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1.  Heterologous Aggregates Promote De Novo Prion Appearance via More than One Mechanism 
PLoS Genetics  2015;11(1):e1004814.
Prions are self-perpetuating conformational variants of particular proteins. In yeast, prions cause heritable phenotypic traits. Most known yeast prions contain a glutamine (Q)/asparagine (N)-rich region in their prion domains. [PSI+], the prion form of Sup35, appears de novo at dramatically enhanced rates following transient overproduction of Sup35 in the presence of [PIN+], the prion form of Rnq1. Here, we establish the temporal de novo appearance of Sup35 aggregates during such overexpression in relation to other cellular proteins. Fluorescently-labeled Sup35 initially forms one or a few dots when overexpressed in [PIN+] cells. One of the dots is perivacuolar, colocalizes with the aggregated Rnq1 dot and grows into peripheral rings/lines, some of which also colocalize with Rnq1. Sup35 dots that are not near the vacuole do not always colocalize with Rnq1 and disappear by the time rings start to grow. Bimolecular fluorescence complementation failed to detect any interaction between Sup35-VN and Rnq1-VC in [PSI+][PIN+] cells. In contrast, all Sup35 aggregates, whether newly induced or in established [PSI+], completely colocalize with the molecular chaperones Hsp104, Sis1, Ssa1 and eukaryotic release factor Sup45. In the absence of [PIN+], overexpressed aggregating proteins such as the Q/N-rich Pin4C or the non-Q/N-rich Mod5 can also promote the de novo appearance of [PSI+]. Similar to Rnq1, overexpressed Pin4C transiently colocalizes with newly appearing Sup35 aggregates. However, no interaction was detected between Mod5 and Sup35 during [PSI+] induction in the absence of [PIN+]. While the colocalization of Sup35 and aggregates of Rnq1 or Pin4C are consistent with the model that the heterologous aggregates cross-seed the de novo appearance of [PSI+], the lack of interaction between Mod5 and Sup35 leaves open the possibility of other mechanisms. We also show that Hsp104 is required in the de novo appearance of [PSI+] aggregates in a [PIN+]-independent pathway.
Author Summary
Certain proteins can misfold into β-sheet-rich, self-seeding aggregates. Such proteins appear to be associated with neurodegenerative diseases such as prion, Alzheimer's and Parkinson's. Yeast prions also misfold into self-seeding aggregates and provide a good model to study how these rogue polymers first appear. De novo prion appearance can be made very frequent in yeast by transient overexpression of the prion protein in the presence of heterologous prions or prion-like aggregates. Here, we show that the aggregates of one such newly induced prion are initially formed in a dot-like structure near the vacuole. These dots then grow into rings at the periphery of the cell prior to becoming smaller rings surrounding the vacuole and maturing into the characteristic heritable prion tiny dots found throughout the cytoplasm. We found considerable colocalization of two heterologous prion/prion-like aggregates with the newly appearing prion protein aggregates, which is consistent with the prevalent model that existing prion aggregates can cross-seed the de novo aggregation of a heterologous prion protein. However, we failed to find any physical interaction between another heterologous aggregating protein and the newly appearing prion aggregates it stimulated to appear, which is inconsistent with cross-seeding.
doi:10.1371/journal.pgen.1004814
PMCID: PMC4287349  PMID: 25568955
2.  Polyglutamine Toxicity Is Controlled by Prion Composition and Gene Dosage in Yeast 
PLoS Genetics  2012;8(4):e1002634.
Polyglutamine expansion causes diseases in humans and other mammals. One example is Huntington's disease. Fragments of human huntingtin protein having an expanded polyglutamine stretch form aggregates and cause cytotoxicity in yeast cells bearing endogenous QN-rich proteins in the aggregated (prion) form. Attachment of the proline(P)-rich region targets polyglutamines to the large perinuclear deposit (aggresome). Aggresome formation ameliorates polyglutamine cytotoxicity in cells containing only the prion form of Rnq1 protein. Here we show that expanded polyglutamines both with (poly-QP) or without (poly-Q) a P-rich stretch remain toxic in the presence of the prion form of translation termination (release) factor Sup35 (eRF3). A Sup35 derivative that lacks the QN-rich domain and is unable to be incorporated into aggregates counteracts cytotoxicity, suggesting that toxicity is due to Sup35 sequestration. Increase in the levels of another release factor, Sup45 (eRF1), due to either disomy by chromosome II containing the SUP45 gene or to introduction of the SUP45-bearing plasmid counteracts poly-Q or poly-QP toxicity in the presence of the Sup35 prion. Protein analysis confirms that polyglutamines alter aggregation patterns of Sup35 and promote aggregation of Sup45, while excess Sup45 counteracts these effects. Our data show that one and the same mode of polyglutamine aggregation could be cytoprotective or cytotoxic, depending on the composition of other aggregates in a eukaryotic cell, and demonstrate that other aggregates expand the range of proteins that are susceptible to sequestration by polyglutamines.
Author Summary
Polyglutamine diseases, including Huntington disease, are associated with expansions of polyglutamine tracts, resulting in aggregation of respective proteins. The severity of Huntington disease is controlled by both DNA and non–DNA factors. Mechanisms of such a control are poorly understood. Polyglutamine may sequester other cellular proteins; however, different experimental models have pointed to different sequestered proteins. By using a yeast model, we demonstrate that the mechanism of polyglutamine toxicity is driven by the composition of other (endogenous) aggregates (for example, yeast prions) present in a eukaryotic cell. Although these aggregates do not necessarily cause significant toxicity on their own, they serve as mediators in protein sequestration and therefore determine which specific proteins are to be sequestered by polyglutamines. We also show that polyglutamine deposition into an aggresome, a perinuclear compartment thought to be cytoprotective, fails to ameliorate cytotoxicity in cells with certain compositions of pre-existing aggregates. Finally, we demonstrate that an increase in the dosage of a sequestered protein due to aneuploidy by a chromosome carrying a respective gene may rescue cytotoxicity. Our data shed light on genetic and epigenetic mechanisms modulating polyglutamine cytotoxicity and establish a new approach for identifying potential therapeutic targets through characterization of the endogenous aggregated proteins.
doi:10.1371/journal.pgen.1002634
PMCID: PMC3334884  PMID: 22536159
3.  Appearance and Propagation of Polyglutamine-based Amyloids in Yeast 
The Journal of Biological Chemistry  2008;283(22):15185-15192.
In yeast, fragmentation of amyloid polymers by the Hsp104 chaperone allows them to propagate as prions. The prion-forming domain of the yeast Sup35 protein is rich in glutamine, asparagine, tyrosine, and glycine residues, which may define its prion properties. Long polyglutamine stretches can also drive amyloid polymerization in yeast, but these polymers are unable to propagate because of poor fragmentation and exist through constant seeding with the Rnq1 prion polymers. We proposed that fragmentation of polyglutamine amyloids may be improved by incorporation of hydrophobic amino acid residues into polyglutamine stretches. To investigate this, we constructed sets of polyglutamine with or without tyrosine stretches fused to the non-prion domains of Sup35. Polymerization of these chimeras started rapidly, and its efficiency increased with stretch size. Polymerization of proteins with polyglutamine stretches shorter than 70 residues required Rnq1 prion seeds. Proteins with longer stretches polymerized independently of Rnq1 and thus could propagate. The presence of tyrosines within polyglutamine stretches dramatically enhanced polymer fragmentation and allowed polymer propagation in the absence of Rnq1 and, in some cases, of Hsp104.
doi:10.1074/jbc.M802071200
PMCID: PMC2397454  PMID: 18381282
4.  Functional Diversification of Hsp40: Distinct J-Protein Functional Requirements for Two Prions Allow for Chaperone-Dependent Prion Selection 
PLoS Genetics  2014;10(7):e1004510.
Yeast prions are heritable amyloid aggregates of functional yeast proteins; their propagation to subsequent cell generations is dependent upon fragmentation of prion protein aggregates by molecular chaperone proteins. Mounting evidence indicates the J-protein Sis1 may act as an amyloid specificity factor, recognizing prion and other amyloid aggregates and enabling Ssa and Hsp104 to act in prion fragmentation. Chaperone interactions with prions, however, can be affected by variations in amyloid-core structure resulting in distinct prion variants or ‘strains’. Our genetic analysis revealed that Sis1 domain requirements by distinct variants of [PSI+] are strongly dependent upon overall variant stability. Notably, multiple strong [PSI+] variants can be maintained by a minimal construct of Sis1 consisting of only the J-domain and glycine/phenylalanine-rich (G/F) region that was previously shown to be sufficient for cell viability and [RNQ+] prion propagation. In contrast, weak [PSI+] variants are lost under the same conditions but maintained by the expression of an Sis1 construct that lacks only the G/F region and cannot support [RNQ+] propagation, revealing mutually exclusive requirements for Sis1 function between these two prions. Prion loss is not due to [PSI+]-dependent toxicity or dependent upon a particular yeast genetic background. These observations necessitate that Sis1 must have at least two distinct functional roles that individual prions differentially require for propagation and which are localized to the glycine-rich domains of the Sis1. Based on these distinctions, Sis1 plasmid-shuffling in a [PSI+]/[RNQ+] strain permitted J-protein-dependent prion selection for either prion. We also found that, despite an initial report to the contrary, the human homolog of Sis1, Hdj1, is capable of [PSI+] prion propagation in place of Sis1. This conservation of function is also prion-variant dependent, indicating that only one of the two Sis1-prion functions may have been maintained in eukaryotic chaperone evolution.
Author Summary
Multiple neurodegenerative disorders such as Alzheimer's, Parkinson's and Creutzfeldt-Jakob disease are associated with the accumulation of fibrous protein aggregates collectively termed ‘amyloid.’ In the baker's yeast Saccharomyces cerevisiae, multiple proteins form intracellular amyloid aggregates known as yeast prions. Yeast prions minimally require a core set of chaperone proteins for stable propagation in yeast, including the J-protein Sis1, which appears to be required for the propagation of all yeast prions and functioning similarly in each case. Here we present evidence which challenges the notion of a universal function for Sis1 in prion propagation and asserts instead that Sis1's function in the maintenance of at least two prions, [RNQ+] and [PSI+], is distinct and mutually exclusive for some prion variants. We also find that the human homolog of Sis1, called Hdj1, has retained the ability to support some, but not all yeast prions, indicating a partial conservation of function. Because yeast chaperones have the ability to both bind and fragment amyloids in vivo, further investigations into these prion-specific properties of Sis1 and Hdj1 will likely lead to new insights into the biological management of protein misfolding.
doi:10.1371/journal.pgen.1004510
PMCID: PMC4109904  PMID: 25058638
5.  Prion and Nonprion Amyloids 
Prion  2007;1(3):179-184.
Yeast prion determinants are related to polymerization of some proteins into amyloid-like fibers. The [PSI+] determinant reflects polymerization of the Sup35 protein. Fragmentation of prion polymers by the Hsp104 chaperone represents a key step of the prion replication cycle. The frequency of fragmentation varies depending on the structure of the prion polymers and defines variation in the prion phenotypes, e.g., the suppressor strength of [PSI+] and stability of its inheritance. Besides [PSI+], overproduction of Sup35 can produce nonheritable phenotypically silent Sup35 amyloid-like polymers. These polymers are fragmented poorly and are present due to efficient seeding with the Rnq1 prion polymers, which occurs by several orders of magnitude more frequently than seeding of [PSI+] appearance. Such Sup35 polymers resemble human nonprion amyloids by their nonheritability, mode of appearance and increased size. Thus, a single protein, Sup35, can model both prion and nonprion amyloids. In yeast, these phenomena are distinguished by the frequency of polymer fragmentation. We argue that in mammals the fragmentation frequency also represents a key factor defining differing properties of prion and nonprion amyloids, including infectivity. By analogy with the Rnq1 seeding of nonheritable Sup35 polymers, the “species barrier” in prion transmission may be due to seeding by heterologous prion of nontransmissible type of amyloid, rather than due to the lack of seeding.
PMCID: PMC2634591  PMID: 19164899
amyloid; prion; Rnq1; Sup35; Ure2; translation termination; yeast
6.  Pathogenic Polyglutamine Tracts Are Potent Inducers of Spontaneous Sup35 and Rnq1 Amyloidogenesis 
PLoS ONE  2010;5(3):e9642.
The glutamine/asparagine (Q/N)-rich yeast prion protein Sup35 has a low intrinsic propensity to spontaneously self-assemble into ordered, β-sheet-rich amyloid fibrils. In yeast cells, de novo formation of Sup35 aggregates is greatly facilitated by high protein concentrations and the presence of preformed Q/N-rich protein aggregates that template Sup35 polymerization. Here, we have investigated whether aggregation-promoting polyglutamine (polyQ) tracts can stimulate the de novo formation of ordered Sup35 protein aggregates in the absence of Q/N-rich yeast prions. Fusion proteins with polyQ tracts of different lengths were produced and their ability to spontaneously self-assemble into amlyloid structures was analyzed using in vitro and in vivo model systems. We found that Sup35 fusions with pathogenic (≥54 glutamines), as opposed to non-pathogenic (19 glutamines) polyQ tracts efficiently form seeding-competent protein aggregates. Strikingly, polyQ-mediated de novo assembly of Sup35 protein aggregates in yeast cells was independent of pre-existing Q/N-rich protein aggregates. This indicates that increasing the content of aggregation-promoting sequences enhances the tendency of Sup35 to spontaneously self-assemble into insoluble protein aggregates. A similar result was obtained when pathogenic polyQ tracts were linked to the yeast prion protein Rnq1, demonstrating that polyQ sequences are generic inducers of amyloidogenesis. In conclusion, long polyQ sequences are powerful molecular tools that allow the efficient production of seeding-competent amyloid structures.
doi:10.1371/journal.pone.0009642
PMCID: PMC2835767  PMID: 20224794
7.  Extensive Diversity of Prion Strains Is Defined by Differential Chaperone Interactions and Distinct Amyloidogenic Regions 
PLoS Genetics  2014;10(5):e1004337.
Amyloidogenic proteins associated with a variety of unrelated diseases are typically capable of forming several distinct self-templating conformers. In prion diseases, these different structures, called prion strains (or variants), confer dramatic variation in disease pathology and transmission. Aggregate stability has been found to be a key determinant of the diverse pathological consequences of different prion strains. Yet, it remains largely unclear what other factors might account for the widespread phenotypic variation seen with aggregation-prone proteins. Here, we examined a set of yeast prion variants of the [RNQ+] prion that differ in their ability to induce the formation of another yeast prion called [PSI+]. Remarkably, we found that the [RNQ+] variants require different, non-contiguous regions of the Rnq1 protein for both prion propagation and [PSI+] induction. This included regions outside of the canonical prion-forming domain of Rnq1. Remarkably, such differences did not result in variation in aggregate stability. Our analysis also revealed a striking difference in the ability of these [RNQ+] variants to interact with the chaperone Sis1. Thus, our work shows that the differential influence of various amyloidogenic regions and interactions with host cofactors are critical determinants of the phenotypic consequences of distinct aggregate structures. This helps reveal the complex interdependent factors that influence how a particular amyloid structure may dictate disease pathology and progression.
Author Summary
Protein conformational disorders, including many neurodegenerative diseases, result when a protein misfolds and undergoes a conformational change to form self-templating aggregates, called amyloid. Interestingly, the proteins that misfold in these diseases tend to form a wide variety of distinct aggregate structures. In prion diseases, these different amyloid conformations are called prion strains. The different conformations of prion strains are responsible for modulating disease progression, pathology, and transmission. Previous work with yeast prions has provided tremendous insight into how distinct prion conformers can cause such phenotypic variability. Here, we used a set of [RNQ+] prion variants to show the complex web of interactions involved in the propagation of distinct aggregate structures. We found that several different non-adjacent regions of Rnq1, even outside the prion-forming domain, make varying contributions to the propagation of distinct variants of the [RNQ+] prion. Moreover, our data support the hypothesis that the [RNQ+] variants differentially interact with the molecular chaperone Sis1. These data strongly suggest that the variable phenotypic manifestations of different aggregate conformations depend upon a unique set of primary structural elements and differential interactions with host cofactors.
doi:10.1371/journal.pgen.1004337
PMCID: PMC4014422  PMID: 24811344
8.  Spatial quality control bypasses cell-based limitations on proteostasis to promote prion curing 
eLife  null;3:e04288.
The proteostasis network has evolved to support protein folding under normal conditions and to expand this capacity in response to proteotoxic stresses. Nevertheless, many pathogenic states are associated with protein misfolding, revealing in vivo limitations on quality control mechanisms. One contributor to these limitations is the physical characteristics of misfolded proteins, as exemplified by amyloids, which are largely resistant to clearance. However, other limitations imposed by the cellular environment are poorly understood. To identify cell-based restrictions on proteostasis capacity, we determined the mechanism by which thermal stress cures the [PSI+]/Sup35 prion. Remarkably, Sup35 amyloid is disassembled at elevated temperatures by the molecular chaperone Hsp104. This process requires Hsp104 engagement with heat-induced non-prion aggregates in late cell-cycle stage cells, which promotes its asymmetric retention and thereby effective activity. Thus, cell division imposes a potent limitation on proteostasis capacity that can be bypassed by the spatial engagement of a quality control factor.
DOI: http://dx.doi.org/10.7554/eLife.04288.001
eLife digest
Proteins must fold into specific shapes to work inside cells, and the misfolding of proteins is associated with a growing number of diseases. For example, prions are misfolded proteins that form insoluble aggregates called amyloids. These aggregates are not easily destroyed and can cause other nearby proteins to misfold and join the amyloid. This process of amyloid assembly leads to progressive diseases such as mad cow disease, Huntington's disease, Alzheimer's disease, and Parkinson's disease, which are collectively known as amyloidoses.
A series of biological pathways called the proteostasis network control protein integrity in a cell. Under normal conditions or even mildly stressful conditions—such as at slightly increased temperatures—the proteostasis network is able to prevent proteins from misfolding. However, if a cell is placed under lots of stress this network may become overwhelmed and misfolded proteins can accumulate. To date, the proteostasis network has not been linked to the clearance of amyloids.
A protein called Sup35, which is found in budding yeast, can exist as two different prion forms. Previous studies have shown that briefly heating the yeast cells can ‘cure’ the so-called ‘weak’ form of the prion. The ‘strong’ prion form, however, was thought to be unaffected by elevated temperature. These previous studies had only tested yeast cells that had been dividing for a few generations; it was unknown if cells that had been dividing for longer might respond differently.
Klaips et al. found that a protein called Hsp104—which helps to fold proteins properly—can break down the amyloid aggregates. This protein is normally only present in small amounts, but heating causes the levels of Hsp104 to rise. Klaips et al. found that the extra Hsp104 protein associated with the aggregates and led to their disassembly. When Hsp104 was prevented from associating with the prions, the aggregates were not cured even if high levels of Hsp104 were present in the cell.
When budding yeast form new cells, a daughter cell ‘buds’ off from the mother cell. Klaips et al. found that mother cells exposed to heat retain most of the Hsp104 when the cell divides, and this retention allowed Hsp104 to accumulate to a level required for the breakdown of amyloid aggregates. Therefore, under normal conditions, amyloids persist because cell division keeps the amount of Hsp104 below this threshold.
Previously it had been thought that the physical characteristics of amyloids accounted for their resilience in the face of the cell mechanisms designed to counteract protein misfolding. However, Klaips et al. show that the balance of the different mechanisms involved in proteostasis can be manipulated to create environments where amyloids are either created and maintained or destroyed. Targeting these mechanisms could therefore present new treatment options for amyloidosis.
DOI: http://dx.doi.org/10.7554/eLife.04288.002
doi:10.7554/eLife.04288
PMCID: PMC4270096  PMID: 25490068
chaperone; protein misfolding; amyloid; prion; S. cerevisiae
9.  Dissection and Design of Yeast Prions 
PLoS Biology  2004;2(4):e86.
Many proteins can misfold into β-sheet-rich, self-seeding polymers (amyloids). Prions are exceptional among such aggregates in that they are also infectious. In fungi, prions are not pathogenic but rather act as epigenetic regulators of cell physiology, providing a powerful model for studying the mechanism of prion replication. We used prion-forming domains from two budding yeast proteins (Sup35p and New1p) to examine the requirements for prion formation and inheritance. In both proteins, a glutamine/asparagine-rich (Q/N-rich) tract mediates sequence-specific aggregation, while an adjacent motif, the oligopeptide repeat, is required for the replication and stable inheritance of these aggregates. Our findings help to explain why although Q/N-rich proteins are relatively common, few form heritable aggregates: prion inheritance requires both an aggregation sequence responsible for self-seeded growth and an element that permits chaperone-dependent replication of the aggregate. Using this knowledge, we have designed novel artificial prions by fusing the replication element of Sup35p to aggregation-prone sequences from other proteins, including pathogenically expanded polyglutamine.
Artificial prions - infectious, misfolded proteins - can be created by fusing the replication element of one prion to aggregation sequences from another
doi:10.1371/journal.pbio.0020086
PMCID: PMC374241  PMID: 15045026
10.  Prion Formation and Polyglutamine Aggregation Are Controlled by Two Classes of Genes 
PLoS Genetics  2011;7(5):e1001386.
Prions are self-perpetuating aggregated proteins that are not limited to mammalian systems but also exist in lower eukaryotes including yeast. While much work has focused around chaperones involved in prion maintenance, including Hsp104, little is known about factors involved in the appearance of prions. De novo appearance of the [PSI+] prion, which is the aggregated form of the Sup35 protein, is dramatically enhanced by transient overexpression of SUP35 in the presence of the prion form of the Rnq1 protein, [PIN+]. When fused to GFP and overexpressed in [ps−] [PIN+] cells, Sup35 forms fluorescent rings, and cells with these rings bud off [PSI+] daughters. We investigated the effects of over 400 gene deletions on this de novo induction of [PSI+]. Two classes of gene deletions were identified. Class I deletions (bug1Δ, bem1Δ, arf1Δ, and hog1Δ) reduced the efficiency of [PSI+] induction, but formed rings normally. Class II deletions (las17Δ, vps5Δ, and sac6Δ) inhibited both [PSI+] induction and ring formation. Furthermore, class II deletions reduced, while class I deletions enhanced, toxicity associated with the expanded glutamine repeats of the huntingtin protein exon 1 that causes Huntington's disease. This suggests that prion formation and polyglutamine aggregation involve a multi-phase process that can be inhibited at different steps.
Author Summary
Certain proteins that exist in functional unaggregated conformers can also form self-perpetuating infectious aggregates called prions. Here we investigate factors involved in the initial switch to the prion form. De novo appearance of the [PSI+] prion, which is the aggregated form of the Sup35 protein, is dramatically enhanced by overexpression of the SUP35 gene in the presence of the prion form of the Rnq1 protein, [PIN+]. When tagged with green fluorescent protein and transiently overexpressed in [psi−] [PIN+] cells, Sup35 forms fluorescent rings, and cells with these rings give rise to daughter cells that are [PSI+]. Here, we investigate factors required for this induction of [PSI+]. Analyses of over 400 gene deletions revealed two classes that reduce [PSI+] induction: one class forms fluorescent rings normally, and the other does not. Interestingly, the former class enhanced, while the latter class reduced, toxicity associated with the expanded polyglutamine repeats of the huntingtin protein exon 1 that causes Huntington's disease. These results suggest that prion formation and polyglutamine aggregation involve a multi-phase process that can be inhibited at different steps.
doi:10.1371/journal.pgen.1001386
PMCID: PMC3098188  PMID: 21625618
11.  Variant-specific [PSI+] Infection Is Transmitted by Sup35 Polymers within [PSI+] Aggregates with Heterogeneous Protein Composition 
Molecular Biology of the Cell  2008;19(6):2433-2443.
The [PSI+] prion is the aggregated self-propagating form of the Sup35 protein from the yeast Saccharomyces cerevisiae. Aggregates of Sup35 in [PSI+] cells exist in different heritable conformations, called “variants,” and they are composed of detergent-resistant Sup35 polymers, which may be closely associated with themselves, other proteins, or both. Here, we report that disassembly of the aggregates into individual Sup35 polymers and non-Sup35 components increases their infectivity while retaining their variant specificity, showing that variant-specific [PSI+] infection can be transmitted by Sup35 polymers alone. Morphological analysis revealed that Sup35 isolated from [PSI+] yeast has the appearance of short barrels, and bundles, which seem to be composed of barrels. We show that the major components of two different variants of [PSI+] are interacting infectious Sup35 polymers and Ssa1/2. Using a candidate approach, we detected Hsp104, Ssb1/2, Sis1, Sse1, Ydj1, and Sla2 among minor components of the aggregates. We demonstrate that Ssa1/2 efficiently binds to the prion domain of Sup35 in [PSI+] cells, but that it interacts poorly with the nonaggregated Sup35 found in [psi−] cells. Hsp104, Sis1, and Sse1 interact preferentially with the prion versus nonprion form of Sup35, whereas Sla2 and Ssb1/2 interact with both forms of Sup35 with similar efficiency.
doi:10.1091/mbc.E08-01-0078
PMCID: PMC2397312  PMID: 18353968
12.  Prion amyloid structure explains templating: how proteins can be genes 
FEMS yeast research  2010;10(8):980-991.
The yeast and fungal prions determine heritable and infectious traits, and are thus genes composed of protein. Most prions are inactive forms of a normal protein as it forms a self-propagating filamentous β – sheet - rich polymer structure called amyloid. Remarkably, a single prion protein sequence can form two or more faithfully inherited prion variants, in effect alleles of these genes. What protein structure explains this protein-based inheritance? Using solid-state NMR, we showed that the infectious amyloids of the prion domains of Ure2p, Sup35p and Rnq1p have an in-register parallel architecture. This structure explains how the amyloid filament ends can template the structure of a new protein as it joins the filament.
The yeast prions [PSI+] and [URE3] are not found in wild strains, indicating they are a disadvantage to the cell. Moreover, the prion domains of Ure2p and Sup35p have functions unrelated to prion formation, indicating that these domains are not present for the purpose of forming prions. Indeed, prion forming ability is not conserved, even within S. cerevisiae, suggesting that the rare formation of prions is a disease. The prion domain sequences generally vary more rapidly in evolution than does the remainder of the molecule, producing a barrier to prion transmission, perhaps selected in evolution by this protection.
doi:10.1111/j.1567-1364.2010.00666.x
PMCID: PMC3025496  PMID: 20726897
prion; amyloid; in-register parallel structure
13.  Amyloid-Mediated Sequestration of Essential Proteins Contributes to Mutant Huntingtin Toxicity in Yeast 
PLoS ONE  2012;7(1):e29832.
Background
Polyglutamine expansion is responsible for several neurodegenerative disorders, among which Huntington disease is the most well-known. Studies in the yeast model demonstrated that both aggregation and toxicity of a huntingtin (htt) protein with an expanded polyglutamine region strictly depend on the presence of the prion form of Rnq1 protein ([PIN+]), which has a glutamine/asparagine-rich domain.
Principal Findings
Here, we showed that aggregation and toxicity of mutant htt depended on [PIN+] only quantitatively: the presence of [PIN+] elevated the toxicity and the levels of htt detergent-insoluble polymers. In cells lacking [PIN+], toxicity of mutant htt was due to the polymerization and inactivation of the essential glutamine/asparagine-rich Sup35 protein and related inactivation of another essential protein, Sup45, most probably via its sequestration into Sup35 aggregates. However, inhibition of growth of [PIN+] cells depended on Sup35/Sup45 depletion only partially, suggesting that there are other sources of mutant htt toxicity in yeast.
Conclusions
The obtained data suggest that induced polymerization of essential glutamine/asparagine-rich proteins and related sequestration of other proteins which interact with these polymers represent an essential source of htt toxicity.
doi:10.1371/journal.pone.0029832
PMCID: PMC3256205  PMID: 22253794
14.  Molecular Basis for Transmission Barrier and Interference between Closely Related Prion Proteins in Yeast* 
The Journal of Biological Chemistry  2011;286(18):15773-15780.
Replicating amyloids, called prions, are responsible for transmissible neurodegenerative diseases in mammals and some heritable phenotypes in fungi. The transmission of prions between species is usually inhibited, being highly sensitive to small differences in amino acid sequence of the prion-forming proteins. To understand the molecular basis of this prion interspecies barrier, we studied the transmission of the [PSI+] prion state from Sup35 of Saccharomyces cerevisiae to hybrid Sup35 proteins with prion-forming domains from four other closely related Saccharomyces species. Whereas all the hybrid Sup35 proteins could adopt a prion form in S. cerevisiae, they could not readily acquire the prion form from the [PSI+] prion of S. cerevisiae. Expression of the hybrid Sup35 proteins in S. cerevisiae [PSI+] cells often resulted in frequent loss of the native [PSI+] prion. Furthermore, all hybrid Sup35 proteins showed different patterns of interaction with the native [PSI+] prion in terms of co-polymerization, acquisition of the prion state, and induced prion loss, all of which were also dependent on the [PSI+] variant. The observed loss of S. cerevisiae [PSI+] can be related to inhibition of prion polymerization of S. cerevisiae Sup35 and formation of a non-heritable form of amyloid. We have therefore identified two distinct molecular origins of prion transmission barriers between closely sequence-related prion proteins: first, the inability of heterologous proteins to co-aggregate with host prion polymers, and second, acquisition by these proteins of a non-heritable amyloid fold.
doi:10.1074/jbc.M110.183889
PMCID: PMC3091186  PMID: 21454674
Amyloid; Prions; Protein Folding; Translation Release Factors; Yeast; Saccharomyces; Sup35; Prion Interference; Prion Species Barrier
15.  Molecular Structures of Amyloid and Prion Fibrils: Consensus vs. Controversy 
Accounts of chemical research  2013;46(7):1487-1496.
Conspectus
Many peptides and proteins self-assemble into amyloid fibrils, including polypeptides that are associated with human amyloid diseases, mammalian and fungal prion proteins, and proteins that are believed to have biologically functional amyloid states. Proper understanding of the common propensity for polypeptides to form amyloid fibrils depends on elucidation of the molecular structures of these fibrils, as does rational design of amyloid inhibitors and imaging agents. Whereas amyloid fibril structures were largely mysterious 15 years ago, a considerable body of reliable structural information now exists, with important contributions from solid state nuclear magnetic resonance (NMR) measurements. This article reviews results from our laboratories and discusses several structural issues that have been sources of controversy.
In many cases, the molecular structures of amyloid fibrils are not determined uniquely by their amino acid sequences. Self-propagating, molecular-level polymorphism complicates the structure determination problem and can lead to apparent disagreements between results from different laboratories, when in fact different laboratories are simply studying different polymorphs. For 40-residue β-amyloid (Aβ1–40) fibrils associated with Alzheimer’s disease, we have developed detailed structural models from solid state NMR and electron microscopy data for two polymorphs, which we found to have similar peptide conformations, identical in-register parallel β-sheet organizations, but different overall symmetry. Other polymorphs have also been partially characterized by solid state NMR, and appear to have similar structures. In contrast, cryo-electron microscopy studies that use significantly different fibril growth conditions have identified structures that appear (at low resolution) to be different from those examined by solid state NMR.
The in-register parallel β-sheet organization found in β-amyloid fibrils has also been found in many other fibril-forming systems by solid state NMR and electron paramagnetic resonance (EPR), and is attributable to stabilization of amyloid structures by intermolecular interactions among like amino acids, including hydrophobic interactions and polar zippers. Surprisingly, antiparallel β-sheets have been identified and characterized by solid state NMR in certain fibrils formed by the D23N mutant of Aβ1–40, which is associated with early-onset, familial neurodegenerative disease. Antiparallel D23N-Aβ1–40 fibrils are metastable with respect to conversion to parallel structures, and therefore represent an off-pathway intermediate in the amyloid fibril formation process. Evidence for antiparallel β-sheets in other amyloid-formation intermediates has been obtained recently by other methods.
As an alternative to simple parallel and antiparallel β-sheet structures, β-helical structural models have been proposed for various fibrils, especially those formed by mammalian and fungal prion proteins. Solid state NMR and EPR data show that fibrils formed in vitro by recombinant PrP have in-register parallel β-sheet structures, but the structure of infectious PrP aggregates is not yet known definitively. The fungal HET-s prion protein has been shown by solid state NMR to have a β-helical structure, but all yeast prions studied by solid state NMR (i.e., Sup35p, Ure2p, and Rnq1p) have in-register parallel β-sheet structures, with the fibril core being formed by their Gln- and Asn-rich N-terminal segments.
doi:10.1021/ar300282r
PMCID: PMC3632659  PMID: 23294335
16.  Distinct Type of Transmission Barrier Revealed by Study of Multiple Prion Determinants of Rnq1 
PLoS Genetics  2010;6(1):e1000824.
Prions are self-propagating protein conformations. Transmission of the prion state between non-identical proteins, e.g. between homologous proteins from different species, is frequently inefficient. Transmission barriers are attributed to sequence differences in prion proteins, but their underlying mechanisms are not clear. Here we use a yeast Rnq1/[PIN+]-based experimental system to explore the nature of transmission barriers. [PIN+], the prion form of Rnq1, is common in wild and laboratory yeast strains, where it facilitates the appearance of other prions. Rnq1's prion domain carries four discrete QN-rich regions. We start by showing that Rnq1 encompasses multiple prion determinants that can independently drive amyloid formation in vitro and transmit the [PIN+] prion state in vivo. Subsequent analysis of [PIN+] transmission between Rnq1 fragments with different sets of prion determinants established that (i) one common QN-rich region is required and usually sufficient for the transmission; (ii) despite identical sequences of the common QNs, such transmissions are impeded by barriers of different strength. Existence of transmission barriers in the absence of amino acid mismatches in transmitting regions indicates that in complex prion domains multiple prion determinants act cooperatively to attain the final prion conformation, and reveals transmission barriers determined by this cooperative fold.
Author Summary
Prions, self-propagating protein conformations and causative agents of lethal neurodegenerative diseases, present a serious public health threat: they can arise sporadically and then spread by transmission to the same, as well as other, species. The risk of infecting humans with prions originating in wild and domestic animals is determined by the so-called transmission barriers. These barriers are attributed to differences in prion proteins from different species, but their underlying mechanisms are not clear. Recent findings that the prion state is transmitted through the interaction between short transmitting regions within prion domains revealed one type of transmission barrier, where productive templating is impeded by non-matching amino acids within transmitting regions. Here we present studies of the prion domain of the [PIN+]-forming protein, Rnq1, and describe a distinct type of transmission barrier not involving individual amino acid mismatches in the transmitting regions. Rnq1's prion domain is complex and encompasses four regions that can independently transmit the prion state. Our data suggest that multiple prion determinants of a complex prion domain act cooperatively to attain the prion conformation, and transmission barriers occur between protein variants that cannot form the same higher order structure, despite the identity of the region(s) driving the transmission.
doi:10.1371/journal.pgen.1000824
PMCID: PMC2809767  PMID: 20107602
17.  The Effects of Amino Acid Composition of Glutamine-Rich Domains on Amyloid Formation and Fragmentation 
PLoS ONE  2012;7(10):e46458.
Fragmentation of amyloid polymers by the chaperone Hsp104 allows them to propagate as prions in yeast. The factors which determine the frequency of fragmentation are unclear, though it is often presumed to depend on the physical strength of prion polymers. Proteins with long polyglutamine stretches represent a tractable model for revealing sequence elements required for polymer fragmentation in yeast, since they form poorly fragmented amyloids. Here we show that interspersion of polyglutamine stretches with various amino acid residues differentially affects the in vivo formation and fragmentation of the respective amyloids. Aromatic residues tyrosine, tryptophan and phenylalanine strongly stimulated polymer fragmentation, leading to the appearance of oligomers as small as dimers. Alanine, methionine, cysteine, serine, threonine and histidine also enhanced fragmentation, while charged residues, proline, glycine and leucine inhibited polymerization. Our data indicate that fragmentation frequency primarily depends on the recognition of fragmentation-promoting residues by Hsp104 and/or its co-chaperones, rather than on the physical stability of polymers. This suggests that differential exposure of such residues to chaperones defines prion variant-specific differences in polymer fragmentation efficiency.
doi:10.1371/journal.pone.0046458
PMCID: PMC3468588  PMID: 23071575
18.  Heterologous Prion Interactions are Altered by Mutations in the Prion Protein Rnq1p 
Journal of molecular biology  2009;388(3):583-596.
Prions in the yeast Saccharomyces cerevisiae show a surprising degree of interdependence. Specifically, the rate of appearance of the [PSI+] prion, which is thought to be an important mechanism to respond to changing environmental conditions, is greatly increased by another prion, [RNQ+]. While the domains of the Rnq1 protein important for formation of the [RNQ+] prion have been defined, the specific residues required remain unknown. Furthermore, residues in Rnq1p that mediate the interaction between [PSI+] and [RNQ+] are unknown. To identify residues important for prion protein interactions, we created a mutant library of Rnq1p clones in the context of a chimera that serves as proxy for [RNQ+] aggregates. Several of the mutant Rnq1p proteins showed structural differences in the aggregates they formed, as revealed by SDD-AGE. Additionally, several of the mutants showed a striking defect in the ability to promote [PSI+] induction. These data indicate that the mutants formed strain variants of [RNQ+]. By dissecting the mutations in the isolated clones, we found five single mutations that caused [PSI+] induction defects, S223P, F184S, Q239R, N297S, and Q298R. These are the first specific mutations characterized in Rnq1p that alter [PSI+] induction. Additionally, we have identified a region important for the propagation of certain strain variants of [RNQ+]. Deletion of this region (amino acids 284–317) affected propagation of the high variant but not medium or low [RNQ+] strain variants. Furthermore, when the low [RNQ+] strain variant was propagated by Δ284–317, [PSI+] induction was greatly increased. These data suggest that this region is important in defining the structure of the [RNQ+] strain variants. These data are consistent with a model of [PSI+] induction caused by physical interactions between Rnq1p and Sup35p.
doi:10.1016/j.jmb.2009.03.036
PMCID: PMC2706087  PMID: 19324054
19.  Yeast prions assembly and propagation 
Prion  2011;5(4):277-284.
Yeast prions are self-perpetuating protein aggregates that are at the origin of heritable and transmissible non-Mendelian phenotypic traits. Among these, [PSI+], [URE3] and [PIN+] are the most well documented prions and arise from the assembly of Sup35p, Ure2p and Rnq1p, respectively, into insoluble fibrillar assemblies. Fibril assembly depends on the presence of N- or C-terminal prion domains (PrDs) which are not homologous in sequence but share unusual amino-acid compositions, such as enrichment in polar residues (glutamines and asparagines) or the presence of oligopeptide repeats. Purified PrDs form amyloid fibrils that can convert prion-free cells to the prion state upon transformation. Nonetheless, isolated PrDs and full-length prion proteins have different aggregation, structural and infectious properties. In addition, mutations in the “non-prion” domains (non-PrDs) of Sup35p, Ure2p and Rnq1p were shown to affect their prion properties in vitro and in vivo. Despite these evidences, the implication of the functional non-PrDs in fibril assembly and prion propagation has been mostly overlooked. In this review, we discuss the contribution of non-PrDs to prion assemblies, and the structure-function relationship in prion infectivity in the light of recent findings on Sup35p and Ure2p assembly into infectious fibrils from our laboratory and others.
doi:10.4161/pri.5.4.18070
PMCID: PMC4012403  PMID: 22052349
prion; Sup35p; Ure2p; Rnq1p; [PSI+]; [URE3]; [PIN+]; amyloid fibrils
20.  The [RNQ+] prion 
Prion  2011;5(4):291-298.
The formation of fibrillar amyloid is most often associated with protein conformational disorders such as prion diseases, Alzheimer disease and Huntington disease. Interestingly, however, an increasing number of studies suggest that amyloid structures can sometimes play a functional role in normal biology. Several proteins form self-propagating amyloids called prions in the budding yeast Saccharomyces cerevisiae. These unique elements operate by creating a reversible, epigenetic change in phenotype. While the function of the non-prion conformation of the Rnq1 protein is unclear, the prion form, [RNQ+], acts to facilitate the de novo formation of other prions to influence cellular phenotypes. The [RNQ+] prion itself does not adversely affect the growth of yeast, but the overexpression of Rnq1p can form toxic aggregated structures that are not necessarily prions. The [RNQ+] prion is also involved in dictating the aggregation and toxicity of polyglutamine proteins ectopically expressed in yeast. Thus, the [RNQ+] prion provides a tractable model that has the potential to reveal significant insight into the factors that dictate how amyloid structures are initiated and propagated in both physiological and pathological contexts.
doi:10.4161/pri.5.4.18213
PMCID: PMC4012398  PMID: 22052347
[RNQ+]; [PSI+]; prion; polyglutamine; functional amyloid; toxic amyloid; chaperones; epigenetic
21.  The [PSI+] Prion Exists as a Dynamic Cloud of Variants 
PLoS Genetics  2013;9(1):e1003257.
[PSI+] is an amyloid-based prion of Sup35p, a subunit of the translation termination factor. Prion “strains” or “variants” are amyloids with different conformations of a single protein sequence, conferring different phenotypes, but each relatively faithfully propagated. Wild Saccharomyces cerevisiae isolates have SUP35 alleles that fall into three groups, called reference, Δ19, and E9, with limited transmissibility of [PSI+] between cells expressing these different polymorphs. Here we show that prion transmission pattern between different Sup35 polymorphs is prion variant-dependent. Passage of one prion variant from one Sup35 polymorph to another need not change the prion variant. Surprisingly, simple mitotic growth of a [PSI+] strain results in a spectrum of variant transmission properties among the progeny clones. Even cells that have grown for >150 generations continue to vary in transmission properties, suggesting that simple variant segregation is insufficient to explain the results. Rather, there appears to be continuous generation of a cloud of prion variants, with one or another becoming stochastically dominant, only to be succeeded by a different mixture. We find that among the rare wild isolates containing [PSI+], all indistinguishably “weak” [PSI+], are several different variants based on their transmission efficiencies to other Sup35 alleles. Most show some limitation of transmission, indicating that the evolved wild Sup35 alleles are effective in limiting the spread of [PSI+]. Notably, a “strong [PSI+]” can have any of several different transmission efficiency patterns, showing that “strong” versus “weak” is insufficient to indicate prion variant uniformity.
Author Summary
The [PSI+] prion (infectious protein) of yeast is a self-propagating amyloid (filamentous protein polymer) of the Sup35 protein, a subunit of the translation termination factor. A single protein can form many biologically distinct prions, called prion variants. Wild yeast strains have three groups of Sup35 sequences (polymorphs), which partially block transmission of the [PSI+] prion from cell to cell. We find that [PSI+] variants (including the rare [PSI+] from wild yeasts) show different transmission patterns from one Sup35 sequence to another. Moreover, we find segregation of different prion variants on mitotic growth and evidence for generation of new variants with growth under non-selective conditions. This data supports the “prion cloud” model, that prions are not uniform structures but have an array of related self-propagating amyloid structures.
doi:10.1371/journal.pgen.1003257
PMCID: PMC3561065  PMID: 23382698
22.  Expanding the yeast prion world 
Prion  2013;7(2):109-113.
Mammalian and fungal prion proteins form self-perpetuating β-sheet-rich fibrillar aggregates called amyloid. Prion inheritance is based on propagation of the regularly oriented amyloid structures of the prion proteins. All yeast prion proteins identified thus far contain aggregation-prone glutamine/asparagine (Gln/Asn)-rich domains, although the mammalian prion protein and fungal prion protein HET-s do not contain such sequences. In order to fill this gap, we searched for novel yeast prion proteins lacking Gln/Asn-rich domains via a genome-wide screen based on cross-seeding between two heterologous proteins and identified Mod5, a yeast tRNA isopentenyltransferase, as a novel non-Gln/Asn-rich yeast prion protein. Mod5 formed self-propagating amyloid fibers in vitro and the introduction of Mod5 amyloids into non-prion yeast induced dominantly and cytoplasmically heritable prion state [MOD+], which harbors aggregates of endogenous Mod5. [MOD+] yeast showed an increased level of membrane lipid ergosterol and acquired resistance to antifungal agents. Importantly, enhanced de novo formation of [MOD+] was observed when non-prion yeast was grown under selective pressures from antifungal drugs. Our findings expand the family of yeast prions to non-Gln/Asn-rich proteins and reveal the acquisition of a fitness advantage for cell survival through active prion conversion.
doi:10.4161/pri.22685
PMCID: PMC3609114  PMID: 23117914
prion; amyloid; Mod5; tRNA isopentenyltransferase; antifungal drug; cellular adaptation
23.  The Hsp70/90 cochaperone, Sti1, suppresses proteotoxicity by regulating spatial quality control of amyloid-like proteins 
Molecular Biology of the Cell  2013;24(23):3588-3602.
Escape of aberrant proteins from protein quality control leads to accumulation of toxic protein species. Sti1 interacts with Hsp70 to mediate spatial PQC of amyloid-like proteins by regulating their distribution in different intracellular protein-handling depots. Sti1 suppresses proteotoxicity by targeting amyloid-like proteins to perinuclear foci.
Conformational diseases are associated with the conversion of normal proteins into aggregation-prone toxic conformers with structures similar to that of β-amyloid. Spatial distribution of amyloid-like proteins into intracellular quality control centers can be beneficial, but cellular mechanisms for protective aggregation remain unclear. We used a high-copy suppressor screen in yeast to identify roles for the Hsp70 system in spatial organization of toxic polyglutamine-expanded Huntingtin (Huntingtin with 103Q glutamine stretch [Htt103Q]) into benign assemblies. Under toxic conditions, Htt103Q accumulates in unassembled states and speckled cytosolic foci. Subtle modulation of Sti1 activity reciprocally affects Htt toxicity and the packaging of Htt103Q into foci. Loss of Sti1 exacerbates Htt toxicity and hinders foci formation, whereas elevation of Sti1 suppresses Htt toxicity while organizing small Htt103Q foci into larger assemblies. Sti1 also suppresses cytotoxicity of the glutamine-rich yeast prion [RNQ+] while reorganizing speckled Rnq1–monomeric red fluorescent protein into distinct foci. Sti1-inducible foci are perinuclear and contain proteins that are bound by the amyloid indicator dye thioflavin-T. Sti1 is an Hsp70 cochaperone that regulates the spatial organization of amyloid-like proteins in the cytosol and thereby buffers proteotoxicity caused by amyloid-like proteins.
doi:10.1091/mbc.E13-06-0315
PMCID: PMC3842988  PMID: 24109600
24.  Heterologous Gln/Asn-Rich Proteins Impede the Propagation of Yeast Prions by Altering Chaperone Availability 
PLoS Genetics  2013;9(1):e1003236.
Prions are self-propagating conformations of proteins that can cause heritable phenotypic traits. Most yeast prions contain glutamine (Q)/asparagine (N)-rich domains that facilitate the accumulation of the protein into amyloid-like aggregates. Efficient transmission of these infectious aggregates to daughter cells requires that chaperones, including Hsp104 and Sis1, continually sever the aggregates into smaller “seeds.” We previously identified 11 proteins with Q/N-rich domains that, when overproduced, facilitate the de novo aggregation of the Sup35 protein into the [PSI +] prion state. Here, we show that overexpression of many of the same 11 Q/N-rich proteins can also destabilize pre-existing [PSI+] or [URE3] prions. We explore in detail the events leading to the loss (curing) of [PSI+] by the overexpression of one of these proteins, the Q/N-rich domain of Pin4, which causes Sup35 aggregates to increase in size and decrease in transmissibility to daughter cells. We show that the Pin4 Q/N-rich domain sequesters Hsp104 and Sis1 chaperones away from the diffuse cytoplasmic pool. Thus, a mechanism by which heterologous Q/N-rich proteins impair prion propagation appears to be the loss of cytoplasmic Hsp104 and Sis1 available to sever [PSI+].
Author Summary
Certain proteins can occasionally misfold into infectious aggregates called prions. Once formed, these aggregates grow by attracting the soluble form of that protein to join them. The presence of these aggregates can cause profound effects on cells and, in humans, can cause diseases such as transmissible spongiform encephalopathies (TSEs). In yeast, the aggregates are efficiently transmitted to daughter cells because they are cut into small pieces by molecular scissors (chaperones). Here we show that heritable prion aggregates are frequently lost when we overproduce certain other proteins with curing activity. We analyzed one such protein in detail and found that when it is overproduced it forms aggregates that sequester chaperones. This sequestration appears to block the ability of the chaperones to cut the prion aggregates. The result is that the prions get too large to be transmitted to daughter cells. Such sequestration of molecular scissors provides a potential approach to thwart the propagation of disease-causing infectious protein aggregates.
doi:10.1371/journal.pgen.1003236
PMCID: PMC3554615  PMID: 23358669
25.  Small Heat Shock Proteins Potentiate Amyloid Dissolution by Protein Disaggregases from Yeast and Humans 
PLoS Biology  2012;10(6):e1001346.
The authors define how small heat-shock proteins synergize to regulate the assembly and disassembly of a beneficial prion, and then they exploit this knowledge to identify the human amyloid depolymerase.
How small heat shock proteins (sHsps) might empower proteostasis networks to control beneficial prions or disassemble pathological amyloid is unknown. Here, we establish that yeast sHsps, Hsp26 and Hsp42, inhibit prionogenesis by the [PSI+] prion protein, Sup35, via distinct and synergistic mechanisms. Hsp42 prevents conformational rearrangements within molten oligomers that enable de novo prionogenesis and collaborates with Hsp70 to attenuate self-templating. By contrast, Hsp26 inhibits self-templating upon binding assembled prions. sHsp binding destabilizes Sup35 prions and promotes their disaggregation by Hsp104, Hsp70, and Hsp40. In yeast, Hsp26 or Hsp42 overexpression prevents [PSI+] induction, cures [PSI+], and potentiates [PSI+]-curing by Hsp104 overexpression. In vitro, sHsps enhance Hsp104-catalyzed disaggregation of pathological amyloid forms of α-synuclein and polyglutamine. Unexpectedly, in the absence of Hsp104, sHsps promote an unprecedented, gradual depolymerization of Sup35 prions by Hsp110, Hsp70, and Hsp40. This unanticipated amyloid-depolymerase activity is conserved from yeast to humans, which lack Hsp104 orthologues. A human sHsp, HspB5, stimulates depolymerization of α-synuclein amyloid by human Hsp110, Hsp70, and Hsp40. Thus, we elucidate a heretofore-unrecognized human amyloid-depolymerase system that could have applications in various neurodegenerative disorders.
Author Summary
Amyloid 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.
doi:10.1371/journal.pbio.1001346
PMCID: PMC3378601  PMID: 22723742

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