Hsp104 Overexpression Research Articles

Unique characteristics of the J-domain proximal regions of Hsp70 cochaperone Apj1 in prion propagation/elimination and its overlap with Sis1 function.

J-domain proteins (JDPs) are obligate cochaperones of Hsp70s. The Class A JDP Apj1 of the yeast cytosol has an unusually complex region between the N-terminal J-domain and the substrate binding region-often called the Grich or GF region in Class A and B JDPs because of its typical abundance of glycine. The N-terminal 161-residue Apj1 fragment is known to be sufficient for Apj1 function in prion curing, driven by the overexpression of Hsp104. Further analyzing the N-terminal segment of Apj1, we found that a 90-residue fragment that includes the 70-residue J-domain and the adjacent 12-residue glutamine/alanine (Q/A) segment is sufficient for curing. Furthermore, the 121-residue fragment that includes the Grich region was sufficient to not only sustain the growth of cells lacking the essential Class B JDP Sis1 but also enabled the maintenance of several prions normally dependent on Sis1 for propagation. A J-domain from another cytosolic JDP could substitute for the Sis1-related functions but not for Apj1 in prion curing. Together, these results separate the functions of JDPs in prion biology and underscore the diverse functionality of multi-domain cytosolic JDPs in yeast.

Overexpression of Hsp104 by Causing Dissolution of the Prion Seeds Cures the Yeast [PSI+] Prion.

The yeast Sup35 protein misfolds into the infectious [PSI+] prion, which is then propagated by the severing activity of the molecular chaperone, Hsp104. Unlike other yeast prions, this prion is unique in that it is efficiently cured by the overexpression as well as the inactivation of Hsp104. However, it is controversial whether curing by overexpression is due to the dissolution of the prion seeds by the trimming activity of Hsp104 or the asymmetric segregation of the prion seeds between mother and daughter cells which requires cell division. To answer this question, we conducted experiments and found no difference in the extent of curing between mother and daughter cells when half of the cells were cured by Hsp104 overexpression in one generation. Furthermore, curing was not affected by the lack of Sir2 expression, which was reported to be required for asymmetric segregation of the [PSI+] seeds. More importantly, when either hydroxyurea or ethanol were used to inhibit cell division, the extent of curing by Hsp104 overexpression was not significantly reduced. Therefore, the curing of [PSI+] by Hsp104 overexpression is not due to asymmetric segregation of the prion seeds, but rather their dissolution by Hsp104.

Hsp70 Binding to the N-terminal Domain of Hsp104 Regulates [PSI+] Curing by Hsp104 Overexpression.

Hsp104 propagates the yeast prion [PSI+], the infectious form of Sup35, by severing the prion seeds, but when Hsp104 is overexpressed, it cures [PSI+] in a process that is not yet understood but may be caused by trimming, which removes monomers from the ends of the amyloid fibers. This curing was shown to depend on both the N-terminal domain of Hsp104 and the expression level of various members of the Hsp70 family, which raises the question as to whether these effects of Hsp70 are due to it binding to the Hsp70 binding site that was identified in the N-terminal domain of Hsp104, a site not involved in prion propagation. Investigating this question, we now find, first, that mutating this site prevents both the curing of [PSI+] by Hsp104 overexpression and the trimming activity of Hsp104. Second, we find that depending on the specific member of the Hsp70 family binding to the N-terminal domain of Hsp104, both trimming and the curing caused by Hsp104 overexpression are either increased or decreased in parallel. Therefore, the binding of Hsp70 to the N-terminal domain of Hsp104 regulates both the rate of [PSI+] trimming by Hsp104 and the rate of [PSI+] curing by Hsp104 overexpression.

The yeast molecular chaperone, Hsp104, influences transthyretin aggregate formation.

Patients with the fatal disorder Transthyretin Amyloidosis (ATTR) experience polyneuropathy through the progressive destruction of peripheral nervous tissue. In these patients, the transthyretin (TTR) protein dissociates from its functional tetrameric structure, misfolds, and aggregates into extracellular amyloid deposits that are associated with disease progression. These aggregates form large fibrillar structures as well as shorter oligomeric aggregates that are suspected to be cytotoxic. Several studies have shown that these extracellular TTR aggregates enter the cell and accumulate intracellularly, which is associated with increased proteostasis response. However, there are limited experimental models to study how proteostasis influences internalized TTR aggregates. Here, we use a humanized yeast system to recapitulate intracellular TTR aggregating protein in vivo. The yeast molecular chaperone Hsp104 is a disaggregase that has been shown to fragment amyloidogenic aggregates associated with certain yeast prions and reduce protein aggregation associated with human neurogenerative diseases. In yeast, we found that TTR forms both SDS-resistant oligomers and SDS-sensitive large molecular weight complexes. In actively dividing cultures, Hsp104 has no impact on oligomeric or large aggregate populations, yet overexpression of Hsp104 is loosely associated with an increase in overall aggregate size. Interestingly, a potentiating mutation in the middle domain of Hsp104 consistently results in an increase in overall TTR aggregate size. These data suggest a novel approach to aggregate management, where the Hsp104 variant shifts aggregate populations away from toxic oligomeric species to more inert larger aggregates. In aged cultures Hsp104 overexpression has no impact on TTR aggregation profiles suggesting that these chaperone approaches to shift aggregate populations are not effective with age, possibly due to proteostasis decline.

The [PSI+] prion modulates cytochrome c oxidase deficiency caused by deletion of COX12.

Cytochrome c oxidase (CcO) is a pivotal enzyme of the mitochondrial respiratory chain, which sustains bioenergetics of eukaryotic cells. Cox12, a peripheral subunit of CcO oxidase, is required for full activity of the enzyme, but its exact function is unknown. Here experimental evolution of a Saccharomyces cerevisiae Δcox12 strain for ∼300 generations allowed to restore the activity of CcO oxidase. In one population, the enhanced bioenergetics was caused by a A375V mutation in the cytosolic AAA+ disaggregase Hsp104. Deletion or overexpression of HSP104 also increased respiration of the Δcox12 ancestor strain. This beneficial effect of Hsp104 was related to the loss of the [PSI+] prion, which forms cytosolic amyloid aggregates of the Sup35 protein. Overall, our data demonstrate that cytosolic aggregation of a prion impairs the mitochondrial metabolism of cells defective for Cox12. These findings identify a new functional connection between cytosolic proteostasis and biogenesis of the mitochondrial respiratory chain.

Structural Elements of ‘Anti‐prion J protein 1’ (Apj1) Required for Efficient Curing of the [PSI+] Prion by Hsp104 Overexpression

Prions are self‐propagating aggregates of misfolded protein that often form ordered amyloids. Amyloids are associated with a variety of human neurodegenerative diseases, including but not limited to Alzheimer’s disease, Huntington’s disease, and Parkinson’s disease. Amyloid‐forming prions are also present in Saccharomyces cerevisiae, where they are propagated through populations by a fragmentation mechanism that requires three chaperone proteins: the disaggregase Hsp104, the Hsp70 Ssa, and the J‐protein Sis1. When Hsp104 is ectopically overexpressed, however, the prion [PSI+] is specifically eliminated from cell populations in a Sis1‐dependent manner. We previously determined that the J‐protein Apj1 is also required for efficient elimination, or curing, of the prion variants [PSI+]STR and [PSI+]Sc4 by Hsp104 overexpression. Additionally, we showed that Apj1 and Sis1 have overlapping functions in the Hsp104‐mediated curing process, as overexpressing one in the absence of a functional version of the other restores curing. A construct bearing the first 161 residues of Apj1 is sufficient to restore efficient curing in Δapj1cells. Residues 1‐161 can be subdivided based on sequence features and homology into: a J‐domain, a polyglutamine track with an imperfect QA repeat region, a region bearing homology to the G/F region of Sis1, and finally a region rich in glycine, serine, phenylalanine, and asparagine which resembles the amino acid content of many prion‐forming domains. Various constructs produced by mutating and truncating regions of Apj1‐161 were ectopically expressed in Δapj1 cells to determine which regions, if any, were sufficient to replace Apj1 in Hsp104‐mediated curing. We found that constructs containing the polyQ region restored efficient curing and those lacking the polyQ region did not support efficient curing, indicating the importance of the polyQ region in the curing process. The Sis1 homologous region and the SFN rich region proved nonessential for Hsp104‐mediated curing. Ongoing experiments are being conducted to determine which regions of Apj1‐161 are required to replace the function of Sis1 in Hsp104‐mediated curing.

DMSO-mediated curing of several yeast prion variants involves Hsp104 expression and protein solubilization, and is decreased in several autophagy related gene (atg) mutants.

Chaperones and autophagy are components of the protein quality control system that contribute to the management of proteins that are misfolded and aggregated. Here, we use yeast prions, which are self-perpetuating aggregating proteins, as a means to understand how these protein quality control systems influence aggregate loss. Chaperones, such as Hsp104, fragment prion aggregates to generate more prion seeds for propagation. While much is known about the role of chaperones, little is known about how other quality control systems contribute to prion propagation. We show that the aprotic solvent dimethyl sulfoxide (DMSO) cures a range of [PSI+] prion variants, which are related to several misfolded aggregated conformations of the Sup35 protein. Our studies show that DMSO-mediated curing is quicker and more efficient than guanidine hydrochloride, a prion curing agent that inactivates the Hsp104 chaperone. Instead, DMSO appears to induce Hsp104 expression. Using the yTRAP system, a recently developed transcriptional reporting system for tracking protein solubility, we found that DMSO also rapidly induces the accumulation of soluble Sup35 protein, suggesting a potential link between Hsp104 expression and disassembly of Sup35 from the prion aggregate. However, DMSO-mediated curing appears to also be associated with other quality control systems. While the induction of autophagy alone does not lead to curing, we found that DMSO-mediated curing is dramatically impaired in autophagy related (atg) gene mutants, suggesting that other factors influence this DMSO mechanism of curing. Our data suggest that DMSO-mediated curing is not simply dependent upon Hsp104 overexpression alone, but may further depend upon other aspects of proteostasis.

Variant-specific and reciprocal Hsp40 functions in Hsp104-mediated prion elimination.

SummaryThe amyloid‐based prions of Saccharomyces cerevisiae are heritable aggregates of misfolded proteins, passed to daughter cells following fragmentation by molecular chaperones including the J‐protein Sis1, Hsp70 and Hsp104. Overexpression of Hsp104 efficiently cures cell populations of the prion [PSI +] by an alternative Sis1‐dependent mechanism that is currently the subject of significant debate. Here, we broadly investigate the role of J‐proteins in this process by determining the impact of amyloid polymorphisms (prion variants) on the ability of well‐studied Sis1 constructs to compensate for Sis1 and ask whether any other S. cerevisiae cytosolic J‐proteins are also required for this process. Our comprehensive screen, examining all 13 members of the yeast cytosolic/nuclear J‐protein complement, uncovered significant variant‐dependent genetic evidence for a role of Apj1 (antiprion DnaJ) in this process. For strong, but not weak [PSI +] variants, depletion of Apj1 inhibits Hsp104‐mediated curing. Overexpression of either Apj1 or Sis1 enhances curing, while overexpression of Ydj1 completely blocks it. We also demonstrated that Sis1 was the only J‐protein necessary for the propagation of at least two weak [PSI +] variants and no J‐protein alteration, or even combination of alterations, affected the curing of weak [PSI +] variants, suggesting the possibility of biochemically distinct, variant‐specific Hsp104‐mediated curing mechanisms.

Complex Effects of J‐protein Alterations on Hsp104‐mediated Curing of Prion [PSI+

The amyloid‐based prions of Saccharomyces cerevisiae are heritable aggregates of misfolded protein, passed to daughter cells following fragmentation by a set of molecular chaperones which includes the J‐protein Sis1, Hsp70, and Hsp104. Overexpression of Hsp104 efficiently cures the prion [PSI+], a phenomenon which has promoted the exploration of Hsp104 as a potential therapeutic agent for neurodegenerative diseases. However, the mechanism of [PSI+] elimination by Hsp104 overexpression has been the subject of significant debate for the past two decades and has garnered significant interest in the recent literature as multiple conflicting models have been proposed. Yeast prion propagation is inexorably reliant on the function of molecular chaperones of the Hsp100, Hsp70, and Hsp40 classes. Specifically, four Hsp40s (also called J‐proteins) have been implicated in various aspects of yeast prion biology: Sis1, Ydj1, Apj1, and Swa2. We found that overexpression of Sis1 or Apj1 accelerates strong [PSI+] elimination by Hsp104 overexpression, yet Ydj1 overexpression has a profound and opposing effect, completely blocking Hsp104‐mediated curing, indicating that Apj1 and Sis1 likely have similar and partially overlapping roles in this process. Interestingly results for weak variants of [PSI+] indicated potentially no role for J‐proteins in curing as no J‐protein alteration whatsoever affected the ability of Hsp104 to cure these variants. Additional experiments to determine the specific J‐protein domains responsible for various effects, as well as J‐protein requirements in cell backgrounds harboring both [PSI+] and [RNQ+] are underway. Overall our data support the hypothesis that Hsp104‐mediated curing may occur by biochemically distinct, variant‐specific mechanisms, only some of which involve J‐proteins.Support or Funding InformationThis work was supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number R15GM110606. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Preliminary X-Ray Crystallographic Studies of the N-Terminal Domains of Hsp104 from Yeast Candida albicans and Saccharomyces cerevisiae

Yeast Hsp104 is an ATP-dependent molecular chaperone, which can solublize and rescue denatured proteins from aggregates into active form by cooperating with Hsp70 and Hsp40 chaperones. Moreover, overexpression of Hsp104 of Saccharomyces cerevisiae (ScHsp104) cures the yeast [PSI+] prion due to the completely dissolution of the prion seeds, demonstrating ScHsp104’s potential to clear amyloid-like protein aggregates, thus making ScHsp104 a promising medication approach for human amyloidogenic neurodegenerative diseases. Because the working mechanisms for ScHsp104’s activities have not been clearly elucidated yet, crystallographic determination of ScHsp104 stands for great significance. Here, the expression, purification and crystallization of the N-terminal domains of Hsp104 from yeast Candida albicans (CaHsp104N) and S. cerevisiae (ScHsp104N) are described. The CaHsp104N crystals diffracted to 1.54 A and belonged to the sp. gr. P3221 or P3121, with unit cell parameters of a = 55.213 A, c = 109.451 A. The data of the ScHsp104N crystals were collected to the resolution of 2.53 A in the sp. gr. C2, with unit cell parameters a = 148.587 A, b = 66.255 A, c = 74.577 A, β = 107.369°. The phase of ScHsp104N is determined by the molecular replacement method using CaHsp104N as the search model.

Overlapping and Specific Functions of the Hsp104 N Domain Define Its Role in Protein Disaggregation

Hsp104 is a ring-forming protein disaggregase that rescues stress-damaged proteins from an aggregated state. To facilitate protein disaggregation, Hsp104 cooperates with Hsp70 and Hsp40 chaperones (Hsp70/40) to form a bi-chaperone system. How Hsp104 recognizes its substrates, particularly the importance of the N domain, remains poorly understood and multiple, seemingly conflicting mechanisms have been proposed. Although the N domain is dispensable for protein disaggregation, it is sensitive to point mutations that abolish the function of the bacterial Hsp104 homolog in vitro, and is essential for curing yeast prions by Hsp104 overexpression in vivo. Here, we present the crystal structure of an N-terminal fragment of Saccharomyces cerevisiae Hsp104 with the N domain of one molecule bound to the C-terminal helix of the neighboring D1 domain. Consistent with mimicking substrate interaction, mutating the putative substrate-binding site in a constitutively active Hsp104 variant impairs the recovery of functional protein from aggregates. We find that the observed substrate-binding defect can be rescued by Hsp70/40 chaperones, providing a molecular explanation as to why the N domain is dispensable for protein disaggregation when Hsp70/40 is present, yet essential for the dissolution of Hsp104-specific substrates, such as yeast prions, which likely depends on a direct N domain interaction.

Heat shock protein 104 (Hsp104)-mediated curing of [PSI+] yeast prions depends on both [PSI+] conformation and the properties of the Hsp104 homologs

Prions arise from proteins that have two possible conformations: properly folded and non-infectious or misfolded and infectious. The [PSI+] yeast prion, which is the misfolded and self-propagating form of the translation termination factor eRF3 (Sup35), can be cured of its infectious conformation by overexpression of Hsp104, which helps dissolve the prion seeds. This dissolution depends on the trimming activity of Hsp104, which reduces the size of the prion seeds without increasing their number. To further understand the relationship between trimming and curing, trimming was followed by measuring the loss of GFP-labeled Sup35 foci from both strong and weak [PSI+] variants; the former variant has more seeds and less soluble Sup35 than the latter. Overexpression of Saccharomyces cerevisiae Hsp104 (Sc-Hsp104) trimmed the weak [PSI+] variants much faster than the strong variants and cured the weak variants an order of magnitude faster than the strong variants. Overexpression of the fungal Hsp104 homologs from Schizosaccharomyces pombe (Sp-Hsp104) or Candida albicans (Ca-Hsp104) also trimmed and cured the weak variants, but interestingly, it neither trimmed nor cured the strong variants. These results show that, because Sc-Hsp104 has greater trimming activity than either Ca-Hsp104 or Sp-Hsp104, it cures both the weak and strong variants, whereas Ca-Hsp104 and Sp-Hsp104 only cure the weak variants. Therefore, curing by Hsp104 overexpression depends on both the trimming ability of the fungal Hsp104 homolog and the strength of the [PSI+] variant: the greater the trimming activity of the Hsp104 homolog and the weaker the variant, the greater the curing.

Over‐expression of the molecular chaperone Hsp104 in Saccharomyces cerevisiae results in the malpartition of [PSI+] propagons

The ability of a yeast cell to propagate [PSI+ ], the prion form of the Sup35 protein, is dependent on the molecular chaperone Hsp104. Inhibition of Hsp104 function in yeast cells leads to a failure to generate new propagons, the molecular entities necessary for [PSI+ ] propagation in dividing cells and they get diluted out as cells multiply. Over-expression of Hsp104 also leads to [PSI+ ] prion loss and this has been assumed to arise from the complete disaggregation of the Sup35 prion polymers. However, in conditions of Hsp104 over-expression in [PSI+ ] cells we find no release of monomers from Sup35 polymers, no monomerization of aggregated Sup35 which is not accounted for by the proportion of prion-free [psi- ] cells present, no change in the molecular weight of Sup35-containing SDS-resistant polymers and no significant decrease in average propagon numbers in the population as a whole. Furthermore, they show that over-expression of Hsp104 does not interfere with the incorporation of newly synthesised Sup35 into polymers, nor with the multiplication of propagons following their depletion in numbers while growing in the presence of guanidine hydrochloride. Rather, they present evidence that over-expression of Hsp104 causes malpartition of [PSI+ ] propagons between mother and daughter cells in a sub-population of cells during cell division thereby generating prion-free [psi- ] cells.

The small heat shock protein Hsp31 cooperates with Hsp104 to modulate Sup35 prion aggregation

ABSTRACTThe yeast homolog of DJ-1, Hsp31, is a multifunctional protein that is involved in several cellular pathways including detoxification of the toxic metabolite methylglyoxal and as a protein deglycase. Prior studies ascribed Hsp31 as a molecular chaperone that can inhibit α-Syn aggregation in vitro and alleviate its toxicity in vivo. It was also shown that Hsp31 inhibits Sup35 aggregate formation in yeast, however, it is unknown if Hsp31 can modulate [PSI+] phenotype and Sup35 prionogenesis. Other small heat shock proteins, Hsp26 and Hsp42 are known to be a part of a synergistic proteostasis network that inhibits Sup35 prion formation and promotes its disaggregation. Here, we establish that Hsp31 inhibits Sup35 [PSI+] prion formation in collaboration with a well-known disaggregase, Hsp104. Hsp31 transiently prevents prion induction but does not suppress induction upon prolonged expression of Sup35 indicating that Hsp31 can be overcome by larger aggregates. In addition, elevated levels of Hsp31 do not cure [PSI+] strains indicating that Hsp31 cannot intervene in a pre-existing prion oligomerization cycle. However, Hsp31 can modulate prion status in cooperation with Hsp104 because it inhibits Sup35 aggregate formation and potentiates [PSI+] prion curing upon overexpression of Hsp104. The absence of Hsp31 reduces [PSI+] prion curing by Hsp104 without influencing its ability to rescue cellular thermotolerance. Hsp31 did not synergize with Hsp42 to modulate the [PSI+] phenotype suggesting that both proteins act on similar stages of the prion cycle. We also showed that Hsp31 physically interacts with Hsp104 and together they prevent Sup35 prion toxicity to greater extent than if they were expressed individually. These results elucidate a mechanism for Hsp31 on prion modulation that suggest it acts at a distinct step early in the Sup35 aggregation process that is different from Hsp104. This is the first demonstration of the modulation of [PSI+] status by the chaperone action of Hsp31. The delineation of Hsp31's role in the chaperone cycle has implications for understanding the role of the DJ-1 superfamily in controlling misfolded proteins in neurodegenerative disease and cancer.

Variant‐specific and reciprocal Hsp40 functions in Hsp104‐mediated prion elimination: A potential role for the ‘Anti‐prion J‐protein 1’ (Apj1)

The prions of Saccharomyces cerevisiae are heritable aggregates of misfolded yeast proteins. Yeast prions are incorporated into daughter cells through the fragmentation of these aggregates by molecular chaperone proteins, including J‐proteins (Hsp40s), Hsp70s, and Hsp104. Previous studies have shown that the overexpression of Hsp104 cures the prion [PSI+], but not other prions, a phenomenon which has promoted the exploration of Hsp104 as a potential therapeutic agent for neurodegenerative diseases, despite the fact that the mechanism of [PSI+] elimination by Hsp104 overabundance is currently the subject of significant debate. We recently reported that the human homolog of Sis1, Hdj1, was capable of substituting for Sis1 in the propagation of strong but not weak [PSI+] variants and was deficient in substituting for Sis1 in the elimination of [PSI+] by Hsp104 overexpression. The objective of this study was to extend that investigation to the Sis1‐domain requirements for Hsp104‐mediated [PSI+] curing, expanding upon another prior investigation by extending the analysis to multiple [PSI+] variants and genetic backgrounds. We also asked whether any of the other 12 J‐proteins located in the S. cerevisiae cytosol are also necessary for Hsp104‐induced [PSI+] curing and if prion amyloid structure (variant identity) affects experimental outcomes. The primary method used in this study was complementation by plasmid‐shuffling in the budding yeast S. cerevisiae.Summary of ResultsOur investigation revealed that at least two weak [PSI+] variants can be maintained in the absence of any of 12 non‐essential J‐proteins, ruling out essential roles for any of these J‐proteins in weak [PSI+] prion propagation. Likewise, elimination of these weak [PSI+] variants by Hsp104 overexpression was ubiquitous, demonstrating that no J‐protein other than Sis1 is necessary for the elimination of weak [PSI+] variants by overexpression of Hsp104. In sharp contrast however, we found that strong variants of [PSI+] exhibited exceptional resistance to Hsp104‐mediated elimination only in strains lacking the J‐protein Apj1 (originally named: Anti‐Yeast Prion J‐protein 1) and conversely Apj1 overexpression accelerates prion elimination. Apj1 has been implicated in prion biology several times; often acting similarly to Ydj1 in prion biology. Here we find a novel genetic interaction for Apj1 and prions that is distinct from Ydj1 function. Additional mutation and truncation experiments revealed domains required for Apj1 function in Hsp104‐mediated [PSI+] curing and may ultimately help to explain the prion‐specificity of this curing mechanism.Support or Funding InformationThis work was supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number R15GM110606. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Hsp104 Overexpression Cures Saccharomyces cerevisiae [ PSI + ] by Causing Dissolution of the Prion Seeds

The [PSI(+)] yeast prion is formed when Sup35 misfolds into amyloid aggregates. [PSI(+)], like other yeast prions, is dependent on the molecular chaperone Hsp104, which severs the prion seeds so that they pass on as the yeast cells divide. Surprisingly, however, overexpression of Hsp104 also cures [PSI(+)]. Several models have been proposed to explain this effect: inhibition of severing, asymmetric segregation of the seeds between mother and daughter cells, and dissolution of the prion seeds. First, we found that neither the kinetics of curing nor the heterogeneity in the distribution of the green fluorescent protein (GFP)-labeled Sup35 foci in partially cured yeast cells is compatible with Hsp104 overexpression curing [PSI(+)] by inhibiting severing. Second, we ruled out the asymmetric segregation model by showing that the extent of curing was essentially the same in mother and daughter cells and that the fluorescent foci did not distribute asymmetrically, but rather, there was marked loss of foci in both mother and daughter cells. These results suggest that Hsp104 overexpression cures [PSI(+)] by dissolution of the prion seeds in a two-step process. First, trimming of the prion seeds by Hsp104 reduces their size, and second, their amyloid core is eliminated, most likely by proteolysis.

Schizosaccharomyces pombe Disaggregation Machinery Chaperones Support Saccharomyces cerevisiae Growth and Prion Propagation

Hsp100 chaperones protect microorganisms and plants from environmental stress by cooperating with Hsp70 and its nucleotide exchange factor (NEF) and Hsp40 cochaperones to resolubilize proteins from aggregates. The Saccharomyces cerevisiae Hsp104 (Sc-Hsp104)-based disaggregation machinery also is essential for replication of amyloid-based prions. Escherichia coli ClpB can substitute for Hsp104 to propagate [PSI(+)] prions in yeast, but only if E. coli DnaK and GrpE (Hsp70 and NEF) are coexpressed. Here, we tested if the reported inability of Schizosaccharomyces pombe Hsp104 (Sp-Hsp104) to support [PSI(+)] propagation was due to similar species-specific chaperone requirements and find that Sp-Hsp104 alone supported propagation of three different yeast prions. Sp-Hsp70 and Sp-Fes1p (NEF) likewise functioned in place of their Sa. cerevisiae counterparts. Thus, chaperones of these long-diverged species possess conserved activities that function in processes essential for both cell growth and prion propagation, suggesting Sc. pombe can propagate its own prions. We show that curing by Hsp104 overexpression and inactivation can be distinguished and confirm the observation that, unlike Sc-Hsp104, Sp-Hsp104 cannot cure yeast of [PSI(+)] when it is overexpressed. These results are consistent with a view that mechanisms underlying prion replication and elimination are distinct.

The Paf1 complex subunit Rtf1 buffers cells against the toxic effects of [PSI+] and defects in Rkr1-dependent protein quality control in Saccharomyces cerevisiae.

The Rtf1 subunit of the Paf1 complex is required for specific histone modifications, including histone H2B lysine 123 monoubiquitylation. In Saccharomyces cerevisiae, deletion of RTF1 is lethal in the absence of Rkr1, a ubiquitin-protein ligase involved in the destruction of nonstop proteins, which arise from mRNAs lacking stop codons or translational readthrough into the poly(A) tail. We performed a transposon-based mutagenesis screen to identify suppressors of rtf1Δ rkr1Δ lethality and found that a mutation in the gene encoding the protein chaperone Hsp104 rescued viability. Hsp104 plays a role in prion propagation, including the maintenance of [PSI+], which contributes to the synthesis of nonstop proteins. We demonstrate that rtf1Δ and rkr1Δ are synthetically lethal only in the presence of [PSI+]. The deletion, inactivation, and overexpression of HSP104 or the overexpression of prion-encoding genes URE2 and LSM4 clear [PSI+] and rescue rtf1Δ rkr1Δ lethality. In addition, the presence of [PSI+] decreases the fitness of rkr1Δ strains. We investigated whether the loss of RTF1 exacerbates an overload in nonstop proteins in rkr1Δ [PSI+] strains but, using reporter plasmids, found that rtf1Δ decreases nonstop protein levels, indicating that excess nonstop proteins may not be the cause of synthetic lethality. Instead, our data suggest that the loss of Rtf1-dependent histone modifications increases the burden on quality control pathways in cells lacking Rkr1 and containing [PSI+].

Small Heat Shock Proteins Potentiate Amyloid Dissolution by Protein Disaggregases from Yeast and Humans

Author SummaryAmyloid fibers are protein aggregates that are associated with numerous neurodegenerative diseases, including Parkinson's disease, for which there are no effective treatments. They can also play beneficial roles; in yeast, for example, they are associated with increased survival and the evolution of new traits. Amyloid fibers are also central to many revolutionary concepts and important questions in biology and nanotechnology, including long-term memory formation and versatile self-organizing nanostructures. Thus, there is an urgent need to understand how we can promote beneficial amyloid assembly, or reverse pathogenic assembly, at will. In this study, we define the mechanisms by which small heat-shock proteins synergize to regulate the assembly and disassembly of a beneficial yeast prion. We then exploit this knowledge to discover an amyloid depolymerase machinery that is conserved from yeast to humans. Remarkably, the human small heat shock protein, HspB5, stimulates Hsp110, Hsp70, and Hsp40 chaperones to gradually depolymerize amyloid fibers formed by α-synuclein (which are implicated in Parkinson's disease) from their ends on a biologically relevant timescale. This newly identified and highly conserved amyloid-depolymerase system could have important therapeutic applications for various neurodegenerative disorders.

Insight into Molecular Basis of Curing of [PSI+] Prion by Overexpression of 104-kDa Heat Shock Protein (Hsp104)

Yeast prions are a powerful model for understanding the dynamics of protein aggregation associated with a number of human neurodegenerative disorders. The AAA+ protein disaggregase Hsp104 can sever the amyloid fibrils produced by yeast prions. This action results in the propagation of "seeds" that are transmitted to daughter cells during budding. Overexpression of Hsp104 eliminates the [PSI+] prion but not other prions. Using biochemical methods we identified Hsp104 binding sites in the highly charged middle domain of Sup35, the protein determinant of [PSI+]. Deletion of a short segment of the middle domain (amino acids 129-148) diminishes Hsp104 binding and strongly affects the ability of the middle domain to stimulate the ATPase activity of Hsp104. In yeast, [PSI+] maintained by Sup35 lacking this segment, like other prions, is propagated by Hsp104 but cannot be cured by Hsp104 overexpression. These results provide new insight into the enigmatic specificity of Hsp104-mediated curing of yeast prions and sheds light on the limitations of the ability of Hsp104 to eliminate aggregates produced by other aggregation-prone proteins.