toxicol. relevant concns. in the submicromolar range, Cd2+ permeates voltage-dependent Ca2+ channels ("T-type" CaV3.1, CatSper), transient receptor potential (TRP) channels (TRPA1, TRPV5/6, TRPML1), solute carriers (SLCs) (DMT1/SLC11A2, ZIP8/SLC39A8, ZIP14/SLC39A14), amino acid/cystine transporters (SLC7A9/SLC3A1, SLC7A9/SLC7A13), and Cd2+-protein complexes are endocytosed by the lipocalin-2/NGAL receptor SLC22A17. Cd2+ transport via the mitochondrial Ca2+ uniporter, ATPases ABCC1/2/5 and transferrin receptor 1 is likely but requires further evidence. Cd2+ flux occurs through the influx carrier OCT2/SLC22A2, efflux MATE proteins SLC47A1/A2, the efflux ATPase ABCB1, and RME of Cd2+-metallothionein by the receptor megalin (low d. lipoprotein receptor-related protein 2, LRP2):cubilin albeit at high concns. thus questioning their relevance in Cd2+ loading. Which Cd2+-protein complexes are internalized by megalin:cubilin in vivo still needs to be detd. A stringent conservative and reductionist approach is mandatory to verify relevance of transport pathways for Cd2+ toxicity and to overcome dissemination of unsubstantiated conjectures.
- 438Moltedo, O.; Verde, C.; Capasso, A.; Parisi, E.; Remondelli, P.; Bonatti, S.; Alvarez-Hernandez, X.; Glass, J.; Alvino, C. G.; Leone, A. Zinc transport and metallothionein secretion in the intestinal human cell line Caco-2. J. Biol. Chem. 2000, 275, 31819– 31825, DOI: 10.1074/jbc.M002907200438Zinc transport and metallothionein secretion in the intestinal human cell line Caco-2Moltedo, Ornella; Verde, Cinzia; Capasso, Antonio; Parisi, Elio; Remondelli, Paolo; Bonatti, Stefano; Alvarez-Hernandez, Xavier; Glass, Jonathan; Alvino, Claudio G.; Leone, ArturoJournal of Biological Chemistry (2000), 275 (41), 31819-31825CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)Caco-2, a human cell line, displays several biochem. and morphol. characteristics of differentiated enterocytes. Among these is the ability to transport zinc from the apical to the basal compartment. This process was enhanced following exposure by the apical compartment to increasing concns. of the metal. High pressure liq. chromatog. fractionation of the media obtained from cells labeled with radioactive zinc showed that metallothioneins (MTs), small metal-binding, cysteine-rich proteins, were present in the apical and basal media of controls as well as in cells grown in the presence of high concns. of zinc. Following exposure to the metal, the levels of Zn-MTs in the apical medium increased, while in the basal compartment the greatest part of zinc appeared in a free form with minor changes in the levels of basal MTs. Metabolic labeling expts. with radioactive cysteine confirmed the apical secretion of MTs. A stable transfectant clone of Caco-2 cells (CL11) was selected for its ability to express constitutively high levels of the mouse metallothionein I protein. This cell line showed an enhanced transport of the metal following exposure to high concns. of zinc and a constitutive secretion of the mouse metallothionein I protein in the apical compartment. Together, these findings strongly support the hypothesis of a functional role between the biosynthesis and secretion of MTs and the transport of zinc in intestinal cells.
- 439Trayhurn, P.; Beattie, J. H. Physiological role of adipose tissue: white adipose tissue as an endocrine and secretory organ. Proc. Nutr. Soc. 2001, 60, 329– 339, DOI: 10.1079/PNS200194439Physiological role of adipose tissue: white adipose tissue as an endocrine and secretory organTrayhurn, Paul; Beattie, John H.Proceedings of the Nutrition Society (2001), 60 (3), 329-339CODEN: PNUSA4; ISSN:0029-6651. (CABI Publishing)A review with refs. The traditional role attributed to white adipose tissue is energy storage, fatty acids being released when fuel is required. The metabolic role of white fat is, however, complex. For example, the tissue is needed for normal glucose homeostasis and a role in inflammatory processes has been proposed. A radical change in perspective followed the discovery of leptin; this crit. hormone in energy balance is produced principally by white fat, giving the tissue an endocrine function. Leptin is one of a no. of proteins secreted from white adipocytes, which include angiotensinogen, adipsin, acylation-stimulating protein, adiponectin, retinol-binding protein, tumor necrosis factor α, interleukin 6, plasminogen activator inhibitor-1 and tissue factor. Some of these proteins are inflammatory cytokines, some play a role in lipid metab., while others are involved in vascular hemostasis or the complement system. The effects of specific proteins may be autocrine or paracrine, or the site of action may be distant from adipose tissue. The most recently described adipocyte secretory proteins are fasting-induced adipose factor, a fibrinogenangiopoietin-related protein, metallothionein and resistin. Resistin is an adipose tissue-specific factor which is reported to induce insulin resistance, linking diabetes to obesity. Metallothionein is a metal-binding and stress-response protein which may have an antioxidant role. The key challenges in establishing the secretory functions of white fat are to identify the complement of secreted proteins, to establish the role of each secreted protein, and to assess the pathophysiol. consequences of changes in adipocyte protein prodn. with alterations in adiposity (obesity, fasting, cachexia). There is already considerable evidence of links between increased prodn. of some adipocyte factors and the metabolic and cardiovascular complications of obesity. In essence, white adipose tissue is a major secretory and endocrine organ involved in a range of functions beyond simple fat storage.
- 440Lynes, M. A.; Zaffuto, K.; Unfricht, D. W.; Marusov, G.; Samson, J. S.; Yin, X. The physiological roles of extracellular metallothionein. Exp. Biol. Med. 2006, 231, 1548– 1554, DOI: 10.1177/153537020623100915440The physiological roles of extracellular metallothioneinLynes, Michael A.; Zaffuto, Kristin; Unfricht, Darryn W.; Marusov, Gregory; Samson, Jacqueline S.; Yin, XiuyunExperimental Biology and Medicine (Maywood, NJ, United States) (2006), 231 (9), 1548-1554CODEN: EBMMBE; ISSN:1535-3702. (Society for Experimental Biology and Medicine)A review. Metallothionein (MT) is a low-mol.-wt. protein with a no. of roles to play in cellular homeostasis. MT is synthesized as a consequence of a variety of cellular stressors, and has been found in both intracellular compartments and in extracellular spaces. The intracellular pool of this cysteine-rich protein can act as a reservoir of essential heavy metals, as a scavenger of reactive O and N species, as an antagonist of toxic metals and org. mols., and as a regulator of transcription factor activity. The presence of MT outside of cells due to the influence of stressors suggests that this protein may make important contributions as a "danger signal" that influences the management of responses to cellular damage. While conventional wisdom has held that extracellular MT is the result of cell death or leakage from stressed cells, there are numerous examples of selective release of proteins by nontraditional mechanisms, including stress response proteins. This suggests that MT may similarly be selectively released, and that the pool of extracellular MT represents an important regulator of various cellular functions. For example, extracellular MT has effects both on the severity of autoimmune disease, and on the development of adaptive immune functions. Extracellular MT may operate as a chemotactic factor that governs the trafficking of inflammatory cells that move to resolve damaged tissues, as a counter to extracellular oxidant-mediated damage, and as a signal that influences the functional behavior of wounded cells. A thorough understanding of the mechanisms of MT release from cells, the conditions under which MT is released to the extracellular environment, and the ways in which MT interacts with sensitive cells may both illuminate the understanding of an important control mechanism that operates in stressful conditions, and should indicate new opportunities for therapeutic management via the manipulation of this pool of extracellular MT.
- 441Liu, H.; Liang, Z.; Wang, F.; Zhou, C.; Zheng, X.; Hu, T.; He, X.; Wu, X.; Lan, P. Exosomes from mesenchymal stromal cells reduce murine colonic inflammation via a macrophage-dependent mechanism. JCI Insight 2019, 4, 131273, DOI: 10.1172/jci.insight.131273There is no corresponding record for this reference.
- 442Atrian, S.; Capdevila, M. Metallothionein–protein interactions. Biomol. Concepts 2013, 4, 143– 160, DOI: 10.1515/bmc-2012-0049442Metallothionein-protein interactionsAtrian, Silvia; Capdevila, MerceBiomolecular Concepts (2013), 4 (2), 143-160CODEN: BCIOB8; ISSN:1868-5021. (Walter de Gruyter GmbH)A review. Metallothioneins (MTs) are a family of universal, small proteins, sharing a high cysteine content and an optimal capacity for metal ion coordination. They take part in a plethora of metal ion-related events (from detoxification to homeostasis, storage, and delivery), in a wide range of stress responses, and in different pathol. processes (tumorigenesis, neurodegeneration, and inflammation). The information on both intracellular and extracellular interactions of MTs with other proteins is here comprehensively reviewed. In mammalian kidney, MT1/MT2 interact with megalin and related receptors, and with the transporter transthyretin. Most of the mammalian MT partners identified concern interactions with central nervous system (mainly brain) proteins, both through phys. contact or metal exchange reactions. Phys. interactions mainly involve neuronal secretion multimers. Regarding metal swap events, brain MT3 appears to control the metal ion load in peptides whose aggregation leads to neurodegenerative disorders, such as Aβ peptide, α-synuclein, and prion proteins (Alzheimer's and Parkinson's diseases, and spongiform encephalopathies, resp.). Interaction with ferritin and bovine serum albumin are also documented. The intercourse of MTs with zinc-dependent enzymes and transcription factors is capable to activate/deactivate them, thus conferring MTs the role of metabolic and gene expression regulators. As some of these proteins are involved in cell cycle and proliferation control (p53, nuclear factor κB, and PKCμ), they are considered in the context of oncogenesis and tumor progression. Only one non-mammalian MT interaction, involving Drosophila MtnA and MtnB major isoforms and peroxiredoxins, has been reported. The prospective use for biomedical applications of the MT-interaction information is finally discussed.
- 443El Ghazi, I.; Martin, B. L.; Armitage, I. M. New proteins found interacting with brain metallothionein-3 are linked to secretion. Int. J. Alzheimer's Dis. 2011, 2011, 208634, DOI: 10.4061/2011/208634There is no corresponding record for this reference.
- 444Binz, P.-A.; Kägi, J. H. R. Metallothionein: Molecular Evolution and Classification. In Metallothionein IV; Klaassen, C. D., Ed.; Birkhäuser: Basel, Switzerland, 1999; pp 7– 14.There is no corresponding record for this reference.
- 445Blindauer, C. Metallothioneins. In Binding, Transport, and Storage of Metal Ions in Biological Cells; Maret, W., Wedd, A. G., Eds.; RSC Cambridge, 2014; pp 606– 665.There is no corresponding record for this reference.
- 446Serén, N.; Glaberman, S.; Carretero, M. A.; Chiari, Y. Molecular evolution and functional divergence of the metallothionein gene family in vertebrates. J. Mol. Evol. 2014, 78, 217– 233, DOI: 10.1007/s00239-014-9612-5446Molecular evolution and functional divergence of the metallothionein gene family in VertebratesSeren, Nina; Glaberman, Scott; Carretero, Miguel A.; Chiari, YleniaJournal of Molecular Evolution (2014), 78 (3-4), 217-233CODEN: JMEVAU; ISSN:0022-2844. (Springer)The metallothionein (MT) gene superfamily consists of metal-binding proteins involved in various metal detoxification and storage mechanisms. The evolution of this gene family in vertebrates has mostly been studied in mammals using sparse taxon or gene sampling. Genomic databases and available data on MT protein function and expression allow a better understanding of the evolution and functional divergence of the different MT types. We recovered 77 MT coding sequences from 20 representative vertebrates with annotated complete genomes. We found multiple MT genes, also in reptiles, which were thought to have only one MT type. Phylogenetic and synteny analyses indicate the existence of a eutherian MT1 and MT2, a tetrapod MT3, an amniote MT4, and fish MT. The optimal gene-tree/species-tree reconciliation analyses identified the best root in the fish clade. Functional analyses reveal variation in hydropathic index among protein domains, likely correlated with their distinct flexibility and metal affinity. Analyses of functional divergence identified amino acid sites correlated with functional divergence among MT types. Uncovering the no. of genes and sites possibly correlated with functional divergence will help to design cost-effective MT functional and gene expression studies. This will permit further understanding of the distinct roles and specificity of these proteins and to properly target specific MT for different types of functional studies. Therefore, this work presents a crit. background on the mol. evolution and functional divergence of vertebrate MTs to carry out further detailed studies on the relationship between heavy metal metab. and tolerances among vertebrates.
- 447Thomas, P. D. The Gene Ontology and the Meaning of Biological Function. Methods Mol. Biol. 2017, 1446, 15– 24, DOI: 10.1007/978-1-4939-3743-1_2447The Gene Ontology and the Meaning of Biological FunctionThomas, Paul D.Methods in Molecular Biology (New York, NY, United States) (2017), 1446 (Gene Ontology Handbook), 15-24CODEN: MMBIED; ISSN:1940-6029. (Springer)The Gene Ontol. (GO) provides a framework and set of concepts for describing the functions of gene products from all organisms. It is specifically designed for supporting the computational representation of biol. systems. A GO annotation is an assocn. between a specific gene product and a GO concept, together making a statement pertinent to the function of that gene. However, the meaning of the term "function" is not as straightforward as it might seem, and has been discussed at length in both philosophical and biol. circles. Here, I first review these discussions. I then present an explicit formulation of the biol. model that underlies the GO and annotations, and discuss how this model relates to the broader debates on the meaning of biol. function.
- 448Abdin, A. Y.; Jacob, C.; Kästner, L. The enigmatic metallothioneins. A case of upward-looking research. Int. J. Mol. Sci. 2021, 22, 5984, DOI: 10.3390/ijms22115984448The enigmatic metallothioneins: a case of upward-looking researchAbdin, Ahmad Yaman; Jacob, Claus; Kaestner, LenaInternational Journal of Molecular Sciences (2021), 22 (11), 5984CODEN: IJMCFK; ISSN:1422-0067. (MDPI AG)In the mid-1950s, Bert Lester Vallee and his colleague Marvin Margoshes discovered a mol. referred to today as metallothionein (MT). Meanwhile, MTs have been shown to be common in many biol. organisms. Despite their prevalence, however, it remains unclear to date what exactly MTs do and how they contribute to the biol. function of an organism or organ. We investigate why biochem. research has not yet been able to pinpoint the function(s) of MTs. We shall systematically examine both the discovery of and recent research on Dr. Vallee's beloved family of MT proteins utilizing tools from philosophy of science. Our anal. highlights that Vallee's initial work exhibited features prototypical of a developing research tradition: it was upward-looking, exploratory, and utilized mere interactions. Since the 1960s, MT research has increasingly become intervention- and hypothesis-based while it remained largely upward-looking in character. While there is no reason to think that upward-looking research cannot successfully yield structure-function mappings, it has not yet been successful in the case of MTs. Thus, we suggest it might be time to change track and consider other research strategies looking into the evolution of MTs. Recent studies in mollusks render research in this direction worthy of pursuit.
- 449Klaassen, C. D.; Liu, J.; Diwan, B. W. Metallothionein protection of cadmium toxicity. Toxicol. Appl. Pharmacol. 2009, 238, 215– 220, DOI: 10.1016/j.taap.2009.03.026449Metallothionein protection of cadmium toxicityKlaassen, Curtis D.; Liu, Jie; Diwan, Bhalchandra A.Toxicology and Applied Pharmacology (2009), 238 (3), 215-220CODEN: TXAPA9; ISSN:0041-008X. (Elsevier B.V.)A review. The discovery of the cadmium (Cd)-binding protein from horse kidney in 1957 marked the birth of research on this low-mol. wt., cysteine-rich protein called metallothionein (MT) in Cd toxicol. MT plays minimal roles in the gastrointestinal absorption of Cd, but MT plays important roles in Cd retention in tissues and dramatically decreases biliary excretion of Cd. Cd-bound to MT is responsible for Cd accumulation in tissues and the long biol. half-life of Cd in the body. Induction of MT protects against acute Cd-induced lethality, as well as acute toxicity to the liver and lung. Intracellular MT also plays important roles in ameliorating Cd toxicity following prolonged exposures, particularly chronic Cd-induced nephrotoxicity, osteotoxicity, and toxicity to the lung, liver, and immune system. There is an assocn. between human and rodent Cd exposure and prostate cancers, esp. in the portions where MT is poorly expressed. MT expression in Cd-induced tumors varies depending on the type and the stage of tumor development. For instance, high levels of MT are detected in Cd-induced sarcomas at the injection site, whereas the sarcoma metastases are devoid of MT. The use of MT-transgenic and MT-null mice has greatly helped define the role of MT in Cd toxicol., with the MT-null mice being hypersensitive and MT-transgenic mice resistant to Cd toxicity. Thus, MT is crit. for protecting human health from Cd toxicity. There are large individual variations in MT expression, which might in turn predispose some people to Cd toxicity.
- 450Calvo, J.; Jung, H.; Meloni, G. Copper metallothioneins. IUBMB Life 2017, 69, 236– 245, DOI: 10.1002/iub.1618450Copper metallothioneinsCalvo, Jenifer; Jung, Hunmin; Meloni, GabrieleIUBMB Life (2017), 69 (4), 236-245CODEN: IULIF8; ISSN:1521-6543. (John Wiley & Sons, Inc.)A review. Metallothioneins (MTs) are a class of low mol. wt. and cysteine-rich metal binding proteins present in all the branches of the tree of life. MTs efficiently bind with high affinity several essential and toxic divalent and monovalent transition metals by forming characteristic polynuclear metal-thiolate clusters within their structure. MTs fulfil multiple biol. functions related to their metal binding properties, with essential roles in both Zn(II) and Cu(I) homeostasis as well as metal detoxification. Depending on the organism considered, the primary sequence, and the specific physiol. and metabolic status, Cu(I)-bound MT isoforms have been isolated, and their chem. and biol. characterized. Besides the recognized role in the biochem. of divalent metals, it is becoming evident that unique biol. functions in selectively controlling copper levels, its reactivity as well as copper-mediated biochem. processes have evolved in some members of the MT superfamily. Selected examples are reviewed to highlight the peculiar chem. properties and biol. functions of copper MTs.
- 451Andrews, G. Regulation of metallothionein gene expression by oxidative stress and metal ions. Biochem. Pharmacol. 2000, 59, 95– 104, DOI: 10.1016/S0006-2952(99)00301-9451Regulation of metallothionein gene expression by oxidative stress and metal ionsAndrews G KBiochemical pharmacology (2000), 59 (1), 95-104 ISSN:0006-2952.The metallothioneins (MT) are small, cysteine-rich heavy metal-binding proteins which participate in an array of protective stress responses. Although a single essential function of MT has not been demonstrated, MT of higher eukaryotes evolved as a mechanism to regulate zinc levels and distribution within cells and organisms. These proteins can also protect against some toxic metals and oxidative stress-inducing agents. In mice, among the four known MT genes, the MT-I and -II genes are most widely expressed. Transcription of these genes is rapidly and dramatically up-regulated in response to zinc and cadmium, as well as in response to agents which cause oxidative stress and/or inflammation. The six zinc-finger metal-responsive transcription factor MTF-1 plays a central role in transcriptional activation of the MT-I gene in response to metals and oxidative stress. Mutation of the MTF-1 gene abolishes these responses, and MTF-1 is induced to bind to the metal response elements in proximal MT promoter in cells treated with zinc or during oxidative stress. The exact molecular mechanisms of action of MTF-1 are not fully understood. Our studies suggest that the DNA-binding activity of MTF-1 in vivo and in vitro is reversibly activated by zinc interactions with the zinc-finger domain. This reflects heterogeneity in the structure and function of the six zinc fingers. We hypothesize that MTF-1 functions as a sensor of free zinc pools in the cell. Changes in free zinc may occur in response to chemically diverse inducers. MTF-1 also exerts effects on MT-I gene transcription which are independent of a large increase in MTF-1 DNA-binding activity. For example, cadmium, which has little effect on the DNA-binding activity of MTF-1 in vivo or in vitro, is a more potent inducer of MT gene expression than is zinc. The basic helix-loop-helix-leucine zipper protein, USF (upstream stimulatory factor family), also plays a role in regulating transcription of the mouse MT-I gene in response to cadmium or H2O2. Expression of dominant negative USF-1 or deletion of its binding site from the proximal promoter attenuates induction of the mouse MT-I gene. USF apparently functions in this context by interacting with as yet unidentified proteins which bind to an antioxidant response element which overlaps the USF-binding site (USF/ARE). Interestingly, this composite element does not participate in the induction of MT-I gene transcription by zinc or redox-cycling quinones. Thus, regulation of the mouse MT-I gene by metals and oxidative stress involves multiple signaling pathways which depend on the species of metal ion and the nature of the oxidative stress.
- 452Vallee, B. L. Introduction to metallothionein. Methods Enzymol. 1991, 205, 3– 7, DOI: 10.1016/0076-6879(91)05077-9452Introduction to metallothioneinVallee, Bert L.Methods in Enzymology (1991), 205 (Metallobiochem. Pt. B), 3-7CODEN: MENZAU; ISSN:0076-6879.A brief description of metallothionein is given, including structural features such as zinc coordination. Possible functional roles of the protein are also discussed.
- 453Palmiter, R. D. The elusive function of metallothioneins. Proc. Natl. Acad. Sci. U. S. A. 1998, 95, 8428– 8430, DOI: 10.1073/pnas.95.15.8428453The elusive function of metallothioneinsPalmiter, Richard D.Proceedings of the National Academy of Sciences of the United States of America (1998), 95 (15), 8428-8430CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)A review with 55 refs. Biochem. and genetics are both required to elucidate the function of macromols. There is no question that metallothioneins (MTs) have unique biochem. properties, but genetic expts. have not substantiated the importance of MTs under physiol. conditions. Even after thousands of studies describing the structure, biochem. characteristics, tissue distribution, induction, and consequences of genetic disruption and deliberate overexpression, the evolutionary forces that led to the initial appearance, gene duplications, and nearly ubiquitous expression of MTs remain enigmatic.
- 454Kelly, E. J.; Palmiter, R. D. A murine model of Menkes disease reveals a physiological function of metallothionein. Nat. Genet. 1996, 13, 219– 222, DOI: 10.1038/ng0696-219454A murine model of Menkes disease reveals a physiological function of metallothioneinKelly, Edward J.; Palmiter, Richard D.Nature Genetics (1996), 13 (2), 219-222CODEN: NGENEC; ISSN:1061-4036. (Nature Publishing Co.)The authors investigated the role of metallothionine in copper homeostasis by using mice bearing inactivated alleles of both Mt1 and Mt2 genes (Mt-/-) which were crossed with Mo-brJ mice. Their results suggest that metallothionine is essential to protect against copper toxicity in the embryonic placenta. They also predict that metallothionine would continue to be required to protect intestinal cells after birth.
- 455Gudekar, N.; Shanbhag, V.; Wang, Y.; Ralle, M.; Weisman, G. A.; Petris, M. J. Metallothioneins regulate ATP7A trafficking and control cell viability during copper deficiency and excess. Sci. Rep. 2020, 10, 7856, DOI: 10.1038/s41598-020-64521-3455Metallothioneins regulate ATP7A trafficking and control cell viability during copper deficiency and excessGudekar, Nikita; Shanbhag, Vinit; Wang, Yanfang; Ralle, Martina; Weisman, Gary A.; Petris, Michael J.Scientific Reports (2020), 10 (1), 7856CODEN: SRCEC3; ISSN:2045-2322. (Nature Research)Abstr.: Copper (Cu) is an essential, yet potentially toxic nutrient, as illustrated by inherited diseases of copper deficiency and excess. Elevated expression of the ATP7A Cu exporter is known to confer copper tolerance, however, the contribution of metal-binding metallothioneins is less clear. In this study, we investigated the relative contributions of ATP7A and the metallothioneins MT-I and MT-II to cell viability under conditions of Cu excess or deficiency. Although the loss of ATP7A increased sensitivity to low Cu concns., the absence of MTs did not significantly affect Cu tolerance. However, the absence of all three proteins caused a synthetic lethal phenotype due to extreme Cu sensitivity, indicating that MTs are crit. for Cu tolerance only in the absence of ATP7A. A lack of MTs resulted in the trafficking of ATP7A from the trans-Golgi complex in a Cu-dependent manner, suggesting that MTs regulate the delivery of Cu to ATP7A. Under Cu deficiency conditions, the absence of MTs and / or ATP7A enhanced cell proliferation compared to wild type cells, suggesting that these proteins compete with essential Cu-dependent pathways when Cu is scarce. These studies reveal new roles for ATP7A and metallothioneins under both Cu deficiency and excess.