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
Abstract
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*
You are free to share(copy and redistribute) this article in any medium or format and to adapt(remix, transform, and build upon) the material for any purpose, even commercially within the parameters below:
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1. Introduction
1.1. Search for a Role of Cadmium in Biology: Discovery of Metallothionein
1.2. MT1 and MT2
1.3. MT1 Proteins
1.4. MT3 (GIF)
1.5. MT4
2. The Structures of Mammalian Zinc and Copper Metallothioneins
2.1. Primary Structures
2.2. Metal Composition: Zn(II), Cd(II), Cu(I)
2.3. 3D Structures
2.3.1. Structures of the Metal Sites
2.3.2. Structures of the Proteins
2.4. From Structure to Reactivity and Regulation
3. Reactivities and Metal Affinities of Metallothioneins
3.1. Redox Chemistry and Biology of MT
3.1.1. TR (Thionein)
3.1.2. TO (Thionin)
3.2. General Notes on Metal Affinities and the Essence of Metal/Thiolate Coordination
3.3. Partially Metalated Zinc MT Species
3.3.1. Partially Metalated MT Species as Components of a Cellular Zinc Buffer
3.3.2. Structures of Partially Metalated MT Species
3.4. Copper MT
3.4.1. Cu(I) Binding Affinity of MT
3.4.2. Cu(II) Interactions with MT
3.5. Cadmium MT
3.6. Metal Ion Selectivity
4. Relating Metallothionein Thermodynamics and Kinetics to Control of Cellular Zn(II) and Cu(I)
4.1. Buffers for the Most Competitive Metal Ions
4.2. Zinc Buffering
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) |
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.
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.
4.3. Zinc Signaling
4.4. Copper Buffering
4.5. Copper Signaling
4.6. Controlling Zn(II)/Cu(I) Ratios
4.7. Metal Transfer and Exchange Reactions
4.7.1. Metal Transfer from MT to Proteins
4.7.2. Metal Transfer from Proteins to Thionein
4.7.3. A Role of ZnATP
5. Regulation of Metallothionein in Biological Space and Time
5.1. Multiplicity of MT Genes and Their Regulation
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 |
5.2. Spatiotemporal Distribution of MT in Cells
5.3. Extracellular MT
5.4. Interaction of MT with Proteins
6. From Metallothionein Structure to Functions, Roles, and Purposes
6.1. MTs in Other Organisms
6.2. The Dilemma of MT’s Biological Functions
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 |
6.3. Challenges for Future Research
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
Biographies
Acknowledgments
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
This article references 455 other publications.
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- 2Margoshes, M.; Vallee, B. L. A Cadmium protein from equine kidney cortex. J. Am. Chem. Soc. 1957, 79, 4813– 4814, DOI: 10.1021/ja01574a064Google Scholar2https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaG2sXpvFKhsQ%253D%253D&md5=2b515b5a86f8996d301338158c7d4127A 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 Scholar3https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaF3MXjvVyhug%253D%253D&md5=2c41635c0735504c82e7a4d9f7e2d5b3Metallothionein: 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 Scholar4https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaF3MXhsVCltbg%253D&md5=6bcfb831275f08cfa40fce72269d872dMetallothionein: 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.
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- 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 Scholar8https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaE1cXosVygtg%253D%253D&md5=e2c6ff819b408667ae5a5a1d21967376Preparation 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 Scholar10https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaE38Xot1CntQ%253D%253D&md5=6afdd0dbad46399556e4621c6565b7d5Separation 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 Scholar11https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL2MXlslKjsr4%253D&md5=3bb9eba70a751aea0594666d56d60585Isolation 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 Scholar12https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL3sXktVWitL8%253D&md5=09cad13e8212f66e12f733cb7ac8158cHuman 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 Scholar13https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3MXmsFehtrs%253D&md5=90422b2f01daec161ef20d779271f6adThe 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 Scholar14https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2MXks1Kis7o%253D&md5=4f1c235c96b4f40ca81ea6223aa95cedBioactivity 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 Scholar15https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3sXktVegsL0%253D&md5=0a3e77a9def4d843619345cdd0468d5dMT-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 Scholar16https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2cXktlWksr0%253D&md5=bf5c862a98e5e1ec6d486af785ec071aInduction 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 Scholar17https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaE28XlvVyjsb8%253D&md5=84c6a3952970d4f1a3e38e2642280355Amino-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 Scholar18https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK38XhsVKnsr8%253D&md5=c7556547e431d9dce1423f9241c56087Definitions 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 Scholar19https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL2cXmtVGhs7o%253D&md5=12b890ee439f73a561f0091d93c131b6Human 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 Scholar20https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXksFWisLc%253D&md5=6a701af0b7ecce400d39f5d25190c891MAFFT 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 Scholar21https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXoslyisLk%253D&md5=539043757a31620d1e125f472b514427Java 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 Scholar22https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XovVGrsro%253D&md5=27c063d3e694cd9d0fe9e3eade880b41Metallothionein 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 Scholar23https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXktVyitbY%253D&md5=ece6310cea25d80c352cbff7ec5d0661The 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 Scholar24https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XhtlGktLzN&md5=530e011782349526f31deffe47918d30Interactive 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 Scholar25https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK38XitFSlt7c%253D&md5=48c7729fc4c6e8e05136a9e458a51a54Quantification 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 Scholar26https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK38XitFSlt7o%253D&md5=d99e66a7f0265acc59b68c8df1fbb54fStandard 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 Scholar27https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK38XitFSltb0%253D&md5=b639921d0d19297ca22aea4c5efeb58cMetal 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 Scholar28https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK38XhvFSmtbY%253D&md5=b14028b34a6c78f1174b02a8e0820d9dMetal 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 Scholar29https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaE2MXlsFymsLw%253D&md5=78a5f7ac08fb3fb59359bcdbb85049a1Metal-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 Scholar30https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL2MXptFGrug%253D%253D&md5=9c0e1d94bbbc606d7b7b332bfe8070fcCadmium-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 Scholar31https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXmslWksL4%253D&md5=8d5f6bfada8f93bae1939a9fdf3af7baThe "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 Scholar32https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK38XhtVWgsb0%253D&md5=c590212917f189dd968071b116cf0a66Nonoxidative 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 Scholar33https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL3MXktVajur4%253D&md5=68e7c1814bd426f93b9aeb2078e252fbZinc(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 Scholar35https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL3MXpvVaqsA%253D%253D&md5=c6fbab6c685c4fc3305ae9a9ed070e09Structure 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 Scholar36https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADyaK387ktVWgtA%253D%253D&md5=c26c01eecb97e0b1ac39c820f0ebd8e1Overview 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 Scholar37https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL38XhslKnu7s%253D&md5=ddda8e2056cddc7b01625250af316285Domain 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 Scholar38https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL38XlvFCrs70%253D&md5=2f3982408e65885803f4efb7364f458dCadmium-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 Scholar39https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38XhvVWnu7c%253D&md5=b541d3ae48375a36064dd7257254c1eeAdvances 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 Scholar40https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL2MXmtV2itrg%253D&md5=f074a733ed1a39da37f952f768a107eePolypeptide-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 Scholar41https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38XnvFCrtbY%253D&md5=6163cad8e4b906d74780bec1228bc4f8Structure 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 Scholar42https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhsVaksbjF&md5=9db541b8c1cc4933e875303cae7cbffbSolution 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 Scholar43https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXislyltbs%253D&md5=a58ce304351f75c61df22c4b9e984616Crystal 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 Scholar44https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL1cXksFSntrc%253D&md5=9faa46d6df73b71995ebcc7d20301139Three-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 Scholar45https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL1cXmt1GjtL0%253D&md5=d4062571adb65bff40dae4149094d08bConformation 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 Scholar46https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3cXlsVOmsr4%253D&md5=0ba576a2b594714fad75007e2d1ee4d9Three-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 Scholar47https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK38XisVym&md5=479db7d762d006383802c28907218081Refined 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 Scholar48https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3sXps1Gjsw%253D%253D&md5=2f71723a812cd14a042116ca2755f80bComparison 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 Scholar49https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BD3Mrht1ymtQ%253D%253D&md5=f6a2b135bc7c556dd6782c79f7ff66d2Three-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 Scholar50https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XhtFejsLc%253D&md5=756cde68ec0e069991fe06cbd0bf220aSolution 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 Scholar51https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXlt1Oqu7o%253D&md5=ef22e4a8b75ada075c4446a82f727c47Coordination 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 Scholar52https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaE2MXjvFyisA%253D%253D&md5=ea9b62f9537a5096008988f679dd535cConversion 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 Scholar53https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2MXktVSjtrc%253D&md5=80d1aa4f8fb41740578c49bce66e3639Copper 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 Scholar54https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXit1GqtbbJ&md5=a4f53836f46b85e01f56aa85ed8cdf77The 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 Scholar55https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXmtlaqug%253D%253D&md5=6d867fadd96e5d49e0b0a15fcbbfa604The 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 Scholar57https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38Xis1OktQ%253D%253D&md5=cab95d968c8f79f6cd1b0002ea602640The 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 Scholar59https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2sXhvF2ltLk%253D&md5=63f51c8902ee4f7df4ae514a79e3de1eCoordination 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 Scholar60https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL28XlsVyhsbc%253D&md5=16e298a861c57c286f8f12696b3f1daaMetallothioneinHamer, 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 Scholar61https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL1MXhvFegsw%253D%253D&md5=814e1f27e7171f5ea6de4fb73b183524A 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 Scholar62https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3cXisVejsbg%253D&md5=f6e201e4592384b87747e60d1411c7ceInduction, 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 Scholar63https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38XpsFWrug%253D%253D&md5=5391d068dd8d1d6111d41fd684e2ef66Functions 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 Scholar64https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2cXhsFKitbY%253D&md5=c8da6fb6fe330f5630f4b807244be2c2Targeted 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 Scholar65https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3sXlslCqtbY%253D&md5=bdc079495aa7d4b9c8bcee56aec06c63Targeting 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 Scholar66https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL2MXktlGiurk%253D&md5=1ab410eb088ce92f2bcbcd22d40aee30Metallothioneins: 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 Scholar67https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL2MXptFKhtA%253D%253D&md5=b8699a417ecaffbcab5aab8a25211382Possible 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 Scholar68https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3sXitlSkt74%253D&md5=2345f878b7fa5c247bdec17a4ba3fb43Oxygen 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 Scholar69https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK38Xot1Sisw%253D%253D&md5=aa698a05fc5f394bacbc6e04062f772aOxidant-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 Scholar70https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL1MXltFWjtLk%253D&md5=6d9552d5c4cb01a1b72e860fe333b45dStructure-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 Scholar71https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL3MXitVCjtL0%253D&md5=e84de780a81012b5c54db0a717e82229On 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 Scholar72https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1cXitlKjs74%253D&md5=54cdf1f20c2e10a1bcd9865de915d234Control 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 Scholar73https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXovF2mu7o%253D&md5=ffdd78e8cbf2b968da10487811d66e79Dual 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 Scholar74https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3MXlslKqtw%253D%253D&md5=f57cc2aa26a72ba50186202bcd71cd79On 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 Scholar75https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3cXitVaitbk%253D&md5=e127fb1f9a79d2acd454ff687ea463a4Zinc 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 Scholar76https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2cXpvFyntA%253D%253D&md5=a2c141d0c9f76680c50b6e94ab016ed2The 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 Scholar77https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2cXpvFyitg%253D%253D&md5=ddf7f7d807067ff6f5abe0d523e5b9fcOxidative 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 Scholar78https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXhtVynsrvJ&md5=31cd58b631f7077cc7d8d59978f5f45dCellular 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 Scholar79https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1cXitlKjsrY%253D&md5=cb67322cbea61fd4d6d651ebf26e295aThiolate 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 Scholar80https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3cXivFKms7o%253D&md5=a556094cbc4fea56191402bb99053cd9The 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 Scholar81https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXjs1Oqsr0%253D&md5=58266391ed41b8653731ab5ed3d802feCellular 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 Scholar83https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXhs1yitLw%253D&md5=0662e3f376af70696acd1b49677b9eacZinc 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 Scholar84https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXmtlCrsbc%253D&md5=d0bb33592bf042a3c30340b7d30c61c0Redox 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 Scholar85https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2MXmsFyltr4%253D&md5=27ce5879d74cf1e2277771a0d2a832efMetallothionein/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 Scholar86https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XpslalsrY%253D&md5=1801e5158c426cfa8e854f2de65bd11eZinc 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 Scholar87https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2MXisV2ltbk%253D&md5=16eb7d366b2e494a0d9d90a8182614ecBasal 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 Scholar88https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXjs1Wnsr8%253D&md5=652ff46db4b893547a0470026d9b0b35Differential 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 Scholar89https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28Xht1entLnE&md5=9c44916a7c25216500f8fa5e74d540a7Zinc-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 Scholar90https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXitVKhtrw%253D&md5=a27e1c946b1f0ccd741214cfba2f5c41Different 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 Scholar91https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXntlGntrs%253D&md5=4bc54b599a608dc1a5c6364e54108db5A 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 Scholar92https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28Xit1SnsQ%253D%253D&md5=2c8f76efe111afc6a82258beaa489b22Metallothionein 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 Scholar93https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXhtFKrsLw%253D&md5=6a514cfff065e88e3eef828a25b40a8cStoichiometry 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 Scholar94https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXhsVyqurjO&md5=bbe2d601d6a7aa8ca007f35c126fd32cPartial 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 Scholar95https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXns1aksg%253D%253D&md5=21665918bde3c8494d7b5bc138f11abcCysteine-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 Scholar96https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK38XitFSlt70%253D&md5=3ac63364ab81135c8adb40299230ea8aLarge-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 Scholar97https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXmsFCguw%253D%253D&md5=b59ad34580cf640840dcebbe351d7aaeHigh 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 Scholar98https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhsFWgt7%252FM&md5=afca1056a6c035cec613f7a29dfdfa7eMetal- 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 Scholar99https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1cXitlKjsrc%253D&md5=53fc9f5f8f75b4efec26929790286e9eThe 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 Scholar101https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3sXltlersbs%253D&md5=c240364d37c619ccf534a548812be2cfA 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 Scholar102https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL1MXhvVOnur0%253D&md5=5b4ea0d1bbb78a325ea6f1cee2927555The 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 Scholar103https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXhslOks70%253D&md5=5de4e1520fa0b79b9d8ca2304aee41d2May 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 Scholar104https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXnslChtA%253D%253D&md5=c326f46d4efe9b5008708ab3e6e532d6Studies 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 Scholar105https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXkvFCqurc%253D&md5=b541daad7e894ac15640fb292ce4af75S-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 Scholar106https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXlvVOrsLw%253D&md5=e160c126df2c4d10836bdb26a81ac084Nitric 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 Scholar107https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BD28jks1Witw%253D%253D&md5=c75432e88436c8d7308bcd07216ce2dfTargeting 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 Scholar108https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3cXns1ejtb8%253D&md5=67052a47b56c104013f6d4e328beb2e5Induction 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 Scholar109https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhtFCqsLfE&md5=780adc1b4182a7b1662bfa2c36ed292dChemistry 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 Scholar110https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1MXotFeit74%253D&md5=d79a52f1eef0736994ba2c580e65b0ceStudy 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 Scholar111https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1MXhvVSrurk%253D&md5=50acd3c930c35f3a109d548313846902Selenium 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 Scholar112https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXmtlCrtrY%253D&md5=190938a6f75dd2afe7d7e972d5afba6bCatalytic 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 Scholar113https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXktlSgtro%253D&md5=2ae4e75853534b1b4c2d52f38aba9b14Catalytic 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 Scholar114https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1MXnslGkur0%253D&md5=999d15a5001ec5325ebdd35b0e1f5b05Metal 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 Scholar115https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XlvVCitbg%253D&md5=f7d989e3f4cc8e69500d46af4bbbb418Thionein 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 Scholar116https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XhtVOrsbbI&md5=ed296258333ef0723e8098626b713a8fSelenocompounds 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 Scholar118https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADyaK2c3jsFSjsw%253D%253D&md5=d67656edbfc94d7c2c6a3a2b7e00596fNitric 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 Scholar119https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38XhtFGhsro%253D&md5=3a87f5c3dee9f20d6c7ac9dbb6aab100Nitric 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 Scholar120https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXpsFGisbg%253D&md5=db5f18d6dfefc85e18b3780da48a3253Regulation 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 Scholar121https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38Xkt1KrtLk%253D&md5=933e2f756e8e1daae569b61cb2f37c07S-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 Scholar122https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XhtVylt7%252FN&md5=0b93e86ac6d7c139c476a9175f8279e7Cell-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 Scholar123https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXhtFahsr3F&md5=f2c5d10b6d268595c03a71d4a053a1f5Thioredoxin 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 Scholar124https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1MXns1yjtLg%253D&md5=a741c9367a04266be4b691a39760f8c1Nitric 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 Scholar125https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2MXhtVSjsL7M&md5=effadf664b38e7b16e38b9a0fe47bd41Evidence 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 Scholar126https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1MXhtlCjur8%253D&md5=1a54da914d22633c071e94af0e62bc71Redox 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 Scholar127https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28Xht1yqsrvK&md5=2e0f11a48d15247b475987800214fe32Aldehydes 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 Scholar128https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXhtFGku7bM&md5=e2b043cfab1ec0ef73ee4e0086967822Protective 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 Scholar129https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXis1eksL4%253D&md5=12b8f3a081380fdab8091a7c194fe888Covalent 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 Scholar130https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXkvFCqtbo%253D&md5=ac43b932d393e02b6e392236d8a93bcfMetallothionein 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.