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Direct Observation of Oligomerization by Single Molecule Fluorescence Reveals a Multistep Aggregation Mechanism for the Yeast Prion Protein Ure2

Jie Yang^†^⊥^¶

Alexander J. Dear^‡^¶

Thomas C. T. Michaels^‡^∥

Christopher M. Dobson^‡

Tuomas P. J. Knowles^*^‡^§

Si Wu^*^†^⊥

, and

Sarah Perrett^*^†^⊥

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† National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Chaoyang District, Beijing 100101, China

‡ Centre for Misfolding Diseases, Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, United Kingdom

§ Cavendish Laboratory, J J Thomson Avenue, Cambridge CB3 1HE, United Kingdom

∥ Paulson School of Engineering and Applied Sciences, Harvard University, Cambridge, Massachusetts 02138, United States

⊥ University of the Chinese Academy of Sciences, 19A Yuquan Road, Shijingshan District, Beijing 100049, China

*[email protected]

Cite this: J. Am. Chem. Soc. 2018, 140, 7, 2493–2503

Publication Date (Web):January 22, 2018

https://doi.org/10.1021/jacs.7b10439

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Abstract

The self-assembly of polypeptides into amyloid structures is associated with a range of increasingly prevalent neurodegenerative diseases as well as with a select set of functional processes in biology. The phenomenon of self-assembly results in species with dramatically different sizes, from small oligomers to large fibrils; however, the kinetic relationship between these species is challenging to characterize. In the case of prion aggregates, these structures can self-replicate and act as infectious agents. Here we use single molecule spectroscopy to obtain quantitative information on the oligomer populations formed during aggregation of the yeast prion protein Ure2. Global analysis of the aggregation kinetics reveals the molecular mechanism underlying oligomer formation and depletion. Quantitative characterization indicates that the majority of Ure2 oligomers are relatively short-lived, and their rate of dissociation is much higher than their rate of conversion into growing fibrils. We identify an initial metastable oligomer, which can subsequently convert into a structurally distinct oligomer, which in turn converts into growing fibrils. We also show that fragmentation is responsible for the autocatalytic self-replication of Ure2 fibrils, but that preformed fibrils do not promote oligomer formation, indicating that secondary nucleation of the type observed for peptides and proteins associated with neurodegenerative disease does not occur at a significant rate for Ure2. These results establish a framework for elucidating the temporal and causal relationship between oligomers and larger fibrillar species in amyloid forming systems, and provide insights into why functional amyloid systems are not toxic to their host organisms.

Introduction

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The self-assembly of soluble proteins into insoluble and highly structured amyloid fibrils rich in β-sheet structure is associated with a variety of human disorders, including Alzheimer’s and Parkinson’s diseases, type II diabetes, and the prion diseases.(1, 2) In addition, the formation of amyloid fibrils has been found to be a common or generic property of polypeptide molecules, and also to be associated with a number of diverse biological functions in living organisms.(1, 3) Over the past decade, oligomeric intermediates that form during the early stages of amyloid fibril formation or dissociate from mature fibrils have become of increasing interest(4) because such species, rather than the fibrils, have been shown to be toxic to cells and are now thought to be the major pathogenic agent in neurodegenerative disease.(1, 2, 5-7) Understanding the nature and dynamics of the oligomers is not only of intrinsic interest, but has the potential to provide a catalyst for the development of therapeutic strategies for protein misfolding diseases.(2, 8, 9)

Conventional biochemical and biophysical methods, such as the thioflavin T (ThT) fluorescence assay,(10) circular dichroism spectroscopy (CD),(11) electron microscopy (EM)(12) and atomic force microscopy (AFM)(13) measurements, are able to provide ensemble information about the aggregation kinetics of amyloidogenic proteins as well as the conformation and morphology of amyloid fibrils and the intermediate prefibrillar species populated during their formation. The metastable and heterogeneous nature of protein oligomers, however, limits the detailed characterization of such prefibrillar species. Recently, a variety of novel approaches have been developed and applied for this purpose. Fluorescence correlation spectroscopy, for example, provides information about the nature and distribution of species formed in the early stages of fibril formation.(14, 15) Additionally, mass spectrometry and NMR spectroscopy have been applied to detect and analyze the properties of oligomers with a range of different structures.(16-18) Single molecule fluorescence spectroscopy, however, offers a particularly powerful approach for exploring the formation and properties of oligomers, as it has the ability to investigate individual molecular species and to reveal conformational dynamics that may be averaged in the ensemble experiment.(19, 20) These techniques have been shown to be able to identify and characterize the low-populated, heterogeneous and transient species formed during fibril assembly of several amyloidogenic proteins.(21-25)

Ure2 from Saccharomyces cerevisiae is the protein determinant of the yeast prion state [URE3](26) and provides an important system for probing amyloid formation and prion propagation. The Ure2 protein is a dimer in solution; each monomer contains 354 amino acids and consists of two domains.(27, 28) The unstructured and flexible N-terminal domain (residues 1–93) is primarily responsible for prion conversion and propagation in vivo and for formation of amyloid fibrils in vitro.(29-31) The globular C-terminal domain (residues 94–354) is similar to glutathione transferases in structure(32, 33) and has both glutathione-dependent peroxidase activity(34) and glutaredoxin activity.(35) Ure2 is also a negative regulatory factor of nitrogen metabolism, as in its native state the protein interacts with the transcription factor Gln3 and represses the uptake of poor nitrogen sources.(36) Therefore, when Ure2 converts into the aggregated prion state, Gln3 is released and activates the expression of the genes related to the metabolism of less favorable nitrogen sources.

While the aggregated prion form of Ure2 is tolerated by yeast cells, precursor aggregates of Ure2 are toxic to mammalian cells,(37, 38) as are amyloid aggregates of other proteins.(39) It is therefore interesting to study the oligomers formed and the structural changes occurring during Ure2 fibril formation, and to compare them to disease-related models. In a previous study we identified by AFM prefibrillar intermediates of Ure2 with a range of different sizes formed during the aggregation process.(31) A soluble oligomeric species formed during the early stages of Ure2 aggregation was separated and characterized by biochemical and spectroscopic methods.(40) Taken together, these results suggest a connection between the population of oligomeric species and the course of Ure2 amyloid assembly into mature fibrils. Theoretical modeling has previously enabled the kinetic parameters that describe the growth and breakage of Ure2 fibrils to be defined, allowing the contribution of individual molecular steps to be correlated with prion propensity;(41) however, data on the oligomeric populations of Ure2, which would allow this type of mechanistic analysis to be carried out, have not previously been available. In the present study, we have applied single molecule fluorescence resonance energy transfer (smFRET) to investigate in detail the intermolecular assembly and aggregation process of Ure2. This approach has enabled oligomerization during the initial lag phase to be observed, and two types of Ure2 oligomers with different assembly modes have been identified. Furthermore, using theoretical analysis combined with single molecule and ensemble kinetic data, we describe the formation and depletion pathway of oligomers, and propose a multistep mechanism for Ure2 fibril formation, in which initial oligomerization is followed by conformational conversion to β-sheet-containing oligomers that are then able to grow to form mature amyloid fibrils.

Methods

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Mutant Construction, Protein Expression, Purification and Labeling

The single point cysteine variants (V9C, S53C and S68C) of Ure2 were obtained by overlapping PCR using a synthetic wild-type URE2 gene as template(42) and ligated into the mini-pRSETa vector. All the mutants constructed in this study were confirmed by DNA sequencing. Full-length Ure2 was expressed in E. coli C41(DE3) cells with a His₆-tag and purified by nickel-affinity chromatography as described previously.(42, 43) Purified Ure2 was dialyzed into 50 mM Tris–HCl (pH 8.4) buffer containing 200 mM NaCl and 500 μM TCEP at 4 °C. Protein purity was checked by SDS-PAGE, and the protein concentration was determined by the absorbance at 280 nm for full-length Ure2 using a molar extinction coefficient of 48,220 M^–1 cm^–1.(42) The details of fluorescence labeling of Ure2 are described in the Supporting Information (SI) Methods.

Single-Molecule FRET Measurement of Ure2 Oligomers

SmFRET experiments were carried out using a home-built confocal microscope or total internal reflection fluorescence (TIRF) microscope based on a Nikon Ti-E inverted microscope similar to that described previously.(44) The details of the instrumentation, experimental procedures and data analysis used for confocal smFRET and TIRF smFRET are described in the SI Methods.

Developing a Model for Kinetic Data Fitting

We set out here to develop a quantitative model that describes the experimental observables: the total fibril mass concentration M(t) and the total oligomer concentration O(t). In addition to M(t) and O(t), the model explicitly considers the concentration of native state dimeric Ure2 m(t), and of fibrils P(t). In particular, the model describes explicitly the formation of oligomers through dimer association with rate constant k_oligo, their conversion to fibrils with rate constant k_c, their destruction (rate constant k_d), and fibril growth and fragmentation (rate constants k₊ and k_–, respectively). Addition of further complexity to this coarse-grained model is in principle readily possible within the master equation formalism; such additional details, including differentiating between multiple structural classes within the oligomer subpopulations, would, however, require further experimental constraints than are currently available, in order to avoid overfitting.(45) We used the Ure2 dimer concentration for m(t) rather than the monomer concentration, as evidence suggests Ure2 remains in its dimeric form throughout the aggregation reaction (see Results).

The rate equations for the model can be written as a master equation:

(1)

(2)

(3)

(4)where we have left out terms with negligible contributions to the overall kinetics, such as the effects of nonelongation steps on monomer depletion.(46) Any larger oligomers are expected to form from growth of smaller oligomers; all oligomers ultimately grow from the initial interaction of a pair of dimeric Ure2 molecules. The physically reasonable choice of overall reaction order for oligomer formation is therefore 2.0. (For further explanation, see SI Methods.)

These equations were solved for early times in the aggregation process, and the solutions used to derive a first-order self-consistent expression for M(t) (see also SI Methods):

(5)with A = αk₊k_c/κ²(k_l + κ), k_l = k_c + k_d,

and α = k_oligom(0)².

The time-dependent evolution of the oligomer population depends only on k_oligo, k_l and m(t). In turn, m(t) depends only on κ and A, or κ, k_l, and αk₊k_c. Overall therefore, the dynamics of the dimer and oligomer populations depend on the following four combinations of rate parameters: k₊k_–, k₊k_c, k_l, and k_oligo. Moreover the fitted values of k_oligo and k_l are approximately independent of the values chosen for k₊k_– and k₊k_c, provided that these two parameter combinations give a reasonable fit to the fibril mass concentration.

Relating Model Rate Constants to Fundamental Reaction Steps

Experiments indicate that we can resolve the observed oligomers into two structurally distinct populations, with a low-FRET oligomer formed initially and subsequently converting into a high-FRET oligomer, which in turn converts to fibrils. The data are not, however, sufficiently detailed to allow a full kinetic analysis to be carried out on both populations individually. The avoidance of overfitting necessitates, therefore, that we consider together the different structural classes of oligomers, and examine the overall fluxes that lead to their generation or depletion. We can, however, incorporate elements of our knowledge of the oligomer subpopulations in the overall interpretation of the results. We did so by determining how each species contributes to the total oligomer formation, depletion and conversion rate constants in our coarse-grained model. The concentration of the later high-FRET oligomer species changes very little over the time course of the aggregation reaction compared to that of the earlier low-FRET species, and is present at significantly lower concentrations than the earlier low-FRET species over the times most relevant to the fitting procedure. Moreover, we show below that high-FRET oligomers are likely to be formed from conversion of low-FRET oligomers. Therefore, the rate constants for total oligomer formation and dissociation obtained from the fitting process can be interpreted as approximately the rate constants for low-FRET oligomer formation and dissociation. The rate constant k_c gives the approximate proportionality between the overall oligomer concentration and the rate of formation of fibrils from oligomers, and so contains information on both the conversion of low-FRET to high-FRET oligomers, as well as the conversion of high-FRET oligomers to fibrils. The concentration data on high-FRET oligomers indicated that the steady-state approximation is likely to be valid here, in which case we can explicitly write k_c in terms of the rate constants of a more detailed kinetic model featuring two separate oligomeric species (see SI Methods). The “conversion” rate constant would then be proportional to the rate constant for transformation of low-FRET oligomers to high-FRET oligomers, as well as to the rate constant for conversion of high-FRET oligomers to fibrillar species.

Fitting the Combined smFRET/ThT Data to the Model

The ThT component of the data was fitted globally to the analytical expression for the full time-course fibril concentration to obtain values for A and κ using the online fitting platform Amylofit.(47) Then, the smFRET component of the data was fitted to our early time expression for O(t) using Mathematica to give approximate values for k_oligo and k_l. Having established that k_d ≫ k_c, we can set k_d = k_l. Combining these conclusions with A and κ yields approximate values for k₊k_– and k₊k_c. The availability of these approximate rate constants as trial parameters enabled a numerical fit of our combined smFRET/ThT data to eqs 1–4 to be carried out, yielding robust rate constants and verifying the consistency of our model with the experimental data. For the fitting of the combined smFRET/ThT data, a ratio of 1.5:1 was chosen for k₊k_–(S68C):k₊k_–(V9C) (see Results). For a full description of the kinetic model and fitting methods, see SI Methods.

Results

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Single Molecule FRET Measurements Can Monitor the Formation of Ure2 Oligomers

Since Ure2 has no intrinsic cysteine residues, single cysteine mutations were introduced to specific sites within the Ure2 N-terminal region to allow covalent linkages to maleimide-functionalized dyes. In order to avoid perturbing the process of Ure2 fibril formation, we chose mutation sites that are located near the turn of the β-strand in the structural models of the Ure2 amyloid fibril core.(48) The residues selected were V9C, S53C and S68C, of which V9C and S53C are near the N-terminal and C-terminal ends of the fibril core (Figure 1A), respectively, and should be sensitive reporters of the conformational changes associated with aggregation. The formation of fibrils by these derivatives was then monitored using ThT fluorescence. The results showed that both Ure2 S53C and S68C formed fibrils at essentially the same rate as that of wild-type Ure2, while V9C formed fibrils more slowly, as reflected in a longer lag time than the wild type (Figure S1A). A possible reason for this change is that V9 is located in one of the regions of the prion domain that have been shown to be important for formation of Ure2 amyloid(31) and may indeed participate in the formation of the first β-strand in Ure2 fibrils;(49) the source of this difference in rate is addressed further in the kinetic analysis below. For the case of the dye-labeled proteins, ThT assays could not be used because of potential FRET effects between ThT and the labeled fluorophores; the kinetics of fibril formation by the labeled proteins were therefore monitored using turbidity measured at OD₄₀₀, which indicates that fluorescence labeling does not significantly perturb the fibril formation rate of the mutants (Figure S1B). Furthermore, the aggregates formed by these unlabeled and labeled Ure2 proteins were imaged using transmission electron microscopy (TEM) and each showed a similar fibrillar morphology to that of wild-type Ure2 fibrils (Figure S1C).

Figure 1. Oligomerization of Ure2 monitored by confocal single molecule FRET. (A) Schematic figure to indicate the cysteine mutations and fluorescence labeling sites that were used in this study, based on a previously suggested structural model of Ure2 fibrils.(48) (B) Scheme for smFRET detection of Ure2 oligomers. (C) The concentration of AF555/AF647 labeled Ure2-S68C oligomers throughout the aggregation reaction. (D) Ensemble kinetics of the aggregation of 15 μM (dimeric concentration) unlabeled Ure2-S68C monitored by ThT fluorescence. All the aggregation reactions were carried out at 18 °C in an Innova 4230 incubator with shaking at 150 rpm in 50 mM Tris–HCl (pH 8.4) buffer containing 200 mM NaCl.

To check the efficiency of fibril formation, we also measured the fraction of labeled protein in the supernatant and the pellet by SDS-PAGE after the aggregation reaction reached a plateau, and most of the soluble protein was found to have converted into the insoluble fibrillar form, with less than 10% of the protein remaining in the supernatant (Figure S1D). Fluorophore attachment on the sites selected here does not, therefore, alter the ability of Ure2 to form fibrils or the nature of the fibrillar products. The self-assembly process of the fluorophore-labeled Ure2, as monitored by single molecule fluorescence experiments, can therefore be considered to be the same as that of unlabeled Ure2. Note that since Ure2 is a highly stable dimer in solution,(42, 50) and the dimeric unit of Ure2 seldom dissociates during fibril formation (Figure S2); we therefore refer to the dimer concentration throughout this study, unless otherwise stated.

Next we used smFRET, which has been used in studies of other amyloidogenic proteins(22-24) to detect the oligomeric species formed during the Ure2 fibril formation process. The soluble oligomer formed between AF555- and AF647-labeled Ure2 gave well-defined FRET signals and could be detected at the single molecule level by monitoring coincident bursts in both donor and acceptor channels when the oligomers diffused across the diffraction-limited focus (Figure 1B). The numbers of selected FRET events were converted to oligomer concentrations (see SI Methods) and plotted against time (Figure 1C). Meanwhile, the progress of fibril formation of the unlabeled Ure2 in bulk solution was monitored by the ThT fluorescence assay under the same incubation conditions (Figure 1D). As shown in the smFRET data (Figure 1C), the number of oligomers rises to its highest level within 5 h, which occurs during the lag phase of the ensemble ThT curve (Figure 1D), reflecting the assembly and increase in the number of oligomers. After 5 h, the soluble oligomeric species were observed to decrease in concentration as the Ure2 becomes sequestered into mature fibrils that are not detected by the confocal smFRET technique. The decrease in the number of oligomers corresponds well with the onset of fibril formation reflected in the bulk ThT assay under the same conditions (Figure 1D).

We assessed the stability of the oligomers formed at different time points by taking an aliquot from the aggregation reaction and diluting it into buffer; we then monitored whether or not there was any decrease in the smFRET burst rate corresponding to the detection of individual oligomers during a 1 h window immediately after dilution. Coverslips were pretreated with unlabeled protein to avoid adsorption of the labeled sample onto the surface. We observed by TIRF imaging that pretreatment of the surface with either BSA or unlabeled Ure2 was equally effective at suppressing the adsorption of the fluorescently labeled sample (Figure S3A). However, when the diluted sample was loaded onto BSA-coated coverslips, oligomer dissociation was evident by confocal smFRET within 1 h (Figure S3B). In contrast, when we used coverslips pretreated with unlabeled Ure2, the oligomers remained at a constant level during the measurement time (Figure S3B–F), a finding that can be rationalized by the desorption of unlabeled protein from the surface into solution, thus stabilizing the oligomers. Therefore, in order to avoid underestimating oligomer concentrations, we used coverslips coated with unlabeled Ure2 when performing smFRET experiments.

These results demonstrate the power of single molecule techniques for the observation of low populations of oligomers. It is evident that aggregates of Ure2 form during the lag phase observed by ThT fluorescence, in agreement with theoretical modeling.(51)

SmFRET Measurements Reveal the Absence of Significant Oligomer Formation via Secondary Nucleation During Ure2 Fibril Formation

The two generic mechanisms that lead to the formation of fibrils are the primary nucleation pathway, during which new oligomers are generated by the direct association of soluble protein or peptide molecules, and secondary pathways, where existing fibrils have the propensity to generate the formation of new fibrils, either through fragmentation or through surface catalyzed secondary nucleation. In the latter case, nucleation of new fibrils takes place on, and is catalyzed by, the surface of existing fibrils,(46) the rate of which therefore depends on the mass concentration of existing fibrils. In the case of Aβ42 aggregation,(52) whose kinetics are dominated by the surface-catalyzed secondary nucleation pathway, it has been shown explicitly that the majority of small oligomers present during the reaction are produced during secondary nucleation.(52) In earlier studies of Ure2, we have shown that fragmentation events are important determinants of the rate of fibril formation under both quiescent and shaking conditions(41, 53, 54) although the production of oligomers through surface-catalyzed secondary nucleation in the case of Ure2 has not previously been discounted.

We measured the generation of Ure2 oligomers directly using smFRET in the presence of 1% preformed mature fibrils to provide a surface for oligomer formation if secondary nucleation were to occur at a significant rate for Ure2. The time course of oligomerization observed by smFRET in the presence of the added fibrils shows a similar initial rate to that of the unseeded system, and the quantity of oligomers detected at each time point is not increased, indicating that the rate of oligomer production during secondary nucleation is insignificant compared to the rate of direct association of dimers to form oligomers during primary nucleation. This was confirmed by explicit fitting to kinetic models featuring oligomer formation during primary nucleation (Figure 2A) and during secondary nucleation (Figure 2B). The former yields a good fit; the latter a poor fit, a result that is very different from the findings for Aβ42(52) (Figure 2C). The quantity of Ure2 oligomers under seeded conditions appears to be lower than in the absence of preformed fibrils, which is likely to be a result of the rapid depletion of native Ure2 by association with, and elongation of, the pre-existing fibril ends. Together with the kinetic analysis of the ensemble fibril formation of Ure2 (SI Methods and Figure S4), this observation confirms that the proliferation of Ure2 fibrils results from fragmentation and not from secondary nucleation.

Figure 2. Absence of a fibril-catalyzed secondary nucleation process for Ure2. (A, B) Ensemble aggregation kinetics of 15 μM unlabeled Ure2-S68C monitored by ThT fluorescence under unseeded (blue) or seeded (red) conditions (*upper panels*). Ure2 oligomers were then detected under unseeded (blue) or seeded (red) conditions by confocal single molecule FRET (*lower panels*). The incubation conditions were the same as in Figure 1. (A) The data fit well to a model that generates oligomers during primary nucleation. (B) A model that generates oligomers during secondary nucleation cannot fit the data. (C) The presence of seeds (*right-hand columns*) drastically increases the concentration of Aβ42 oligomers measured at a single time point in the lag phase of an Aβ42 aggregation experiment(52) (*right panel*), but do not increase the production of Ure2 oligomers in this study (*left panel*), indicating fundamentally different mechanisms of oligomer formation for these two systems.

Analysis of Oligomer Populations Reveals the Existence of an Oligomer Conformational Conversion Step

The results so far have established that Ure2 oligomers are formed predominantly from the free association of dimers during primary nucleation. We can demonstrate using the following simple argument that only a minority of these oligomers ultimately become fibrils, and that both an oligomer dissociation pathway and a conformational conversion step are needed. Taking a conservative estimate of the initial oligomer formation rate of 20 nM/h (Figure 1C), and noting that minimal native Ure2 depletion occurs over the first 4 h of aggregation, a concentration of oligomers of at least 80 nM will be formed in the first 4 h. The rate of oligomer formation declines subsequently, but does not cease until all Ure2 is depleted from solution at ∼10–12 h; we therefore estimate the lower bound on the total concentration of oligomers that form during primary nucleation to be 100 nM. Mature Ure2 fibrils are observed to have lengths typically greater than 100 nm, and mostly on the micrometer scale. Given that the interchain distance within an amyloid fibril is ca. 0.5 nm, fibrils must typically contain at least 100 Ure2 dimers. In the smFRET experiments the concentration of Ure2 dimers incorporated into fibrils is approximately 15 μM, so the final concentration of fibrils is at most 150 nM. Ure2 has also been shown to follow fragmentation-dominant kinetics, so fibril formation through primary nucleation is insignificant compared to the total formation of fibrils through fragmentation. The total concentration of fibrils formed through primary nucleation must therefore be at least an order of magnitude less than the total concentration of fibrils formed, and so is significantly less than 15 nM. This value is far lower than the concentration of oligomers formed during primary nucleation observed in our smFRET experiments, and leads us to conclude that most oligomers must dissociate rather than elongate. Given that oligomers undergo faster dissociation than the fibrils, the oligomers must be structurally distinct from fibrils. In the following two sections we demonstrate that new fibrils are likely to originate from structural conversion of these oligomers, although this must occur much more slowly than dissociation. This result was confirmed by comparing fitted values for the oligomer conversion and dissociation rate constants (see kinetic analysis below).

Two Types of Oligomeric Species with Different Structures Can Be Observed

To investigate directly the possibility of structurally distinct subpopulations of Ure2 oligomers, potentially related by additional conformational conversion steps, we performed an smFRET efficiency distribution analysis. Here we used S53C rather than S68C Ure2, because both these variants show similar fibril formation kinetics to the wild-type protein, but the S53 residue is closer to the fibril core than is S68 and is thus expected to be more sensitive to structural changes within the oligomers. The experiment was carried out in a similar manner to that described above except that AF488 instead of AF555 was used to label Ure2 in order to reduce cross talk between the donor and acceptor fluorophore in the smFRET experiments, allowing detection of population distributions not apparent when using the AF555 dye as donor. We calculated the FRET efficiency of selected oligomers at different time points during fibril formation to obtain a FRET efficiency distribution histogram (Figure 3). At the early stages within the lag time, for example after 1 h of aggregation in the case of S53C, only one broad, low FRET efficiency distribution with a maximum of 0.40 was observed. As the aggregation reaction progressed, but before significant fibril mass had formed, an additional higher FRET efficiency distribution with a maximum of 0.68 appeared, suggesting these later oligomers contain a more compact assembly of Ure2 molecules than the initial low-FRET oligomers (Figure 3A). Despite the slow kinetics of fibril formation, the existence of two different types of oligomers was also observed for the V9C mutant (Figure 3B), where the low and high FRET distributions peaked at 0.46 and 0.66, respectively. High-FRET oligomers appear after low-FRET oligomers but before fibrils, suggesting that they are formed by conversion of the low-FRET oligomers. Additionally, the lack of an increase in the later high-FRET oligomer population as the fibrils start to form indicates that there is no significant production of surface-catalyzed secondary nuclei (Figure 3C,D). It should be noted that FRET efficiency is not only related to the distance between the donor and acceptor but also to their dipole orientation. The similar change in the FRET efficiency when dyes were introduced at different sites (i.e., S53C and V9C) indicates that the signal reflects principally the change in the average distances between dyes, and not the change of orientation of dyes, as there is little probability of the latter occurring simultaneously at two different residue sites.

Figure 3. Different types of Ure2 oligomers revealed by confocal single molecule FRET. (A,B) SmFRET efficiency distribution of selected oligomers of AF488/AF647-labeled Ure2 at different incubation times. The FRET distributions at different time points were fitted globally to double Gaussian functions giving the average peak positions indicated. (C,D) Population of low- and high-FRET oligomers of Ure2 at different incubation times. The incubation conditions were the same as in Figure 1. (A,C) Ure2-S53C. (B,D) Ure2-V9C.

An interesting finding in our smFRET study is the observation of a lower FRET efficiency for the initially formed oligomers for S53C than for V9C but a similar FRET efficiency of the late phase oligomers for the two variants. This observation provides clues as to the structures of the two types of oligomers. Since the V9 residue is located in the hydrophobic region (residues 9–21) of the N-terminal domain and the S53 residue is in the Q/N rich region (residues 44 to 80), the closer intermolecular distances within the early stage oligomers of V9C than S53C strongly suggests that the initial intermolecular oligomerization involves hydrophobic interactions. After the reorganization of the initial oligomers to β-sheet-containing oligomers, both V9 and S53 sites would be included in the amyloid structure and should have similar interchain distances between the same residues aligned along the fibril axis, thus explaining the similarity of the intermolecular FRET efficiency of the late phase oligomers (the high-FRET species) of the two mutants.

To obtain further evidence to support the coexistence and interconversion of two types of oligomers, we carried out a dot blot assay using the conformation-specific antibodies A11 and OC that have been used previously for identification of specific types of oligomers.(55, 56) We first probed the two types of oligomers formed during aggregation of full-length Ure2, but the signals obtained in the experiments were very weak, probably caused by the blocking effect of the globular C-terminal domain surrounding the N-terminal prion domain of Ure2. Therefore, we used the N-terminal prion domain fragment (residues 1–93) to perform the dot blot assay (see SI Methods). The results (Figure S5) show that two types of oligomers exist during aggregation of the Ure2 prion domain and that the concentration of relatively disordered oligomers (A11 reactive species) reaches a maximum earlier than the β-sheet rich oligomers (OC reactive species), indicating a possible conversion from the former to the latter, consistent with our conclusions based on the smFRET results. Taken together, these results indicate that the two oligomeric intermediates have distinct structural properties, consistent with the existence of a conformational conversion step between the earlier and later appearing oligomers.

Ure2 Oligomers That Disaggregate from Mature Fibrils Have Structures Similar to Those of the Oligomers Appearing Later in the Aggregation Reaction

We next probed by smFRET the structures of the Ure2 species dissociated from fibrils. The mature fibrils formed from an equimolar mixture of AF488-Ure2 and AF647-Ure2 were incubated in fresh buffer for at least 1 h before detection by confocal smFRET, under the same conditions as for the aggregation reaction. Upon incubation, the oligomers dissociating from fibrils reached a concentration of 0.1–0.2% of the total Ure2 fibril mass concentration, the same proportion as observed in the combined smFRET/ThT data set at the end of the aggregation reaction. These oligomers showed a broad FRET distribution, mainly between 0.2 and 0.8, and could not be precisely fitted by a Gaussian function due to the extremely low occurrence of such species. In order to detect large or insoluble species, which may not be observed during the confocal smFRET experiment, we applied TIRF with smFRET to probe the dissociated oligomers in order to increase the detection efficiency for larger species (see SI Methods). In addition, in order to obtain a higher number of disaggregated oligomers, we sonicated the AF488/AF647 fibrils before carrying out the smFRET measurements. The resulting mixture of oligomers and small fibrils in TIRF images showed coincident signals in both AF488 and AF647 channels, and the FRET distribution of the samples of Ure2 S53C and V9C could be fitted by double Gaussian functions (Figure 4). Two major populations centered at around 0.64 and around 0.8 were observed, the lower of which was similar to the high-FRET distribution observed during the aggregation reaction (Figure 3), and can be attributed to disaggregated oligomers. The species showing higher FRET values, with a maximum at around 0.8, can be attributed to small fibrils, indicating the more compact structure within amyloid fibrils. This observation is remarkably similar to that in previous studies of α-synuclein.(22) The results of TIRF experiments, therefore, demonstrate that the oligomers that disaggregate from Ure2 fibrils have the same structural properties as the oligomeric species formed in the later stages of the aggregation reaction. This finding strongly suggests that the high-FRET oligomers contain β-sheet structure similar to that found in mature amyloid fibrils, and that this type of oligomeric species is able to convert to elongation-competent fibril-type species.

Figure 4. Single molecule TIRF measurements of disaggregated fibrils. (A,B) SmFRET distribution histogram of Ure2 oligomers disaggregated from AF488/AF647-labeled fibrils. Data were fitted to a double Gaussian function (continuous line) to obtain the FRET values of the two species. (A) Ure2-S53C. (B) Ure2-V9C.

Kinetic Analysis of Combined smFRET and ThT Data Yields a Quantitative Understanding of Oligomer Formation, Dissociation and Conversion

Analysis of the kinetics of the aggregation reaction is a crucial step in understanding the microscopic mechanism of amyloid formation. Previous theoretical work has provided an analytical solution to the kinetics of fibril formation involving fragmentation,(57) and by globally fitting the ThT curves over a range of concentrations(58) to this expression, two combined kinetic parameters, k_nk₊ and k₊k_– can be obtained, where k_n, k₊ and k_– represent the amyloid nucleation, elongation and fragmentation rates, respectively. To study how the mutations affect these rates, and thus to gain further structural and mechanistic insight into the nucleation process, we performed smFRET experiments to compare the oligomer formation of the two Ure2 mutants, V9C and S68C.

Previous kinetic modeling of amyloid aggregation used a single coarse-grained reaction step to represent the “primary nucleation” pathway by which new fibrils are generated via an initial association step. Here, the availability of accurate kinetic data on the total concentration of oligomeric intermediates allows us to devise a less coarse-grained kinetic model that explicitly includes intermediates in the nucleation step. The model remains partly coarse-grained, however, as it makes no distinction between different oligomer types; nevertheless, it provides additional insights into the nature of the nucleation process. In this model, oligomers are formed through an initial assembly process, occurring with rate constant k_oligo, and subsequently convert into growth-competent fibril-type species with a rate constant k_c. These species can then elongate by dimer addition with rate constant k₊, and fragment with rate constant k_–. The oligomers can also dissociate with rate constant k_d (that we have shown above is much larger than k_c). Note that we can approximately interpret oligomerization and dissociation as fundamental reaction steps; however, the conversion step is in fact a coarse-grained step that contains information on the transformation of low-FRET to high-FRET oligomers, as well as on the subsequent conversion of the latter species to fibrils (see Methods and SI for full details). The accurate determination of reaction orders with respect to dimers requires kinetic data for a range of initial dimer concentrations. Given just one initial dimer concentration, however, we can make the reasonable assumption of a reaction order of 0 for conversion and 2 for oligomer formation. Any inaccuracy in these reaction orders is effectively incorporated into our definitions of k_c and k_oligo, and does not significantly affect the quality of the fitting (see SI Methods).

An accurate analytical solution for the time dependence of the fibril mass concentration in our model can be derived by extension of previous approaches.(57, 59) The solution is identical to the analytical solution for the kinetics of a fragmenting system, upon which the kinetic analysis of the bulk ThT experiments in the SI is based, except that the fibril nucleation rate k_nm(0)^n_c is resolved in terms of the microscopic processes introduced in our oligomer model. Specifically, we obtain

(6)where

and α = k_oligom(0)^n_c. This result, combined with an analysis of the equation governing oligomer kinetics, reveals that the kinetics of this system are controlled by the parameter combinations k_oligo, k_d, k₊k_c, and k₊k_–. The combined rate parameters k₊k_c and k₊k_– can be determined with order-of-magnitude accuracy.

A numerical procedure was used to fit the combined smFRET and bulk ThT data to this model (see Methods for further details). The numerical fits are reasonable given the accuracy of the data, and show that our coarse-grained model provides a good description of the system (Figure 5). The minor divergence between the fitted curve and the data for S68C oligomers at the latest times is consistent with the fact that disaggregation from fibrils yields a small population of oligomers at equilibrium, yet to avoid overfitting there is no explicit fibril disaggregation step in the model. Fitting gives a value for k_oligo of 1.6 × 10^–3 μM^–1 h^–1 for both variants (Figure 5A,B), and a value of k_d of 0.60 h^–1 for S68C, and 0.45 h^–1 for V9C. This result indicates that the kinetics of oligomer formation by S68C and V9C are broadly similar, but that the S68C oligomers dissociate more readily by a factor of approximately 1.3. Given that the rates of oligomer formation are very similar, it is likely that the reaction order of oligomerization is approximately the same in the two mutants at this concentration range.

Figure 5. Fitting of combined smFRET/ThT data to models indicates a probable effect of mutations on the dissociation of oligomers, but not on their formation. (A–C) The bulk aggregation kinetics of 15 μM unlabeled Ure2-V9C (red) and Ure2-S68C (blue) monitored by ThT fluorescence (*left panels*) and the concentration of AF555/AF647 labeled Ure2-V9C (red) and Ure2-S68C (blue) oligomers throughout the aggregation reaction monitored by confocal smFRET (*right panels*) were globally fitted to a theoretical model (see Methods) including the formation, dissociation, and conversion of oligomers, and the elongation and fragmentation of fibrils. The incubation conditions were the same as in Figure 1. (A) Allowing both k_oligo and k_d to differ for each mutant gives good fits, with a mean squared error of 1.54. (B) If k_oligo is constrained to be the same for both mutants, the model fits the data equally well, with a mean squared error of 1.58. (C) If neither k_oligo nor k_d is allowed to differ, the fit is less good, especially around the time when the oligomer concentration is at a maximum, with a mean squared error of 1.87. This result therefore implies that k_oligo is the same for the two variants, while the values of k_d may differ slightly.

The differences in k₊k_c and k₊k_– between the V9C and S68C variants are less than an order of magnitude, indicating that these parameters are similar for both variants. An order-of-magnitude estimate for k₊ of 40 μM^–1 h^–1 was obtained from analysis of the seeded aggregation experiment monitored by ThT fluorescence (see SI Methods and Figure S7 for details). This value allows us to calculate order-of-magnitude estimates for k_– and k_c, of 1 × 10^–4 h^–1 and 2 × 10^–3 h^–1 respectively. We note that k_c is 2 orders of magnitude smaller than k_d, as expected from our analysis of oligomer dissociation. To estimate the differences in these parameters between each variant, we used a ratio k₊(S68C):k₊(V9C) of 1.5:1, as indicated by seeded bulk experiments (see SI Methods and Figure S7), and a ratio k_–(S68C):k_–(V9C) of 1:1 as indicated by fragmentation rate measurements (see SI Methods and Figure S8). This series of steps then allows us to calculate a ratio k_c(S68C):k_c(V9C) of 1.4:1. Although this value is rather sensitive both to experimental error and to errors in the parameter ratios that are used, it can be interpreted as demonstrating that the conversion rates are similar, although possibly somewhat larger in S68C. Taken together, these results indicate that the V9C mutation decreases the fibril elongation rate and oligomer dissociation rate, leaving the fibril fragmentation rate and oligomer formation rate unaffected. Furthermore, they suggest that the oligomer conversion rate may also be slightly decreased by the V9C mutation.

Discussion

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We have applied single molecule FRET measurements to investigate the aggregation behavior of the yeast prion protein Ure2 and to observe the low populations of transient oligomers formed during the aggregation reaction that are challenging to detect by ensemble methods. The single molecule FRET observations indicate that the majority of the oligomers formed during the initial step of the nucleation process dissociate back to the native dimeric state, but a small population of oligomers undergoes a conformational conversion step leading to formation of elongation-competent species. Quantitative analysis of a combination of bulk and single molecule data has provided detailed information about the rates of the microscopic kinetic steps in the formation of amyloid fibrils, and how these rates are altered by point mutations in the prion domain. Based on our observations, a mechanism has been proposed for the formation of Ure2 amyloid fibrils in which native Ure2 forms relatively disordered oligomers, probably driven by hydrophobic intermolecular interactions, only a small proportion of which then rearrange to form structurally more compact β-sheet containing oligomers that are able to convert further to elongation-competent fibrillar species and grow by addition of native dimers to form mature amyloid fibrils (Figure 6).

Figure 6. Proposed model for the aggregation pathway of Ure2. Native dimeric Ure2 forms relatively disordered oligomers driven by hydrophobic interactions and either dissociates back to the native state or undergoes conformational conversion to form more compact oligomers containing β-sheet structure, which can in turn convert into growth-competent fibrillar species. Fragmentation of fibrils then contributes to their proliferation.

Primary nucleation of amyloidogenic proteins, in which a native protein converts into an elongation-competent species, is a crucial step in fibril formation. Molecular simulations of the aggregation of Aβ42 indicate that primary nucleation occurs via intermediate disordered non-β oligomers, which not only facilitates encounters between the monomeric proteins but also provides an environment that facilitates their conversion to fibrillar β-structure.(60, 61) Nonspecific intermolecular interactions, such as hydrophobic interactions, play a crucial role in the formation of the initial disordered oligomers,(60) while the intra- and intermolecular hydrogen bonding within β-sheets is considered to be the driving force for the subsequent conformational conversion.(62) This formation of hydrogen bonds compensates for the disruption of the hydrophobic interactions in the initial disordered oligomers, thus favoring conformational reorganization to β-sheet structure in order to reach the lowest energy state. The conformational reorganization between early relatively disordered oligomers and β-rich elongation-competent species during amyloid formation has also been suggested by experimental studies of other amyloidogenic proteins such as α-synuclein,(22) Aβ40,(63) and the yeast prion protein Sup35.(64) In agreement with theoretical and experimental results for other amyloid proteins, both theoretical analysis of oligomer concentrations and direct observation of two types of Ure2 oligomers possessing different assembly and emergence times (Figure 3) provide additional evidence for the oligomerization/conversion model as a generic feature of amyloid nucleation.

In order to obtain a quantitative understanding of the fundamental reaction steps that contribute to the amyloid formation process, analytical methods have been used to describe fibril growth and to obtain the microscopic kinetic parameters.(46, 57) Since the advent of single molecule techniques, it has become feasible to probe the nucleation process directly in real time throughout the aggregation reaction.(22-25) However, the kinetic analysis in previous studies has either focused on the early stage of oligomer formation(22, 25) or made use of a highly coarse-grained nucleated polymerization model in which secondary processes were not considered and in which the fitted rate constants were difficult to relate to specific reaction steps.(24) In this study, we provide a kinetic analysis of the combined single molecule FRET and ensemble ThT data (Figure 5 and Figure 2) that considers not only the oligomer formation process but also the elongation and fragmentation processes, providing quantitative information about the complete aggregation pathway of an amyloid protein. Another advance in the present study is the determination of an expression for the bulk primary nucleation rate k_n, which is found to depend not only on the rate constants of oligomer formation, dissociation and conversion, but also on the rate constants for fibril elongation and fragmentation. Global analysis of the oligomer formation kinetics, measured by smFRET, and the fibril formation kinetics, monitored by the ensemble ThT assay, results in two independent parameters k_oligo and k_d and two combined parameters k_ck₊ and k₊k₋, where the latter two can be decomposed by direct measurement of k₊ or of k_–. Thus, the theoretical analysis of the smFRET data provides detailed quantitative information about the fundamental steps in the process of amyloid nucleation.

Our study also provides new structural insights into the aggregation mechanism of Ure2. The disordered N-terminal domain consists of a hydrophobic region (residues 15–42) and two Q/N repeat regions (residues 1–14 and 43–89).(31) In a previous study, we have demonstrated that deletion of residues 1–42 eliminates the ability of Ure2 to form fibrils at measurable rates, while deletion of either residues 1–14 or residues 15–42 increases the lag time significantly,(31) indicating the importance of this region in the aggregation reaction. We observed that the V9C mutation causes an increase in the length of the lag phase for fibril formation compared with that of wild-type Ure2. Kinetic analysis of the bulk ThT variable-concentration data under both unseeded and cross-seeded conditions reveals that this effect is predominantly due to a decrease in the elongation rate caused by the V9C mutation. We then investigated the effect of the mutation on the nucleation process in detail by kinetic analysis of the combined smFRET and bulk ThT data. The rates of both oligomer dissociation and oligomer conversion are slightly decreased in the V9C mutant relative to WT Ure2, while the rate of oligomer formation is unaffected, indicating that V9C forms a more stable oligomer. However, the V9C mutation has a negligible effect on the overall rate of formation of elongation-competent oligomers (k_n). Taken together, these results show that the V9 residue plays an important role in the elongation of Ure2 fibrils, consistent with a recent structural study using site-directed spin labeling and electron paramagnetic resonance (EPR), which demonstrated that the residues 8–12 form the first β-strand in the fibrils.(49)

The toxicity of amyloid oligomers has been demonstrated to be correlated with the degree of exposure of hydrophobic surface.(65, 66) Since oligomers assembled by functional amyloidogenic proteins, such as Ure2 in this study, and Sup35 in a previous study,(64) have similar conformational features (for example, reactivity with A11 and OC antibodies) and a similar formation and conversion pathway to that of toxic amyloidogenic proteins such as α-synuclein(22) and Aβ,(60, 63) the difference between functional and disease-related amyloid remains to be elucidated. In this study, we observed a gradual decrease in the concentration of oligomers accompanying the onset of ThT fluorescence. Calculations and theoretical analysis of the smFRET data has led to the conclusion that the majority of Ure2 oligomers formed do not ultimately become fibrils but are depleted by dissociation. In contrast, the populations of Aβ40 and α-synuclein oligomers have been observed to follow a single exponential process and to remain at a significant equilibrium concentration even after all the monomers are depleted.(22, 23, 25) A possible explanation for the accumulation of Aβ40 and α-synuclein oligomers is that they convert very slowly relative to their formation, and become kinetically trapped once monomers are depleted, as the residual population of oligomers can no longer grow through monomer addition into fibrils. Another possible explanation for the difference is that the fibrils of Aβ40 and α-synuclein may not be as stable as the Ure2 fibrils, such that the equilibrium between oligomers and fibrils favors oligomers to a much greater extent than is the case with Ure2. In either of the above cases, Aβ40 and α-synuclein oligomers should have higher stability relative to the native state than Ure2 oligomers, and therefore would not be expected to dissociate significantly. The accumulation of relatively high concentrations of oligomers formed by disease-related amyloidogenic proteins could therefore be the cause of their toxicity.

A further potential reason for the higher toxicity of disease-related amyloidogenic proteins is that oligomers are generated not only during primary nucleation but also during secondary nucleation. For the case of Ure2, we have been able to demonstrate directly the lack of formation of surface-catalyzed secondary nuclei using smFRET measurements (Figure 2). Several structural studies indicate that the N-terminal domain of Ure2 forms an in-register parallel β-sheet structure with its C-terminal domain decorating the fibril core.(67, 68) Compared with other amyloidogenic proteins which show monomer-dependent secondary nucleation, such as Aβ42,(52) α-synuclein(69) and IAPP,(70) the surface of Ure2 fibrils is likely to be blocked by the presence of its C-terminal globular domain, thus restricting the access of native Ure2 to the fibrillar structure and inhibiting the ability of the pre-existing aggregates to catalyze the nucleation of the native protein. The absence of surface-catalyzed secondary nucleation will greatly reduce the generation of oligomers,(52) which is another possible reason for the lower toxicity of functional amyloids. Taken together, the low stability of Ure2 oligomers and the absence of secondary nucleation suggests that the functional yeast prions may replicate and propagate by fragmentation rather than secondary nucleation and hence avoid significant populations of potentially toxic oligomers, as occurs in the aggregation of neurodegenerative disease-related proteins. This study paves the way for the detailed study and comparison of further examples of both disease-related and functional amyloid systems, particularly the variety of amyloidogenic proteins that play structural and functional roles in bacteria.(71, 72) The understanding gained from such studies will not only shed light on the mechanisms by which amyloid structures are harnessed for functional roles, but may also provide clues as to possible new therapeutic strategies to combat amyloid disease.

Supporting Information

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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/jacs.7b10439.

Methods, Figures, Tables (PDF)

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¶Author Contributions

These authors contributed equally.

Funding Information

The Perrett group acknowledges support from the Chinese Ministry of Science and Technology [2017YFA0504000], the National Natural Science Foundation of China [31110103914, 21673278, 31300631], the National Laboratory of Biomacromolecules, and the CAS Center of Excellence in Biomacromolecules. T.P.J.K. acknowledges support from the Engineering and Physical Sciences Research Council, the Biotechnology and Biological Sciences Research Council, the European Research Council and the Frances and Augustus Newman Foundation. A.J.D. was supported by the Schiff Foundation. T.C.T.M. was supported by Peterhouse and St John’s College, Cambridge; and the Swiss National Science Foundation. C.M.D. and T.P.J.K. acknowledge support from the Cambridge Centre for Misfolding Diseases.

The authors declare no competing financial interest.

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Acknowledgment

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We thank Dr. Xun Li (Nikon Instruments Co. Ltd. Beijing Branch) and Dr. Zeyong Zhi (Beijing Coolight Technology) for helpful discussions about single molecule instrumentation. We thank David Klenerman and George Meisl (University of Cambridge) for helpful discussions regarding data analysis. Our electron microscopy work was performed at the Center for Biological Imaging (Institute of Biophysics, Chinese Academy of Sciences).

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Guerrero-Munoz, Marcos J.; Castillo-Carranza, Diana L.; Krishnamurthy, Shashirekha; Paulucci-Holthauzen, Adriana A.; Sengupta, Urmi; Lasagna-Reeves, Cristian A.; Ahmad, Yembur; Jackson, George R.; Kayed, Rakez
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Alzheimer's disease is a complex disease characterized by overlapping phenotypes with different neurodegenerative disorders. Oligomers are considered the most toxic species in amyloid pathologies. We examd. human AD brain samples using an anti-oligomer antibody generated in our lab. and detected potential hybrid oligomers composed of amyloid-β, prion protein, α-synuclein, and TDP-43 phosphorylated at serines 409 and 410. These data and in vitro results suggest that Aβ oligomer seeds act as a template for the aggregation of other proteins and generate an overlapping phenotype with other neuronal disorders. Furthermore, these results could explain why anti-amyloid-β therapy has been unsuccessful.
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Cheng, B.; Gong, H.; Xiao, H.; Petersen, R. B.; Zheng, L.; Huang, K. Biochim. Biophys. Acta, Gen. Subj. 2013, 1830, 4860– 4871 DOI: 10.1016/j.bbagen.2013.06.029
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8
Inhibiting toxic aggregation of amyloidogenic proteins: A therapeutic strategy for protein misfolding diseases
Cheng, Biao; Gong, Hao; Xiao, Hongwen; Petersen, Robert B.; Zheng, Ling; Huang, Kun
Biochimica et Biophysica Acta, General Subjects (2013), 1830 (10), 4860-4871CODEN: BBGSB3; ISSN:0304-4165. (Elsevier B.V.)
A review. The deposition of self-assembled amyloidogenic proteins is assocd. with multiple diseases, including Alzheimer's disease, Parkinson's disease and type 2 diabetes mellitus. The toxic misfolding and self-assembling of amyloidogenic proteins are believed to underlie protein misfolding diseases. Novel drug candidates targeting self-assembled amyloidogenic proteins represent a potential therapeutic approach for protein misfolding diseases. In this perspective review, the authors provide an overview of the recent progress in identifying inhibitors that block the aggregation of amyloidogenic proteins and the clin. applications thereof. Compds. such as polyphenols, certain short peptides, and monomer- or oligomer-specific antibodies, can interfere with the self-assembly of amyloidogenic proteins, prevent the formation of oligomers, amyloid fibrils and the consequent cytotoxicity. Some inhibitors have been tested in clin. trials for treating protein misfolding diseases. Inhibitors that target the aggregation of amyloidogenic proteins bring new hope to therapy for protein misfolding diseases.
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Guerrero-Munoz, M. J.; Castillo-Carranza, D. L.; Kayed, R. Biochem. Pharmacol. 2014, 88, 468– 478 DOI: 10.1016/j.bcp.2013.12.023
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9
Therapeutic approaches against common structural features of toxic oligomers shared by multiple amyloidogenic proteins
Guerrero-Munoz, Marcos J.; Castillo-Carranza, Diana L.; Kayed, Rakez
Biochemical Pharmacology (Amsterdam, Netherlands) (2014), 88 (4), 468-478CODEN: BCPCA6; ISSN:0006-2952. (Elsevier B.V.)
A review. Impaired proteostasis is one of the main features of all amyloid diseases, which are assocd. with the formation of insol. aggregates from amyloidogenic proteins. The aggregation process can be caused by overprodn. or poor clearance of these proteins. However, numerous reports suggest that amyloid oligomers are the most toxic species, rather than insol. fibrillar material, in Alzheimer's, Parkinson's, and Prion diseases, among others. Although the exact protein that aggregates varies between amyloid disorders, they all share common structural features that can be used as therapeutic targets. In this review, we focus on therapeutic approaches against shared features of toxic oligomeric structures and future directions.
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10
LeVine, H., 3rd Methods Enzymol. 1999, 309, 274– 284 DOI: 10.1016/S0076-6879(99)09020-5
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10
Quantification of β-sheet amyloid fibril structures with thioflavin T
LeVine, Harry, III
Methods in Enzymology (1999), 309 (Amyloid, Prions, and Other Protein Aggregates), 274-284CODEN: MENZAU; ISSN:0076-6879. (Academic Press)
A spectrochem. method for the detn. of β-amyloid using thioflavin T was presented. (c) 1999 Academic Press.
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Bartolini, M.; Bertucci, C.; Bolognesi, M. L.; Cavalli, A.; Melchiorre, C.; Andrisano, V. ChemBioChem 2007, 8, 2152– 2161 DOI: 10.1002/cbic.200700427
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11
Insight into the kinetic of amyloid β (1-42) peptide self-aggregation: elucidation of inhibitors' mechanism of action
Bartolini, Manuela; Bertucci, Carlo; Bolognesi, Maria Laura; Cavalli, Andrea; Melchiorre, Carlo; Andrisano, Vincenza
ChemBioChem (2007), 8 (17), 2152-2161CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)
The initial transition of amyloid β (1-42) (Aβ42) sol. monomers/small oligomers from unordered/α-helix to a β-sheet-rich conformation represents a suitable target to design new potent inhibitors and to obtain effective therapeutics for Alzheimer's disease. Under optimized conditions, this reliable and reproducible CD kinetic study showed a 3-step sigmoid profile that was characterized by a lag phase (prevailing unordered/α-helix conformation), an exponential growth phase (increasing β-sheet secondary structure) and a plateau phase (prevailing β-sheet secondary structure). This kinetic anal. brought insight into the inhibitors' mechanism of action. In fact, an increase in the duration of the lag phase can be related to the formation of an inhibitor-Aβ complex, in which the non-amyloidogenic conformation is stabilized. When the exponential rate is affected exclusively, such as in the case of Congo red and tetracycline, then the inhibitor affinity might be higher for the pleated β-sheet structure. Finally, by adding the inhibitor at the end of the exponential phase, the sol. protofibrils can be disrupted and the Aβ amyloidogenic structure can revert into monomers/small oligomers. Congo red and tetracycline preferentially bind to amyloid in the β-sheet conformation because both decreased the slope of the exponential growth, even if to a different extent, whereas no effect was obsd. for tacrine and galantamine. Some very preliminary indications can be derived about the structural requirements for binding to nonamyloidogenic or β-sheet amyloid secondary structure for the development of potent antiaggregating agents. On these premises, memoquin, a multifunctional mol. that was designed to become a drug candidate for the treatment of Alzheimer's disease, was investigated under the reported CD assay and its anti-amyloidogenic mechanism of action was elucidated.
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Newcomb, C. J.; Moyer, T. J.; Lee, S. S.; Stupp, S. I. Curr. Opin. Colloid Interface Sci. 2012, 17, 350– 359 DOI: 10.1016/j.cocis.2012.09.004
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12
Advances in cryogenic transmission electron microscopy for the characterization of dynamic self-assembling nanostructures
Newcomb, Christina J.; Moyer, Tyson J.; Lee, Sungsoo S.; Stupp, Samuel I.
Current Opinion in Colloid & Interface Science (2012), 17 (6), 350-359CODEN: COCSFL; ISSN:1359-0294. (Elsevier Ltd.)
A review. Elucidating the structural information of nanoscale materials in their solvent-exposed state is crucial, as a result, cryogenic transmission electron microscopy (cryo-TEM) has become an increasingly popular technique in the materials science, chem., and biol. communities. Cryo-TEM provides a method to directly visualize the specimen structure in a soln.-state through a thin film of vitrified solvent. This technique complements x-ray, neutron, and light scattering methods that probe the statistical av. of all species present; furthermore, cryo-TEM can be used to observe changes in structure over time. In the area of self-assembly, this tool was particularly powerful for the characterization of natural and synthetic small mol. assemblies, as well as hybrid org.-inorg. composites. In this review, recent advances in cryogenic TEM are discussed in the context of self-assembling systems with emphasis on characterization of transitions obsd. in response to external stimuli.
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Smith, J. F.; Knowles, T. P.; Dobson, C. M.; Macphee, C. E.; Welland, M. E. Proc. Natl. Acad. Sci. U. S. A. 2006, 103, 15806– 15811 DOI: 10.1073/pnas.0604035103
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13
Characterization of the nanoscale properties of individual amyloid fibrils
Smith, Jeffrey F.; Knowles, Tuomas P. J.; Dobson, Christopher M.; MacPhee, Cait E.; Welland, Mark E.
Proceedings of the National Academy of Sciences of the United States of America (2006), 103 (43), 15806-15811CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
We report the detailed mech. characterization of individual amyloid fibrils by at. force microscopy and spectroscopy. These self-assembling materials, formed here from the protein insulin, were shown to have a strength of 0.6 ± 0.4 GPa, comparable to that of steel (0.6-1.8 GPa), and a mech. stiffness, as measured by Young's modulus, of 3.3 ± 0.4 GPa, comparable to that of silk (1-10 GPa). The values of these parameters reveal that the fibrils possess properties that make these structures highly attractive for future technol. applications. In addn., anal. of the soln. state growth kinetics indicated a breakage rate const. of 1.7 ± 1.3 × 10-8 s-1, which reveals that a fibril 10 μm in length breaks spontaneously on av. every 47 min, suggesting that internal fracturing is likely to be of fundamental importance in the proliferation of amyloid fibrils and therefore for understanding the progression of their assocd. pathogenic disorders.
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Nath, S.; Meuvis, J.; Hendrix, J.; Carl, S. A.; Engelborghs, Y. Biophys. J. 2010, 98, 1302– 1311 DOI: 10.1016/j.bpj.2009.12.4290
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14
Early aggregation steps in α-synuclein as measured by FCS and FRET: evidence for a contagious conformational change
Nath, Sangeeta; Meuvis, Jessika; Hendrix, Jelle; Carl, Shaun A.; Engelborghs, Yves
Biophysical Journal (2010), 98 (7), 1302-1311CODEN: BIOJAU; ISSN:0006-3495. (Cell Press)
The kinetics of aggregation of α-synuclein are usually studied by turbidity or thioflavin T fluorescence. Here, the authors followed the disappearance of monomers and the formation of early oligomers using fluorescence correlation spectroscopy (FCS). Alexa488-labeled A140C-α-synuclein was used as a fluorescent probe in trace amts. in the presence of excess unlabeled α-synuclein. Repeated short measurements produced a distribution of diffusion coeffs. Initially, a sharp peak was obtained corresponding to monomers, followed by a distinct transient population and the gradual formation of broader-sized distributions of higher oligomers. The kinetics of aggregation could be followed by the decreasing no. of fast-diffusing species. Both the disappearance of fast-diffusing species and the appearance of turbidity could be fitted to the Finke-Watzky equation, but the apparent rate consts. obtained were different. This reflected the fact that the disappearance of fast species occurred largely during the lag phase of turbidity development, due to the limited sensitivity of turbidity to the early aggregation process. The nucleation of the early oligomers was concn.-dependent and accompanied by a conformational change that preceded β-structure formation, and could be visualized using FRET between the donor-labeled N-terminus and the acceptor-labeled Cys residue in the A140C mutant.
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Paredes, J. M.; Casares, S.; Ruedas-Rama, M. J.; Fernandez, E.; Castello, F.; Varela, L.; Orte, A. Int. J. Mol. Sci. 2012, 13, 9400– 9418 DOI: 10.3390/ijms13089400
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15
Early amyloidogenic oligomerization studied through fluorescence lifetime correlation spectroscopy
Paredes, Jose M.; Casares, Salvador; Ruedas-Rama, Maria J.; Fernandez, Elena; Castello, Fabio; Varela-Alvarez, Lorena; Orte, Angel
International Journal of Molecular Sciences (2012), 13 (), 9400-9418CODEN: IJMCFK; ISSN:1422-0067. (MDPI AG)
Amyloidogenic protein aggregation is a persistent biomedical problem. Despite active research in disease-related aggregation, the need for multidisciplinary approaches to the problem is evident. Recent advances in single-mol. fluorescence spectroscopy are valuable for examg. heterogenic biomol. systems. In this work, we have explored the initial stages of amyloidogenic aggregation by employing fluorescence lifetime correlation spectroscopy (FLCS), an advanced modification of conventional fluorescence correlation spectroscopy (FCS) that utilizes time-resolved information. FLCS provides size distributions and kinetics for the oligomer growth of the SH3 domain of α-spectrin, whose N47A mutant forms amyloid fibrils at pH 3.2 and 37 °C in the presence of salt. The combination of FCS with addnl. fluorescence lifetime information provides an exciting approach to focus on the initial aggregation stages, allowing a better understanding of the fibrillization process, by providing multidimensional information, valuable in combination with other conventional methodologies.
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Carulla, N.; Zhou, M.; Arimon, M.; Gairi, M.; Giralt, E.; Robinson, C. V.; Dobson, C. M. Proc. Natl. Acad. Sci. U. S. A. 2009, 106, 7828– 7833 DOI: 10.1073/pnas.0812227106
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Experimental characterization of disordered and ordered aggregates populated during the process of amyloid fibril formation
Carulla, Natalia; Zhou, Min; Arimon, Muriel; Gairi, Margarida; Giralt, Ernest; Robinson, Carol V.; Dobson, Christopher M.
Proceedings of the National Academy of Sciences of the United States of America (2009), 106 (19), 7828-7833, S7828/1-S7828/11CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
Recent exptl. evidence points to intermediates populated during the process of amyloid fibril formation as the toxic moieties primarily responsible for the development of increasingly common disorders such as Alzheimer's disease and type II diabetes. We describe here the application of a pulse-labeling hydrogen-deuterium (HD) exchange strategy monitored by mass spectrometry (MS) and NMR spectroscopy (NMR) to characterize the aggregation process of an SH3 domain under 2 different conditions, both of which ultimately lead to well-defined amyloid fibrils. Under one condition, the intermediates appear to be largely amorphous in nature, whereas under the other condition protofibrillar species are clearly evident. Under the conditions favoring amorphous-like intermediates, only species having no protection against HD exchange can be detected in addn. to the mature fibrils that show a high degree of protection. By contrast, under the conditions favoring protofibrillar-like intermediates, MS reveals that multiple species are present with different degrees of HD exchange protection, indicating that aggregation occurs initially through relatively disordered species that subsequently evolve to form ordered aggregates that eventually lead to amyloid fibrils. Further anal. using NMR provides residue-specific information on the structural reorganizations that take place during aggregation, as well as on the time scales by which they occur.
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Paslawski, W.; Mysling, S.; Thomsen, K.; Jorgensen, T. J.; Otzen, D. E. Angew. Chem., Int. Ed. 2014, 53, 7560– 7563 DOI: 10.1002/anie.201400491
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17
Co-existence of Two Different α-Synuclein Oligomers with Different Core Structures Determined by Hydrogen/Deuterium Exchange Mass Spectrometry
Paslawski, Wojciech; Mysling, Simon; Thomsen, Karen; Jorgensen, Thomas J. D.; Otzen, Daniel E.
Angewandte Chemie, International Edition (2014), 53 (29), 7560-7563CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
Neurodegenerative disorders are characterized by the formation of protein oligomers and amyloid fibrils, which in the case of Parkinson's disease involves the protein α-synuclein (αSN). Cytotoxicity is mainly assocd. with the oligomeric species, but we still know little about their assembly and structure. Hydrogen/deuterium exchange (HDX) monitored by mass spectrometry is used to analyze oligomers formed by wild-type (wt) αSN and also three familial αSN mutants (A30P, E46K, and A53T). All four variants show co-existence of two different oligomers. The backbone amides of oligomer type I are protected from exchange with D2O until they dissoc. into monomeric αSN by EX1 exchange kinetics. Fewer residues are protected against exchange in oligomer type II, but this type does not revert to αSN monomers. Both oligomers are protected in the core sequence Y39-A89. Based on incubation studies, oligomer type I appears to form straight fibrils, while oligomer type II forms amorphous clusters that do not directly contribute to the fibrillation process.
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Fusco, G.; Chen, S. W.; Williamson, P. T. F.; Cascella, R.; Perni, M.; Jarvis, J. A.; Cecchi, C.; Vendruscolo, M.; Chiti, F.; Cremades, N.; Ying, L.; Dobson, C. M.; De Simone, A. Science 2017, 358, 1440– 1443 DOI: 10.1126/science.aan6160
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Structural basis of membrane disruption and cellular toxicity by α-synuclein oligomers
Fusco, Giuliana; Chen, Serene W.; Williamson, Philip T. F.; Cascella, Roberta; Perni, Michele; Jarvis, James A.; Cecchi, Cristina; Vendruscolo, Michele; Chiti, Fabrizio; Cremades, Nunilo; Ying, Liming; Dobson, Christopher M.; De Simone, Alfonso
Science (Washington, DC, United States) (2017), 358 (6369), 1440-1443CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
Oligomeric species populated during the aggregation process of α-synuclein have been linked to neuronal impairment in Parkinson's disease and related neurodegenerative disorders. By using soln. and solid-state NMR techniques in conjunction with other structural methods, we identified the fundamental characteristics that enable toxic α-synuclein oligomers to perturb biol. membranes and disrupt cellular function; these include a highly lipophilic element that promotes strong membrane interactions and a structured region that inserts into lipid bilayers and disrupts their integrity. In support of these conclusions, mutations that target the region that promotes strong membrane interactions by α-synuclein oligomers suppressed their toxicity in neuroblastoma cells and primary cortical neurons.
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Banerjee, P. R.; Deniz, A. A. Chem. Soc. Rev. 2014, 43, 1172– 1188 DOI: 10.1039/C3CS60311C
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19
Shedding light on protein folding landscapes by single-molecule fluorescence
Banerjee, Priya R.; Deniz, Ashok A.
Chemical Society Reviews (2014), 43 (4), 1172-1188CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)
A review. Single-mol. (SM) fluorescence methods have been increasingly instrumental in our current understanding of a no. of key aspects of protein folding and aggregation landscapes over the past decade. With the advantage of a model free approach and the power of probing multiple subpopulations and stochastic dynamics directly in a heterogeneous structural ensemble, SM methods have emerged as a principle technique for studying complex systems such as intrinsically disordered proteins (IDPs), globular proteins in the unfolded basin and during folding, and early steps of protein aggregation in amyloidogenesis. This review highlights the application of these methods in investigating the free energy landscapes, folding properties and dynamics of individual protein mols. and their complexes, with an emphasis on inherently flexible systems such as IDPs.
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Schuler, B.; Hofmann, H. Curr. Opin. Struct. Biol. 2013, 23, 36– 47 DOI: 10.1016/j.sbi.2012.10.008
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20
Single-molecule spectroscopy of protein folding dynamics-expanding scope and timescales
Schuler, Benjamin; Hofmann, Hagen
Current Opinion in Structural Biology (2013), 23 (1), 36-47CODEN: COSBEF; ISSN:0959-440X. (Elsevier Ltd.)
A review. Single-mol. spectroscopy has developed into an important method for probing protein structure and dynamics, esp. in structurally heterogeneous systems. A broad range of questions in the diversifying field of protein folding have been addressed with single-mol. Foerster resonance energy transfer (FRET) and photo-induced electron transfer (PET). Building on more than a decade of rapid method development, these techniques can now be used to investigate a wide span of timescales, an aspect that we focus on in this review. Important current topics range from the structure and dynamics of unfolded and intrinsically disordered proteins, including the coupling of folding and binding, to transition path times, the folding and misfolding of larger proteins, and their interactions with mol. chaperones.
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Orte, A.; Birkett, N. R.; Clarke, R. W.; Devlin, G. L.; Dobson, C. M.; Klenerman, D. Proc. Natl. Acad. Sci. U. S. A. 2008, 105, 14424– 14429 DOI: 10.1073/pnas.0803086105
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21
Direct characterization of amyloidogenic oligomers by single-molecule fluorescence
Orte, Angel; Birkett, Neil R.; Clarke, Richard W.; Devlin, Glyn L.; Dobson, Christopher M.; Klenerman, David
Proceedings of the National Academy of Sciences of the United States of America (2008), 105 (38), 14424-14429,S14424/1-S14424/8CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
A key issue in understanding the pathogenic conditions assocd. with the aberrant aggregation of misfolded proteins is the identification and characterization of species formed during the aggregation process. Probing the nature of such species has, however, proved to be extremely challenging to conventional techniques because of their transient and heterogeneous character. We describe here the application of a two-color single-mol. fluorescence technique to examine the assembly of oligomeric species formed during the aggregation of the SH3 domain of PI3 kinase. The single-mol. expts. show that the species formed at the stage of the reaction where aggregates have previously been found to be maximally cytotoxic are a heterogeneous ensemble of oligomers with a median size of 38 ± 10 mols. This no. is remarkably similar to ests. from bulk measurements of the critial size of species obsd. to seed ordered fibril formation and of the most infective form of prion particle. Moreover, although the size distribution of the SH3 oligomers remains virtually const. as the time of aggregation increases, their stability increases substantially. These findings together provide direct evidence for a general mechanism of amyloid aggregation in which the stable cross-β structure emerges via internal reorganization of disordered oligomers formed during the lag phase of the self-assembly reaction.
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Cremades, N.; Cohen, S. I.; Deas, E.; Abramov, A. Y.; Chen, A. Y.; Orte, A.; Sandal, M.; Clarke, R. W.; Dunne, P.; Aprile, F. A.; Bertoncini, C. W.; Wood, N. W.; Knowles, T. P.; Dobson, C. M.; Klenerman, D. Cell 2012, 149, 1048– 1059 DOI: 10.1016/j.cell.2012.03.037
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22
Direct Observation of the Interconversion of Normal and Toxic Forms of α-Synuclein
Cremades, Nunilo; Cohen, Samuel I. A.; Deas, Emma; Abramov, Andrey Y.; Chen, Allen Y.; Orte, Angel; Sandal, Massimo; Clarke, Richard W.; Dunne, Paul; Aprile, Francesco A.; Bertoncini, Carlos W.; Wood, Nicholas W.; Knowles, Tuomas P. J.; Dobson, Christopher M.; Klenerman, David
Cell (Cambridge, MA, United States) (2012), 149 (5), 1048-1059CODEN: CELLB5; ISSN:0092-8674. (Cell Press)
Here, we use single-mol. techniques to study the aggregation of α-synuclein, the protein whose misfolding and deposition is assocd. with Parkinson's disease. We identify a conformational change from the initially formed oligomers to stable, more compact proteinase-K-resistant oligomers as the key step that leads ultimately to fibril formation. The oligomers formed as a result of the structural conversion generate much higher levels of oxidative stress in rat primary neurons than do the oligomers formed initially, showing that they are more damaging to cells. The structural conversion is remarkably slow, indicating a high kinetic barrier for the conversion and suggesting that there is a significant period of time for the cellular protective machinery to operate and potentially for therapeutic intervention, prior to the onset of cellular damage. In the absence of added sol. protein, the assembly process is reversed and fibrils disaggregate to form stable oligomers, hence acting as a source of cytotoxic species.
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23
Narayan, P.; Orte, A.; Clarke, R. W.; Bolognesi, B.; Hook, S.; Ganzinger, K. A.; Meehan, S.; Wilson, M. R.; Dobson, C. M.; Klenerman, D. Nat. Struct. Mol. Biol. 2012, 19, 79– 83 DOI: 10.1038/nsmb.2191
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23
The extracellular chaperone clusterin sequesters oligomeric forms of the amyloid-β1-40 peptide
Narayan, Priyanka; Orte, Angel; Clarke, Richard W.; Bolognesi, Benedetta; Hook, Sharon; Ganzinger, Kristina A.; Meehan, Sarah; Wilson, Mark R.; Dobson, Christopher M.; Klenerman, David
Nature Structural & Molecular Biology (2012), 19 (1), 79-83CODEN: NSMBCU; ISSN:1545-9993. (Nature Publishing Group)
In recent genome-wide assocn. studies, the extracellular chaperone protein, clusterin, has been identified as a newly-discovered risk factor in Alzheimer's disease. We have examd. the interactions between human clusterin and the Alzheimer's disease-assocd. amyloid-β1-40 peptide (Aβ1-40), which is prone to aggregate into an ensemble of oligomeric intermediates implicated in both the proliferation of amyloid fibrils and in neuronal toxicity. Using highly sensitive single-mol. fluorescence methods, we have found that Aβ1-40 forms a heterogeneous distribution of small oligomers (from dimers to 50-mers), all of which interact with clusterin to form long-lived, stable complexes. Consequently, clusterin is able to influence both the aggregation and disaggregation of Aβ1-40 by sequestration of the Aβ oligomers. These results not only elucidate the protective role of clusterin but also provide a mol. basis for the genetic link between clusterin and Alzheimer's disease.
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Shammas, S. L.; Garcia, G. A.; Kumar, S.; Kjaergaard, M.; Horrocks, M. H.; Shivji, N.; Mandelkow, E.; Knowles, T. P.; Mandelkow, E.; Klenerman, D. Nat. Commun. 2015, 6, 7025 DOI: 10.1038/ncomms8025
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24
A mechanistic model of tau amyloid aggregation based on direct observation of oligomers
Shammas, Sarah L.; Garcia, Gonzalo A.; Kumar, Satish; Kjaergaard, Magnus; Horrocks, Mathew H.; Shivji, Nadia; Mandelkow, Eva; Knowles, Tuomas P. J.; Mandelkow, Eckhard; Klenerman, David
Nature Communications (2015), 6 (), 7025CODEN: NCAOBW; ISSN:2041-1723. (Nature Publishing Group)
Protein aggregation plays a key role in neurodegenerative disease, giving rise to small oligomers that may become cytotoxic to cells. The fundamental microscopic reactions taking place during aggregation, and their rate consts., have been difficult to det. due to lack of suitable methods to identify and follow the low concn. of oligomers over time. Here we use single-mol. fluorescence to study the aggregation of the repeat domain of tau (K18), and two mutant forms linked with familial frontotemporal dementia, the deletion mutant ΔK280 and the point mutant P301L. Our kinetic anal. reveals that aggregation proceeds via monomeric assembly into small oligomers, and a subsequent slow structural conversion step before fibril formation. Using this approach, we have been able to quant. det. how these mutations alter the aggregation energy landscape.
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Iljina, M.; Garcia, G. A.; Horrocks, M. H.; Tosatto, L.; Choi, M. L.; Ganzinger, K. A.; Abramov, A. Y.; Gandhi, S.; Wood, N. W.; Cremades, N.; Dobson, C. M.; Knowles, T. P.; Klenerman, D. Proc. Natl. Acad. Sci. U. S. A. 2016, 113, E1206– 1215 DOI: 10.1073/pnas.1524128113
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25
Kinetic model of the aggregation of alpha-synuclein provides insights into prion-like spreading
Iljina, Marija; Garcia, Gonzalo A.; Horrocks, Mathew H.; Tosatto, Laura; Choi, Minee L.; Ganzinger, Kristina A.; Abramov, Andrey Y.; Gandhi, Sonia; Wood, Nicholas W.; Cremades, Nunilo; Dobson, Christopher M.; Knowles, Tuomas P. J.; Klenerman, David
Proceedings of the National Academy of Sciences of the United States of America (2016), 113 (9), E1206-E1215CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
α-Synuclein (αS) self-assembles into small oligomeric species and subsequently into amyloid fibrils that accumulate and proliferate during the development of Parkinson's disease. However, the quant. characterization of the aggregation and spreading of αS remains challenging to achieve. Previously, the authors identified a conformational conversion step leading from the initially formed oligomers to more compact oligomers preceding fibril formation. Here, by a combination of single-mol. FRET measurements and kinetic anal., the authors found that the reaction in soln. involves 2 unimol. structural conversion steps, from the disordered to more compact oligomers and then to fibrils, which can elongate by further monomer addn. The authors obtained individual rate consts. for these key microscopic steps by applying a global kinetic anal. to both the decrease in the concn. of monomeric protein mols. and the increase in oligomer concns. over a 0.5-140-μM range of αS. The resulting explicit kinetic model of αS aggregation was used to quant. explore seeding the reaction by either the compact oligomers or fibrils. The authors' predictions revealed that, although fibrils were more effective at seeding than oligomers, very high nos. of seeds of either type, of the order of 104, were required to achieve efficient seeding and bypass the slow generation of aggregates through primary nucleation. Complementary cellular expts. demonstrated that 2 orders of magnitude lower nos. of oligomers were sufficient to generate high levels of reactive O species (ROS), suggesting that effective templated seeding is likely to require both the presence of template aggregates and conditions of cellular stress.
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26
Wickner, R. B. Science 1994, 264, 566– 569 DOI: 10.1126/science.7909170
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26
[URE3] as an altered URE2 protein: evidence for a prion analog in Saccharomyces cerevisiae
Wickner, Reed B.
Science (Washington, DC, United States) (1994), 264 (5158), 566-9CODEN: SCIEAS; ISSN:0036-8075.
A cytoplasmically inherited element, [URE3], allows yeast to use ureidosuccinate in the presence of NH4+. Chromosomal mutations in the URE2 gene produce the same phenotype. [URE3] depends for its propagation on the URE2 product (Ure2p), a neg. regulator of enzymes of N metab. S. cerevisiae strains cured of [URE3] with guanidium chloride returned to the [URE3]-carrying state without its introduction from other cells. Overprodn. of Ure2p increased the frequency with which a strain became [URE3] by 100-fold. In analogy to mammalian prions, [URE3] may be an altered form of Ure2p that is inactive for its normal function but can convert normal Ure2p to the altered form. The genetic evidence presented here suggests that protein-based inheritance, involving a protein unrelated to the mammalian prion protein, can occur in a microorganism.
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Lian, H. Y.; Jiang, Y.; Zhang, H.; Jones, G. W.; Perrett, S. Biochim. Biophys. Acta, Proteins Proteomics 2006, 1764, 535– 545 DOI: 10.1016/j.bbapap.2005.11.016
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27
The yeast prion protein Ure2: structure, function and folding
Lian, Hui-Yong; Jiang, Yi; Zhang, Hong; Jones, Gary W.; Perrett, Sarah
Biochimica et Biophysica Acta, Proteins and Proteomics (2006), 1764 (3), 535-545CODEN: BBAPBW; ISSN:1570-9639. (Elsevier B.V.)
A review. The Saccharomyces cerevisiae protein Ure2 functions as a regulator of nitrogen metab. and as a glutathione-dependent peroxidase. Ure2 also has the characteristics of a prion, in that it can undergo a heritable conformational change to an aggregated state; the prion form of Ure2 loses the regulatory function, but the enzymic function appears to be maintained. A no. of factors are found to affect the prion properties of Ure2, including mutation and expression levels of mol. chaperones, and the effect of these factors on structure and stability are being investigated. The relationship between structure, function and folding for the yeast prion Ure2 are discussed.
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Thual, C.; Komar, A. A.; Bousset, L.; Fernandez-Bellot, E.; Cullin, C.; Melki, R. J. Biol. Chem. 1999, 274, 13666– 13674 DOI: 10.1074/jbc.274.19.13666
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28
Structural characterization of Saccharomyces cerevisiae prion-like protein Ure2
Thual, Carine; Komar, Anton A.; Bousset, Luc; Fernandez-Bellot, Eric; Cullin, Christophe; Melki, Ronald
Journal of Biological Chemistry (1999), 274 (19), 13666-13674CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
Saccharomyces cerevisiae prion-like protein Ure2 was expressed in Escherichia coli and was purified to homogeneity. We show here that Ure2p is a sol. protein that can assemble into fibers that are similar to the fibers obsd. in the case of PrP in its scrapie prion filaments form or that form on Sup35 self-assembly. Ure2p self-assembly is a cooperative process where one can distinguish a lag phase followed by an elongation phase preceding a plateau. A combination of size exclusion chromatog., sedimentation velocity, and electron microscopy demonstrates that the sol. form of Ure2p consists at least of three forms of the protein as follows: a monomeric, dimeric, and tetrameric form whose abundance is concn.-dependent. By the use of limited proteolysis, intrinsic fluorescence, and CD measurements, we bring strong evidence for the existence of at least two structural domains in Ure2p mols. Indeed, Ure2p NH2-terminal region is found poorly structured, whereas its COOH-terminal domain appears to be compactly folded. Finally, we show that only slight conformational changes accompany Ure2p assembly into insol. high mol. wt. oligomers. These changes essentially affect the COOH-terminal part of the mol. The properties of Ure2p are compared in the discussion to that of other prion-like proteins such as Sup35 and mammalian prion protein PrP.
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Taylor, K. L.; Cheng, N.; Williams, R. W.; Steven, A. C.; Wickner, R. B. Science 1999, 283, 1339– 1343 DOI: 10.1126/science.283.5406.1339
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29
Prion domain initiation of amyloid formation in vitro from native Ure2p
Taylor, Kimberly L.; Cheng, Naiqian; Williams, Robert W.; Steven, Alasdair C.; Wickner, Reed B.
Science (Washington, D. C.) (1999), 283 (5406), 1339-1343CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
The [URE3] non-Mendelian genetic element of Saccharomyces cerevisiae is an infectious protein (prion) form of Ure2p, a regulator of nitrogen catabolism. Here, synthetic Ure2p1-65 were shown to polymerize to form filaments 40 to 45 angstroms in diam. with more than 60 % β sheet. Ure2p1-65 specifically induced full-length native Ure2p to copolymerize under conditions where native Ure2p alone did not polymerize. Like Ure2p in exts. of [URE3] strains, these 180- to 220-angstrom-diam. filaments were protease resistant. The Ure2p1-65-Ure2p cofilaments could seed polymn. of native Ure2p to form thicker, less regular filaments. All filaments stained with Congo Red to produce the green birefringence typical of amyloid. This self-propagating amyloid formation can explain the properties of [URE3].
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Masison, D. C.; Wickner, R. B. Science 1995, 270, 93– 95 DOI: 10.1126/science.270.5233.93
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30
Prion-inducing domain of yeast Ure2p and protease resistance of Ure2p in prion-containing cells
Masison, Daniel C.; Wickner, Reed B.
Science (Washington, D. C.) (1995), 270 (5233), 93-5CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
The genetic properties of the [URE3] non-Mendelian element of Saccharomyces cerevisiae suggest that it is a prion (infectious protein) form of Ure2p, a regulator of nitrogen catabolism. In exts. from [URE3] strains, Ure2p was partially resistant to proteinase K compared with Ure2p from wild-type exts. Overexpression of Ure2p in wild-type strains induced a 20- to 200-fold increase in the frequency with which [URE3] arose. Overexpression of just the amino-terminal 65 residues of Ure2p increased the frequency of [URE3] induction 6000-fold. Without this "prion-inducing domain" the carboxyl-terminal domain performed the nitrogen regulation function of Ure2p, but could not be changed to the [URE3] prion state. Thus, this domain induced the prion state in trans, whereas in cis it conferred susceptibility of the adjoining nitrogen regulatory domain to prion infections.
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Jiang, Y.; Li, H.; Zhu, L.; Zhou, J. M.; Perrett, S. J. Biol. Chem. 2004, 279, 3361– 3369 DOI: 10.1074/jbc.M310494200
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31
Amyloid Nucleation and Hierarchical Assembly of Ure2p Fibrils: Role of asparagine/glutamine repeat and nonrepeat regions of the prion domain
Jiang, Yi; Li, Hui; Zhu, Li; Zhou, Jun-Mei; Perrett, Sarah
Journal of Biological Chemistry (2004), 279 (5), 3361-3369CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
The yeast prion protein Ure2 forms amyloid-like filaments in vivo and in vitro. This ability depends on the N-terminal prion domain, which contains Asn/Gln repeats, a motif thought to cause human disease by forming stable protein aggregates. The Asn/Gln region of the Ure2p prion domain extends to residue 89, but residues 15-42 represent an island of "normal" random sequence, which is highly conserved in related species and is relatively hydrophobic. We compare the time course of structural changes monitored by thioflavin T (ThT) binding fluorescence and at. force microscopy for Ure2 and a series of prion domain mutants under a range of conditions. At. force microscopy height images at successive time points during a single growth expt. showed the sequential appearance of at least four fibril types that could be readily differentiated by height (5, 8, 12, or 9 nm), morphol. (twisted or smooth), and/or time of appearance (early or late in the plateau phase of ThT binding). The Ure2 dimer (h = 2.6±0.5 nm) and granular particles corresponding to higher order oligomers (h = 4-12 nm) could also be detected. The mutants 15Ure2 and Δ15-42Ure2 showed the same time-dependent variation in fibril types but with an increased lag time detected by ThT binding compared with wild-type Ure2. In addn., Δ15-42Ure2 showed reduced binding to ThT. The results imply a role of the conserved region in both amyloid nucleation and formation of the binding surface recognized by ThT. Further, Ure2 amyloid formation is a multistep process via a series of fibrillar intermediates.
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Bousset, L.; Belrhali, H.; Janin, J.; Melki, R.; Morera, S. Structure 2001, 9, 39– 46 DOI: 10.1016/S0969-2126(00)00553-0
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Structure of the Globular Region of the Prion Protein Ure2 from the Yeast Saccharomyces cerevisiae
Bousset, L.; Belrhali, H.; Janin, J.; Melki, R.; Morera, S.
Structure (Cambridge, MA, United States) (2001), 9 (1), 39-46CODEN: STRUE6; ISSN:0969-2126. (Cell Press)
Background: The [URE3] non-Mendelian element of the yeast S. cerevisiae is due to the propagation of a transmissible form of the protein Ure2. The infectivity of Ure2p is thought to originate from a conformational change of the normal form of the prion protein. This conformational change generates a form of Ure2p that assembles into amyloid fibrils. Hence, knowledge of the three-dimensional structure of prion proteins such as Ure2p should help in understanding the mechanism of amyloid formation assocd. with a no. of neurodegenerative diseases. Results: Here we report the three-dimensional crystal structure of the globular region of Ure2p (residues 95-354), also called the functional region, solved at 2.5 A resoln. by the MAD method. The structure of Ure2p 95-354 shows a two-domain protein forming a globular dimer. The N-terminal domain is composed of a central 4 strand β sheet flanked by four α helixes, two on each side. In contrast, the C-terminal domain is entirely α-helical. The fold of Ure2p 95-354 resembles that of the β class glutathione S-transferases (GST), in line with a weak similarity in the amino acid sequence that exists between these proteins. Ure2p dimerizes as GST does and possesses a potential ligand binding site, although it lacks GST activity. Conclusions: The structure of the functional region of Ure2p is the first crystal structure of a prion protein. Structure comparisons between Ure2p 95-354 and GST identified a 32 amino acid residues cap region in Ure2p exposed to the solvent. The cap region is highly flexible and may interact with the N-terminal region of the partner subunit in the dimer. The implication of this interaction in the assembly of Ure2p into amyloid fibrils is discussed.
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Umland, T. C.; Taylor, K. L.; Rhee, S.; Wickner, R. B.; Davies, D. R. Proc. Natl. Acad. Sci. U. S. A. 2001, 98, 1459– 1464 DOI: 10.1073/pnas.98.4.1459
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33
The crystal structure of the nitrogen regulation fragment of the yeast prion protein Ure2p
Umland, Timothy C.; Taylor, Kimberly L.; Rhee, Sangkee; Wickner, Reed B.; Davies, David R.
Proceedings of the National Academy of Sciences of the United States of America (2001), 98 (4), 1459-1464CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
The yeast nonchromosomal gene [URE3] is due to a prion form of the nitrogen regulatory protein Ure2p. It is a neg. regulator of nitrogen catabolism and acts by inhibiting the transcription factor Gln3p. Ure2p residues 1-80 are necessary for prion generation and propagation. The C-terminal fragment retains nitrogen regulatory activity, albeit somewhat less efficiently than the full-length protein, and it also lowers the frequency of prion generation. The crystal structure of this C-terminal fragment, Ure2p(97-354), at 2.3 Å resoln. is described here. It adopts the same fold as the glutathione S-transferase superfamily, consistent with their sequence similarity. However, Ure2p(97-354) lacks a properly positioned catalytic residue that is required for S-transferase activity. Residues within this regulatory fragment that have been indicated by mutational studies to influence prion generation have been mapped onto the three-dimensional structure, and possible implications for prion activity are discussed.
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Bai, M.; Zhou, J. M.; Perrett, S. J. Biol. Chem. 2004, 279, 50025– 50030 DOI: 10.1074/jbc.M406612200
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34
The Yeast Prion Protein Ure2 Shows Glutathione Peroxidase Activity in Both Native and Fibrillar Forms
Bai, Ming; Zhou, Jun-Mei; Perrett, Sarah
Journal of Biological Chemistry (2004), 279 (48), 50025-50030CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
Ure2p is the precursor protein of the Saccharomyces cerevisiae prion [URE3]. Ure2p shows homol. to glutathione transferases but lacks typical glutathione transferase activity. A recent study found that deletion of the Ure2 gene causes increased sensitivity to heavy metal ions and oxidants, whereas prion strains show normal sensitivity. To demonstrate that protection against oxidant toxicity is an inherent property of native and prion Ure2p requires biochem. characterization of the purified protein. Here the authors use steady-state kinetic methods to characterize the multisubstrate peroxidase activity of Ure2p using GSH with cumene hydroperoxide, hydrogen peroxide, or tert-Bu hydroperoxide as substrates. Glutathione-dependent peroxidase activity was proportional to the Ure2p concn. and showed optima at pH 8 and 40°. Michaelis-Menten behavior with convergent straight lines in double reciprocal plots was obsd. This excludes a ping-pong mechanism and implies either a rapid-equil. random or a steady-state ordered sequential mechanism for Ure2p, consistent with its classification as a glutathione transferase. The mutant 90Ure2, which lacks the unstructured N-terminal prion domain, showed kinetic parameters identical to wild type. Fibrillar aggregates showed the same level of activity as native protein. Demonstration of peroxidase activity for Ure2 represents important progress in elucidation of its role in vivo. Further, establishment of an in vitro activity assay provides a valuable tool for the study of structure-function relationships of the Ure2 protein as both a prion and an enzyme.
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Zhang, Z. R.; Perrett, S. J. Biol. Chem. 2009, 284, 14058– 14067 DOI: 10.1074/jbc.M901189200
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35
Novel Glutaredoxin Activity of the Yeast Prion Protein Ure2 Reveals a Native-like Dimer within Fibrils
Zhang, Zai-Rong; Perrett, Sarah
Journal of Biological Chemistry (2009), 284 (21), 14058-14067CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
Ure2 is the protein determinant of the Saccharomyces cerevisiae prion [URE3]. Ure2 has structural similarity to glutathione transferases, protects cells against heavy metal and oxidant toxicity in vivo, and shows glutathione-dependent peroxidase activity in vitro. Here we report that Ure2 (which has no cysteine residues) also shows thiol-disulfide oxidoreductase activity similar to that of glutaredoxin enzymes. This demonstrates that disulfide reductase activity can be independent of the classical glutaredoxin CXXC/CXXS motif or indeed an intrinsic catalytic cysteine residue. The kinetics of the glutaredoxin activity of Ure2 showed pos. cooperativity for the substrate glutathione in both the sol. native state and in amyloid-like fibrils, indicating native-like dimeric structure within Ure2 fibrils. Characterization of the glutaredoxin activity of Ure2 sheds light on its ability to protect yeast from heavy metal ions and oxidant toxicity and suggests a role in reversible protein glutathionylation signal transduction. Observation of allosteric enzyme behavior within amyloid-like Ure2 fibrils not only provides insight into the mol. structure of the fibrils but also has implications for the mechanism of [URE3] prion formation.
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36
Blinder, D.; Coschigano, P. W.; Magasanik, B. J. Bacteriol. 1996, 178, 4734– 4736 DOI: 10.1128/jb.178.15.4734-4736.1996
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36
Interaction of the GATA factor Gln3p with the nitrogen regulator Ure2p in Saccharomyces cerevisiae
Blinder, Dmitry; Coshigano, Peter W.; Magasanik, Boris
Journal of Bacteriology (1996), 178 (15), 4734-4736CODEN: JOBAAY; ISSN:0021-9193. (American Society for Microbiology)
We used cells carrying plasmids causing the overprodn. of Gln3p, Ure2p, or both of these proteins to elucidate the ability of Ure2p to prevent the activation of gene expression by Gln3p in cells growing in a glutamine-contg. medium. Our results indicate that Ure2p probably does not interfere with the binding of the GATA factor Gln3p to GATAAG sites but acts directly on Gln3p to block its ability to activate transcription.
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Pieri, L.; Bucciantini, M.; Nosi, D.; Formigli, L.; Savistchenko, J.; Melki, R.; Stefani, M. J. Biol. Chem. 2006, 281, 15337– 15344 DOI: 10.1074/jbc.M511647200
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37
The Yeast Prion Ure2p Native-like Assemblies Are Toxic to Mammalian Cells Regardless of Their Aggregation State
Pieri, Laura; Bucciantini, Monica; Nosi, Daniele; Formigli, Lucia; Savistchenko, Jimmy; Melki, Ronald; Stefani, Massimo
Journal of Biological Chemistry (2006), 281 (22), 15337-15344CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
The yeast prion Ure2p assembles in vitro into oligomers and fibrils retaining the α-helix content and binding properties of the sol. protein. Here we show that the different forms of Ure2p native-like assemblies (dimers, oligomers, and fibrils) are similarly toxic to murine H-END cells when added to the culture medium. Interestingly, the amyloid fibrils obtained by heat treatment of the toxic native-like fibrils appear harmless. Moreover, the Ure2p C-terminal domain, lacking the N-terminal segment necessary for aggregation but contg. the glutathione binding site, is not cytotoxic. This finding strongly supports the idea that Ure2p toxicity depends on the structural properties of the flexible N-terminal prion domain and can therefore be considered as an inherent feature of the protein, unrelated to its aggregation state but rather assocd. with a basic toxic fold shared by all of the Ure2p native-like assemblies. Indeed, the latter are able to interact with the cell surface, leading to alteration of calcium homeostasis, membrane permeabilization, and oxidative stress, whereas the heat-treated amyloid fibrils do not. Our results support the idea of a general mechanism of toxicity of any protein/peptide aggregate endowed with structural features, making it able to interact with cell membranes and to destabilize them. This evidence extends the widely accepted view that the toxicity by protein aggregates is restricted to amyloid prefibrillar aggregates and provides new insights into the mechanism by which native-like oligomers compromise cell viability.
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Zhang, C.; Jackson, A. P.; Zhang, Z. R.; Han, Y.; Yu, S.; He, R. Q.; Perrett, S. PLoS One 2010, 5e12529 DOI: 10.1371/journal.pone.0012529
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There is no corresponding record for this reference.
39
Bucciantini, M.; Giannoni, E.; Chiti, F.; Baroni, F.; Formigli, L.; Zurdo, J.; Taddei, N.; Ramponi, G.; Dobson, C. M.; Stefani, M. Nature 2002, 416, 507– 511 DOI: 10.1038/416507a
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39
Inherent toxicity of aggregates implies a common mechanism for protein misfolding diseases
Bucciantini, Monica; Giannoni, Elisa; Chiti, Fabrizio; Baroni, Fabiana; Formigli, Lucia; Zurdo, Jesus; Taddei, Niccolo; Ramponi, Giampietro; Dobson, Christopher M.; Stefani, Massimo
Nature (London, United Kingdom) (2002), 416 (6880), 507-511CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
A range of human degenerative conditions, including Alzheimer's disease, light-chain amyloidosis and the spongiform encephalopathies, is assocd. with the deposition in tissue of proteinaceous aggregates known as amyloid fibrils or plaques. It has been shown previously that fibrillar aggregates that are closely similar to those assocd. with clin. amyloidoses can be formed in vitro from proteins not connected with these diseases, including the SH3 domain from bovine phosphatidylinositol 3'-kinase and the amino-terminal domain of the Escherichia coli HypF protein. Here we show that species formed early in the aggregation of these non-disease-assocd. proteins can be inherently highly cytotoxic. This finding provides added evidence that avoidance of protein aggregation is crucial for the preservation of biol. function and suggests common features in the origins of this family of protein deposition diseases.
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40
Catharino, S.; Buchner, J.; Walter, S. Biol. Chem. 2005, 386, 633– 641 DOI: 10.1515/BC.2005.074
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40
Characterization of oligomeric species in the fibrillization pathway of the yeast prion Ure2p
Catharino, Silvia; Buchner, Johannes; Walter, Stefan
Biological Chemistry (2005), 386 (7), 633-641CODEN: BICHF3; ISSN:1431-6730. (Walter de Gruyter GmbH & Co. KG)
The [URE3] prion of Saccharomyces cerevisiae shares many features with mammalian prions and poly-glutamine related disorders and has become a model for studying amyloid diseases. The development of the [URE3] phenotype is thought to be caused by a structural switch in the Ure2p protein. In [URE3] cells, Ure2p is found predominantly in an aggregated state, while it is a sol. dimer in wild-type cells. In vitro, Ure2p forms fibrils with amyloid-like properties. Several studies suggest that the N-terminal domain of Ure2p is essential for prion formation. In this work, we investigated the fibril formation of Ure2p by isolating sol. oligomeric species, which are generated during fibrillization, and characterized them with respect to size and structure. Our data support the crit. role of the N-terminal domain for fibril formation, as we obsd. fibrils in the presence of 5 M guanidinium chloride, conditions at which the C-terminal domain is completely unfolded. Based on fluorescence measurements, we conclude that the structure of the C-terminal domain is very similar in dimeric and fibrillar Ure2p. When studying the time course of fibrillization, we detected the formation of small, sol. oligomeric species during the early stages of the process. Their remarkable resistance against denaturants, their increased content of β-structure, and their ability to 'seed' Ure2p fibrillization suggest that conversion to the amyloid-like conformation has already occurred. Thus, they likely represent crit. intermediates in the fibrillization pathway of Ure2p.
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Wang, Y. Q.; Buell, A. K.; Wang, X. Y.; Welland, M. E.; Dobson, C. M.; Knowles, T. P.; Perrett, S. J. Biol. Chem. 2011, 286, 12101– 12107 DOI: 10.1074/jbc.M110.208934
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Relationship between Prion Propensity and the Rates of Individual Molecular Steps of Fibril Assembly
Wang, Yi-Qian; Buell, Alexander K.; Wang, Xin-Yu; Welland, Mark E.; Dobson, Christopher M.; Knowles, Tuomas P. J.; Perrett, Sarah
Journal of Biological Chemistry (2011), 286 (14), 12101-12107CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
Peptides and proteins possess an inherent propensity to self-assemble into generic fibrillar nanostructures known as amyloid fibrils, some of which are involved in medical conditions such as Alzheimer disease. In certain cases, such structures can self-propagate in living systems as prions and transmit characteristic traits to the host organism. The mechanisms that allow certain amyloid species but not others to function as prions are not fully understood. Much progress in understanding the prion phenomenon has been achieved through the study of prions in yeast as this system has proved to be exptl. highly tractable; but quant. understanding of the biophysics and kinetics of the assembly process has remained challenging. Here, we explore the assembly of two closely related homologues of the Ure2p protein from Saccharomyces cerevisiae and Saccharomyces paradoxus, and by using a combination of kinetic theory with soln. and biosensor assays, we are able to compare the rates of the individual microscopic steps of prion fibril assembly. We find that for these proteins the fragmentation rate is encoded in the structure of the seed fibrils, whereas the elongation rate is principally detd. by the nature of the sol. precursor protein. Our results further reveal that fibrils that elongate faster but fracture less frequently can lose their ability to propagate as prions. These findings illuminate the connections between the in vitro aggregation of proteins and the in vivo proliferation of prions, and provide a framework for the quant. understanding of the parameters governing the behavior of amyloid fibrils in normal and aberrant biol. pathways.
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42
Perrett, S.; Freeman, S. J.; Butler, P. J.; Fersht, A. R. J. Mol. Biol. 1999, 290, 331– 345 DOI: 10.1006/jmbi.1999.2872
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42
Equilibrium Folding Properties of the Yeast Prion Protein Determinant Ure2
Perrett, Sarah; Freeman, Samantha J.; Butler, P. Jonathan G.; Fersht, Alan R.
Journal of Molecular Biology (1999), 290 (1), 331-345CODEN: JMOBAK; ISSN:0022-2836. (Academic Press)
The yeast non-Mendelian factor [URE3] propagates by a prion-like mechanism, involving aggregation of the chromosomally encoded protein Ure2. The [URE3] phenotype is equiv. to loss of function of Ure2, a protein involved in regulation of nitrogen metab. The prion-like behavior of Ure2 in vivo is dependent on the first 65 amino acid residues of its N-terminal region which contains a highly repetitive sequence rich in asparagine. This region has been termed the prion-detg. domain (PrD). Removal of as little as residues 2-20 of the protein is sufficient to prevent occurrence of the [URE3] phenotype. Removal of the PrD does not affect the regulatory activity of Ure2. The C-terminal portion of the protein has homol. to glutathione S -transferases, which are dimeric proteins. We have produced the Ure2 protein to high yield in Escherichia coli from a synthetic gene. The recombinant purified protein is shown to be a dimer. The stability, folding and oligomeric state of Ure2 and a series of N-terminally truncated or deleted variants were studied and compared. The stability of Ure2, ΔGD-N, H2O, detd. by chem. denaturation and monitored by fluorescence, is 12.1(±0.4) kcal mol-1at 25° and pH 8.4. A range of structural probes show a single, coincident unfolding transition, which is invariant over a 550-fold change in protein concn. The stability is the same within error for Ure2 variants lacking all or part of the prion-detg. domain. The data indicate that in the folded protein the PrD is in an unstructured conformation and does not form specific intra- or intermol. interactions at micromolar protein concns. This suggests that the C-terminal domain may stabilize the PrD against prion formation by steric means, and implies that the PrD does not induce prion formation by altering the thermodn. stability of the folded protein. (c) 1999 Academic Press.
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Fei, L.; Perrett, S. J. Biol. Chem. 2009, 284, 11134– 11141 DOI: 10.1074/jbc.M809673200
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43
Disulfide Bond Formation Significantly Accelerates the Assembly of Ure2p Fibrils because of the Proximity of a Potential Amyloid Stretch
Fei, Li; Perrett, Sarah
Journal of Biological Chemistry (2009), 284 (17), 11134-11141CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
Aggregation of the Ure2 protein is at the origin of the [URE3] prion trait in the yeast Saccharomyces cerevisiae. The N-terminal region of Ure2p is necessary and sufficient to induce the [URE3] phenotype in vivo and to polymerize into amyloid-like fibrils in vitro. However, as the N-terminal region is poorly ordered in the native state, making it difficult to detect structural changes in this region by spectroscopic methods, detailed information about the fibril assembly process is therefore lacking. Short fibril-forming peptide regions (4-7 residues) have been identified in a no. of prion and other amyloid-related proteins, but such short regions have not yet been identified in Ure2p. In this study, we identify a unique cysteine mutant (R17C) that can greatly accelerate the fibril assembly kinetics of Ure2p under oxidizing conditions. We found that the segment QVNI, corresponding to residues 18-21 in Ure2p, plays a crit. role in the fast assembly properties of R17C, suggesting that this segment represents a potential amyloid-forming region. A series of peptides contg. the QVNI segment were found to form fibrils in vitro. Furthermore, the peptide fibrils could seed fibril formation for wild-type Ure2p. Preceding the QVNI segment with a cysteine or a hydrophobic residue, instead of a charged residue, caused the rate of assembly into fibrils to increase greatly for both peptides and full-length Ure2p. Our results indicate that the potential amyloid stretch and its preceding residue can modulate the fibril assembly of Ure2p to control the initiation of prion formation.
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Wu, S.; Ge, X.; Lv, Z.; Zhi, Z.; Chang, Z.; Zhao, X. S. Biochem. J. 2011, 438, 505– 511 DOI: 10.1042/BJ20110264
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44
Interaction between bacterial outer membrane proteins and periplasmic quality control factors: a kinetic partitioning mechanism
Wu, Si; Ge, Xi; Lv, Zhixin; Zhi, Zeyong; Chang, Zengyi; Zhao, Xin Sheng
Biochemical Journal (2011), 438 (3), 505-511CODEN: BIJOAK; ISSN:0264-6021. (Portland Press Ltd.)
The outer membrane proteins (OMPs) of Gram-neg. bacteria have to be translocated through the periplasmic space before reaching their final destination. The aq. environment of the periplasmic space and high permeability of the outer membrane engender such a translocation process inevitably challenging. In Escherichia coli, although periplasmic chaperones SurA, Skp, and DegP have been identified to function in translocating OMPs across the periplasm, their precise roles and their relations remain to be elucidated. Here, the authors studied the interaction between the OMP, OmpC, and these periplasmic quality control factors by using FRET and single-mol. detection methods. The results revealed that the binding rate of OmpC to SurA or Skp was much faster than that to DegP, which may lead to sequential interaction between OMPs and different quality control factors. Such a kinetic partitioning mechanism for the chaperone-substrate interaction may be essential for the quality control of the biogenesis of OMPs.
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45
Burnham, K. P.; Anderson, D. R. Model selection and multimodel inference: a practical information-theoretic approach; Springer: New York, 2003.
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46
Cohen, S. I.; Vendruscolo, M.; Welland, M. E.; Dobson, C. M.; Terentjev, E. M.; Knowles, T. P. J. Chem. Phys. 2011, 135, 065105 DOI: 10.1063/1.3608916
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46
Nucleated polymerization with secondary pathways. I. Time evolution of the principal moments
Cohen, Samuel I. A.; Vendruscolo, Michele; Welland, Mark E.; Dobson, Christopher M.; Terentjev, Eugene M.; Knowles, Tuomas P. J.
Journal of Chemical Physics (2011), 135 (6), 065105/1-065105/16CODEN: JCPSA6; ISSN:0021-9606. (American Institute of Physics)
Self-assembly processes resulting in linear structures are often obsd. in mol. biol., and include the formation of functional filaments such as actin and tubulin, as well as generally dysfunctional ones such as amyloid aggregates. Although the basic kinetic equations describing these phenomena are well-established, it has proved to be challenging, due to their non-linear nature, to derive solns. to these equations except for special cases. The availability of general anal. solns. provides a route for detg. the rates of mol. level processes from the anal. of macroscopic exptl. measurements of the growth kinetics, in addn. to the phenomenol. parameters, such as lag times and maximal growth rates that are already obtainable from std. fitting procedures. We describe here an anal. approach based on fixed-point anal., which provides self-consistent solns. for the growth of filamentous structures that can, in addn. to elongation, undergo internal fracturing and monomer-dependent nucleation as mechanisms for generating new free ends acting as growth sites. Our results generalize the anal. expression for sigmoidal growth kinetics from the Oosawa theory for nucleated polymn. to the case of fragmenting filaments. We det. the corresponding growth laws in closed form and derive from first principles a no. of relationships which have been empirically established for the kinetics of the self-assembly of amyloid fibrils. (c) 2011 American Institute of Physics.
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Meisl, G.; Kirkegaard, J. B.; Arosio, P.; Michaels, T. C.; Vendruscolo, M.; Dobson, C. M.; Linse, S.; Knowles, T. P. Nat. Protoc. 2016, 11, 252– 272 DOI: 10.1038/nprot.2016.010
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47
Molecular mechanisms of protein aggregation from global fitting of kinetic models
Meisl, Georg; Kirkegaard, Julius B.; Arosio, Paolo; Michaels, Thomas C. T.; Vendruscolo, Michele; Dobson, Christopher M.; Linse, Sara; Knowles, Tuomas P. J.
Nature Protocols (2016), 11 (2), 252-272CODEN: NPARDW; ISSN:1750-2799. (Nature Publishing Group)
The elucidation of the mol. mechanisms by which sol. proteins convert into their amyloid forms is a fundamental prerequisite for understanding and controlling disorders that are linked to protein aggregation, such as Alzheimer's and Parkinson's diseases. However, because of the complexity assocd. with aggregation reaction networks, the anal. of kinetic data of protein aggregation to obtain the underlying mechanisms represents a complex task. Here we describe a framework, using quant. kinetic assays and global fitting, to det. and to verify a mol. mechanism for aggregation reactions that is compatible with exptl. kinetic data. We implement this approach in a web-based software, AmyloFit. Our procedure starts from the results of kinetic expts. that measure the concn. of aggregate mass as a function of time. We illustrate the approach with results from the aggregation of the β-amyloid (Aβ) peptides measured using thioflavin T, but the method is suitable for data from any similar kinetic expt. measuring the accumulation of aggregate mass as a function of time; the input data are in the form of a tab-sepd. text file. We also outline general exptl. strategies and practical considerations for obtaining kinetic data of sufficient quality to draw detailed mechanistic conclusions, and the procedure starts with instructions for extensive data quality control. For the core part of the anal., we provide an online platform (http://www.amylofit.ch.cam.ac.uk) that enables robust global anal. of kinetic data without the need for extensive programming or detailed math. knowledge. The software automates repetitive tasks and guides users through the key steps of kinetic anal.: detn. of constraints to be placed on the aggregation mechanism based on the concn. dependence of the aggregation reaction, choosing from several fundamental models describing assembly into linear aggregates and fitting the chosen models using an advanced minimization algorithm to yield the reaction orders and rate consts. Finally, we outline how to use this approach to investigate which targets potential inhibitors of amyloid formation bind to and where in the reaction mechanism they act. The protocol, from processing data to detg. mechanisms, can be completed in <1 d.
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Kajava, A. V.; Baxa, U.; Wickner, R. B.; Steven, A. C. Proc. Natl. Acad. Sci. U. S. A. 2004, 101, 7885– 7890 DOI: 10.1073/pnas.0402427101
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48
A model for Ure2p prion filaments and other amyloids: The parallel superpleated β-structure
Kajava, Andrey V.; Baxa, Ulrich; Wickner, Reed B.; Steven, Alasdair C.
Proceedings of the National Academy of Sciences of the United States of America (2004), 101 (21), 7885-7890CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
In its prion form, Ure2p, a regulator of nitrogen catabolism in Saccharomyces cerevisiae, polymerizes into filaments whereby its C-terminal regulatory domain is inactivated but retains its native fold. The filament has an amyloid fibril backbone formed by the Asn-rich, N-terminal, "prion" domain. The prion domain is also capable of forming fibrils when alone or when fused to other proteins. We have developed a model for the fibril that we call a parallel superpleated β-structure. In this model, the prion domain is divided into nine seven-residue segments, each with a four-residue strand and a three-residue turn, that zig-zag in a planar serpentine arrangement. Serpentines are stacked axially, in register, generating an array of parallel β-sheets, with a small and potentially variable left-hand twist. The interior of the filament is mostly stabilized not by packing of apolar side chains but by H-bond networks generated by the stacking of Asn side chains: charged residues are excluded. The model is consistent with current biophys., biochem., and structural data (notably, mass-per-unit-length measurements by scanning transmission electron microscopy that gave one subunit rise per 0.47 nm) and is readily adaptable to other amyloids, for instance the core of Sup35p filaments and glutamine expansions in huntingtin.
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49
Ngo, S.; Chiang, V.; Guo, Z. J. Struct. Biol. 2012, 180, 374– 381 DOI: 10.1016/j.jsb.2012.08.008
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49
Quantitative analysis of spin exchange interactions to identify β strand and turn regions in Ure2 prion domain fibrils with site-directed spin labeling
Ngo, Sam; Chiang, Vicky; Guo, Zhefeng
Journal of Structural Biology (2012), 180 (2), 374-381CODEN: JSBIEM; ISSN:1047-8477. (Elsevier Inc.)
Amyloid formation is assocd. with a range of debilitating human disorders including Alzheimer's and prion diseases. The amyloid structure is essential for understanding the role of amyloids in these diseases. Amyloid formation of Ure2 protein underlies the yeast prion [URE3]. Here we use site-directed spin labeling and ESR (EPR) spectroscopy to investigate the structure of amyloid fibrils formed by the Ure2 prion domain. The Ure2 prion domain under study contains a Sup35M domain at C-terminus as a solubilization element. We introduced spin labels at every residue from positions 2-15, and every 5th residue from positions 20-80 in Ure2 prion domain. EPR spectra at most labeling sites show strong spin exchange interactions, suggesting a parallel in-register β structure. With quant. anal. of spin exchange interactions, we show that residues 8-12 form the first β strand, followed by a turn at residues 13-14, and then the second β strand from residue 15 to at least residue 20. Comparison of the spin exchange frequency for the fibrils formed under quiescent and agitated conditions also revealed differences in the fibril structures. Currently there is a lack of techniques for in-depth structural studies of amyloid fibrils. Detailed structural information is obtained almost exclusively from solid-state NMR. The identification of β-strand and turn regions in this work suggests that quant. anal. of spin exchange interactions in spin-labeled amyloid fibrils is a powerful approach for identifying the β-strand and turn/loop residues and for studying structural differences of different fibril polymorphs.
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50
Galani, D.; Fersht, A. R.; Perrett, S. J. Mol. Biol. 2002, 315, 213– 227 DOI: 10.1006/jmbi.2001.5234
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50
Folding of the Yeast Prion Protein Ure2: Kinetic Evidence for Folding and Unfolding Intermediates
Galani, Despina; Fersht, Alan R.; Perrett, Sarah
Journal of Molecular Biology (2002), 315 (2), 213-227CODEN: JMOBAK; ISSN:0022-2836. (Academic Press)
The Saccharomyces cerevisiae non-Mendelian factor [URE3] propagates by a prion-like mechanism, involving aggregation of the chromosomally encoded protein Ure2. The N-terminal prion domain (PrD) of Ure2 is required for prion activity in vivo and amyloid formation in vitro. However, the mol. mechanism of the prion-like activity remains obscure. Here we measure the kinetics of folding of Ure2 and two N-terminal variants that lack all or part of the PrD. The kinetic folding behavior of the three proteins is identical, indicating that the PrD does not change the stability, rates of folding or folding pathway of Ure2. Both unfolding and refolding kinetics are multiphasic. An intermediate is populated during unfolding at high denaturant concns. resulting in the appearance of an unfolding burst phase and "roll-over" in the denaturant dependence of the unfolding rate consts. During refolding the appearance of a burst phase indicates formation of an intermediate during the dead-time of stopped-flow mixing. A further fast phase shows second-order kinetics, indicating formation of a dimeric intermediate. Regain of native-like fluorescence displays a distinct lag due to population of this on-pathway dimeric intermediate. Double-jump expts. indicate that isomerization of Pro166, which is cis in the native state, occurs late in refolding after regain of native-like fluorescence. During protein refolding there is kinetic partitioning between productive folding via the dimeric intermediate and a non-productive side reaction via an aggregation prone monomeric intermediate. In the light of this and other studies, schemes for folding, aggregation and prion formation are proposed. (c) 2002 Academic Press.
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Arosio, P.; Knowles, T. P.; Linse, S. Phys. Chem. Chem. Phys. 2015, 17, 7606– 7618 DOI: 10.1039/C4CP05563B
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51
On the lag phase in amyloid fibril formation
Arosio, Paolo; Knowles, Tuomas P. J.; Linse, Sara
Physical Chemistry Chemical Physics (2015), 17 (12), 7606-7618CODEN: PPCPFQ; ISSN:1463-9076. (Royal Society of Chemistry)
A review. The formation of nanoscale amyloid fibrils from normally sol. peptides and proteins is a common form of self-assembly phenomenon that has fundamental connections with biol. functions and human diseases. The kinetics of this process has been widely studied and exhibits on a macroscopic level three characteristic stages: (1) a lag phase; (2) a growth phase; and (3) a final plateau regime. The question of which mol. events take place during each one of these phases has been a central element in the quest for a mechanism of amyloid formation. Here, the authors discuss the nature and mol. origin of the lag-phase in amyloid formation by making use of tools and concepts from phys. chem., in particular from chem. reaction kinetics. The authors discuss how, in macroscopic samples, it has become apparent that the lag-phase is not a waiting time for nuclei to form. Rather, multiple parallel processes exist and typically millions of primary nuclei form during the lag phase from monomers in soln. Thus, the lag-time represents a time that is required for the nuclei that are formed early on in the reaction to grow and proliferate in order to reach an aggregate concn. that is readily detected in bulk assays. In many cases, this proliferation takes place through secondary nucleation, where fibrils may present a catalytic surface for the formation of new aggregates. Fibrils may also break (fragmentation) and thereby provide new ends for elongation. Thus, at least 2 (primary nucleation and elongation) and in many systems at least 4 (primary nucleation, elongation, secondary nucleation, and fragmentation) microscopic processes occur during the lag phase. Moreover, these same processes occur during all 3 phases of the macroscopic aggregation process, albeit at different rates as governed by rate consts. and by the concn. of reacting species at each point in time.
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Cohen, S. I.; Linse, S.; Luheshi, L. M.; Hellstrand, E.; White, D. A.; Rajah, L.; Otzen, D. E.; Vendruscolo, M.; Dobson, C. M.; Knowles, T. P. Proc. Natl. Acad. Sci. U. S. A. 2013, 110, 9758– 9763 DOI: 10.1073/pnas.1218402110
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52
Proliferation of amyloid-β42 aggregates occurs through a secondary nucleation mechanism
Cohen, Samuel I. A.; Linse, Sara; Luheshi, Leila M.; Hellstrand, Erik; White, Duncan A.; Rajah, Luke; Otzen, Daniel E.; Vendruscolo, Michele; Dobson, Christopher M.; Knowles, Tuomas P. J.
Proceedings of the National Academy of Sciences of the United States of America (2013), 110 (24), 9758-9763, S9758/1-S9758/11CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
The generation of toxic oligomers during the aggregation of the amyloid-β (Aβ) peptide Aβ42 into amyloid fibrils and plaques has emerged as a central feature of the onset and progression of Alzheimer's disease, but the mol. pathways that control pathol. aggregation have proved challenging to identify. Here, the authors used a combination of kinetic studies, selective radiolabeling expts., and cell viability assays to detect directly the rates of formation of both fibrils and oligomers and the resulting cytotoxic effects. The results showed that once a small but crit. concn. of amyloid fibrils had accumulated, the toxic oligomeric species were predominantly formed from monomeric peptide mols. through a fibril-catalyzed secondary nucleation reaction, rather than through a classical mechanism of homogeneous primary nucleation. This catalytic mechanism coupled together the growth of insol. amyloid fibrils and the generation of diffusible oligomeric aggregates that are implicated as neurotoxic agents in Alzheimer's disease. These results revealed that the aggregation of Aβ42 is promoted by a pos. feedback loop that originates from the interactions between the monomeric and fibrillar forms of this peptide. These findings bring together the main mol. species implicated in the Aβ aggregation cascade and suggest that perturbation of the secondary nucleation pathway identified in this study could be an effective strategy to control the proliferation of neurotoxic Aβ42 oligomers.
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Chen, L.; Chen, L. J.; Wang, H. Y.; Wang, Y. Q.; Perrett, S. Protein Eng., Des. Sel. 2011, 24, 69– 78 DOI: 10.1093/protein/gzq100
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53
Deletion of a Ure2 C-terminal prion-inhibiting region promotes the rate of fibril seed formation and alters interaction with Hsp40
Chen Li; Chen Li-Jun; Wang Hai-Yan; Wang Yi-Qian; Perrett Sarah
Protein engineering, design & selection : PEDS (2011), 24 (1-2), 69-78 ISSN:.
Prions are proteins that can undergo a heritable conformational change to an aggregated amyloid-like state, which is then transmitted to other similar molecules. Ure2, the nitrogen metabolism regulation factor of Saccharomyces cerevisiae, shows prion properties in vivo and forms amyloid fibrils in vitro. Ure2 consists of an N-terminal prion-inducing domain and a C-terminal functional domain. Previous studies have shown that mutations affecting the prion properties of Ure2 are not restricted to the N-terminal prion domain: the deletion of residues 151-158 in the C-domain increases the in vivo prion-inducing propensity of Ure2. Here, we characterized this mutant in vitro and found that the 151-158 deletion has minimal effect on the thermodynamic stability or folding properties of the protein. However, deletion of residues 151-158 accelerates the nucleation, growth and fragmentation of amyloid-like aggregates in vitro, and the aggregates formed are able to seed formation of fibrils of the wild-type protein. In addition, the absence of 151-158 was found to disrupt the inhibitory effect of the Hsp40 chaperone Ydj1 on Ure2 fibril formation. These results suggest that the enhanced in vivo prion-inducing ability of the 151-158 deletion mutant is due to its enhanced ability to generate prion seeds.
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Xu, L. Q.; Wu, S.; Buell, A. K.; Cohen, S. I.; Chen, L. J.; Hu, W. H.; Cusack, S. A.; Itzhaki, L. S.; Zhang, H.; Knowles, T. P.; Dobson, C. M.; Welland, M. E.; Jones, G. W.; Perrett, S. Philos. Trans. R. Soc., B 2013, 368, 20110410 DOI: 10.1098/rstb.2011.0410
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Influence of specific HSP70 domains on fibril formation of the yeast prion protein Ure2
Xu, Li-Qiong; Wu, Si; Buell, Alexander K.; Cohen, Samuel I. A.; Chen, Li-Jun; Hu, Wan-Hui; Cusack, Sarah A.; Itzhaki, Laura S.; Zhang, Hong; Knowles, Tuomas P. J.; Dobson, Christopher M.; Welland, Mark E.; Jones, Gary W.; Perrett, Sarah
Philosophical Transactions of the Royal Society, B: Biological Sciences (2013), 368 (1617), 20110410/1-20110410/13CODEN: PTRBAE; ISSN:0962-8436. (Royal Society)
Ure2p is the protein determinant of the Saccharomyces cerevisiae prion state [URE3]. Constitutive overexpression of the HSP70 family member SSA1 cures cells of [URE3]. Here, we show that Ssa1p increases the lag time of Ure2p fibril formation in vitro in the presence or absence of nucleotide. The presence of the HSP40 co-chaperone Ydj1p has an additive effect on the inhibition of Ure2p fibril formation, whereas the Ydj1p H34Q mutant shows reduced inhibition alone and in combination with Ssa1p. In order to investigate the structural basis of these effects, we constructed and tested an Ssa1p mutant lacking the ATPase domain, as well as a series of C-terminal truncation mutants. The results indicate that Ssa1p can bind to Ure2p and delay fibril formation even in the absence of the ATPase domain, but interaction of Ure2p with the substrate-binding domain is strongly influenced by the C-terminal lid region. Dynamic light scattering, quartz crystal microbalance assays, pull-down assays and kinetic anal. indicate that Ssa1p interacts with both native Ure2p and fibril seeds, and reduces the rate of Ure2p fibril elongation in a concn.-dependent manner. These results provide new insights into the structural and mechanistic basis for inhibition of Ure2p fibril formation by Ssa1p and Ydj1p.
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Glabe, C. G. J. Biol. Chem. 2008, 283, 29639– 29643 DOI: 10.1074/jbc.R800016200
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Structural Classification of Toxic Amyloid Oligomers
Glabe, Charles G.
Journal of Biological Chemistry (2008), 283 (44), 29639-29643CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
A review. Amyloid oligomers are believed to play important causal roles in many types of amyloid-related degenerative diseases. Many different labs. have reported amyloid oligomers that differ in size, morphol., toxicity, and method of prepn. or purifn., raising the question of the structural relationships among these oligomer prepns. The structural plasticity that has been reported to occur in amyloids formed from the same protein sequence indicates that it is quite possible that different oligomer prepns. may represent distinct structural variants. In view of the difficulty in detg. the precise structure of amyloids, conformation- and epitope-specific antibodies may provide a facile means of classifying amyloid oligomer structures. Conformation-dependent antibodies that recognize generic epitopes that are specifically assocd. with distinct aggregation states of many different amyloid-forming sequences indicate that there are at least two fundamentally distinct types of amyloid oligomers: fibrillar and prefibrillar oligomers. Classification of amyloid oligomers according to their underlying structures may be a more useful and rational approach than relying on differences in size and morphol.
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Kayed, R.; Head, E.; Thompson, J. L.; McIntire, T. M.; Milton, S. C.; Cotman, C. W.; Glabe, C. G. Science 2003, 300, 486– 489 DOI: 10.1126/science.1079469
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Common Structure of Soluble Amyloid Oligomers Implies Common Mechanism of Pathogenesis
Kayed, Rakez; Head, Elizabeth; Thompson, Jennifer L.; McIntire, Theresa M.; Milton, Saskia C.; Cotman, Carl W.; Glabe, Charles G.
Science (Washington, DC, United States) (2003), 300 (5618), 486-489CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
Sol. oligomers are common to most amyloids and may represent the primary toxic species of amyloids, like the Aβ peptide in Alzheimer's disease (AD). Here the authors show that all of the sol. oligomers tested display a common conformation-dependent structure that is unique to sol. oligomers regardless of sequence. The in vitro toxicity of sol. oligomers is inhibited by oligomer-specific antibody. Sol. oligomers have a unique distribution in human AD brain that is distinct from fibrillar amyloid. These results indicate that different types of sol. amyloid oligomers have a common structure and suggest they share a common mechanism of toxicity.
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Knowles, T. P.; Waudby, C. A.; Devlin, G. L.; Cohen, S. I.; Aguzzi, A.; Vendruscolo, M.; Terentjev, E. M.; Welland, M. E.; Dobson, C. M. Science 2009, 326, 1533– 1537 DOI: 10.1126/science.1178250
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An Analytical Solution to the Kinetics of Breakable Filament Assembly
Knowles, Tuomas P. J.; Waudby, Christopher A.; Devlin, Glyn L.; Cohen, Samuel I. A.; Aguzzi, Adriano; Vendruscolo, Michele; Terentjev, Eugene M.; Welland, Mark E.; Dobson, Christopher M.
Science (Washington, DC, United States) (2009), 326 (5959), 1533-1537CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
We present an anal. treatment of a set of coupled kinetic equations that governs the self-assembly of filamentous mol. structures. Application to the case of protein aggregation demonstrates that the kinetics of amyloid growth can often be dominated by secondary rather than by primary nucleation events. Our results further reveal a range of general features of the growth kinetics of fragmenting filamentous structures, including the existence of generic scaling laws that provide mechanistic information in contexts ranging from in vitro amyloid growth to the in vivo development of mammalian prion diseases.
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Cohen, S. I.; Vendruscolo, M.; Dobson, C. M.; Knowles, T. P. J. Mol. Biol. 2012, 421, 160– 171 DOI: 10.1016/j.jmb.2012.02.031
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From Macroscopic Measurements to Microscopic Mechanisms of Protein Aggregation
Cohen, Samuel I. A.; Vendruscolo, Michele; Dobson, Christopher M.; Knowles, Tuomas P. J.
Journal of Molecular Biology (2012), 421 (2-3), 160-171CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Ltd.)
A review. The ability to relate bulk exptl. measurements of amyloid formation to the microscopic assembly processes that underlie protein aggregation is crit. to achieve a quant. understanding of this complex phenomenon. In this review, the authors focus on the insights from classical and modern theories of linear growth phenomena and discuss how theory allows the roles of growth and nucleation processes to be defined through the anal. of exptl. in vitro time courses of amyloid formation. Moreover, the authors discuss the specific signatures in the time course of the reactions that correspond to the actions of primary and secondary nucleation processes and outline strategies for identifying and characterizing the nature of the dominant process responsible in each case for the generation of new aggregates. The authors highlight the power of a global anal. of exptl. time courses acquired under different conditions, and discuss how such an anal. allows a rigorous connection to be established between the macroscopic measurements and the rates of the individual microscopic processes that underlie the phenomenon of amyloid formation.
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Garcia, G. A.; Cohen, S. I.; Dobson, C. M.; Knowles, T. P. Phys. Rev. E Stat Nonlin Soft Matter Phys. 2014, 89, 032712 DOI: 10.1103/PhysRevE.89.032712
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Nucleation-conversion-polymerization reactions of biological macromolecules with prenucleation clusters
Garcia, Gonzalo A.; Cohen, Samuel I. A.; Dobson, Christopher M.; Knowles, Tuomas P. J.
Physical Review E: Statistical, Nonlinear, and Soft Matter Physics (2014), 89 (3-A), 032712/1-032712/6CODEN: PRESCM; ISSN:1539-3755. (American Physical Society)
The self-assembly of biomols., such as peptides and proteins, into filaments is conventionally understood as a nucleated polymn. reaction. However, detailed anal. of exptl. observation has revealed recently that nucleation pathways generate growth-competent nuclei via a cascade of metastable intermediate species, which are omitted in conventional models of filamentous growth based on classical nucleation theory. Here we take an anal. approach to generalizing the classical theory of nucleated polymn. to include the formation of these prenucleation clusters, providing a quant. general classification of the behavior exhibited by these nucleation-conversion-polymn. reactions. A phase diagram is constructed, and anal. predictions are derived for key exptl. observables. Using this approach, we delineate the characteristic time scales that det. the nature of biopolymer growth phenomena.
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Saric, A.; Chebaro, Y. C.; Knowles, T. P.; Frenkel, D. Proc. Natl. Acad. Sci. U. S. A. 2014, 111, 17869– 17874 DOI: 10.1073/pnas.1410159111
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Crucial role of nonspecific interactions in amyloid nucleation
Saric, Andjela; Chebaro, Yassmine C.; Knowles, Tuomas P. J.; Frenkel, Daan
Proceedings of the National Academy of Sciences of the United States of America (2014), 111 (50), 17869-17874CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
Protein oligomers have been implicated as toxic agents in a wide range of amyloid-related diseases. However, it has remained unsolved whether the oligomers are a necessary step in the formation of amyloid fibrils or just a dangerous byproduct. Analogously, it has not been resolved if the amyloid nucleation process is a classical one-step nucleation process or a two-step process involving prenucleation clusters. We use coarse-grained computer simulations to study the effect of nonspecific attractions between peptides on the primary nucleation process underlying amyloid fibrillization. We find that, for peptides that do not attract, the classical one-step nucleation mechanism is possible but only at nonphysiol. high peptide concns. At low peptide concns., which mimic the physiol. relevant regime, attractive interpeptide interactions are essential for fibril formation. Nucleation then inevitably takes place through a two-step mechanism involving prefibrillar oligomers. We show that oligomers not only help peptides meet each other but also, create an environment that facilitates the conversion of monomers into the β-sheet-rich form characteristic of fibrils. Nucleation typically does not proceed through the most prevalent oligomers but through an oligomer size that is only obsd. in rare fluctuations, which is why such aggregates might be hard to capture exptl. Finally, we find that the nucleation of amyloid fibrils cannot be described by classical nucleation theory: in the two-step mechanism, the crit. nucleus size increases with increases in both concn. and interpeptide interactions, which is in direct contrast with predictions from classical nucleation theory.
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Saric, A.; Michaels, T. C. T.; Zaccone, A.; Knowles, T. P. J.; Frenkel, D. J. Chem. Phys. 2016, 145, 211926 DOI: 10.1063/1.4965040
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Kinetics of spontaneous filament nucleation via oligomers: Insights from theory and simulation
Saric, Andjela; Michaels, Thomas C. T.; Zaccone, Alessio; Knowles, Tuomas P. J.; Frenkel, Daan
Journal of Chemical Physics (2016), 145 (21), 211926/1-211926/9CODEN: JCPSA6; ISSN:0021-9606. (American Institute of Physics)
Nucleation processes are at the heart of a large no. of phenomena, from cloud formation to protein crystn. A recently emerging area where nucleation is highly relevant is the initiation of filamentous protein self-assembly, a process that has broad implications in many research areas ranging from medicine to nanotechnol. As such, spontaneous nucleation of protein fibrils has received much attention in recent years with many theor. and exptl. studies focussing on the underlying phys. principles. In this paper we make a step forward in this direction and explore the early time behavior of filamentous protein growth in the context of nucleation theory. We first provide an overview of the thermodn. and kinetics of spontaneous nucleation of protein filaments in the presence of one relevant degree of freedom, namely the cluster size. In this case, we review how key kinetic observables, such as the reaction order of spontaneous nucleation, are directly related to the phys. size of the crit. nucleus. We then focus on the increasingly prominent case of filament nucleation that includes a conformational conversion of the nucleating building-block as an addnl. slow step in the nucleation process. Using computer simulations, we study the concn. dependence of the nucleation rate. We find that, under these circumstances, the reaction order of spontaneous nucleation with respect to the free monomer does no longer relate to the overall phys. size of the nucleating aggregate but rather to the portion of the aggregate that actively participates in the conformational conversion. Our results thus provide a novel interpretation of the common kinetic descriptors of protein filament formation, including the reaction order of the nucleation step or the scaling exponent of lag times, and put into perspective current theor. descriptions of protein aggregation. (c) 2016 American Institute of Physics.
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Auer, S.; Meersman, F.; Dobson, C. M.; Vendruscolo, M. PLoS Comput. Biol. 2008, 4, e1000222 DOI: 10.1371/journal.pcbi.1000222
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A generic mechanism of emergence of amyloid protofilaments from disordered oligomeric aggregates
Auer Stefan; Meersman Filip; Dobson Christopher M; Vendruscolo Michele
PLoS computational biology (2008), 4 (11), e1000222 ISSN:.
The presence of oligomeric aggregates, which is often observed during the process of amyloid formation, has recently attracted much attention because it has been associated with a range of neurodegenerative conditions including Alzheimer's and Parkinson's diseases. We provide a description of a sequence-indepedent mechanism by which polypeptide chains aggregate by forming metastable oligomeric intermediate states prior to converting into fibrillar structures. Our results illustrate that the formation of ordered arrays of hydrogen bonds drives the formation of beta-sheets within the disordered oligomeric aggregates that form early under the effect of hydrophobic forces. Individual beta-sheets initially form with random orientations and subsequently tend to align into protofilaments as their lengths increase. Our results suggest that amyloid aggregation represents an example of the Ostwald step rule of first-order phase transitions by showing that ordered cross-beta structures emerge preferentially from disordered compact dynamical intermediate assemblies.
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Lee, J.; Culyba, E. K.; Powers, E. T.; Kelly, J. W. Nat. Chem. Biol. 2011, 7, 602– 609 DOI: 10.1038/nchembio.624
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Amyloid-β forms fibrils by nucleated conformational conversion of oligomers
Lee, Jiyong; Culyba, Elizabeth K.; Powers, Evan T.; Kelly, Jeffery W.
Nature Chemical Biology (2011), 7 (9), 602-609CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)
Amyloid-β amyloidogenesis is reported to occur via a nucleated polymn. mechanism. If this is true, the energetically unfavorable oligomeric nucleus should be very hard to detect. However, many labs. have detected early nonfibrillar amyloid-β oligomers without observing amyloid fibrils, suggesting that a mechanistic revision may be needed. Here the authors introduce Cys-Cys-amyloid-β1-40, which cannot bind to the latent fluorophore FlAsH as a monomer, but can bind FlAsH as an nonfibrillar oligomer or as a fibril, rendering the conjugates fluorescent. Through FlAsH monitoring of Cys-Cys-amyloid-β1-40 aggregation, the authors found that amyloid-β1-40 rapidly and efficiently forms spherical oligomers in vitro (85% yield) that are kinetically competent to slowly convert to amyloid fibrils by a nucleated conformational conversion mechanism. This methodol. was used to show that plasmalogen ethanolamine vesicles eliminate the proteotoxicity-assocd. oligomerization phase of amyloid-β amyloidogenesis while allowing fibril formation, rationalizing how low concns. of plasmalogen ethanolamine in the brain are epidemiol. linked to Alzheimer's disease.
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Krishnan, R.; Goodman, J. L.; Mukhopadhyay, S.; Pacheco, C. D.; Lemke, E. A.; Deniz, A. A.; Lindquist, S. Proc. Natl. Acad. Sci. U. S. A. 2012, 109, 11172– 11177 DOI: 10.1073/pnas.1209527109
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Conserved features of intermediates in amyloid assembly determine their benign or toxic states
Krishnan, Rajaraman; Goodman, Jessica L.; Mukhopadhyay, Samrat; Pacheco, Chris D.; Lemke, Edward A.; Deniz, Ashok A.; Lindquist, Susan
Proceedings of the National Academy of Sciences of the United States of America (2012), 109 (28), 11172-11177, S11172/1-S11172/10CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
Some amyloid-forming polypeptides are assocd. with devastating human diseases and others provide important biol. functions. For both, oligomeric intermediates appear during amyloid assembly. Currently we have few tools for characterizing these conformationally labile intermediates and discerning what governs their benign vs. toxic states. Here, we examine intermediates in the assembly of a normal, functional amyloid, the prion-detg. region of yeast Sup35 (NM). During assembly, NM formed a variety of oligomers with different sizes and conformation-specific antibody reactivities. Earlier oligomers were less compact and reacted with the conformational antibody A11. More mature oligomers were more compact and reacted with conformational antibody OC. We found we could arrest NM in either of these two distinct oligomeric states with small mols. or crosslinking. The A11-reactive oligomers were more hydrophobic (as measured by Nile Red binding) and were highly toxic to neuronal cells, while OC-reactive oligomers were less hydrophobic and were not toxic. The A11 and OC antibodies were originally raised against oligomers of Aβ, an amyloidogenic peptide implicated in Alzheimer's disease (AD) that is completely unrelated to NM in sequence. Thus, this natural yeast prion samples two conformational states similar to those sampled by Al, and when assembly stalls at one of these two states, but not the other, it becomes extremely toxic. Our results have implications for selective pressures operating on the evolution of amyloid folds across a billion years of evolution. Understanding the features that govern such conformational transitions will shed light on human disease and evolution alike.
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Bolognesi, B.; Kumita, J. R.; Barros, T. P.; Esbjorner, E. K.; Luheshi, L. M.; Crowther, D. C.; Wilson, M. R.; Dobson, C. M.; Favrin, G.; Yerbury, J. J. ACS Chem. Biol. 2010, 5, 735– 740 DOI: 10.1021/cb1001203
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ANS Binding Reveals Common Features of Cytotoxic Amyloid Species
Bolognesi, Benedetta; Kumita, Janet R.; Barros, Teresa P.; Esbjoerner, Elin K.; Luheshi, Leila M.; Crowther, Damian C.; Wilson, Mark R.; Dobson, Christopher M.; Favrin, Giorgio; Yerbury, Justin J.
ACS Chemical Biology (2010), 5 (8), 735-740CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)
Oligomeric assemblies formed from a variety of disease-assocd. peptides and proteins have been strongly assocd. with toxicity in many neurodegenerative conditions, such as Alzheimer's disease. The precise nature of the toxic agents, however, remains still to be established. We show that prefibrillar aggregates of E22G (arctic) variant of the Aβ1-42 peptide bind strongly to 1-anilinonaphthalene 8-sulfonate and that changes in this property correlate significantly with changes in its cytotoxicity. Moreover, we show that this phenomenon is common to other amyloid systems, such as wild-type Aβ1-42, the I59T variant of human lysozyme and an SH3 domain. These findings are consistent with a model in which the exposure of hydrophobic surfaces as a result of the aggregation of misfolded species is a crucial and common feature of these pathogenic species.
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Campioni, S.; Mannini, B.; Zampagni, M.; Pensalfini, A.; Parrini, C.; Evangelisti, E.; Relini, A.; Stefani, M.; Dobson, C. M.; Cecchi, C.; Chiti, F. Nat. Chem. Biol. 2010, 6, 140– 147 DOI: 10.1038/nchembio.283
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A causative link between the structure of aberrant protein oligomers and their toxicity
Campioni, Silvia; Mannini, Benedetta; Zampagni, Mariagioia; Pensalfini, Anna; Parrini, Claudia; Evangelisti, Elisa; Relini, Annalisa; Stefani, Massimo; Dobson, Christopher M.; Cecchi, Cristina; Chiti, Fabrizio
Nature Chemical Biology (2010), 6 (2), 140-147CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)
The aberrant assembly of peptides and proteins into fibrillar aggregates proceeds through oligomeric intermediates that are thought to be the primary pathogenic species in many protein deposition diseases. We describe two types of oligomers formed by the HypF-N protein that are morphol. and tinctorially similar, as detected with at. force microscopy (AFM) and thioflavin T (ThT) assays, though one is benign when added to cell cultures whereas the other is toxic. Structural investigation at a residue-specific level using site-directed labeling with pyrene indicated differences in the hydrophobic packing between adjacent protein mols. in the oligomers. A lower degree of hydrophobic packing was found to correlate with a higher ability to penetrate the cell membrane and cause an influx of Ca2+ ions. Our findings suggest that structural flexibility and hydrophobic exposure are primary determinants of the ability of oligomeric assemblies to cause cellular dysfunction and its consequences, such as neurodegeneration.
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Baxa, U.; Wickner, R. B.; Steven, A. C.; Anderson, D. E.; Marekov, L. N.; Yau, W. M.; Tycko, R. Biochemistry 2007, 46, 13149– 13162 DOI: 10.1021/bi700826b
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67
Characterization of β-Sheet Structure in Ure2p1-89 Yeast Prion Fibrils by Solid-State Nuclear Magnetic Resonance
Baxa, Ulrich; Wickner, Reed B.; Steven, Alasdair C.; Anderson, D. Eric; Marekov, Lyuben N.; Yau, Wai-Ming; Tycko, Robert
Biochemistry (2007), 46 (45), 13149-13162CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
Residues 1-89 constitute the Asn- and Gln-rich segment of the Ure2p protein and produce the [URE3] prion of Saccharomyces cerevisiae by forming the core of intracellular Ure2p amyloid. We report the results of solid-state NMR (NMR) measurements that probe the mol. structure of amyloid fibrils formed by Ure2p1-89 in vitro. Data include measurements of intermol. magnetic dipole-dipole couplings in samples that are 13C-labeled at specific sites and two-dimensional 15N-13C and 13C-13C NMR spectra of samples that are uniformly 15N- and 13C-labeled. Intermol. dipole-dipole couplings indicate that the β-sheets in Ure2p1-89 fibrils have an in-register parallel structure. An in-register parallel β-sheet structure permits polar zipper interactions among side chains of Gln and Asn residues and explains the tolerance of [URE3] to scrambling of the sequence in residues 1-89. Two-dimensional NMR spectra of uniformly labeled Ure2p1-89 fibrils, even when fully hydrated, show NMR linewidths that exceed those in solid-state NMR spectra of fibrils formed by residues 218-289 of the HET-s prion protein of Podospora anserina by factors of three or more, indicating a lower degree of structural order at the mol. level in Ure2p1-89 fibrils. The very high degree of structural order in HET-s fibrils indicated by solid-state NMR data is therefore not a universal characteristic of prion proteins, and is likely to be a consequence of the evolved biol. function of HET-s in heterokaryon incompatibility. Anal. of cross peak intensities in two-dimensional NMR spectra of uniformly labeled Ure2p1-89 fibrils suggests that certain portions of the amino acid sequence may not participate in a rigid β-sheet structure, possibly including portions of the Asn-rich segment between residues 44 and 76.
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Kryndushkin, D. S.; Wickner, R. B.; Tycko, R. J. Mol. Biol. 2011, 409, 263– 277 DOI: 10.1016/j.jmb.2011.03.067
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The Core of Ure2p Prion Fibrils Is Formed by the N-Terminal Segment in a Parallel Cross-β Structure: Evidence from Solid-State NMR
Kryndushkin, Dmitry S.; Wickner, Reed B.; Tycko, Robert
Journal of Molecular Biology (2011), 409 (2), 263-277CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Ltd.)
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Abstract
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Figure 1
Figure 1. Oligomerization of Ure2 monitored by confocal single molecule FRET. (A) Schematic figure to indicate the cysteine mutations and fluorescence labeling sites that were used in this study, based on a previously suggested structural model of Ure2 fibrils.(48) (B) Scheme for smFRET detection of Ure2 oligomers. (C) The concentration of AF555/AF647 labeled Ure2-S68C oligomers throughout the aggregation reaction. (D) Ensemble kinetics of the aggregation of 15 μM (dimeric concentration) unlabeled Ure2-S68C monitored by ThT fluorescence. All the aggregation reactions were carried out at 18 °C in an Innova 4230 incubator with shaking at 150 rpm in 50 mM Tris–HCl (pH 8.4) buffer containing 200 mM NaCl.
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Figure 2
Figure 2. Absence of a fibril-catalyzed secondary nucleation process for Ure2. (A, B) Ensemble aggregation kinetics of 15 μM unlabeled Ure2-S68C monitored by ThT fluorescence under unseeded (blue) or seeded (red) conditions (upper panels). Ure2 oligomers were then detected under unseeded (blue) or seeded (red) conditions by confocal single molecule FRET (lower panels). The incubation conditions were the same as in Figure 1. (A) The data fit well to a model that generates oligomers during primary nucleation. (B) A model that generates oligomers during secondary nucleation cannot fit the data. (C) The presence of seeds (right-hand columns) drastically increases the concentration of Aβ42 oligomers measured at a single time point in the lag phase of an Aβ42 aggregation experiment(52) (right panel), but do not increase the production of Ure2 oligomers in this study (left panel), indicating fundamentally different mechanisms of oligomer formation for these two systems.
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Figure 3
Figure 3. Different types of Ure2 oligomers revealed by confocal single molecule FRET. (A,B) SmFRET efficiency distribution of selected oligomers of AF488/AF647-labeled Ure2 at different incubation times. The FRET distributions at different time points were fitted globally to double Gaussian functions giving the average peak positions indicated. (C,D) Population of low- and high-FRET oligomers of Ure2 at different incubation times. The incubation conditions were the same as in Figure 1. (A,C) Ure2-S53C. (B,D) Ure2-V9C.
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Figure 4
Figure 4. Single molecule TIRF measurements of disaggregated fibrils. (A,B) SmFRET distribution histogram of Ure2 oligomers disaggregated from AF488/AF647-labeled fibrils. Data were fitted to a double Gaussian function (continuous line) to obtain the FRET values of the two species. (A) Ure2-S53C. (B) Ure2-V9C.
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Figure 5
Figure 5. Fitting of combined smFRET/ThT data to models indicates a probable effect of mutations on the dissociation of oligomers, but not on their formation. (A–C) The bulk aggregation kinetics of 15 μM unlabeled Ure2-V9C (red) and Ure2-S68C (blue) monitored by ThT fluorescence (left panels) and the concentration of AF555/AF647 labeled Ure2-V9C (red) and Ure2-S68C (blue) oligomers throughout the aggregation reaction monitored by confocal smFRET (right panels) were globally fitted to a theoretical model (see Methods) including the formation, dissociation, and conversion of oligomers, and the elongation and fragmentation of fibrils. The incubation conditions were the same as in Figure 1. (A) Allowing both k_oligo and k_d to differ for each mutant gives good fits, with a mean squared error of 1.54. (B) If k_oligo is constrained to be the same for both mutants, the model fits the data equally well, with a mean squared error of 1.58. (C) If neither k_oligo nor k_d is allowed to differ, the fit is less good, especially around the time when the oligomer concentration is at a maximum, with a mean squared error of 1.87. This result therefore implies that k_oligo is the same for the two variants, while the values of k_d may differ slightly.
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Figure 6
Figure 6. Proposed model for the aggregation pathway of Ure2. Native dimeric Ure2 forms relatively disordered oligomers driven by hydrophobic interactions and either dissociates back to the native state or undergoes conformational conversion to form more compact oligomers containing β-sheet structure, which can in turn convert into growth-competent fibrillar species. Fragmentation of fibrils then contributes to their proliferation.
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  Alzheimer's disease is a complex disease characterized by overlapping phenotypes with different neurodegenerative disorders. Oligomers are considered the most toxic species in amyloid pathologies. We examd. human AD brain samples using an anti-oligomer antibody generated in our lab. and detected potential hybrid oligomers composed of amyloid-β, prion protein, α-synuclein, and TDP-43 phosphorylated at serines 409 and 410. These data and in vitro results suggest that Aβ oligomer seeds act as a template for the aggregation of other proteins and generate an overlapping phenotype with other neuronal disorders. Furthermore, these results could explain why anti-amyloid-β therapy has been unsuccessful.
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8. 8
  Cheng, B.; Gong, H.; Xiao, H.; Petersen, R. B.; Zheng, L.; Huang, K. Biochim. Biophys. Acta, Gen. Subj. 2013, 1830, 4860– 4871 DOI: 10.1016/j.bbagen.2013.06.029
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  8
  Inhibiting toxic aggregation of amyloidogenic proteins: A therapeutic strategy for protein misfolding diseases
  Cheng, Biao; Gong, Hao; Xiao, Hongwen; Petersen, Robert B.; Zheng, Ling; Huang, Kun
  Biochimica et Biophysica Acta, General Subjects (2013), 1830 (10), 4860-4871CODEN: BBGSB3; ISSN:0304-4165. (Elsevier B.V.)
  A review. The deposition of self-assembled amyloidogenic proteins is assocd. with multiple diseases, including Alzheimer's disease, Parkinson's disease and type 2 diabetes mellitus. The toxic misfolding and self-assembling of amyloidogenic proteins are believed to underlie protein misfolding diseases. Novel drug candidates targeting self-assembled amyloidogenic proteins represent a potential therapeutic approach for protein misfolding diseases. In this perspective review, the authors provide an overview of the recent progress in identifying inhibitors that block the aggregation of amyloidogenic proteins and the clin. applications thereof. Compds. such as polyphenols, certain short peptides, and monomer- or oligomer-specific antibodies, can interfere with the self-assembly of amyloidogenic proteins, prevent the formation of oligomers, amyloid fibrils and the consequent cytotoxicity. Some inhibitors have been tested in clin. trials for treating protein misfolding diseases. Inhibitors that target the aggregation of amyloidogenic proteins bring new hope to therapy for protein misfolding diseases.
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9. 9
  Guerrero-Munoz, M. J.; Castillo-Carranza, D. L.; Kayed, R. Biochem. Pharmacol. 2014, 88, 468– 478 DOI: 10.1016/j.bcp.2013.12.023
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  Therapeutic approaches against common structural features of toxic oligomers shared by multiple amyloidogenic proteins
  Guerrero-Munoz, Marcos J.; Castillo-Carranza, Diana L.; Kayed, Rakez
  Biochemical Pharmacology (Amsterdam, Netherlands) (2014), 88 (4), 468-478CODEN: BCPCA6; ISSN:0006-2952. (Elsevier B.V.)
  A review. Impaired proteostasis is one of the main features of all amyloid diseases, which are assocd. with the formation of insol. aggregates from amyloidogenic proteins. The aggregation process can be caused by overprodn. or poor clearance of these proteins. However, numerous reports suggest that amyloid oligomers are the most toxic species, rather than insol. fibrillar material, in Alzheimer's, Parkinson's, and Prion diseases, among others. Although the exact protein that aggregates varies between amyloid disorders, they all share common structural features that can be used as therapeutic targets. In this review, we focus on therapeutic approaches against shared features of toxic oligomeric structures and future directions.
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10. 10
  LeVine, H., 3rd Methods Enzymol. 1999, 309, 274– 284 DOI: 10.1016/S0076-6879(99)09020-5
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  10
  Quantification of β-sheet amyloid fibril structures with thioflavin T
  LeVine, Harry, III
  Methods in Enzymology (1999), 309 (Amyloid, Prions, and Other Protein Aggregates), 274-284CODEN: MENZAU; ISSN:0076-6879. (Academic Press)
  A spectrochem. method for the detn. of β-amyloid using thioflavin T was presented. (c) 1999 Academic Press.
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  https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3cXksVKhuw%253D%253D&md5=e319fb304f4f80f7212dde361c4c7416
11. 11
  Bartolini, M.; Bertucci, C.; Bolognesi, M. L.; Cavalli, A.; Melchiorre, C.; Andrisano, V. ChemBioChem 2007, 8, 2152– 2161 DOI: 10.1002/cbic.200700427
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  11
  Insight into the kinetic of amyloid β (1-42) peptide self-aggregation: elucidation of inhibitors' mechanism of action
  Bartolini, Manuela; Bertucci, Carlo; Bolognesi, Maria Laura; Cavalli, Andrea; Melchiorre, Carlo; Andrisano, Vincenza
  ChemBioChem (2007), 8 (17), 2152-2161CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)
  The initial transition of amyloid β (1-42) (Aβ42) sol. monomers/small oligomers from unordered/α-helix to a β-sheet-rich conformation represents a suitable target to design new potent inhibitors and to obtain effective therapeutics for Alzheimer's disease. Under optimized conditions, this reliable and reproducible CD kinetic study showed a 3-step sigmoid profile that was characterized by a lag phase (prevailing unordered/α-helix conformation), an exponential growth phase (increasing β-sheet secondary structure) and a plateau phase (prevailing β-sheet secondary structure). This kinetic anal. brought insight into the inhibitors' mechanism of action. In fact, an increase in the duration of the lag phase can be related to the formation of an inhibitor-Aβ complex, in which the non-amyloidogenic conformation is stabilized. When the exponential rate is affected exclusively, such as in the case of Congo red and tetracycline, then the inhibitor affinity might be higher for the pleated β-sheet structure. Finally, by adding the inhibitor at the end of the exponential phase, the sol. protofibrils can be disrupted and the Aβ amyloidogenic structure can revert into monomers/small oligomers. Congo red and tetracycline preferentially bind to amyloid in the β-sheet conformation because both decreased the slope of the exponential growth, even if to a different extent, whereas no effect was obsd. for tacrine and galantamine. Some very preliminary indications can be derived about the structural requirements for binding to nonamyloidogenic or β-sheet amyloid secondary structure for the development of potent antiaggregating agents. On these premises, memoquin, a multifunctional mol. that was designed to become a drug candidate for the treatment of Alzheimer's disease, was investigated under the reported CD assay and its anti-amyloidogenic mechanism of action was elucidated.
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12. 12
  Newcomb, C. J.; Moyer, T. J.; Lee, S. S.; Stupp, S. I. Curr. Opin. Colloid Interface Sci. 2012, 17, 350– 359 DOI: 10.1016/j.cocis.2012.09.004
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  12
  Advances in cryogenic transmission electron microscopy for the characterization of dynamic self-assembling nanostructures
  Newcomb, Christina J.; Moyer, Tyson J.; Lee, Sungsoo S.; Stupp, Samuel I.
  Current Opinion in Colloid & Interface Science (2012), 17 (6), 350-359CODEN: COCSFL; ISSN:1359-0294. (Elsevier Ltd.)
  A review. Elucidating the structural information of nanoscale materials in their solvent-exposed state is crucial, as a result, cryogenic transmission electron microscopy (cryo-TEM) has become an increasingly popular technique in the materials science, chem., and biol. communities. Cryo-TEM provides a method to directly visualize the specimen structure in a soln.-state through a thin film of vitrified solvent. This technique complements x-ray, neutron, and light scattering methods that probe the statistical av. of all species present; furthermore, cryo-TEM can be used to observe changes in structure over time. In the area of self-assembly, this tool was particularly powerful for the characterization of natural and synthetic small mol. assemblies, as well as hybrid org.-inorg. composites. In this review, recent advances in cryogenic TEM are discussed in the context of self-assembling systems with emphasis on characterization of transitions obsd. in response to external stimuli.
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13. 13
  Smith, J. F.; Knowles, T. P.; Dobson, C. M.; Macphee, C. E.; Welland, M. E. Proc. Natl. Acad. Sci. U. S. A. 2006, 103, 15806– 15811 DOI: 10.1073/pnas.0604035103
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  13
  Characterization of the nanoscale properties of individual amyloid fibrils
  Smith, Jeffrey F.; Knowles, Tuomas P. J.; Dobson, Christopher M.; MacPhee, Cait E.; Welland, Mark E.
  Proceedings of the National Academy of Sciences of the United States of America (2006), 103 (43), 15806-15811CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
  We report the detailed mech. characterization of individual amyloid fibrils by at. force microscopy and spectroscopy. These self-assembling materials, formed here from the protein insulin, were shown to have a strength of 0.6 ± 0.4 GPa, comparable to that of steel (0.6-1.8 GPa), and a mech. stiffness, as measured by Young's modulus, of 3.3 ± 0.4 GPa, comparable to that of silk (1-10 GPa). The values of these parameters reveal that the fibrils possess properties that make these structures highly attractive for future technol. applications. In addn., anal. of the soln. state growth kinetics indicated a breakage rate const. of 1.7 ± 1.3 × 10-8 s-1, which reveals that a fibril 10 μm in length breaks spontaneously on av. every 47 min, suggesting that internal fracturing is likely to be of fundamental importance in the proliferation of amyloid fibrils and therefore for understanding the progression of their assocd. pathogenic disorders.
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14. 14
  Nath, S.; Meuvis, J.; Hendrix, J.; Carl, S. A.; Engelborghs, Y. Biophys. J. 2010, 98, 1302– 1311 DOI: 10.1016/j.bpj.2009.12.4290
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  Early aggregation steps in α-synuclein as measured by FCS and FRET: evidence for a contagious conformational change
  Nath, Sangeeta; Meuvis, Jessika; Hendrix, Jelle; Carl, Shaun A.; Engelborghs, Yves
  Biophysical Journal (2010), 98 (7), 1302-1311CODEN: BIOJAU; ISSN:0006-3495. (Cell Press)
  The kinetics of aggregation of α-synuclein are usually studied by turbidity or thioflavin T fluorescence. Here, the authors followed the disappearance of monomers and the formation of early oligomers using fluorescence correlation spectroscopy (FCS). Alexa488-labeled A140C-α-synuclein was used as a fluorescent probe in trace amts. in the presence of excess unlabeled α-synuclein. Repeated short measurements produced a distribution of diffusion coeffs. Initially, a sharp peak was obtained corresponding to monomers, followed by a distinct transient population and the gradual formation of broader-sized distributions of higher oligomers. The kinetics of aggregation could be followed by the decreasing no. of fast-diffusing species. Both the disappearance of fast-diffusing species and the appearance of turbidity could be fitted to the Finke-Watzky equation, but the apparent rate consts. obtained were different. This reflected the fact that the disappearance of fast species occurred largely during the lag phase of turbidity development, due to the limited sensitivity of turbidity to the early aggregation process. The nucleation of the early oligomers was concn.-dependent and accompanied by a conformational change that preceded β-structure formation, and could be visualized using FRET between the donor-labeled N-terminus and the acceptor-labeled Cys residue in the A140C mutant.
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15. 15
  Paredes, J. M.; Casares, S.; Ruedas-Rama, M. J.; Fernandez, E.; Castello, F.; Varela, L.; Orte, A. Int. J. Mol. Sci. 2012, 13, 9400– 9418 DOI: 10.3390/ijms13089400
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  15
  Early amyloidogenic oligomerization studied through fluorescence lifetime correlation spectroscopy
  Paredes, Jose M.; Casares, Salvador; Ruedas-Rama, Maria J.; Fernandez, Elena; Castello, Fabio; Varela-Alvarez, Lorena; Orte, Angel
  International Journal of Molecular Sciences (2012), 13 (), 9400-9418CODEN: IJMCFK; ISSN:1422-0067. (MDPI AG)
  Amyloidogenic protein aggregation is a persistent biomedical problem. Despite active research in disease-related aggregation, the need for multidisciplinary approaches to the problem is evident. Recent advances in single-mol. fluorescence spectroscopy are valuable for examg. heterogenic biomol. systems. In this work, we have explored the initial stages of amyloidogenic aggregation by employing fluorescence lifetime correlation spectroscopy (FLCS), an advanced modification of conventional fluorescence correlation spectroscopy (FCS) that utilizes time-resolved information. FLCS provides size distributions and kinetics for the oligomer growth of the SH3 domain of α-spectrin, whose N47A mutant forms amyloid fibrils at pH 3.2 and 37 °C in the presence of salt. The combination of FCS with addnl. fluorescence lifetime information provides an exciting approach to focus on the initial aggregation stages, allowing a better understanding of the fibrillization process, by providing multidimensional information, valuable in combination with other conventional methodologies.
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16. 16
  Carulla, N.; Zhou, M.; Arimon, M.; Gairi, M.; Giralt, E.; Robinson, C. V.; Dobson, C. M. Proc. Natl. Acad. Sci. U. S. A. 2009, 106, 7828– 7833 DOI: 10.1073/pnas.0812227106
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  16
  Experimental characterization of disordered and ordered aggregates populated during the process of amyloid fibril formation
  Carulla, Natalia; Zhou, Min; Arimon, Muriel; Gairi, Margarida; Giralt, Ernest; Robinson, Carol V.; Dobson, Christopher M.
  Proceedings of the National Academy of Sciences of the United States of America (2009), 106 (19), 7828-7833, S7828/1-S7828/11CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
  Recent exptl. evidence points to intermediates populated during the process of amyloid fibril formation as the toxic moieties primarily responsible for the development of increasingly common disorders such as Alzheimer's disease and type II diabetes. We describe here the application of a pulse-labeling hydrogen-deuterium (HD) exchange strategy monitored by mass spectrometry (MS) and NMR spectroscopy (NMR) to characterize the aggregation process of an SH3 domain under 2 different conditions, both of which ultimately lead to well-defined amyloid fibrils. Under one condition, the intermediates appear to be largely amorphous in nature, whereas under the other condition protofibrillar species are clearly evident. Under the conditions favoring amorphous-like intermediates, only species having no protection against HD exchange can be detected in addn. to the mature fibrils that show a high degree of protection. By contrast, under the conditions favoring protofibrillar-like intermediates, MS reveals that multiple species are present with different degrees of HD exchange protection, indicating that aggregation occurs initially through relatively disordered species that subsequently evolve to form ordered aggregates that eventually lead to amyloid fibrils. Further anal. using NMR provides residue-specific information on the structural reorganizations that take place during aggregation, as well as on the time scales by which they occur.
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17. 17
  Paslawski, W.; Mysling, S.; Thomsen, K.; Jorgensen, T. J.; Otzen, D. E. Angew. Chem., Int. Ed. 2014, 53, 7560– 7563 DOI: 10.1002/anie.201400491
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  17
  Co-existence of Two Different α-Synuclein Oligomers with Different Core Structures Determined by Hydrogen/Deuterium Exchange Mass Spectrometry
  Paslawski, Wojciech; Mysling, Simon; Thomsen, Karen; Jorgensen, Thomas J. D.; Otzen, Daniel E.
  Angewandte Chemie, International Edition (2014), 53 (29), 7560-7563CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
  Neurodegenerative disorders are characterized by the formation of protein oligomers and amyloid fibrils, which in the case of Parkinson's disease involves the protein α-synuclein (αSN). Cytotoxicity is mainly assocd. with the oligomeric species, but we still know little about their assembly and structure. Hydrogen/deuterium exchange (HDX) monitored by mass spectrometry is used to analyze oligomers formed by wild-type (wt) αSN and also three familial αSN mutants (A30P, E46K, and A53T). All four variants show co-existence of two different oligomers. The backbone amides of oligomer type I are protected from exchange with D2O until they dissoc. into monomeric αSN by EX1 exchange kinetics. Fewer residues are protected against exchange in oligomer type II, but this type does not revert to αSN monomers. Both oligomers are protected in the core sequence Y39-A89. Based on incubation studies, oligomer type I appears to form straight fibrils, while oligomer type II forms amorphous clusters that do not directly contribute to the fibrillation process.
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18. 18
  Fusco, G.; Chen, S. W.; Williamson, P. T. F.; Cascella, R.; Perni, M.; Jarvis, J. A.; Cecchi, C.; Vendruscolo, M.; Chiti, F.; Cremades, N.; Ying, L.; Dobson, C. M.; De Simone, A. Science 2017, 358, 1440– 1443 DOI: 10.1126/science.aan6160
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  18
  Structural basis of membrane disruption and cellular toxicity by α-synuclein oligomers
  Fusco, Giuliana; Chen, Serene W.; Williamson, Philip T. F.; Cascella, Roberta; Perni, Michele; Jarvis, James A.; Cecchi, Cristina; Vendruscolo, Michele; Chiti, Fabrizio; Cremades, Nunilo; Ying, Liming; Dobson, Christopher M.; De Simone, Alfonso
  Science (Washington, DC, United States) (2017), 358 (6369), 1440-1443CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
  Oligomeric species populated during the aggregation process of α-synuclein have been linked to neuronal impairment in Parkinson's disease and related neurodegenerative disorders. By using soln. and solid-state NMR techniques in conjunction with other structural methods, we identified the fundamental characteristics that enable toxic α-synuclein oligomers to perturb biol. membranes and disrupt cellular function; these include a highly lipophilic element that promotes strong membrane interactions and a structured region that inserts into lipid bilayers and disrupts their integrity. In support of these conclusions, mutations that target the region that promotes strong membrane interactions by α-synuclein oligomers suppressed their toxicity in neuroblastoma cells and primary cortical neurons.
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19. 19
  Banerjee, P. R.; Deniz, A. A. Chem. Soc. Rev. 2014, 43, 1172– 1188 DOI: 10.1039/C3CS60311C
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  19
  Shedding light on protein folding landscapes by single-molecule fluorescence
  Banerjee, Priya R.; Deniz, Ashok A.
  Chemical Society Reviews (2014), 43 (4), 1172-1188CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)
  A review. Single-mol. (SM) fluorescence methods have been increasingly instrumental in our current understanding of a no. of key aspects of protein folding and aggregation landscapes over the past decade. With the advantage of a model free approach and the power of probing multiple subpopulations and stochastic dynamics directly in a heterogeneous structural ensemble, SM methods have emerged as a principle technique for studying complex systems such as intrinsically disordered proteins (IDPs), globular proteins in the unfolded basin and during folding, and early steps of protein aggregation in amyloidogenesis. This review highlights the application of these methods in investigating the free energy landscapes, folding properties and dynamics of individual protein mols. and their complexes, with an emphasis on inherently flexible systems such as IDPs.
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20. 20
  Schuler, B.; Hofmann, H. Curr. Opin. Struct. Biol. 2013, 23, 36– 47 DOI: 10.1016/j.sbi.2012.10.008
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  20
  Single-molecule spectroscopy of protein folding dynamics-expanding scope and timescales
  Schuler, Benjamin; Hofmann, Hagen
  Current Opinion in Structural Biology (2013), 23 (1), 36-47CODEN: COSBEF; ISSN:0959-440X. (Elsevier Ltd.)
  A review. Single-mol. spectroscopy has developed into an important method for probing protein structure and dynamics, esp. in structurally heterogeneous systems. A broad range of questions in the diversifying field of protein folding have been addressed with single-mol. Foerster resonance energy transfer (FRET) and photo-induced electron transfer (PET). Building on more than a decade of rapid method development, these techniques can now be used to investigate a wide span of timescales, an aspect that we focus on in this review. Important current topics range from the structure and dynamics of unfolded and intrinsically disordered proteins, including the coupling of folding and binding, to transition path times, the folding and misfolding of larger proteins, and their interactions with mol. chaperones.
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21. 21
  Orte, A.; Birkett, N. R.; Clarke, R. W.; Devlin, G. L.; Dobson, C. M.; Klenerman, D. Proc. Natl. Acad. Sci. U. S. A. 2008, 105, 14424– 14429 DOI: 10.1073/pnas.0803086105
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  21
  Direct characterization of amyloidogenic oligomers by single-molecule fluorescence
  Orte, Angel; Birkett, Neil R.; Clarke, Richard W.; Devlin, Glyn L.; Dobson, Christopher M.; Klenerman, David
  Proceedings of the National Academy of Sciences of the United States of America (2008), 105 (38), 14424-14429,S14424/1-S14424/8CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
  A key issue in understanding the pathogenic conditions assocd. with the aberrant aggregation of misfolded proteins is the identification and characterization of species formed during the aggregation process. Probing the nature of such species has, however, proved to be extremely challenging to conventional techniques because of their transient and heterogeneous character. We describe here the application of a two-color single-mol. fluorescence technique to examine the assembly of oligomeric species formed during the aggregation of the SH3 domain of PI3 kinase. The single-mol. expts. show that the species formed at the stage of the reaction where aggregates have previously been found to be maximally cytotoxic are a heterogeneous ensemble of oligomers with a median size of 38 ± 10 mols. This no. is remarkably similar to ests. from bulk measurements of the critial size of species obsd. to seed ordered fibril formation and of the most infective form of prion particle. Moreover, although the size distribution of the SH3 oligomers remains virtually const. as the time of aggregation increases, their stability increases substantially. These findings together provide direct evidence for a general mechanism of amyloid aggregation in which the stable cross-β structure emerges via internal reorganization of disordered oligomers formed during the lag phase of the self-assembly reaction.
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22. 22
  Cremades, N.; Cohen, S. I.; Deas, E.; Abramov, A. Y.; Chen, A. Y.; Orte, A.; Sandal, M.; Clarke, R. W.; Dunne, P.; Aprile, F. A.; Bertoncini, C. W.; Wood, N. W.; Knowles, T. P.; Dobson, C. M.; Klenerman, D. Cell 2012, 149, 1048– 1059 DOI: 10.1016/j.cell.2012.03.037
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  22
  Direct Observation of the Interconversion of Normal and Toxic Forms of α-Synuclein
  Cremades, Nunilo; Cohen, Samuel I. A.; Deas, Emma; Abramov, Andrey Y.; Chen, Allen Y.; Orte, Angel; Sandal, Massimo; Clarke, Richard W.; Dunne, Paul; Aprile, Francesco A.; Bertoncini, Carlos W.; Wood, Nicholas W.; Knowles, Tuomas P. J.; Dobson, Christopher M.; Klenerman, David
  Cell (Cambridge, MA, United States) (2012), 149 (5), 1048-1059CODEN: CELLB5; ISSN:0092-8674. (Cell Press)
  Here, we use single-mol. techniques to study the aggregation of α-synuclein, the protein whose misfolding and deposition is assocd. with Parkinson's disease. We identify a conformational change from the initially formed oligomers to stable, more compact proteinase-K-resistant oligomers as the key step that leads ultimately to fibril formation. The oligomers formed as a result of the structural conversion generate much higher levels of oxidative stress in rat primary neurons than do the oligomers formed initially, showing that they are more damaging to cells. The structural conversion is remarkably slow, indicating a high kinetic barrier for the conversion and suggesting that there is a significant period of time for the cellular protective machinery to operate and potentially for therapeutic intervention, prior to the onset of cellular damage. In the absence of added sol. protein, the assembly process is reversed and fibrils disaggregate to form stable oligomers, hence acting as a source of cytotoxic species.
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23. 23
  Narayan, P.; Orte, A.; Clarke, R. W.; Bolognesi, B.; Hook, S.; Ganzinger, K. A.; Meehan, S.; Wilson, M. R.; Dobson, C. M.; Klenerman, D. Nat. Struct. Mol. Biol. 2012, 19, 79– 83 DOI: 10.1038/nsmb.2191
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  23
  The extracellular chaperone clusterin sequesters oligomeric forms of the amyloid-β1-40 peptide
  Narayan, Priyanka; Orte, Angel; Clarke, Richard W.; Bolognesi, Benedetta; Hook, Sharon; Ganzinger, Kristina A.; Meehan, Sarah; Wilson, Mark R.; Dobson, Christopher M.; Klenerman, David
  Nature Structural & Molecular Biology (2012), 19 (1), 79-83CODEN: NSMBCU; ISSN:1545-9993. (Nature Publishing Group)
  In recent genome-wide assocn. studies, the extracellular chaperone protein, clusterin, has been identified as a newly-discovered risk factor in Alzheimer's disease. We have examd. the interactions between human clusterin and the Alzheimer's disease-assocd. amyloid-β1-40 peptide (Aβ1-40), which is prone to aggregate into an ensemble of oligomeric intermediates implicated in both the proliferation of amyloid fibrils and in neuronal toxicity. Using highly sensitive single-mol. fluorescence methods, we have found that Aβ1-40 forms a heterogeneous distribution of small oligomers (from dimers to 50-mers), all of which interact with clusterin to form long-lived, stable complexes. Consequently, clusterin is able to influence both the aggregation and disaggregation of Aβ1-40 by sequestration of the Aβ oligomers. These results not only elucidate the protective role of clusterin but also provide a mol. basis for the genetic link between clusterin and Alzheimer's disease.
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24. 24
  Shammas, S. L.; Garcia, G. A.; Kumar, S.; Kjaergaard, M.; Horrocks, M. H.; Shivji, N.; Mandelkow, E.; Knowles, T. P.; Mandelkow, E.; Klenerman, D. Nat. Commun. 2015, 6, 7025 DOI: 10.1038/ncomms8025
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  A mechanistic model of tau amyloid aggregation based on direct observation of oligomers
  Shammas, Sarah L.; Garcia, Gonzalo A.; Kumar, Satish; Kjaergaard, Magnus; Horrocks, Mathew H.; Shivji, Nadia; Mandelkow, Eva; Knowles, Tuomas P. J.; Mandelkow, Eckhard; Klenerman, David
  Nature Communications (2015), 6 (), 7025CODEN: NCAOBW; ISSN:2041-1723. (Nature Publishing Group)
  Protein aggregation plays a key role in neurodegenerative disease, giving rise to small oligomers that may become cytotoxic to cells. The fundamental microscopic reactions taking place during aggregation, and their rate consts., have been difficult to det. due to lack of suitable methods to identify and follow the low concn. of oligomers over time. Here we use single-mol. fluorescence to study the aggregation of the repeat domain of tau (K18), and two mutant forms linked with familial frontotemporal dementia, the deletion mutant ΔK280 and the point mutant P301L. Our kinetic anal. reveals that aggregation proceeds via monomeric assembly into small oligomers, and a subsequent slow structural conversion step before fibril formation. Using this approach, we have been able to quant. det. how these mutations alter the aggregation energy landscape.
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25. 25
  Iljina, M.; Garcia, G. A.; Horrocks, M. H.; Tosatto, L.; Choi, M. L.; Ganzinger, K. A.; Abramov, A. Y.; Gandhi, S.; Wood, N. W.; Cremades, N.; Dobson, C. M.; Knowles, T. P.; Klenerman, D. Proc. Natl. Acad. Sci. U. S. A. 2016, 113, E1206– 1215 DOI: 10.1073/pnas.1524128113
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  25
  Kinetic model of the aggregation of alpha-synuclein provides insights into prion-like spreading
  Iljina, Marija; Garcia, Gonzalo A.; Horrocks, Mathew H.; Tosatto, Laura; Choi, Minee L.; Ganzinger, Kristina A.; Abramov, Andrey Y.; Gandhi, Sonia; Wood, Nicholas W.; Cremades, Nunilo; Dobson, Christopher M.; Knowles, Tuomas P. J.; Klenerman, David
  Proceedings of the National Academy of Sciences of the United States of America (2016), 113 (9), E1206-E1215CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
  α-Synuclein (αS) self-assembles into small oligomeric species and subsequently into amyloid fibrils that accumulate and proliferate during the development of Parkinson's disease. However, the quant. characterization of the aggregation and spreading of αS remains challenging to achieve. Previously, the authors identified a conformational conversion step leading from the initially formed oligomers to more compact oligomers preceding fibril formation. Here, by a combination of single-mol. FRET measurements and kinetic anal., the authors found that the reaction in soln. involves 2 unimol. structural conversion steps, from the disordered to more compact oligomers and then to fibrils, which can elongate by further monomer addn. The authors obtained individual rate consts. for these key microscopic steps by applying a global kinetic anal. to both the decrease in the concn. of monomeric protein mols. and the increase in oligomer concns. over a 0.5-140-μM range of αS. The resulting explicit kinetic model of αS aggregation was used to quant. explore seeding the reaction by either the compact oligomers or fibrils. The authors' predictions revealed that, although fibrils were more effective at seeding than oligomers, very high nos. of seeds of either type, of the order of 104, were required to achieve efficient seeding and bypass the slow generation of aggregates through primary nucleation. Complementary cellular expts. demonstrated that 2 orders of magnitude lower nos. of oligomers were sufficient to generate high levels of reactive O species (ROS), suggesting that effective templated seeding is likely to require both the presence of template aggregates and conditions of cellular stress.
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26. 26
  Wickner, R. B. Science 1994, 264, 566– 569 DOI: 10.1126/science.7909170
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  [URE3] as an altered URE2 protein: evidence for a prion analog in Saccharomyces cerevisiae
  Wickner, Reed B.
  Science (Washington, DC, United States) (1994), 264 (5158), 566-9CODEN: SCIEAS; ISSN:0036-8075.
  A cytoplasmically inherited element, [URE3], allows yeast to use ureidosuccinate in the presence of NH4+. Chromosomal mutations in the URE2 gene produce the same phenotype. [URE3] depends for its propagation on the URE2 product (Ure2p), a neg. regulator of enzymes of N metab. S. cerevisiae strains cured of [URE3] with guanidium chloride returned to the [URE3]-carrying state without its introduction from other cells. Overprodn. of Ure2p increased the frequency with which a strain became [URE3] by 100-fold. In analogy to mammalian prions, [URE3] may be an altered form of Ure2p that is inactive for its normal function but can convert normal Ure2p to the altered form. The genetic evidence presented here suggests that protein-based inheritance, involving a protein unrelated to the mammalian prion protein, can occur in a microorganism.
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27. 27
  Lian, H. Y.; Jiang, Y.; Zhang, H.; Jones, G. W.; Perrett, S. Biochim. Biophys. Acta, Proteins Proteomics 2006, 1764, 535– 545 DOI: 10.1016/j.bbapap.2005.11.016
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  The yeast prion protein Ure2: structure, function and folding
  Lian, Hui-Yong; Jiang, Yi; Zhang, Hong; Jones, Gary W.; Perrett, Sarah
  Biochimica et Biophysica Acta, Proteins and Proteomics (2006), 1764 (3), 535-545CODEN: BBAPBW; ISSN:1570-9639. (Elsevier B.V.)
  A review. The Saccharomyces cerevisiae protein Ure2 functions as a regulator of nitrogen metab. and as a glutathione-dependent peroxidase. Ure2 also has the characteristics of a prion, in that it can undergo a heritable conformational change to an aggregated state; the prion form of Ure2 loses the regulatory function, but the enzymic function appears to be maintained. A no. of factors are found to affect the prion properties of Ure2, including mutation and expression levels of mol. chaperones, and the effect of these factors on structure and stability are being investigated. The relationship between structure, function and folding for the yeast prion Ure2 are discussed.
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28. 28
  Thual, C.; Komar, A. A.; Bousset, L.; Fernandez-Bellot, E.; Cullin, C.; Melki, R. J. Biol. Chem. 1999, 274, 13666– 13674 DOI: 10.1074/jbc.274.19.13666
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  28
  Structural characterization of Saccharomyces cerevisiae prion-like protein Ure2
  Thual, Carine; Komar, Anton A.; Bousset, Luc; Fernandez-Bellot, Eric; Cullin, Christophe; Melki, Ronald
  Journal of Biological Chemistry (1999), 274 (19), 13666-13674CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
  Saccharomyces cerevisiae prion-like protein Ure2 was expressed in Escherichia coli and was purified to homogeneity. We show here that Ure2p is a sol. protein that can assemble into fibers that are similar to the fibers obsd. in the case of PrP in its scrapie prion filaments form or that form on Sup35 self-assembly. Ure2p self-assembly is a cooperative process where one can distinguish a lag phase followed by an elongation phase preceding a plateau. A combination of size exclusion chromatog., sedimentation velocity, and electron microscopy demonstrates that the sol. form of Ure2p consists at least of three forms of the protein as follows: a monomeric, dimeric, and tetrameric form whose abundance is concn.-dependent. By the use of limited proteolysis, intrinsic fluorescence, and CD measurements, we bring strong evidence for the existence of at least two structural domains in Ure2p mols. Indeed, Ure2p NH2-terminal region is found poorly structured, whereas its COOH-terminal domain appears to be compactly folded. Finally, we show that only slight conformational changes accompany Ure2p assembly into insol. high mol. wt. oligomers. These changes essentially affect the COOH-terminal part of the mol. The properties of Ure2p are compared in the discussion to that of other prion-like proteins such as Sup35 and mammalian prion protein PrP.
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29. 29
  Taylor, K. L.; Cheng, N.; Williams, R. W.; Steven, A. C.; Wickner, R. B. Science 1999, 283, 1339– 1343 DOI: 10.1126/science.283.5406.1339
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  29
  Prion domain initiation of amyloid formation in vitro from native Ure2p
  Taylor, Kimberly L.; Cheng, Naiqian; Williams, Robert W.; Steven, Alasdair C.; Wickner, Reed B.
  Science (Washington, D. C.) (1999), 283 (5406), 1339-1343CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
  The [URE3] non-Mendelian genetic element of Saccharomyces cerevisiae is an infectious protein (prion) form of Ure2p, a regulator of nitrogen catabolism. Here, synthetic Ure2p1-65 were shown to polymerize to form filaments 40 to 45 angstroms in diam. with more than 60 % β sheet. Ure2p1-65 specifically induced full-length native Ure2p to copolymerize under conditions where native Ure2p alone did not polymerize. Like Ure2p in exts. of [URE3] strains, these 180- to 220-angstrom-diam. filaments were protease resistant. The Ure2p1-65-Ure2p cofilaments could seed polymn. of native Ure2p to form thicker, less regular filaments. All filaments stained with Congo Red to produce the green birefringence typical of amyloid. This self-propagating amyloid formation can explain the properties of [URE3].
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30. 30
  Masison, D. C.; Wickner, R. B. Science 1995, 270, 93– 95 DOI: 10.1126/science.270.5233.93
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  30
  Prion-inducing domain of yeast Ure2p and protease resistance of Ure2p in prion-containing cells
  Masison, Daniel C.; Wickner, Reed B.
  Science (Washington, D. C.) (1995), 270 (5233), 93-5CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
  The genetic properties of the [URE3] non-Mendelian element of Saccharomyces cerevisiae suggest that it is a prion (infectious protein) form of Ure2p, a regulator of nitrogen catabolism. In exts. from [URE3] strains, Ure2p was partially resistant to proteinase K compared with Ure2p from wild-type exts. Overexpression of Ure2p in wild-type strains induced a 20- to 200-fold increase in the frequency with which [URE3] arose. Overexpression of just the amino-terminal 65 residues of Ure2p increased the frequency of [URE3] induction 6000-fold. Without this "prion-inducing domain" the carboxyl-terminal domain performed the nitrogen regulation function of Ure2p, but could not be changed to the [URE3] prion state. Thus, this domain induced the prion state in trans, whereas in cis it conferred susceptibility of the adjoining nitrogen regulatory domain to prion infections.
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31. 31
  Jiang, Y.; Li, H.; Zhu, L.; Zhou, J. M.; Perrett, S. J. Biol. Chem. 2004, 279, 3361– 3369 DOI: 10.1074/jbc.M310494200
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  31
  Amyloid Nucleation and Hierarchical Assembly of Ure2p Fibrils: Role of asparagine/glutamine repeat and nonrepeat regions of the prion domain
  Jiang, Yi; Li, Hui; Zhu, Li; Zhou, Jun-Mei; Perrett, Sarah
  Journal of Biological Chemistry (2004), 279 (5), 3361-3369CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
  The yeast prion protein Ure2 forms amyloid-like filaments in vivo and in vitro. This ability depends on the N-terminal prion domain, which contains Asn/Gln repeats, a motif thought to cause human disease by forming stable protein aggregates. The Asn/Gln region of the Ure2p prion domain extends to residue 89, but residues 15-42 represent an island of "normal" random sequence, which is highly conserved in related species and is relatively hydrophobic. We compare the time course of structural changes monitored by thioflavin T (ThT) binding fluorescence and at. force microscopy for Ure2 and a series of prion domain mutants under a range of conditions. At. force microscopy height images at successive time points during a single growth expt. showed the sequential appearance of at least four fibril types that could be readily differentiated by height (5, 8, 12, or 9 nm), morphol. (twisted or smooth), and/or time of appearance (early or late in the plateau phase of ThT binding). The Ure2 dimer (h = 2.6±0.5 nm) and granular particles corresponding to higher order oligomers (h = 4-12 nm) could also be detected. The mutants 15Ure2 and Δ15-42Ure2 showed the same time-dependent variation in fibril types but with an increased lag time detected by ThT binding compared with wild-type Ure2. In addn., Δ15-42Ure2 showed reduced binding to ThT. The results imply a role of the conserved region in both amyloid nucleation and formation of the binding surface recognized by ThT. Further, Ure2 amyloid formation is a multistep process via a series of fibrillar intermediates.
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32. 32
  Bousset, L.; Belrhali, H.; Janin, J.; Melki, R.; Morera, S. Structure 2001, 9, 39– 46 DOI: 10.1016/S0969-2126(00)00553-0
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  Structure of the Globular Region of the Prion Protein Ure2 from the Yeast Saccharomyces cerevisiae
  Bousset, L.; Belrhali, H.; Janin, J.; Melki, R.; Morera, S.
  Structure (Cambridge, MA, United States) (2001), 9 (1), 39-46CODEN: STRUE6; ISSN:0969-2126. (Cell Press)
  Background: The [URE3] non-Mendelian element of the yeast S. cerevisiae is due to the propagation of a transmissible form of the protein Ure2. The infectivity of Ure2p is thought to originate from a conformational change of the normal form of the prion protein. This conformational change generates a form of Ure2p that assembles into amyloid fibrils. Hence, knowledge of the three-dimensional structure of prion proteins such as Ure2p should help in understanding the mechanism of amyloid formation assocd. with a no. of neurodegenerative diseases. Results: Here we report the three-dimensional crystal structure of the globular region of Ure2p (residues 95-354), also called the functional region, solved at 2.5 A resoln. by the MAD method. The structure of Ure2p 95-354 shows a two-domain protein forming a globular dimer. The N-terminal domain is composed of a central 4 strand β sheet flanked by four α helixes, two on each side. In contrast, the C-terminal domain is entirely α-helical. The fold of Ure2p 95-354 resembles that of the β class glutathione S-transferases (GST), in line with a weak similarity in the amino acid sequence that exists between these proteins. Ure2p dimerizes as GST does and possesses a potential ligand binding site, although it lacks GST activity. Conclusions: The structure of the functional region of Ure2p is the first crystal structure of a prion protein. Structure comparisons between Ure2p 95-354 and GST identified a 32 amino acid residues cap region in Ure2p exposed to the solvent. The cap region is highly flexible and may interact with the N-terminal region of the partner subunit in the dimer. The implication of this interaction in the assembly of Ure2p into amyloid fibrils is discussed.
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33. 33
  Umland, T. C.; Taylor, K. L.; Rhee, S.; Wickner, R. B.; Davies, D. R. Proc. Natl. Acad. Sci. U. S. A. 2001, 98, 1459– 1464 DOI: 10.1073/pnas.98.4.1459
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  The crystal structure of the nitrogen regulation fragment of the yeast prion protein Ure2p
  Umland, Timothy C.; Taylor, Kimberly L.; Rhee, Sangkee; Wickner, Reed B.; Davies, David R.
  Proceedings of the National Academy of Sciences of the United States of America (2001), 98 (4), 1459-1464CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
  The yeast nonchromosomal gene [URE3] is due to a prion form of the nitrogen regulatory protein Ure2p. It is a neg. regulator of nitrogen catabolism and acts by inhibiting the transcription factor Gln3p. Ure2p residues 1-80 are necessary for prion generation and propagation. The C-terminal fragment retains nitrogen regulatory activity, albeit somewhat less efficiently than the full-length protein, and it also lowers the frequency of prion generation. The crystal structure of this C-terminal fragment, Ure2p(97-354), at 2.3 Å resoln. is described here. It adopts the same fold as the glutathione S-transferase superfamily, consistent with their sequence similarity. However, Ure2p(97-354) lacks a properly positioned catalytic residue that is required for S-transferase activity. Residues within this regulatory fragment that have been indicated by mutational studies to influence prion generation have been mapped onto the three-dimensional structure, and possible implications for prion activity are discussed.
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34. 34
  Bai, M.; Zhou, J. M.; Perrett, S. J. Biol. Chem. 2004, 279, 50025– 50030 DOI: 10.1074/jbc.M406612200
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  The Yeast Prion Protein Ure2 Shows Glutathione Peroxidase Activity in Both Native and Fibrillar Forms
  Bai, Ming; Zhou, Jun-Mei; Perrett, Sarah
  Journal of Biological Chemistry (2004), 279 (48), 50025-50030CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
  Ure2p is the precursor protein of the Saccharomyces cerevisiae prion [URE3]. Ure2p shows homol. to glutathione transferases but lacks typical glutathione transferase activity. A recent study found that deletion of the Ure2 gene causes increased sensitivity to heavy metal ions and oxidants, whereas prion strains show normal sensitivity. To demonstrate that protection against oxidant toxicity is an inherent property of native and prion Ure2p requires biochem. characterization of the purified protein. Here the authors use steady-state kinetic methods to characterize the multisubstrate peroxidase activity of Ure2p using GSH with cumene hydroperoxide, hydrogen peroxide, or tert-Bu hydroperoxide as substrates. Glutathione-dependent peroxidase activity was proportional to the Ure2p concn. and showed optima at pH 8 and 40°. Michaelis-Menten behavior with convergent straight lines in double reciprocal plots was obsd. This excludes a ping-pong mechanism and implies either a rapid-equil. random or a steady-state ordered sequential mechanism for Ure2p, consistent with its classification as a glutathione transferase. The mutant 90Ure2, which lacks the unstructured N-terminal prion domain, showed kinetic parameters identical to wild type. Fibrillar aggregates showed the same level of activity as native protein. Demonstration of peroxidase activity for Ure2 represents important progress in elucidation of its role in vivo. Further, establishment of an in vitro activity assay provides a valuable tool for the study of structure-function relationships of the Ure2 protein as both a prion and an enzyme.
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35. 35
  Zhang, Z. R.; Perrett, S. J. Biol. Chem. 2009, 284, 14058– 14067 DOI: 10.1074/jbc.M901189200
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  Novel Glutaredoxin Activity of the Yeast Prion Protein Ure2 Reveals a Native-like Dimer within Fibrils
  Zhang, Zai-Rong; Perrett, Sarah
  Journal of Biological Chemistry (2009), 284 (21), 14058-14067CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
  Ure2 is the protein determinant of the Saccharomyces cerevisiae prion [URE3]. Ure2 has structural similarity to glutathione transferases, protects cells against heavy metal and oxidant toxicity in vivo, and shows glutathione-dependent peroxidase activity in vitro. Here we report that Ure2 (which has no cysteine residues) also shows thiol-disulfide oxidoreductase activity similar to that of glutaredoxin enzymes. This demonstrates that disulfide reductase activity can be independent of the classical glutaredoxin CXXC/CXXS motif or indeed an intrinsic catalytic cysteine residue. The kinetics of the glutaredoxin activity of Ure2 showed pos. cooperativity for the substrate glutathione in both the sol. native state and in amyloid-like fibrils, indicating native-like dimeric structure within Ure2 fibrils. Characterization of the glutaredoxin activity of Ure2 sheds light on its ability to protect yeast from heavy metal ions and oxidant toxicity and suggests a role in reversible protein glutathionylation signal transduction. Observation of allosteric enzyme behavior within amyloid-like Ure2 fibrils not only provides insight into the mol. structure of the fibrils but also has implications for the mechanism of [URE3] prion formation.
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36. 36
  Blinder, D.; Coschigano, P. W.; Magasanik, B. J. Bacteriol. 1996, 178, 4734– 4736 DOI: 10.1128/jb.178.15.4734-4736.1996
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  Interaction of the GATA factor Gln3p with the nitrogen regulator Ure2p in Saccharomyces cerevisiae
  Blinder, Dmitry; Coshigano, Peter W.; Magasanik, Boris
  Journal of Bacteriology (1996), 178 (15), 4734-4736CODEN: JOBAAY; ISSN:0021-9193. (American Society for Microbiology)
  We used cells carrying plasmids causing the overprodn. of Gln3p, Ure2p, or both of these proteins to elucidate the ability of Ure2p to prevent the activation of gene expression by Gln3p in cells growing in a glutamine-contg. medium. Our results indicate that Ure2p probably does not interfere with the binding of the GATA factor Gln3p to GATAAG sites but acts directly on Gln3p to block its ability to activate transcription.
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37. 37
  Pieri, L.; Bucciantini, M.; Nosi, D.; Formigli, L.; Savistchenko, J.; Melki, R.; Stefani, M. J. Biol. Chem. 2006, 281, 15337– 15344 DOI: 10.1074/jbc.M511647200
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  The Yeast Prion Ure2p Native-like Assemblies Are Toxic to Mammalian Cells Regardless of Their Aggregation State
  Pieri, Laura; Bucciantini, Monica; Nosi, Daniele; Formigli, Lucia; Savistchenko, Jimmy; Melki, Ronald; Stefani, Massimo
  Journal of Biological Chemistry (2006), 281 (22), 15337-15344CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
  The yeast prion Ure2p assembles in vitro into oligomers and fibrils retaining the α-helix content and binding properties of the sol. protein. Here we show that the different forms of Ure2p native-like assemblies (dimers, oligomers, and fibrils) are similarly toxic to murine H-END cells when added to the culture medium. Interestingly, the amyloid fibrils obtained by heat treatment of the toxic native-like fibrils appear harmless. Moreover, the Ure2p C-terminal domain, lacking the N-terminal segment necessary for aggregation but contg. the glutathione binding site, is not cytotoxic. This finding strongly supports the idea that Ure2p toxicity depends on the structural properties of the flexible N-terminal prion domain and can therefore be considered as an inherent feature of the protein, unrelated to its aggregation state but rather assocd. with a basic toxic fold shared by all of the Ure2p native-like assemblies. Indeed, the latter are able to interact with the cell surface, leading to alteration of calcium homeostasis, membrane permeabilization, and oxidative stress, whereas the heat-treated amyloid fibrils do not. Our results support the idea of a general mechanism of toxicity of any protein/peptide aggregate endowed with structural features, making it able to interact with cell membranes and to destabilize them. This evidence extends the widely accepted view that the toxicity by protein aggregates is restricted to amyloid prefibrillar aggregates and provides new insights into the mechanism by which native-like oligomers compromise cell viability.
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38. 38
  Zhang, C.; Jackson, A. P.; Zhang, Z. R.; Han, Y.; Yu, S.; He, R. Q.; Perrett, S. PLoS One 2010, 5e12529 DOI: 10.1371/journal.pone.0012529
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39. 39
  Bucciantini, M.; Giannoni, E.; Chiti, F.; Baroni, F.; Formigli, L.; Zurdo, J.; Taddei, N.; Ramponi, G.; Dobson, C. M.; Stefani, M. Nature 2002, 416, 507– 511 DOI: 10.1038/416507a
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  39
  Inherent toxicity of aggregates implies a common mechanism for protein misfolding diseases
  Bucciantini, Monica; Giannoni, Elisa; Chiti, Fabrizio; Baroni, Fabiana; Formigli, Lucia; Zurdo, Jesus; Taddei, Niccolo; Ramponi, Giampietro; Dobson, Christopher M.; Stefani, Massimo
  Nature (London, United Kingdom) (2002), 416 (6880), 507-511CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
  A range of human degenerative conditions, including Alzheimer's disease, light-chain amyloidosis and the spongiform encephalopathies, is assocd. with the deposition in tissue of proteinaceous aggregates known as amyloid fibrils or plaques. It has been shown previously that fibrillar aggregates that are closely similar to those assocd. with clin. amyloidoses can be formed in vitro from proteins not connected with these diseases, including the SH3 domain from bovine phosphatidylinositol 3'-kinase and the amino-terminal domain of the Escherichia coli HypF protein. Here we show that species formed early in the aggregation of these non-disease-assocd. proteins can be inherently highly cytotoxic. This finding provides added evidence that avoidance of protein aggregation is crucial for the preservation of biol. function and suggests common features in the origins of this family of protein deposition diseases.
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40. 40
  Catharino, S.; Buchner, J.; Walter, S. Biol. Chem. 2005, 386, 633– 641 DOI: 10.1515/BC.2005.074
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  Characterization of oligomeric species in the fibrillization pathway of the yeast prion Ure2p
  Catharino, Silvia; Buchner, Johannes; Walter, Stefan
  Biological Chemistry (2005), 386 (7), 633-641CODEN: BICHF3; ISSN:1431-6730. (Walter de Gruyter GmbH & Co. KG)
  The [URE3] prion of Saccharomyces cerevisiae shares many features with mammalian prions and poly-glutamine related disorders and has become a model for studying amyloid diseases. The development of the [URE3] phenotype is thought to be caused by a structural switch in the Ure2p protein. In [URE3] cells, Ure2p is found predominantly in an aggregated state, while it is a sol. dimer in wild-type cells. In vitro, Ure2p forms fibrils with amyloid-like properties. Several studies suggest that the N-terminal domain of Ure2p is essential for prion formation. In this work, we investigated the fibril formation of Ure2p by isolating sol. oligomeric species, which are generated during fibrillization, and characterized them with respect to size and structure. Our data support the crit. role of the N-terminal domain for fibril formation, as we obsd. fibrils in the presence of 5 M guanidinium chloride, conditions at which the C-terminal domain is completely unfolded. Based on fluorescence measurements, we conclude that the structure of the C-terminal domain is very similar in dimeric and fibrillar Ure2p. When studying the time course of fibrillization, we detected the formation of small, sol. oligomeric species during the early stages of the process. Their remarkable resistance against denaturants, their increased content of β-structure, and their ability to 'seed' Ure2p fibrillization suggest that conversion to the amyloid-like conformation has already occurred. Thus, they likely represent crit. intermediates in the fibrillization pathway of Ure2p.
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41. 41
  Wang, Y. Q.; Buell, A. K.; Wang, X. Y.; Welland, M. E.; Dobson, C. M.; Knowles, T. P.; Perrett, S. J. Biol. Chem. 2011, 286, 12101– 12107 DOI: 10.1074/jbc.M110.208934
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  Relationship between Prion Propensity and the Rates of Individual Molecular Steps of Fibril Assembly
  Wang, Yi-Qian; Buell, Alexander K.; Wang, Xin-Yu; Welland, Mark E.; Dobson, Christopher M.; Knowles, Tuomas P. J.; Perrett, Sarah
  Journal of Biological Chemistry (2011), 286 (14), 12101-12107CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
  Peptides and proteins possess an inherent propensity to self-assemble into generic fibrillar nanostructures known as amyloid fibrils, some of which are involved in medical conditions such as Alzheimer disease. In certain cases, such structures can self-propagate in living systems as prions and transmit characteristic traits to the host organism. The mechanisms that allow certain amyloid species but not others to function as prions are not fully understood. Much progress in understanding the prion phenomenon has been achieved through the study of prions in yeast as this system has proved to be exptl. highly tractable; but quant. understanding of the biophysics and kinetics of the assembly process has remained challenging. Here, we explore the assembly of two closely related homologues of the Ure2p protein from Saccharomyces cerevisiae and Saccharomyces paradoxus, and by using a combination of kinetic theory with soln. and biosensor assays, we are able to compare the rates of the individual microscopic steps of prion fibril assembly. We find that for these proteins the fragmentation rate is encoded in the structure of the seed fibrils, whereas the elongation rate is principally detd. by the nature of the sol. precursor protein. Our results further reveal that fibrils that elongate faster but fracture less frequently can lose their ability to propagate as prions. These findings illuminate the connections between the in vitro aggregation of proteins and the in vivo proliferation of prions, and provide a framework for the quant. understanding of the parameters governing the behavior of amyloid fibrils in normal and aberrant biol. pathways.
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42. 42
  Perrett, S.; Freeman, S. J.; Butler, P. J.; Fersht, A. R. J. Mol. Biol. 1999, 290, 331– 345 DOI: 10.1006/jmbi.1999.2872
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  Equilibrium Folding Properties of the Yeast Prion Protein Determinant Ure2
  Perrett, Sarah; Freeman, Samantha J.; Butler, P. Jonathan G.; Fersht, Alan R.
  Journal of Molecular Biology (1999), 290 (1), 331-345CODEN: JMOBAK; ISSN:0022-2836. (Academic Press)
  The yeast non-Mendelian factor [URE3] propagates by a prion-like mechanism, involving aggregation of the chromosomally encoded protein Ure2. The [URE3] phenotype is equiv. to loss of function of Ure2, a protein involved in regulation of nitrogen metab. The prion-like behavior of Ure2 in vivo is dependent on the first 65 amino acid residues of its N-terminal region which contains a highly repetitive sequence rich in asparagine. This region has been termed the prion-detg. domain (PrD). Removal of as little as residues 2-20 of the protein is sufficient to prevent occurrence of the [URE3] phenotype. Removal of the PrD does not affect the regulatory activity of Ure2. The C-terminal portion of the protein has homol. to glutathione S -transferases, which are dimeric proteins. We have produced the Ure2 protein to high yield in Escherichia coli from a synthetic gene. The recombinant purified protein is shown to be a dimer. The stability, folding and oligomeric state of Ure2 and a series of N-terminally truncated or deleted variants were studied and compared. The stability of Ure2, ΔGD-N, H2O, detd. by chem. denaturation and monitored by fluorescence, is 12.1(±0.4) kcal mol-1at 25° and pH 8.4. A range of structural probes show a single, coincident unfolding transition, which is invariant over a 550-fold change in protein concn. The stability is the same within error for Ure2 variants lacking all or part of the prion-detg. domain. The data indicate that in the folded protein the PrD is in an unstructured conformation and does not form specific intra- or intermol. interactions at micromolar protein concns. This suggests that the C-terminal domain may stabilize the PrD against prion formation by steric means, and implies that the PrD does not induce prion formation by altering the thermodn. stability of the folded protein. (c) 1999 Academic Press.
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43. 43
  Fei, L.; Perrett, S. J. Biol. Chem. 2009, 284, 11134– 11141 DOI: 10.1074/jbc.M809673200
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  Disulfide Bond Formation Significantly Accelerates the Assembly of Ure2p Fibrils because of the Proximity of a Potential Amyloid Stretch
  Fei, Li; Perrett, Sarah
  Journal of Biological Chemistry (2009), 284 (17), 11134-11141CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
  Aggregation of the Ure2 protein is at the origin of the [URE3] prion trait in the yeast Saccharomyces cerevisiae. The N-terminal region of Ure2p is necessary and sufficient to induce the [URE3] phenotype in vivo and to polymerize into amyloid-like fibrils in vitro. However, as the N-terminal region is poorly ordered in the native state, making it difficult to detect structural changes in this region by spectroscopic methods, detailed information about the fibril assembly process is therefore lacking. Short fibril-forming peptide regions (4-7 residues) have been identified in a no. of prion and other amyloid-related proteins, but such short regions have not yet been identified in Ure2p. In this study, we identify a unique cysteine mutant (R17C) that can greatly accelerate the fibril assembly kinetics of Ure2p under oxidizing conditions. We found that the segment QVNI, corresponding to residues 18-21 in Ure2p, plays a crit. role in the fast assembly properties of R17C, suggesting that this segment represents a potential amyloid-forming region. A series of peptides contg. the QVNI segment were found to form fibrils in vitro. Furthermore, the peptide fibrils could seed fibril formation for wild-type Ure2p. Preceding the QVNI segment with a cysteine or a hydrophobic residue, instead of a charged residue, caused the rate of assembly into fibrils to increase greatly for both peptides and full-length Ure2p. Our results indicate that the potential amyloid stretch and its preceding residue can modulate the fibril assembly of Ure2p to control the initiation of prion formation.
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44. 44
  Wu, S.; Ge, X.; Lv, Z.; Zhi, Z.; Chang, Z.; Zhao, X. S. Biochem. J. 2011, 438, 505– 511 DOI: 10.1042/BJ20110264
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  Interaction between bacterial outer membrane proteins and periplasmic quality control factors: a kinetic partitioning mechanism
  Wu, Si; Ge, Xi; Lv, Zhixin; Zhi, Zeyong; Chang, Zengyi; Zhao, Xin Sheng
  Biochemical Journal (2011), 438 (3), 505-511CODEN: BIJOAK; ISSN:0264-6021. (Portland Press Ltd.)
  The outer membrane proteins (OMPs) of Gram-neg. bacteria have to be translocated through the periplasmic space before reaching their final destination. The aq. environment of the periplasmic space and high permeability of the outer membrane engender such a translocation process inevitably challenging. In Escherichia coli, although periplasmic chaperones SurA, Skp, and DegP have been identified to function in translocating OMPs across the periplasm, their precise roles and their relations remain to be elucidated. Here, the authors studied the interaction between the OMP, OmpC, and these periplasmic quality control factors by using FRET and single-mol. detection methods. The results revealed that the binding rate of OmpC to SurA or Skp was much faster than that to DegP, which may lead to sequential interaction between OMPs and different quality control factors. Such a kinetic partitioning mechanism for the chaperone-substrate interaction may be essential for the quality control of the biogenesis of OMPs.
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45. 45
  Burnham, K. P.; Anderson, D. R. Model selection and multimodel inference: a practical information-theoretic approach; Springer: New York, 2003.
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  Cohen, S. I.; Vendruscolo, M.; Welland, M. E.; Dobson, C. M.; Terentjev, E. M.; Knowles, T. P. J. Chem. Phys. 2011, 135, 065105 DOI: 10.1063/1.3608916
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  Nucleated polymerization with secondary pathways. I. Time evolution of the principal moments
  Cohen, Samuel I. A.; Vendruscolo, Michele; Welland, Mark E.; Dobson, Christopher M.; Terentjev, Eugene M.; Knowles, Tuomas P. J.
  Journal of Chemical Physics (2011), 135 (6), 065105/1-065105/16CODEN: JCPSA6; ISSN:0021-9606. (American Institute of Physics)
  Self-assembly processes resulting in linear structures are often obsd. in mol. biol., and include the formation of functional filaments such as actin and tubulin, as well as generally dysfunctional ones such as amyloid aggregates. Although the basic kinetic equations describing these phenomena are well-established, it has proved to be challenging, due to their non-linear nature, to derive solns. to these equations except for special cases. The availability of general anal. solns. provides a route for detg. the rates of mol. level processes from the anal. of macroscopic exptl. measurements of the growth kinetics, in addn. to the phenomenol. parameters, such as lag times and maximal growth rates that are already obtainable from std. fitting procedures. We describe here an anal. approach based on fixed-point anal., which provides self-consistent solns. for the growth of filamentous structures that can, in addn. to elongation, undergo internal fracturing and monomer-dependent nucleation as mechanisms for generating new free ends acting as growth sites. Our results generalize the anal. expression for sigmoidal growth kinetics from the Oosawa theory for nucleated polymn. to the case of fragmenting filaments. We det. the corresponding growth laws in closed form and derive from first principles a no. of relationships which have been empirically established for the kinetics of the self-assembly of amyloid fibrils. (c) 2011 American Institute of Physics.
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47. 47
  Meisl, G.; Kirkegaard, J. B.; Arosio, P.; Michaels, T. C.; Vendruscolo, M.; Dobson, C. M.; Linse, S.; Knowles, T. P. Nat. Protoc. 2016, 11, 252– 272 DOI: 10.1038/nprot.2016.010
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  Molecular mechanisms of protein aggregation from global fitting of kinetic models
  Meisl, Georg; Kirkegaard, Julius B.; Arosio, Paolo; Michaels, Thomas C. T.; Vendruscolo, Michele; Dobson, Christopher M.; Linse, Sara; Knowles, Tuomas P. J.
  Nature Protocols (2016), 11 (2), 252-272CODEN: NPARDW; ISSN:1750-2799. (Nature Publishing Group)
  The elucidation of the mol. mechanisms by which sol. proteins convert into their amyloid forms is a fundamental prerequisite for understanding and controlling disorders that are linked to protein aggregation, such as Alzheimer's and Parkinson's diseases. However, because of the complexity assocd. with aggregation reaction networks, the anal. of kinetic data of protein aggregation to obtain the underlying mechanisms represents a complex task. Here we describe a framework, using quant. kinetic assays and global fitting, to det. and to verify a mol. mechanism for aggregation reactions that is compatible with exptl. kinetic data. We implement this approach in a web-based software, AmyloFit. Our procedure starts from the results of kinetic expts. that measure the concn. of aggregate mass as a function of time. We illustrate the approach with results from the aggregation of the β-amyloid (Aβ) peptides measured using thioflavin T, but the method is suitable for data from any similar kinetic expt. measuring the accumulation of aggregate mass as a function of time; the input data are in the form of a tab-sepd. text file. We also outline general exptl. strategies and practical considerations for obtaining kinetic data of sufficient quality to draw detailed mechanistic conclusions, and the procedure starts with instructions for extensive data quality control. For the core part of the anal., we provide an online platform (http://www.amylofit.ch.cam.ac.uk) that enables robust global anal. of kinetic data without the need for extensive programming or detailed math. knowledge. The software automates repetitive tasks and guides users through the key steps of kinetic anal.: detn. of constraints to be placed on the aggregation mechanism based on the concn. dependence of the aggregation reaction, choosing from several fundamental models describing assembly into linear aggregates and fitting the chosen models using an advanced minimization algorithm to yield the reaction orders and rate consts. Finally, we outline how to use this approach to investigate which targets potential inhibitors of amyloid formation bind to and where in the reaction mechanism they act. The protocol, from processing data to detg. mechanisms, can be completed in <1 d.
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48. 48
  Kajava, A. V.; Baxa, U.; Wickner, R. B.; Steven, A. C. Proc. Natl. Acad. Sci. U. S. A. 2004, 101, 7885– 7890 DOI: 10.1073/pnas.0402427101
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  48
  A model for Ure2p prion filaments and other amyloids: The parallel superpleated β-structure
  Kajava, Andrey V.; Baxa, Ulrich; Wickner, Reed B.; Steven, Alasdair C.
  Proceedings of the National Academy of Sciences of the United States of America (2004), 101 (21), 7885-7890CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
  In its prion form, Ure2p, a regulator of nitrogen catabolism in Saccharomyces cerevisiae, polymerizes into filaments whereby its C-terminal regulatory domain is inactivated but retains its native fold. The filament has an amyloid fibril backbone formed by the Asn-rich, N-terminal, "prion" domain. The prion domain is also capable of forming fibrils when alone or when fused to other proteins. We have developed a model for the fibril that we call a parallel superpleated β-structure. In this model, the prion domain is divided into nine seven-residue segments, each with a four-residue strand and a three-residue turn, that zig-zag in a planar serpentine arrangement. Serpentines are stacked axially, in register, generating an array of parallel β-sheets, with a small and potentially variable left-hand twist. The interior of the filament is mostly stabilized not by packing of apolar side chains but by H-bond networks generated by the stacking of Asn side chains: charged residues are excluded. The model is consistent with current biophys., biochem., and structural data (notably, mass-per-unit-length measurements by scanning transmission electron microscopy that gave one subunit rise per 0.47 nm) and is readily adaptable to other amyloids, for instance the core of Sup35p filaments and glutamine expansions in huntingtin.
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49. 49
  Ngo, S.; Chiang, V.; Guo, Z. J. Struct. Biol. 2012, 180, 374– 381 DOI: 10.1016/j.jsb.2012.08.008
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  49
  Quantitative analysis of spin exchange interactions to identify β strand and turn regions in Ure2 prion domain fibrils with site-directed spin labeling
  Ngo, Sam; Chiang, Vicky; Guo, Zhefeng
  Journal of Structural Biology (2012), 180 (2), 374-381CODEN: JSBIEM; ISSN:1047-8477. (Elsevier Inc.)
  Amyloid formation is assocd. with a range of debilitating human disorders including Alzheimer's and prion diseases. The amyloid structure is essential for understanding the role of amyloids in these diseases. Amyloid formation of Ure2 protein underlies the yeast prion [URE3]. Here we use site-directed spin labeling and ESR (EPR) spectroscopy to investigate the structure of amyloid fibrils formed by the Ure2 prion domain. The Ure2 prion domain under study contains a Sup35M domain at C-terminus as a solubilization element. We introduced spin labels at every residue from positions 2-15, and every 5th residue from positions 20-80 in Ure2 prion domain. EPR spectra at most labeling sites show strong spin exchange interactions, suggesting a parallel in-register β structure. With quant. anal. of spin exchange interactions, we show that residues 8-12 form the first β strand, followed by a turn at residues 13-14, and then the second β strand from residue 15 to at least residue 20. Comparison of the spin exchange frequency for the fibrils formed under quiescent and agitated conditions also revealed differences in the fibril structures. Currently there is a lack of techniques for in-depth structural studies of amyloid fibrils. Detailed structural information is obtained almost exclusively from solid-state NMR. The identification of β-strand and turn regions in this work suggests that quant. anal. of spin exchange interactions in spin-labeled amyloid fibrils is a powerful approach for identifying the β-strand and turn/loop residues and for studying structural differences of different fibril polymorphs.
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50. 50
  Galani, D.; Fersht, A. R.; Perrett, S. J. Mol. Biol. 2002, 315, 213– 227 DOI: 10.1006/jmbi.2001.5234
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  50
  Folding of the Yeast Prion Protein Ure2: Kinetic Evidence for Folding and Unfolding Intermediates
  Galani, Despina; Fersht, Alan R.; Perrett, Sarah
  Journal of Molecular Biology (2002), 315 (2), 213-227CODEN: JMOBAK; ISSN:0022-2836. (Academic Press)
  The Saccharomyces cerevisiae non-Mendelian factor [URE3] propagates by a prion-like mechanism, involving aggregation of the chromosomally encoded protein Ure2. The N-terminal prion domain (PrD) of Ure2 is required for prion activity in vivo and amyloid formation in vitro. However, the mol. mechanism of the prion-like activity remains obscure. Here we measure the kinetics of folding of Ure2 and two N-terminal variants that lack all or part of the PrD. The kinetic folding behavior of the three proteins is identical, indicating that the PrD does not change the stability, rates of folding or folding pathway of Ure2. Both unfolding and refolding kinetics are multiphasic. An intermediate is populated during unfolding at high denaturant concns. resulting in the appearance of an unfolding burst phase and "roll-over" in the denaturant dependence of the unfolding rate consts. During refolding the appearance of a burst phase indicates formation of an intermediate during the dead-time of stopped-flow mixing. A further fast phase shows second-order kinetics, indicating formation of a dimeric intermediate. Regain of native-like fluorescence displays a distinct lag due to population of this on-pathway dimeric intermediate. Double-jump expts. indicate that isomerization of Pro166, which is cis in the native state, occurs late in refolding after regain of native-like fluorescence. During protein refolding there is kinetic partitioning between productive folding via the dimeric intermediate and a non-productive side reaction via an aggregation prone monomeric intermediate. In the light of this and other studies, schemes for folding, aggregation and prion formation are proposed. (c) 2002 Academic Press.
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51. 51
  Arosio, P.; Knowles, T. P.; Linse, S. Phys. Chem. Chem. Phys. 2015, 17, 7606– 7618 DOI: 10.1039/C4CP05563B
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  On the lag phase in amyloid fibril formation
  Arosio, Paolo; Knowles, Tuomas P. J.; Linse, Sara
  Physical Chemistry Chemical Physics (2015), 17 (12), 7606-7618CODEN: PPCPFQ; ISSN:1463-9076. (Royal Society of Chemistry)
  A review. The formation of nanoscale amyloid fibrils from normally sol. peptides and proteins is a common form of self-assembly phenomenon that has fundamental connections with biol. functions and human diseases. The kinetics of this process has been widely studied and exhibits on a macroscopic level three characteristic stages: (1) a lag phase; (2) a growth phase; and (3) a final plateau regime. The question of which mol. events take place during each one of these phases has been a central element in the quest for a mechanism of amyloid formation. Here, the authors discuss the nature and mol. origin of the lag-phase in amyloid formation by making use of tools and concepts from phys. chem., in particular from chem. reaction kinetics. The authors discuss how, in macroscopic samples, it has become apparent that the lag-phase is not a waiting time for nuclei to form. Rather, multiple parallel processes exist and typically millions of primary nuclei form during the lag phase from monomers in soln. Thus, the lag-time represents a time that is required for the nuclei that are formed early on in the reaction to grow and proliferate in order to reach an aggregate concn. that is readily detected in bulk assays. In many cases, this proliferation takes place through secondary nucleation, where fibrils may present a catalytic surface for the formation of new aggregates. Fibrils may also break (fragmentation) and thereby provide new ends for elongation. Thus, at least 2 (primary nucleation and elongation) and in many systems at least 4 (primary nucleation, elongation, secondary nucleation, and fragmentation) microscopic processes occur during the lag phase. Moreover, these same processes occur during all 3 phases of the macroscopic aggregation process, albeit at different rates as governed by rate consts. and by the concn. of reacting species at each point in time.
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52. 52
  Cohen, S. I.; Linse, S.; Luheshi, L. M.; Hellstrand, E.; White, D. A.; Rajah, L.; Otzen, D. E.; Vendruscolo, M.; Dobson, C. M.; Knowles, T. P. Proc. Natl. Acad. Sci. U. S. A. 2013, 110, 9758– 9763 DOI: 10.1073/pnas.1218402110
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  52
  Proliferation of amyloid-β42 aggregates occurs through a secondary nucleation mechanism
  Cohen, Samuel I. A.; Linse, Sara; Luheshi, Leila M.; Hellstrand, Erik; White, Duncan A.; Rajah, Luke; Otzen, Daniel E.; Vendruscolo, Michele; Dobson, Christopher M.; Knowles, Tuomas P. J.
  Proceedings of the National Academy of Sciences of the United States of America (2013), 110 (24), 9758-9763, S9758/1-S9758/11CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
  The generation of toxic oligomers during the aggregation of the amyloid-β (Aβ) peptide Aβ42 into amyloid fibrils and plaques has emerged as a central feature of the onset and progression of Alzheimer's disease, but the mol. pathways that control pathol. aggregation have proved challenging to identify. Here, the authors used a combination of kinetic studies, selective radiolabeling expts., and cell viability assays to detect directly the rates of formation of both fibrils and oligomers and the resulting cytotoxic effects. The results showed that once a small but crit. concn. of amyloid fibrils had accumulated, the toxic oligomeric species were predominantly formed from monomeric peptide mols. through a fibril-catalyzed secondary nucleation reaction, rather than through a classical mechanism of homogeneous primary nucleation. This catalytic mechanism coupled together the growth of insol. amyloid fibrils and the generation of diffusible oligomeric aggregates that are implicated as neurotoxic agents in Alzheimer's disease. These results revealed that the aggregation of Aβ42 is promoted by a pos. feedback loop that originates from the interactions between the monomeric and fibrillar forms of this peptide. These findings bring together the main mol. species implicated in the Aβ aggregation cascade and suggest that perturbation of the secondary nucleation pathway identified in this study could be an effective strategy to control the proliferation of neurotoxic Aβ42 oligomers.
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53. 53
  Chen, L.; Chen, L. J.; Wang, H. Y.; Wang, Y. Q.; Perrett, S. Protein Eng., Des. Sel. 2011, 24, 69– 78 DOI: 10.1093/protein/gzq100
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  53
  Deletion of a Ure2 C-terminal prion-inhibiting region promotes the rate of fibril seed formation and alters interaction with Hsp40
  Chen Li; Chen Li-Jun; Wang Hai-Yan; Wang Yi-Qian; Perrett Sarah
  Protein engineering, design & selection : PEDS (2011), 24 (1-2), 69-78 ISSN:.
  Prions are proteins that can undergo a heritable conformational change to an aggregated amyloid-like state, which is then transmitted to other similar molecules. Ure2, the nitrogen metabolism regulation factor of Saccharomyces cerevisiae, shows prion properties in vivo and forms amyloid fibrils in vitro. Ure2 consists of an N-terminal prion-inducing domain and a C-terminal functional domain. Previous studies have shown that mutations affecting the prion properties of Ure2 are not restricted to the N-terminal prion domain: the deletion of residues 151-158 in the C-domain increases the in vivo prion-inducing propensity of Ure2. Here, we characterized this mutant in vitro and found that the 151-158 deletion has minimal effect on the thermodynamic stability or folding properties of the protein. However, deletion of residues 151-158 accelerates the nucleation, growth and fragmentation of amyloid-like aggregates in vitro, and the aggregates formed are able to seed formation of fibrils of the wild-type protein. In addition, the absence of 151-158 was found to disrupt the inhibitory effect of the Hsp40 chaperone Ydj1 on Ure2 fibril formation. These results suggest that the enhanced in vivo prion-inducing ability of the 151-158 deletion mutant is due to its enhanced ability to generate prion seeds.
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54. 54
  Xu, L. Q.; Wu, S.; Buell, A. K.; Cohen, S. I.; Chen, L. J.; Hu, W. H.; Cusack, S. A.; Itzhaki, L. S.; Zhang, H.; Knowles, T. P.; Dobson, C. M.; Welland, M. E.; Jones, G. W.; Perrett, S. Philos. Trans. R. Soc., B 2013, 368, 20110410 DOI: 10.1098/rstb.2011.0410
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  Influence of specific HSP70 domains on fibril formation of the yeast prion protein Ure2
  Xu, Li-Qiong; Wu, Si; Buell, Alexander K.; Cohen, Samuel I. A.; Chen, Li-Jun; Hu, Wan-Hui; Cusack, Sarah A.; Itzhaki, Laura S.; Zhang, Hong; Knowles, Tuomas P. J.; Dobson, Christopher M.; Welland, Mark E.; Jones, Gary W.; Perrett, Sarah
  Philosophical Transactions of the Royal Society, B: Biological Sciences (2013), 368 (1617), 20110410/1-20110410/13CODEN: PTRBAE; ISSN:0962-8436. (Royal Society)
  Ure2p is the protein determinant of the Saccharomyces cerevisiae prion state [URE3]. Constitutive overexpression of the HSP70 family member SSA1 cures cells of [URE3]. Here, we show that Ssa1p increases the lag time of Ure2p fibril formation in vitro in the presence or absence of nucleotide. The presence of the HSP40 co-chaperone Ydj1p has an additive effect on the inhibition of Ure2p fibril formation, whereas the Ydj1p H34Q mutant shows reduced inhibition alone and in combination with Ssa1p. In order to investigate the structural basis of these effects, we constructed and tested an Ssa1p mutant lacking the ATPase domain, as well as a series of C-terminal truncation mutants. The results indicate that Ssa1p can bind to Ure2p and delay fibril formation even in the absence of the ATPase domain, but interaction of Ure2p with the substrate-binding domain is strongly influenced by the C-terminal lid region. Dynamic light scattering, quartz crystal microbalance assays, pull-down assays and kinetic anal. indicate that Ssa1p interacts with both native Ure2p and fibril seeds, and reduces the rate of Ure2p fibril elongation in a concn.-dependent manner. These results provide new insights into the structural and mechanistic basis for inhibition of Ure2p fibril formation by Ssa1p and Ydj1p.
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55. 55
  Glabe, C. G. J. Biol. Chem. 2008, 283, 29639– 29643 DOI: 10.1074/jbc.R800016200
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  55
  Structural Classification of Toxic Amyloid Oligomers
  Glabe, Charles G.
  Journal of Biological Chemistry (2008), 283 (44), 29639-29643CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
  A review. Amyloid oligomers are believed to play important causal roles in many types of amyloid-related degenerative diseases. Many different labs. have reported amyloid oligomers that differ in size, morphol., toxicity, and method of prepn. or purifn., raising the question of the structural relationships among these oligomer prepns. The structural plasticity that has been reported to occur in amyloids formed from the same protein sequence indicates that it is quite possible that different oligomer prepns. may represent distinct structural variants. In view of the difficulty in detg. the precise structure of amyloids, conformation- and epitope-specific antibodies may provide a facile means of classifying amyloid oligomer structures. Conformation-dependent antibodies that recognize generic epitopes that are specifically assocd. with distinct aggregation states of many different amyloid-forming sequences indicate that there are at least two fundamentally distinct types of amyloid oligomers: fibrillar and prefibrillar oligomers. Classification of amyloid oligomers according to their underlying structures may be a more useful and rational approach than relying on differences in size and morphol.
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56. 56
  Kayed, R.; Head, E.; Thompson, J. L.; McIntire, T. M.; Milton, S. C.; Cotman, C. W.; Glabe, C. G. Science 2003, 300, 486– 489 DOI: 10.1126/science.1079469
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  56
  Common Structure of Soluble Amyloid Oligomers Implies Common Mechanism of Pathogenesis
  Kayed, Rakez; Head, Elizabeth; Thompson, Jennifer L.; McIntire, Theresa M.; Milton, Saskia C.; Cotman, Carl W.; Glabe, Charles G.
  Science (Washington, DC, United States) (2003), 300 (5618), 486-489CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
  Sol. oligomers are common to most amyloids and may represent the primary toxic species of amyloids, like the Aβ peptide in Alzheimer's disease (AD). Here the authors show that all of the sol. oligomers tested display a common conformation-dependent structure that is unique to sol. oligomers regardless of sequence. The in vitro toxicity of sol. oligomers is inhibited by oligomer-specific antibody. Sol. oligomers have a unique distribution in human AD brain that is distinct from fibrillar amyloid. These results indicate that different types of sol. amyloid oligomers have a common structure and suggest they share a common mechanism of toxicity.
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57. 57
  Knowles, T. P.; Waudby, C. A.; Devlin, G. L.; Cohen, S. I.; Aguzzi, A.; Vendruscolo, M.; Terentjev, E. M.; Welland, M. E.; Dobson, C. M. Science 2009, 326, 1533– 1537 DOI: 10.1126/science.1178250
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  57
  An Analytical Solution to the Kinetics of Breakable Filament Assembly
  Knowles, Tuomas P. J.; Waudby, Christopher A.; Devlin, Glyn L.; Cohen, Samuel I. A.; Aguzzi, Adriano; Vendruscolo, Michele; Terentjev, Eugene M.; Welland, Mark E.; Dobson, Christopher M.
  Science (Washington, DC, United States) (2009), 326 (5959), 1533-1537CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
  We present an anal. treatment of a set of coupled kinetic equations that governs the self-assembly of filamentous mol. structures. Application to the case of protein aggregation demonstrates that the kinetics of amyloid growth can often be dominated by secondary rather than by primary nucleation events. Our results further reveal a range of general features of the growth kinetics of fragmenting filamentous structures, including the existence of generic scaling laws that provide mechanistic information in contexts ranging from in vitro amyloid growth to the in vivo development of mammalian prion diseases.
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58. 58
  Cohen, S. I.; Vendruscolo, M.; Dobson, C. M.; Knowles, T. P. J. Mol. Biol. 2012, 421, 160– 171 DOI: 10.1016/j.jmb.2012.02.031
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  58
  From Macroscopic Measurements to Microscopic Mechanisms of Protein Aggregation
  Cohen, Samuel I. A.; Vendruscolo, Michele; Dobson, Christopher M.; Knowles, Tuomas P. J.
  Journal of Molecular Biology (2012), 421 (2-3), 160-171CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Ltd.)
  A review. The ability to relate bulk exptl. measurements of amyloid formation to the microscopic assembly processes that underlie protein aggregation is crit. to achieve a quant. understanding of this complex phenomenon. In this review, the authors focus on the insights from classical and modern theories of linear growth phenomena and discuss how theory allows the roles of growth and nucleation processes to be defined through the anal. of exptl. in vitro time courses of amyloid formation. Moreover, the authors discuss the specific signatures in the time course of the reactions that correspond to the actions of primary and secondary nucleation processes and outline strategies for identifying and characterizing the nature of the dominant process responsible in each case for the generation of new aggregates. The authors highlight the power of a global anal. of exptl. time courses acquired under different conditions, and discuss how such an anal. allows a rigorous connection to be established between the macroscopic measurements and the rates of the individual microscopic processes that underlie the phenomenon of amyloid formation.
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59. 59
  Garcia, G. A.; Cohen, S. I.; Dobson, C. M.; Knowles, T. P. Phys. Rev. E Stat Nonlin Soft Matter Phys. 2014, 89, 032712 DOI: 10.1103/PhysRevE.89.032712
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  59
  Nucleation-conversion-polymerization reactions of biological macromolecules with prenucleation clusters
  Garcia, Gonzalo A.; Cohen, Samuel I. A.; Dobson, Christopher M.; Knowles, Tuomas P. J.
  Physical Review E: Statistical, Nonlinear, and Soft Matter Physics (2014), 89 (3-A), 032712/1-032712/6CODEN: PRESCM; ISSN:1539-3755. (American Physical Society)
  The self-assembly of biomols., such as peptides and proteins, into filaments is conventionally understood as a nucleated polymn. reaction. However, detailed anal. of exptl. observation has revealed recently that nucleation pathways generate growth-competent nuclei via a cascade of metastable intermediate species, which are omitted in conventional models of filamentous growth based on classical nucleation theory. Here we take an anal. approach to generalizing the classical theory of nucleated polymn. to include the formation of these prenucleation clusters, providing a quant. general classification of the behavior exhibited by these nucleation-conversion-polymn. reactions. A phase diagram is constructed, and anal. predictions are derived for key exptl. observables. Using this approach, we delineate the characteristic time scales that det. the nature of biopolymer growth phenomena.
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60. 60
  Saric, A.; Chebaro, Y. C.; Knowles, T. P.; Frenkel, D. Proc. Natl. Acad. Sci. U. S. A. 2014, 111, 17869– 17874 DOI: 10.1073/pnas.1410159111
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  60
  Crucial role of nonspecific interactions in amyloid nucleation
  Saric, Andjela; Chebaro, Yassmine C.; Knowles, Tuomas P. J.; Frenkel, Daan
  Proceedings of the National Academy of Sciences of the United States of America (2014), 111 (50), 17869-17874CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
  Protein oligomers have been implicated as toxic agents in a wide range of amyloid-related diseases. However, it has remained unsolved whether the oligomers are a necessary step in the formation of amyloid fibrils or just a dangerous byproduct. Analogously, it has not been resolved if the amyloid nucleation process is a classical one-step nucleation process or a two-step process involving prenucleation clusters. We use coarse-grained computer simulations to study the effect of nonspecific attractions between peptides on the primary nucleation process underlying amyloid fibrillization. We find that, for peptides that do not attract, the classical one-step nucleation mechanism is possible but only at nonphysiol. high peptide concns. At low peptide concns., which mimic the physiol. relevant regime, attractive interpeptide interactions are essential for fibril formation. Nucleation then inevitably takes place through a two-step mechanism involving prefibrillar oligomers. We show that oligomers not only help peptides meet each other but also, create an environment that facilitates the conversion of monomers into the β-sheet-rich form characteristic of fibrils. Nucleation typically does not proceed through the most prevalent oligomers but through an oligomer size that is only obsd. in rare fluctuations, which is why such aggregates might be hard to capture exptl. Finally, we find that the nucleation of amyloid fibrils cannot be described by classical nucleation theory: in the two-step mechanism, the crit. nucleus size increases with increases in both concn. and interpeptide interactions, which is in direct contrast with predictions from classical nucleation theory.
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61. 61
  Saric, A.; Michaels, T. C. T.; Zaccone, A.; Knowles, T. P. J.; Frenkel, D. J. Chem. Phys. 2016, 145, 211926 DOI: 10.1063/1.4965040
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  61
  Kinetics of spontaneous filament nucleation via oligomers: Insights from theory and simulation
  Saric, Andjela; Michaels, Thomas C. T.; Zaccone, Alessio; Knowles, Tuomas P. J.; Frenkel, Daan
  Journal of Chemical Physics (2016), 145 (21), 211926/1-211926/9CODEN: JCPSA6; ISSN:0021-9606. (American Institute of Physics)
  Nucleation processes are at the heart of a large no. of phenomena, from cloud formation to protein crystn. A recently emerging area where nucleation is highly relevant is the initiation of filamentous protein self-assembly, a process that has broad implications in many research areas ranging from medicine to nanotechnol. As such, spontaneous nucleation of protein fibrils has received much attention in recent years with many theor. and exptl. studies focussing on the underlying phys. principles. In this paper we make a step forward in this direction and explore the early time behavior of filamentous protein growth in the context of nucleation theory. We first provide an overview of the thermodn. and kinetics of spontaneous nucleation of protein filaments in the presence of one relevant degree of freedom, namely the cluster size. In this case, we review how key kinetic observables, such as the reaction order of spontaneous nucleation, are directly related to the phys. size of the crit. nucleus. We then focus on the increasingly prominent case of filament nucleation that includes a conformational conversion of the nucleating building-block as an addnl. slow step in the nucleation process. Using computer simulations, we study the concn. dependence of the nucleation rate. We find that, under these circumstances, the reaction order of spontaneous nucleation with respect to the free monomer does no longer relate to the overall phys. size of the nucleating aggregate but rather to the portion of the aggregate that actively participates in the conformational conversion. Our results thus provide a novel interpretation of the common kinetic descriptors of protein filament formation, including the reaction order of the nucleation step or the scaling exponent of lag times, and put into perspective current theor. descriptions of protein aggregation. (c) 2016 American Institute of Physics.
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62. 62
  Auer, S.; Meersman, F.; Dobson, C. M.; Vendruscolo, M. PLoS Comput. Biol. 2008, 4, e1000222 DOI: 10.1371/journal.pcbi.1000222
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  A generic mechanism of emergence of amyloid protofilaments from disordered oligomeric aggregates
  Auer Stefan; Meersman Filip; Dobson Christopher M; Vendruscolo Michele
  PLoS computational biology (2008), 4 (11), e1000222 ISSN:.
  The presence of oligomeric aggregates, which is often observed during the process of amyloid formation, has recently attracted much attention because it has been associated with a range of neurodegenerative conditions including Alzheimer's and Parkinson's diseases. We provide a description of a sequence-indepedent mechanism by which polypeptide chains aggregate by forming metastable oligomeric intermediate states prior to converting into fibrillar structures. Our results illustrate that the formation of ordered arrays of hydrogen bonds drives the formation of beta-sheets within the disordered oligomeric aggregates that form early under the effect of hydrophobic forces. Individual beta-sheets initially form with random orientations and subsequently tend to align into protofilaments as their lengths increase. Our results suggest that amyloid aggregation represents an example of the Ostwald step rule of first-order phase transitions by showing that ordered cross-beta structures emerge preferentially from disordered compact dynamical intermediate assemblies.
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63. 63
  Lee, J.; Culyba, E. K.; Powers, E. T.; Kelly, J. W. Nat. Chem. Biol. 2011, 7, 602– 609 DOI: 10.1038/nchembio.624
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  63
  Amyloid-β forms fibrils by nucleated conformational conversion of oligomers
  Lee, Jiyong; Culyba, Elizabeth K.; Powers, Evan T.; Kelly, Jeffery W.
  Nature Chemical Biology (2011), 7 (9), 602-609CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)
  Amyloid-β amyloidogenesis is reported to occur via a nucleated polymn. mechanism. If this is true, the energetically unfavorable oligomeric nucleus should be very hard to detect. However, many labs. have detected early nonfibrillar amyloid-β oligomers without observing amyloid fibrils, suggesting that a mechanistic revision may be needed. Here the authors introduce Cys-Cys-amyloid-β1-40, which cannot bind to the latent fluorophore FlAsH as a monomer, but can bind FlAsH as an nonfibrillar oligomer or as a fibril, rendering the conjugates fluorescent. Through FlAsH monitoring of Cys-Cys-amyloid-β1-40 aggregation, the authors found that amyloid-β1-40 rapidly and efficiently forms spherical oligomers in vitro (85% yield) that are kinetically competent to slowly convert to amyloid fibrils by a nucleated conformational conversion mechanism. This methodol. was used to show that plasmalogen ethanolamine vesicles eliminate the proteotoxicity-assocd. oligomerization phase of amyloid-β amyloidogenesis while allowing fibril formation, rationalizing how low concns. of plasmalogen ethanolamine in the brain are epidemiol. linked to Alzheimer's disease.
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64. 64
  Krishnan, R.; Goodman, J. L.; Mukhopadhyay, S.; Pacheco, C. D.; Lemke, E. A.; Deniz, A. A.; Lindquist, S. Proc. Natl. Acad. Sci. U. S. A. 2012, 109, 11172– 11177 DOI: 10.1073/pnas.1209527109
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  64
  Conserved features of intermediates in amyloid assembly determine their benign or toxic states
  Krishnan, Rajaraman; Goodman, Jessica L.; Mukhopadhyay, Samrat; Pacheco, Chris D.; Lemke, Edward A.; Deniz, Ashok A.; Lindquist, Susan
  Proceedings of the National Academy of Sciences of the United States of America (2012), 109 (28), 11172-11177, S11172/1-S11172/10CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
  Some amyloid-forming polypeptides are assocd. with devastating human diseases and others provide important biol. functions. For both, oligomeric intermediates appear during amyloid assembly. Currently we have few tools for characterizing these conformationally labile intermediates and discerning what governs their benign vs. toxic states. Here, we examine intermediates in the assembly of a normal, functional amyloid, the prion-detg. region of yeast Sup35 (NM). During assembly, NM formed a variety of oligomers with different sizes and conformation-specific antibody reactivities. Earlier oligomers were less compact and reacted with the conformational antibody A11. More mature oligomers were more compact and reacted with conformational antibody OC. We found we could arrest NM in either of these two distinct oligomeric states with small mols. or crosslinking. The A11-reactive oligomers were more hydrophobic (as measured by Nile Red binding) and were highly toxic to neuronal cells, while OC-reactive oligomers were less hydrophobic and were not toxic. The A11 and OC antibodies were originally raised against oligomers of Aβ, an amyloidogenic peptide implicated in Alzheimer's disease (AD) that is completely unrelated to NM in sequence. Thus, this natural yeast prion samples two conformational states similar to those sampled by Al, and when assembly stalls at one of these two states, but not the other, it becomes extremely toxic. Our results have implications for selective pressures operating on the evolution of amyloid folds across a billion years of evolution. Understanding the features that govern such conformational transitions will shed light on human disease and evolution alike.
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65. 65
  Bolognesi, B.; Kumita, J. R.; Barros, T. P.; Esbjorner, E. K.; Luheshi, L. M.; Crowther, D. C.; Wilson, M. R.; Dobson, C. M.; Favrin, G.; Yerbury, J. J. ACS Chem. Biol. 2010, 5, 735– 740 DOI: 10.1021/cb1001203
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  65
  ANS Binding Reveals Common Features of Cytotoxic Amyloid Species
  Bolognesi, Benedetta; Kumita, Janet R.; Barros, Teresa P.; Esbjoerner, Elin K.; Luheshi, Leila M.; Crowther, Damian C.; Wilson, Mark R.; Dobson, Christopher M.; Favrin, Giorgio; Yerbury, Justin J.
  ACS Chemical Biology (2010), 5 (8), 735-740CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)
  Oligomeric assemblies formed from a variety of disease-assocd. peptides and proteins have been strongly assocd. with toxicity in many neurodegenerative conditions, such as Alzheimer's disease. The precise nature of the toxic agents, however, remains still to be established. We show that prefibrillar aggregates of E22G (arctic) variant of the Aβ1-42 peptide bind strongly to 1-anilinonaphthalene 8-sulfonate and that changes in this property correlate significantly with changes in its cytotoxicity. Moreover, we show that this phenomenon is common to other amyloid systems, such as wild-type Aβ1-42, the I59T variant of human lysozyme and an SH3 domain. These findings are consistent with a model in which the exposure of hydrophobic surfaces as a result of the aggregation of misfolded species is a crucial and common feature of these pathogenic species.
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66. 66
  Campioni, S.; Mannini, B.; Zampagni, M.; Pensalfini, A.; Parrini, C.; Evangelisti, E.; Relini, A.; Stefani, M.; Dobson, C. M.; Cecchi, C.; Chiti, F. Nat. Chem. Biol. 2010, 6, 140– 147 DOI: 10.1038/nchembio.283
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  66
  A causative link between the structure of aberrant protein oligomers and their toxicity
  Campioni, Silvia; Mannini, Benedetta; Zampagni, Mariagioia; Pensalfini, Anna; Parrini, Claudia; Evangelisti, Elisa; Relini, Annalisa; Stefani, Massimo; Dobson, Christopher M.; Cecchi, Cristina; Chiti, Fabrizio
  Nature Chemical Biology (2010), 6 (2), 140-147CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)
  The aberrant assembly of peptides and proteins into fibrillar aggregates proceeds through oligomeric intermediates that are thought to be the primary pathogenic species in many protein deposition diseases. We describe two types of oligomers formed by the HypF-N protein that are morphol. and tinctorially similar, as detected with at. force microscopy (AFM) and thioflavin T (ThT) assays, though one is benign when added to cell cultures whereas the other is toxic. Structural investigation at a residue-specific level using site-directed labeling with pyrene indicated differences in the hydrophobic packing between adjacent protein mols. in the oligomers. A lower degree of hydrophobic packing was found to correlate with a higher ability to penetrate the cell membrane and cause an influx of Ca2+ ions. Our findings suggest that structural flexibility and hydrophobic exposure are primary determinants of the ability of oligomeric assemblies to cause cellular dysfunction and its consequences, such as neurodegeneration.
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67. 67
  Baxa, U.; Wickner, R. B.; Steven, A. C.; Anderson, D. E.; Marekov, L. N.; Yau, W. M.; Tycko, R. Biochemistry 2007, 46, 13149– 13162 DOI: 10.1021/bi700826b
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  67
  Characterization of β-Sheet Structure in Ure2p1-89 Yeast Prion Fibrils by Solid-State Nuclear Magnetic Resonance
  Baxa, Ulrich; Wickner, Reed B.; Steven, Alasdair C.; Anderson, D. Eric; Marekov, Lyuben N.; Yau, Wai-Ming; Tycko, Robert
  Biochemistry (2007), 46 (45), 13149-13162CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
  Residues 1-89 constitute the Asn- and Gln-rich segment of the Ure2p protein and produce the [URE3] prion of Saccharomyces cerevisiae by forming the core of intracellular Ure2p amyloid. We report the results of solid-state NMR (NMR) measurements that probe the mol. structure of amyloid fibrils formed by Ure2p1-89 in vitro. Data include measurements of intermol. magnetic dipole-dipole couplings in samples that are 13C-labeled at specific sites and two-dimensional 15N-13C and 13C-13C NMR spectra of samples that are uniformly 15N- and 13C-labeled. Intermol. dipole-dipole couplings indicate that the β-sheets in Ure2p1-89 fibrils have an in-register parallel structure. An in-register parallel β-sheet structure permits polar zipper interactions among side chains of Gln and Asn residues and explains the tolerance of [URE3] to scrambling of the sequence in residues 1-89. Two-dimensional NMR spectra of uniformly labeled Ure2p1-89 fibrils, even when fully hydrated, show NMR linewidths that exceed those in solid-state NMR spectra of fibrils formed by residues 218-289 of the HET-s prion protein of Podospora anserina by factors of three or more, indicating a lower degree of structural order at the mol. level in Ure2p1-89 fibrils. The very high degree of structural order in HET-s fibrils indicated by solid-state NMR data is therefore not a universal characteristic of prion proteins, and is likely to be a consequence of the evolved biol. function of HET-s in heterokaryon incompatibility. Anal. of cross peak intensities in two-dimensional NMR spectra of uniformly labeled Ure2p1-89 fibrils suggests that certain portions of the amino acid sequence may not participate in a rigid β-sheet structure, possibly including portions of the Asn-rich segment between residues 44 and 76.
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68. 68
  Kryndushkin, D. S.; Wickner, R. B.; Tycko, R. J. Mol. Biol. 2011, 409, 263– 277 DOI: 10.1016/j.jmb.2011.03.067
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  68
  The Core of Ure2p Prion Fibrils Is Formed by the N-Terminal Segment in a Parallel Cross-β Structure: Evidence from Solid-State NMR
  Kryndushkin, Dmitry S.; Wickner, Reed B.; Tycko, Robert
  Journal of Molecular Biology (2011), 409 (2), 263-277CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Ltd.)
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Direct Observation of Oligomerization by Single Molecule Fluorescence Reveals a Multistep Aggregation Mechanism for the Yeast Prion Protein Ure2

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Abstract

Introduction

Methods

Mutant Construction, Protein Expression, Purification and Labeling

Single-Molecule FRET Measurement of Ure2 Oligomers

Developing a Model for Kinetic Data Fitting

Relating Model Rate Constants to Fundamental Reaction Steps

Fitting the Combined smFRET/ThT Data to the Model

Results

Single Molecule FRET Measurements Can Monitor the Formation of Ure2 Oligomers

Figure 1

SmFRET Measurements Reveal the Absence of Significant Oligomer Formation via Secondary Nucleation During Ure2 Fibril Formation

Figure 2

Analysis of Oligomer Populations Reveals the Existence of an Oligomer Conformational Conversion Step

Two Types of Oligomeric Species with Different Structures Can Be Observed

Figure 3

Ure2 Oligomers That Disaggregate from Mature Fibrils Have Structures Similar to Those of the Oligomers Appearing Later in the Aggregation Reaction

Figure 4

Kinetic Analysis of Combined smFRET and ThT Data Yields a Quantitative Understanding of Oligomer Formation, Dissociation and Conversion

Figure 5

Discussion

Figure 6

Supporting Information

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Terms & Conditions

Acknowledgment

References

Cited By

Abstract

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

References

Supporting Information

Supporting Information

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