Alternative assembly pathways of the amyloidogenic yeast prion determinant Sup35-NM

The self-perpetuating conformational change of the translation termination factor Sup35 is associated with a prion phenomenon of Saccharomyces cerevisiae. In vitro, the prion-determining region (NM) of Sup35 assembles into amyloid-like fibres through a mechanism of nucleated conformational conversion. Here, we describe an alternative assembly pathway of NM that produces filaments that are composed of beta-strands and random coiled regions with several-fold smaller diameters than the amyloid fibres. NM filaments are not detectable with either thioflavin T or Congo Red and do not show SDS or protease resistance. As filaments do not self-convert into fibres and do not act as seed, they are not intermediates of amyloid fibre formation. Instead, they represent a stable off-pathway form. Similar to mammalian prion proteins, Sup35 contains oligopeptide repeats located in the NM region. We found that the number of repeats determines the partitioning of the protein between filaments and amyloid-like fibres. Low numbers of repeats favour the formation of the filamentous structure, whereas high numbers of repeats favour the formation of amyloid-like fibres.     

Origins and kinetic consequences of diversity in Sup35 yeast prion fibers

A remarkable feature of prions is that infectious particles composed of the same prion protein can give rise to different phenotypes. This strain phenomenon suggests that a single prion protein can adopt multiple infectious conformations. Here we use a novel single fiber growth assay to examine the heterogeneity of amyloid fibers formed by the yeast Sup35 prion protein. Sup35 spontaneously forms multiple, distinct and faithfully propagating fiber types, which differ dramatically both in their degrees of polarity and overall growth rates. Both in terms of the number of distinct self-propagating fiber types, as well as the ability of these differences to dictate the rate of prion growth, this diversity is well suited to account for the range of prion strain phenotypes observed in vivo.     

Oligopeptide repeats in the yeast protein Sup35p stabilize intermolecular prion interactions

The nuclear-encoded Sup35p protein is responsible for the prion-like [PSI(+)] determinant of yeast, with Sup35p existing largely as a high molecular weight aggregate in [PSI(+)] strains. Here we show that the five oligopeptide repeats present at the N-terminus of Sup35p are responsible for stabilizing aggregation of Sup35p in vivo. Sequential deletion of the oligopeptide repeats prevented the maintenance of [PSI(+)] by the truncated Sup35p, although deletants containing only two repeats could be incorporated into pre-existing aggregates of wild-type Sup35p. The mammalian prion protein PrP also contains similar oligopeptide repeats and we show here that a human PrP repeat (PHGGGWGQ) is able functionally to replace a Sup35p oligopeptide repeat to allow stable [PSI(+)] propagation in vivo. Our data suggest a model in which the oligopeptide repeats in Sup35p stabilize intermolecular interactions between Sup35p proteins that initiate establishment of the aggregated state. Modulating repeat number therefore alters the rate of yeast prion conversion in vivo. Furthermore, there appears to be evolutionary conservation of function of the N-terminally located oligopeptide repeats in prion propagation.     

beta-Helix is a likely core structure of yeast prion Sup35 amyloid fibers

Helicobacter hepaticus, a causal agent of hepatocarcinoma in mice, exhibits a cytolethal distending toxin activity. The three subunits of this holotoxin, CdtA, CdtB, and CdtC, and three CdtB mutants were produced as recombinant histidine-tagged proteins by using an in vitro cell-free protein expression system. We found that the presence of the three H. hepaticus Cdt subunits is required for cellular toxicity and that only a C-terminal CdtB mutation abolishes the activity of the complex. In vitro, H. hepaticus CdtB exhibits a DNase activity which is also abolished by this C-terminal CdtB mutation. These results suggest that the effect of H. hepaticus CDT probably involves the DNase activity of CdtB.     

酿酒酵母Sup35朊蛋白结构域的自组装机理及其应用的研究进展

Sup35是酿酒酵母的翻译终止因子,其朊蛋白结构域在体内外都能形成淀粉样蛋白纤维。由于其高度有序的交叉β-片层构象与其他物种中的淀粉样蛋白纤维相似,因此,Sup35的分子自组装机理的研究可以作为蛋白质错误折叠性疾病及朊病毒生物学等相关研究的理想模型。而Sup35朊蛋白结构域自组装成纳米线的能力在生物技术和纳米材料等方面已得到广泛的应用。     

The role of the N-terminal oligopeptide repeats of the yeast Sup35 prion protein in propagation and transmission of prion variants

The cytoplasmic [PSI+] determinant of Saccharomyces cerevisiae is the prion form of the Sup35 protein. Oligopeptide repeats within the Sup35 N-terminal domain (PrD) presumably are required for the stable [PSI+] inheritance that in turn involves fragmentation of Sup35 polymers by the chaperone Hsp104. The nonsense suppressor [PSI+] phenotype can vary in efficiency probably due to different inheritable Sup35 polymer structures. Here we study the ability of Sup35 mutants with various deletions of the oligopeptide repeats to support [PSI+] propagation. We define the minimal region of the Sup35-PrD necessary to support [PSI+] as amino acids 1-64, which include the first two repeats, although a longer fragment, 1-83, is required to maintain weak [PSI+] variants. Replacement of wild-type Sup35 with deletion mutants decreases the strength of the [PSI+] phenotype. However, with one exception, reintroducing the wild-type Sup35 restores the original phenotype. Thus, the specific prion fold defining the [PSI+] variant can be preserved by the mutant Sup35 protein despite the change of phenotype. Coexpression of wild-type and mutant Sup35 containing three, two, one, or no oligopeptide repeats causes variant-specific [PSI+] elimination. These data suggest that [PSI+] variability is primarily defined by differential folding of the Sup35-PrD oligopeptide-repeat region.     

Monitoring polyglutamine toxicity in yeast

Experiments in yeast have significantly contributed to our understanding of general aspects of biochemistry, genetics, and cell biology. Yeast models have also delivered deep insights in to the molecular mechanism underpinning human diseases, including neurodegenerative diseases. Many neurodegenerative diseases are associated with the conversion of a protein from a normal and benign conformation into a disease-associated and toxic conformation - a process called protein misfolding. The misfolding of proteins with abnormally expanded polyglutamine (polyQ) regions causes several neurodegenerative diseases, such as Huntington's disease and the Spinocerebellar Ataxias. Yeast cells expressing polyQ expansion proteins recapitulate polyQ length-dependent aggregation and toxicity, which are hallmarks of all polyQ-expansion diseases. The identification of modifiers of polyQ toxicity in yeast revealed molecular mechanisms and cellular pathways that contribute to polyQ toxicity. Notably, several of these findings in yeast were reproduced in other model organisms and in human patients, indicating the validity of the yeast polyQ model. Here, we describe different expression systems for polyQ-expansion proteins in yeast and we outline experimental protocols to reliably and quantitatively monitor polyQ toxicity in yeast.     

A variational model for oligomer-formation process of GNNQQNY peptide from yeast prion protein Sup35

Many human neurodegenerative diseases are associated with the aggregation of insoluble amyloid-like fibrous proteins. However, the processes by which the randomly diffused monomer peptides aggregate into the highly regulated amyloid fibril structures are largely unknown. We proposed a residue-level coarse-grained variational model for the investigation of the aggregation pathway for a small assembly of amyloid proteins, the peptide GNNQQNY from yeast prion protein Sup35. By examining the free energy surface, we identified the residue-level sequential pathways for double parallel and antiparallel β-peptides, which show that the central dry polar zipper structure is the major folding core in both cases. The critical nucleus size is determined to be three peptides for the homogeneous nucleation process, whereas the zig-zag growth pattern appears most favorably for heterogeneous nucleation. Consistent with the dock-and-lock mechanism, the aggregation process of free peptides to the fibril core was found to be highly cooperative. The quantitative validation with the computational simulations and experimental data demonstrated the usefulness of the proposed model in understanding the general mechanism of the amyloid fibril system.     

Visualization of aggregation of the Rnq1 prion domain and cross-seeding interactions with Sup35NM

Factors triggering the de novo appearance of prions are still poorly understood. In yeast, the appearance of one prion, [PSI(+)], is enhanced by the presence of another prion, [PIN(+)]. The [PSI(+)] and [PIN(+)] prion-forming proteins are, respectively, the translational termination factor Sup35 and the yet poorly characterized Rnq1 protein that is rich in glutamines and asparagines. The prion domain of Rnq1 (RnqPD) polymerizes more readily in vitro than the full-length protein. As is typical for amyloidogenic proteins, the reaction begins with a lag phase, followed by exponential growth. Seeding with pre-formed aggregates significantly shortens the lag. A generic antibody against pre-amyloid oligomer inhibits the unseeded but not the self-seeded reaction. As revealed by electron microscopy, RnqPD polymerizes predominantly into spherical species that eventually agglomerate. We observed infrequent fiber-like structures in samples taken at 4 h of polymerization, but in overnight samples SDS treatment was required to reveal fibers among agglomerates. Polymerization reactions in which RnqPD and the prion domain of Sup35 (Sup35NM) cross-seed each other proceeded with a shortened lag that only depends weakly on the protein concentration. Cross-seeded Sup35NM fibers appear to sprout from globular RnqPD aggregates as seen by electron microscopy. RnqPD spherical aggregates appear to associate with and, later occlude, Sup35NM seed fibers. Our kinetic and morphological analyses suggest that, upon cross-seeding, the aggregate provides the surface on which oligomers of the heterologous protein nucleate their subsequent amyloid formation.     

Effects of randomizing the Sup35NM prion domain sequence on formation of amyloid fibrils in vitro

The mechanism by which proteins aggregate and form amyloid fibrils is still elusive. In order to preclude interference by cellular factors and to clarify the role of the primary sequence of Sup35p prion domain in formation of amyloid fibrils, we generated five Sup35NM variants by randomizing amino acid sequences in PrDs without altering the amino acid composition and analyzed the in vitro process of amyloid fibril formation. The results showed that each of the five Sup35NM variants polymerized into amyloid fibrils in vitro under native conditions. Furthermore, the Sup35NM variants showed differences in their aggregation time courses. These findings indicate that specific amino acid sequence features in PrD can modify the rate of conversion of Sup35p into amyloid fibrils in vitro.     

Sequestration of Sup35 by aggregates of huntingtin fragments causes toxicity of [PSI+] yeast

Expression of huntingtin fragments with 103 glutamines (HttQ103) is toxic in yeast containing either the [PIN(+)] prion, which is the amyloid form of Rnq1, or [PSI(+)] prion, which is the amyloid form of Sup35. We find that HttQP103, which has a polyproline region at the C-terminal end of the polyQ repeat region, is significantly more toxic in [PSI(+)] yeast than in [PIN(+)], even though HttQP103 formed multiple aggregates in both [PSI(+)] and [PIN(+)] yeast. This toxicity was only observed in the strong [PSI(+)] variant, not the weak [PSI(+)] variant, which has more soluble Sup35 present than the strong variant. Furthermore, expression of the MC domains of Sup35, which retains the C-terminal domain of Sup35, but lacks the N-terminal prion domain, almost completely rescued HttQP103 toxicity, but was less effective in rescuing HttQ103 toxicity. Therefore, the toxicity of HttQP103 in yeast containing the [PSI(+)] prion is primarily due to sequestration of the essential protein, Sup35.     

Molecular dynamics simulations on the oligomer-formation process of the GNNQQNY peptide from yeast prion protein Sup35

Oligomeric intermediates are possible cytotoxic species in diseases associated with amyloid deposits. Understanding the early steps of fibril formation at atomic details may provide useful information for the rational therapeutic design. In this study, using the heptapeptide GNNQQNY from the yeast prion-like protein Sup35 as a model system, for which a detailed atomic structure of the fibril formed has been determined by x-ray microcrystallography, we investigated its oligomer-formation process from monomer to tetramer at the atomistic level by means of a molecular dynamics simulation with explicit water. Although the number of simulations was limited, the qualitative statistical data gave some interesting results, which indicated that the oligomer formation might start from antiparallel beta-sheet-like dimers. When a new single peptide strand was added to the preformed dimers to form trimers and then tetramers, the transition time from disorder aggregates to regular ones for the parallel alignment was found to be obviously much less than for the antiparallel one. Moreover, the parallel pattern also statistically stayed longer, providing more chances for oligomer extending, although the number of parallel stack events was almost equal to antiparallel ones. Therefore, our simulations showed that new strands might prefer to extend in a parallel arrangement to form oligomers, which agrees with the microcrystal structure of the amyloid fibril formed by this peptide. In addition, analysis of the pi-pi stacking of aromatic residues showed that this type of interaction did not play an important role in giving directionality for beta-strand alignment but played a great influence on stabilizing the structures formed in the oligomer-formation process.     

Mass spectroscopic analysis of Sup35NM prion polymerization

Sup35NM, the prion determining domain of the protein responsible for the yeast prion phenomenon [Psi], has become a powerful model for studying key processes in amyloid-related human diseases. One of these processes is a conformational conversion of soluble precursor protein into insoluble fibrillar structures. In this study, we created a set of Sup35NM mutants and used proteolytic digestion coupled with mass spectroscopy to monitor local structure of the protein during polymerization. Experimental data were compared to a network model and showed that during the conformational conversion residue Arg-28 became highly protected from cleavage, residue Arg-98 remained partially solvent exposed, and residues between 28 and 98 showed an intermediate degree of protection. In addition, we found that a distinct subset of proteolytic polypeptides spanning 28-98 residues segment spontaneously formed stable dimers. This finding suggests that the [29-98] region is the key interacting region of Sup35NM responsible for amyloid conversion.     

Substoichiometric Hsp104 regulates the genesis and persistence of self-replicable amyloid seeds of Sup35 prion domain

Prion-like self-perpetuating conformational conversion of proteins is involved in both transmissible neurodegenerative diseases in mammals and non-Mendelian inheritance in yeast. The transmissibility of amyloid-like aggregates is dependent on the stoichiometry of chaperones such as heat shock proteins (Hsps), including disaggregases. To provide the mechanistic underpinnings of the formation and persistence of prefibrillar amyloid seeds, we investigated the role of substoichiometric Hsp104 on the in vitro amyloid aggregation of the prion domain (NM-domain) of Saccharomyces cerevisiae Sup35. At low substoichiometric concentrations, we show Hsp104 exhibits a dual role: it considerably accelerates the formation of prefibrillar species by shortening the lag phase but also prolongs their persistence by introducing unusual kinetic halts and delaying their conversion into mature amyloid fibers. Additionally, Hsp104-modulated amyloid species displayed a better seeding capability compared to NM-only amyloids. Using biochemical and biophysical tools coupled with site-specific dynamic readouts, we characterized the distinct structural and dynamical signatures of these amyloids. We reveal that Hsp104-remodeled amyloidogenic species are compositionally diverse in prefibrillar aggregates and are packed in a more ordered fashion compared to NM-only amyloids. Finally, we show these Hsp104-remodeled, conformationally distinct NM aggregates display an enhanced autocatalytic self-templating ability that might be crucial for phenotypic outcomes. Taken together, our results demonstrate that substoichiometric Hsp104 promotes compositional diversity and conformational modulations during amyloid formation, yielding effective prefibrillar seeds that are capable of driving prion-like Sup35 propagation. Our findings underscore the key functional and pathological roles of substoichiometric chaperones in prion-like propagation.     

Modification of [PSI +] prion properties by combining amino acid changes in N-terminal domain of Sup35 protein

The prion [PSI ⁺] is an amyloid isoform of the release factor eRF3 encoded by the SUP35 gene in Saccharomyces cerevisiae yeast. Naturally occurring amyloid complexes have been studied for a long time, yet their structural organization is still not well understood. The formation of amyloid forms of the wild-type Sup35 protein (Sup35p) is directed by its N-terminal portion, which forms a superpleated β-sheet structure. We previously constructed five mutants, each of which carried a replacement in two consecutive amino acids, one in each of the oligopeptide repeats (OR) and in the Sup35p N-terminal region. Mutations sup35-M1 (YQ46-47KK) and sup35-M2 (QQ61-62KK) lead to the compete loss of prion conformation. Three other mutants, i.e., sup35-M3 (QQ70-71KK), sup35-M4 (QQ80-81KK), and sup35-M5 (QQ89-90KK), formed functional prions. In the current study, we investigated the contribution of each mutant peptide to the stability of the prion and aggregation properties, and compared the effects of single mutants and combinations of different mutant alleles. Studies were carried out in yeast strains designed to carry single or a combination of different SUP35 alleles. Based on our analysis, we propose a model that clarifies the 3D organization of the β-sheet within the prion. We also provide evidence that sup35-M2 and sup35-M4 mutations change the 3D structure of prion complexes. We propose that the destabilization of prion complexes in these mutants is due to the decreased efficiency of the fragmentation of the prion aggregates by chaperone complexes.     

A mutation within the C-terminal domain of Sup35p that affects [PSI+] prion propagation

The epigenetic factor [PSI+] in the yeast Saccharomyces cerevisiae is due to the prion form of Sup35p. The N-terminal domain of Sup35p (N), alone or together with the middle-domain (NM), assembles in vitro into fibrils that induce [PSI+] when introduced into yeast cells. The Sup35p C-terminal domain (C), involved in translation termination, is essential for growth. The involvement of Sup35p C-terminal domain into [PSI+] propagation is subject to debate. We previously showed that mutation of threonine 341 within Sup35p C-domain affects translation termination efficiency. Here, we demonstrate that mutating threonine 341 to aspartate or alanine results in synthetic lethality with [PSI+] and weakening of [PSI+] respectively. The corresponding Sup35D and Sup35A proteins assemble into wild-type like fibrils in vitro, but with a slower elongation rate. Moreover, cross-seeding between Sup35p and Sup35A is inefficient both in vivo and in vitro, suggesting that the point mutation alters the structural properties of Sup35p within the fibrils. Thus, Sup35p C-terminal domain modulates [PSI+] prion propagation, possibly through a functional interaction with the N and/or M domains of the protein. Our results clearly demonstrate that Sup35p C-terminal domain plays a critical role in prion propagation and provide new insights into the mechanism of prion conversion.     

Methionine oxidation of Sup35 protein induces formation of the [PSI+] prion in a yeast peroxiredoxin mutant

The frequency with which the yeast [PSI(+)] prion form of Sup35 arises de novo is controlled by a number of genetic and environmental factors. We have previously shown that in cells lacking the antioxidant peroxiredoxin proteins Tsa1 and Tsa2, the frequency of de novo formation of [PSI(+)] is greatly elevated. We show here that Tsa1/Tsa2 also function to suppress the formation of the [PIN(+)] prion form of Rnq1. However, although oxidative stress increases the de novo formation of both [PIN(+)] and [PSI(+)], it does not overcome the requirement of cells being [PIN(+)] to form the [PSI(+)] prion. We use an anti-methionine sulfoxide antibody to show that methionine oxidation is elevated in Sup35 during oxidative stress conditions. Abrogating Sup35 methionine oxidation by overexpressing methionine sulfoxide reductase (MSRA) prevents [PSI(+)] formation, indicating that Sup35 oxidation may underlie the switch from a soluble to an aggregated form of Sup35. In contrast, we were unable to detect methionine oxidation of Rnq1, and MSRA overexpression did not affect [PIN(+)] formation in a tsa1 tsa2 mutant. The molecular basis of how yeast and mammalian prions form infectious amyloid-like structures de novo is poorly understood. Our data suggest a causal link between Sup35 protein oxidation and de novo [PSI(+)] prion formation.     

Qualitative and quantitative multiplexed proteomic analysis of complex yeast protein fractions that modulate the assembly of the yeast prion Sup35p

Background

The aggregation of the baker''s yeast prion Sup35p is at the origin of the transmissible [PSI⁺] trait. We and others have shown that molecular chaperones modulate Sup35p aggregation. However, other protein classes might be involved in [PSI⁺] formation.

Results

We designed a functional proteomic study that combines two techniques to identify modulators of Sup35p aggregation and describe the changes associated to [PSI⁺] formation. The first allows measuring the effect of fractionated Saccharomyces cerevisiae cytosolic extracts from [PSI⁺] and [psi⁻] yeast cells on Sup35p assembly. The second is a multiplex qualitative and quantitative comparison of protein composition of active and inactive fractions using a gel-free and label-free LC-MS approach. We identify changes in proteins involved in translation, folding, degradation, oxido-reduction and metabolic processes.

Conclusion

Our functional proteomic study provides the first inventory list of over 300 proteins that directly or indirectly affect Sup35p aggregation and [PSI⁺] formation. Our results highlight the complexity of the cellular changes accompanying [PSI⁺] formation and pave the way for in vitro studies aimed to document the effect of individual and/or combinations of proteins identified here, susceptible of affecting Sup35p assembly.     

Mechanism of inhibition of Psi+ prion determinant propagation by a mutation of the N-terminus of the yeast Sup35 protein.

The SUP35 gene of Saccharomyces cerevisiae encodes the polypeptide chain release factor eRF3. This protein (also called Sup35p) is thought to be able to undergo a heritable conformational switch, similarly to mammalian prions, giving rise to the cytoplasmically inherited Psi+ determinant. A dominant mutation (PNM2 allele) in the SUP35 gene causing a Gly58-->Asp change in the Sup35p N-terminal domain eliminates Psi+. Here we observed that the mutant Sup35p can be converted to the prion-like form in vitro, but such conversion proceeds slower than that of wild-type Sup35p. The overexpression of mutant Sup35p induced the de novo appearance of Psi+ cells containing the prion-like form of mutant Sup35p, which was able to transmit its properties to wild-type Sup35p both in vitro and in vivo. Our data indicate that this Psi+-eliminating mutation does not alter the initial binding of Sup35p molecules to the Sup35p Psi+-specific aggregates, but rather inhibits its subsequent prion-like rearrangement and/or binding of the next Sup35p molecule to the growing prion-like Sup35p aggregate.     

Protein misfolding and amyloid formation for the peptide GNNQQNY from yeast prion protein Sup35: simulation by reaction path annealing

We study the early steps of amyloid formation of the seven residue peptide GNNQQNY from yeast prion-like protein Sup35 by simulating the random coil to beta-sheet and alpha-helix to beta-sheet transition both in the absence and presence of a cross-beta amyloid nucleus. The simulation method at atomic resolution employs a new implementation of a Langevin dynamics "reaction path annealing" algorithm. The results indicate that the presence of amyloid-like cross-beta-sheet strands both facilitates the transition into the cross-beta conformation and substantially lowers the free energy barrier for this transition. This model systems allows us to investigate the energetic and kinetic details of this transition, which is consistent with an auto-catalyzed, nucleation-like mechanism for the formation of beta-amyloid. In particular, we find that electrostatic interactions of peptide backbone dipoles contribute significantly to the stability of the beta-amyloid state. Furthermore, we find water exclusion and interactions of polar side-chains to be driving forces of amyloid formation: the cross-beta conformation is stabilized by burial of polar side-chains and inter-residue hydrogen bonds in the presence of an amyloid-like "seed". These findings are in support of a "dry, polar zipper model" of amyloid formation.