The effects of glutamine/asparagine content on aggregation and heterologous prion induction by yeast prion-like domains

Prion-like domains are low complexity, intrinsically disordered domains that compositionally resemble yeast prion domains. Many prion-like domains are involved in the formation of either functional or pathogenic protein aggregates. These aggregates range from highly dynamic liquid droplets to highly ordered detergent-insoluble amyloid-like aggregates. To better understand the amino acid sequence features that promote conversion to stable, detergent-insoluble aggregates, we used the prediction algorithm PAPA to identify predicted aggregation-prone prion-like domains with a range of compositions. While almost all of the predicted aggregation-prone domains formed foci when expressed in cells, the ability to form the detergent-insoluble aggregates was highly correlated with glutamine/asparagine (Q/N) content, suggesting that high Q/N content may specifically promote conversion to the amyloid state in vivo. We then used this data set to examine cross-seeding between prion-like proteins. The prion protein Sup35 requires the presence of a second prion, [PIN⁺], to efficiently form prions, but this requirement can be circumvented by the expression of various Q/N-rich protein fragments. Interestingly, almost all of the Q/N-rich domains that formed SDS-insoluble aggregates were able to promote prion formation by Sup35, highlighting the highly promiscuous nature of these interactions.     

Extra N-Terminal Residues Have a Profound Effect on the Aggregation Properties of the Potential Yeast Prion Protein Mca1

The metacaspase Mca1 from Saccharomyces cerevisiae displays a Q/N-rich region at its N-terminus reminiscent of yeast prion proteins. In this study, we show that the ability of Mca1 to form insoluble aggregates is modulated by a peptide stretch preceding its putative prion-forming domain. Based on its genomic locus, three potential translational start sites of Mca1 can give rise to two slightly different long Mca1 proteins or a short version, Mca1_451/453 and Mca1_432, respectively, although under normal physiological conditions Mca1₄₃₂ is the predominant form expressed. All Mca1 variants exhibit the Q/N-rich regions, while only the long variants Mca1_451/453 share an extra stretch of 19 amino acids at their N-terminal end. Strikingly, only long versions of Mca1 but not Mca1₄₃₂ revealed pronounced aggregation in vivo and displayed prion-like properties when fused to the C-terminal domain of Sup35 suggesting that the N-terminal peptide element promotes the conformational switch of Mca1 protein into an insoluble state. Transfer of the 19 N-terminal amino acid stretch of Mca1₄₅₁ to the N-terminus of firefly luciferase resulted in increased aggregation of luciferase, suggesting a protein destabilizing function of the peptide element. We conclude that the aggregation propensity of the potential yeast prion protein Mca1 in vivo is strongly accelerated by a short peptide segment preceding its Q/N-rich region and we speculate that such a conformational switch might occur in vivo via the usage of alternative translational start sites.     

Compositional Determinants of Prion Formation in Yeast

Numerous prions (infectious proteins) have been identified in yeast that result from the conversion of soluble proteins into β-sheet-rich amyloid-like protein aggregates. Yeast prion formation is driven primarily by amino acid composition. However, yeast prion domains are generally lacking in the bulky hydrophobic residues most strongly associated with amyloid formation and are instead enriched in glutamines and asparagines. Glutamine/asparagine-rich domains are thought to be involved in both disease-related and beneficial amyloid formation. These domains are overrepresented in eukaryotic genomes, but predictive methods have not yet been developed to efficiently distinguish between prion and nonprion glutamine/asparagine-rich domains. We have developed a novel in vivo assay to quantitatively assess how composition affects prion formation. Using our results, we have defined the compositional features that promote prion formation, allowing us to accurately distinguish between glutamine/asparagine-rich domains that can form prion-like aggregates and those that cannot. Additionally, our results explain why traditional amyloid prediction algorithms fail to accurately predict amyloid formation by the glutamine/asparagine-rich yeast prion domains.Amyloid fibers are associated with a large number of neurodegenerative diseases and systemic amyloidoses. Amyloid fibrils are rich in a cross-beta quaternary structure in which β-strands are perpendicular to the long axis of the fibril (8).[URE3] and [PSI⁺] are the prion (infectious protein) forms of the Saccharomyces cerevisiae proteins Ure2 and Sup35, respectively (61). Formation of both prions involves conversion of the native proteins into an infectious, amyloid form. Ure2 and Sup35 have served as powerful model systems for examining the basis for amyloid formation and propagation. Both proteins possess a well-ordered functional domain responsible for the normal function of the protein, while a functionally and structurally separate glutamine/asparagine (Q/N)-rich intrinsically disordered domain is necessary and sufficient for prion aggregation and propagation (4, 26, 27, 52, 53). Both proteins can form multiple prion variants, which are distinguished by the efficiency of prion propagation and by the precise structure of the amyloid core (14, 54).Five other prion proteins have also been identified in yeast: Rnq1 (13, 46), Swi1 (15), Cyc8 (33), Mca1 (30), and Mot3 (1). Numerous other proteins, including New1, contain domains that show prion activity when inserted in place of the Sup35 prion-forming domain (PFD) (1, 42). Each of these prion proteins contains a Q/N-rich PFD. Similar Q/N-rich domains are overrepresented in eukaryotic genomes (28), raising the intriguing possibility that prion-like structural conversions by Q/N-rich domains may be common in other eukaryotes. However, we currently have little ability to predict whether a given Q/N-rich domain can form prions.A variety of algorithms have been developed to predict a peptide''s propensity to form amyloid fibrils based on its amino acid sequence, including BETASCAN (6), TANGO (17), Zyggregator (51), SALSA (62), and PASTA (55). These algorithms have been successful at identifying regions prone to amyloid aggregation and predicting the effects of mutations on aggregation propensity for many amyloid-forming proteins. However, they have generally been quite ineffective for Q/N-rich amyloid domains such as the yeast PFDs. For example, using the statistical mechanics-based algorithm TANGO (17), which predicts aggregation propensity based on a peptide''s physicochemical properties, Linding et al. found that the Sup35 and Ure2 PFDs both completely lack predicted β-aggregation nuclei (24). Similarly, yeast PFDs are generally lacking in the hydrophobic residues predicted by algorithms such as Zyggregator to nucleate amyloid formation.Why are these algorithms so effective for many amyloid-forming proteins but not for yeast PFDs? For most amyloid proteins, amyloid formation is driven by short hydrophobic protein stretches, and increased hydrophobicity is correlated with an increased amyloid aggregation propensity (34). In contrast, the yeast PFDs are all highly polar domains, due largely to the high concentration of Q/N residues and the lack of hydrophobic residues. High Q/N content is clearly not a requirement for a domain to act as a prion in yeast, since neither the mammalian prion protein PrP nor the Podospora anserina prion protein HET-s is Q/N rich, yet fragments from both proteins can act as prions in yeast (49, 50). However, the significant compositional differences between the yeast PFDs and most other amyloid/prion proteins suggest that there may be two distinct classes of amyloid-forming proteins driven by different types of interactions. Specifically, Q/N residues, which are predicted to have a relatively low amyloid propensity in the context of hydrophobic amyloid domains (34), may promote amyloid formation when present at sufficiently high density. Stacking of Q/N residues to form polar zippers has been proposed to stabilize amyloid fibrils (35). Consistent with this hypothesis, mutational studies of Sup35 indicate that Q/N residues are critical for driving [PSI⁺] formation (12), and expanded poly-Q or poly-N tracts are sufficient to drive amyloid aggregation (36, 63). Therefore, this paper examines the sequence features that allow the polar, Q/N-rich yeast PFDs to form prions.Mutational studies of the PFDs of Ure2 and Sup35 have shown that amino acid composition is the predominant feature driving prion formation (40, 41). Due to the unique compositional biases observed in the yeast PFDs, algorithms have been developed to identify potential PFDs based solely on amino acid composition (19, 28, 42). These algorithms are designed to produce a list of potential prion proteins that meet a specific set of criteria (such as high Q/N content) but are not able to predict the prion propensity of each member of the list or to predict the effects of mutations on prion formation. A recent study by Alberti et al. was the first to systematically test whether compositional similarity to known PFDs is sufficient to distinguish between Q/N-rich proteins that form prions and those that do not. They developed a hidden Markov model to identify domains that are compositionally similar to known PFDs and then analyzed the 100 highest-scoring Q/N-rich domains in a series of in vivo and in vitro assays (1). Remarkably, they discovered 18 proteins with prion-like activity in all assays. However, an equal number, including some of the domains with greatest compositional similarity to known PFDs, showed no prion-like activity.This inability to distinguish between Q/N-rich proteins that form prions and those that do not might seem to suggest that amino acid composition is not an accurate predictor of prion propensity. However, an alternative explanation is that known yeast PFDs are not an ideal training set for a composition-based prediction algorithm, since yeast prions are likely not optimized for maximal prion propensity. It is unclear whether yeast prion formation is a beneficial phenomenon providing a mechanism to regulate protein activity or a detrimental phenomenon analogous to human amyloid disease. [PSI⁺] can increase resistance to certain stress conditions (56), but the failure to observe [PSI⁺] in wild yeast strains (29) argues that beneficial [PSI⁺] formation is at most a rare event. If yeast prions are diseases, the PFDs certainly would not be optimized for maximum prion potential. If prion formation is a beneficial event allowing for rapid conversion between active and inactive states, the prion potential of the PFD would be optimized such that the frequencies of prion formation and loss would yield the optimal balance of prion and nonprion cells (25). Thus, specific residues might be excluded from yeast PFDs either because they inhibit prion formation or because they too strongly promote prion formation; bioinformatic analysis can reveal which residues are excluded from yeast PFDs but not why they are excluded. Accurate prediction of prion propensity requires understanding which deviations from known prion-forming compositions will promote prion formation and which will inhibit.We have therefore developed the first in vivo method to quantitatively determine the prion propensity for each amino acid in the context of a Q/N-rich PFD. As expected, we found proline and charged residues to be strongly inhibitory to prion formation; but surprisingly, despite being largely underrepresented in yeast PFDs, hydrophobic residues strongly promoted prion formation. Furthermore, although Q/N residues dominate yeast PFDs, prion propensity appears relatively insensitive to the exact number of Q/N residues. Using these data, we were able to distinguish with approximately 90% accuracy between Q/N-rich domains that can form prion-like aggregates and those that cannot. These experiments provide the first detailed insight into the compositional requirements for yeast prion formation and illuminate the different methods by which Q/N- and non-Q/N-rich amyloidogenic proteins aggregate.     

Manipulating the aggregation activity of human prion-like proteins

Considerable advances in understanding the protein features favoring prion formation in yeast have facilitated the development of effective yeast prion prediction algorithms. Here we discuss a recent study in which we systematically explored the utility of the yeast prion prediction algorithm PAPA for designing mutations to modulate the aggregation activity of the human prion-like protein hnRNPA2B1. Mutations in hnRNPA2B1 cause multisystem proteinopathy in humans, and accelerate aggregation of the protein in vitro. Additionally, mutant hnRNPA2B1 forms cytoplasmic inclusions when expressed in Drosophila, and the mutant prion-like domain can substitute for a portion of a yeast prion domain in supporting prion activity in yeast. PAPA was quite successful at predicting the effects of PrLD mutations on prion activity in yeast and on in vitro aggregation propensity. Additionally, PAPA successfully predicted the effects of most, but not all, mutations in the PrLD of the hnRNPA2B1 protein when expressed in Drosophila. These results suggest that PAPA is quite effective at predicting the effects of mutations on intrinsic aggregation propensity, but that intracellular factors can influence aggregation and prion-like activity in vivo. A more complete understanding of these intracellular factors may inform the next generation of prion prediction algorithms.     

Scrambled prion domains form prions and amyloid

The [URE3] prion of Saccharomyces cerevisiae is a self-propagating amyloid form of Ure2p. The amino-terminal prion domain of Ure2p is necessary and sufficient for prion formation and has a high glutamine (Q) and asparagine (N) content. Such Q/N-rich domains are found in two other yeast prion proteins, Sup35p and Rnq1p, although none of the many other yeast Q/N-rich domain proteins have yet been found to be prions. To examine the role of amino acid sequence composition in prion formation, we used Ure2p as a model system and generated five Ure2p variants in which the order of the amino acids in the prion domain was randomly shuffled while keeping the amino acid composition and C-terminal domain unchanged. Surprisingly, all five formed prions in vivo, with a range of frequencies and stabilities, and the prion domains of all five readily formed amyloid fibers in vitro. Although it is unclear whether other amyloid-forming proteins would be equally resistant to scrambling, this result demonstrates that [URE3] formation is driven primarily by amino acid composition, largely independent of primary sequence.     

Expanding the yeast prion world

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.     

The story of stolen chaperones

Prions are self-seeding alternate protein conformations. Most yeast prions contain glutamine/asparagine (Q/N)-rich domains that promote the formation of amyloid-like prion aggregates. Chaperones, including Hsp104 and Sis1, are required to continually break these aggregates into smaller “seeds.” Decreasing aggregate size and increasing the number of growing aggregate ends facilitates both aggregate transmission and growth. Our previous work showed that overexpression of 11 proteins with Q/N-rich domains facilitates the de novo aggregation of Sup35 into the [PSI⁺] prion, presumably by a cross-seeding mechanism. We now discuss our recent paper, in which we showed that overexpression of most of these same 11 Q/N-rich proteins, including Pin4C and Cyc8, destabilized pre-existing Q/N rich prions. Overexpression of both Pin4C and Cyc8 caused [PSI⁺] aggregates to enlarge. This is incompatible with a previously proposed “capping” model where the overexpressed Q/N-rich protein poisons, or “caps,” the growing aggregate ends. Rather the data match what is expected of a reduction in prion severing by chaperones. Indeed, while Pin4C overexpression does not alter chaperone levels, Pin4C aggregates sequester chaperones away from the prion aggregates. Cyc8 overexpression cures [PSI⁺] by inducing an increase in Hsp104 levels, as excess Hsp104 binds to [PSI⁺] aggregates in a way that blocks their shearing.     

Heterologous Gln/Asn-Rich Proteins Impede the Propagation of Yeast Prions by Altering Chaperone Availability

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 ⁺].     

Suppression of polyglutamine toxicity by the yeast Sup35 prion domain in Drosophila

The propensity of proteins to form beta-sheet-rich amyloid fibrils is related to a variety of biological phenomena, including a number of human neurodegenerative diseases and prions. A subset of amyloidogenic proteins forms amyloid fibrils through glutamine/asparagine (Q/N)-rich domains, such as pathogenic polyglutamine (poly(Q)) proteins involved in neurodegenerative disease, as well as yeast prions. In the former, the propensity of an expanded poly(Q) tract to abnormally fold confers toxicity on the respective protein, leading to neuronal dysfunction. In the latter, Q/N-rich prion domains mediate protein aggregation important for epigenetic regulation. Here, we investigated the relationship between the pathogenic ataxin-3 protein of the human disease spinocerebellar ataxia type 3 (SCA3) and the yeast prion Sup35, using Drosophila as a model system. We found that the capacity of the Sup35 prion domain to mediate protein aggregation is conserved in Drosophila. Although select yeast prions enhance poly(Q) toxicity in yeast, the Sup35N prion domain suppressed poly(Q) toxicity in the fly. Suppression required the oligopeptide repeat of the Sup35N prion domain, which is critical for prion properties in yeast. These results suggest a trans effect of prion domains on pathogenic poly(Q) disease proteins in a multicellular environment and raise the possibility that Drosophila may allow studies of prion mechanisms.     

Dissection and design of yeast prions

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.     

The effects of amino acid composition on yeast prion formation and prion domain interactions

Yeast prions provide a powerful model system for examining prion formation and propagation in vivo. Yeast prion formation is driven primarily by amino acid composition, not by primary amino acid sequence. However, although yeast prion domains are consistently glutamine/asparagine-rich, they otherwise vary significantly in their compositions. Therefore, elucidating the exact compositional requirements for yeast prion formation has proven challenging. We have developed an in vivo method that allows for estimation of the prion propensity of each amino acid within the context of a yeast prion domain.¹ Using these values, we are able to predict the prion-propensity of various glutamine/asparagine-rich yeast domains. These results provide insight into the basis for yeast prion formation, and may aid in the discovery of additional novel prion domains. Additionally, we examined whether amino acid composition could drive interactions between heterologous glutamine/asparagine-rich proteins.² Although inefficient interactions between yeast prion domains have previously been observed, we found that one prion protein, Ure², is able to interact with compositionally similar domains with unprecedented efficiency. This observation, combined with the growing number of yeast prions, suggests that a broad network of interactions between heterologous glutamine/asparagine-rich proteins may affect yeast prion formation.Key words: yeast, prion, amyloid, Ure2, Sup35The highly-studied yeast prion proteins Sup35, Ure2 and Rnq1, which form the [PSI⁺], [URE3] and [PIN⁺] prions, respectively, each contain a glutamine/asparagine (Q/N) rich domain that drives prion formation. ³ Prion formation is thought to involve conversion of the soluble proteins into an insoluble amyloid form.³ Four additional yeast prion proteins containing Q/N-rich prion-forming domains have recently been discovered, and domains with similar Q/N-content are over-represented in various eukaryotic genomes.^4–8 This suggests that numerous other prion proteins may remain to be discovered, yet predicting which of these Q/N-rich domains is likely to form prions has proven difficult.Randomizing the order of the amino acids in either the Sup35 or Ure2 prion domains does not block prion formation, demonstrating that prion formation is driven primarily by amino acid composition, not primary sequence.^9,10 We therefore explored two related questions. First, can amino acid composition be used to accurately predict prion propensity?¹ Second, to what extent can compositionally similar Q/N-rich proteins interact during prion formation?²Surprisingly, although composition drives yeast prion formation, compositional similarity to known prion domains is a poor predictor of prion propensity. In addition to high Q/N content, yeast prion domains show an under-representation of both charged and hydrophobic residues (1^,11 Alberti et al. recently identified the 100 yeast domains with greatest compositional similarity to the known prion domains, and tested each domain in four different assays for prion-like activity.⁴ This remarkable effort revealed numerous new potential prion domains; however, there was very little correlation between a domain’s compositional similarity to known prion domains and its prion-like activity.^1,4 The most likely explanation for this unexpected finding is that because the yeast prion domains are likely not optimized for maximum prion propensity, some compositional deviations will increase prion propensity and some will decrease it. However, algorithms that predict prion propensity based on compositional similarity to known prion domains are predicated on the assumption that all compositional changes will reduce prion propensity. Instead, accurate prediction of prion propensity requires understanding how changes in amino acid composition affect prion propensity.

Table 1

Prion propensity, order propensity and prevalence for each amino acid

Protein misfolding in Dictyostelium: Using a freak of nature to gain insight into a universal problem

Abstract

Prion-like proteins can undergo conformational rearrangements from an intrinsically disordered to a highly ordered amyloid state. This ability to change conformation is encoded in distinctive domains, termed prion domains (PrDs). Previous work suggests that PrDs change conformation to affect protein function and create phenotypic diversity. More recent work shows that PrDs can also undergo many weak interactions when disordered, allowing them to organize the intracellular space into dynamic compartments. However, mutations within PrDs and altered aggregation properties have also been linked to age-related diseases in humans. Thus, the physiological role of prion-like proteins, the mechanisms regulating their conformational promiscuity and the links to disease are still unclear. Here, we summarize recent work with prion-like proteins in Dictyostelium discoideum. This work was motivated by the finding that D. discoideum has the highest content of prion-like proteins of all organisms investigated to date. Surprisingly, we find that endogenous and exogenous prion-like proteins remain soluble in D. discoideum and do not misfold and aggregate. We provide evidence that this is due to specific adaptations in the protein quality control machinery, which may allow D. discoideum to tolerate its highly aggregation-prone proteome. We predict that D. discoideum will be an important model to study the function of prion-like proteins and their mechanistic links to disease.     

Essential role of coiled coils for aggregation and activity of Q/N-rich prions and PolyQ proteins

The functional switch of glutamine/asparagine (Q/N)-rich prions and the neurotoxicity of polyQ-expanded proteins involve complex aggregation-prone structural transitions, commonly presumed to be forming β sheets. By analyzing sequences of interaction partners of these proteins, we discovered a recurrent presence of coiled-coil domains both in the partners and in segments that flank or overlap Q/N-rich and polyQ domains. Since coiled coils can mediate protein interactions and multimerization, we studied their possible involvement in Q/N-rich and polyQ aggregations. Using circular dichroism and chemical crosslinking, we found that Q/N-rich and polyQ peptides form α-helical coiled coils in?vitro and assemble into multimers. Using structure-guided mutagenesis, we found that coiled-coil domains modulate in?vivo properties of two Q/N-rich prions and polyQ-expanded huntingtin. Mutations that disrupt coiled coils impair aggregation and activity, whereas mutations that enhance coiled-coil propensity promote aggregation. These findings support a coiled-coil model for the functional switch of Q/N-rich prions and for the pathogenesis of polyQ-expansion diseases.     

What Makes a Protein Sequence a Prion?

Typical amyloid diseases such as Alzheimer''s and Parkinson''s were thought to exclusively result from de novo aggregation, but recently it was shown that amyloids formed in one cell can cross-seed aggregation in other cells, following a prion-like mechanism. Despite the large experimental effort devoted to understanding the phenomenon of prion transmissibility, it is still poorly understood how this property is encoded in the primary sequence. In many cases, prion structural conversion is driven by the presence of relatively large glutamine/asparagine (Q/N) enriched segments. Several studies suggest that it is the amino acid composition of these regions rather than their specific sequence that accounts for their priogenicity. However, our analysis indicates that it is instead the presence and potency of specific short amyloid-prone sequences that occur within intrinsically disordered Q/N-rich regions that determine their prion behaviour, modulated by the structural and compositional context. This provides a basis for the accurate identification and evaluation of prion candidate sequences in proteomes in the context of a unified framework for amyloid formation and prion propagation.     

Interdependence of amyloid formation in yeast

In eukaryotic cells amyloid aggregates may incorporate various functionally unrelated proteins. In mammalian diseases this may cause amyloid toxicity, while in yeast this could contribute to prion phenotypes. Insolubility of amyloids in the presence of strong ionic detergents, such as SDS or sarcosyl, allows discrimination between amorphous and amyloid aggregates. Here, we used this property of amyloids to study the interdependence of their formation in yeast. We observed that SDS-resistant polymers of proteins with extended polyglutamine domains caused the appearance of SDS or sarcosyl-insoluble polymers of three tested chromosomally-encoded Q/N-rich proteins, Sup35, Rnq1 and Pub1. These polymers were non-heritable, since they could not propagate in the absence of polyglutamine polymers. Sup35 prion polymers caused the appearance of non-heritable sarcosyl-resistant polymers of Pub1. Since eukaryotic genomes encode hundreds of proteins with long Q/N-rich regions, polymer interdependence suggests that conversion of a single protein into polymer form may significantly affect cell physiology by causing partial transfer of other Q/N-rich proteins into a non-functional polymer state.     

Protein misfolding in Dictyostelium: Using a freak of nature to gain insight into a universal problem

Prion-like proteins can undergo conformational rearrangements from an intrinsically disordered to a highly ordered amyloid state. This ability to change conformation is encoded in distinctive domains, termed prion domains (PrDs). Previous work suggests that PrDs change conformation to affect protein function and create phenotypic diversity. More recent work shows that PrDs can also undergo many weak interactions when disordered, allowing them to organize the intracellular space into dynamic compartments. However, mutations within PrDs and altered aggregation properties have also been linked to age-related diseases in humans. Thus, the physiological role of prion-like proteins, the mechanisms regulating their conformational promiscuity and the links to disease are still unclear. Here, we summarize recent work with prion-like proteins in Dictyostelium discoideum. This work was motivated by the finding that D. discoideum has the highest content of prion-like proteins of all organisms investigated to date. Surprisingly, we find that endogenous and exogenous prion-like proteins remain soluble in D. discoideum and do not misfold and aggregate. We provide evidence that this is due to specific adaptations in the protein quality control machinery, which may allow D. discoideum to tolerate its highly aggregation-prone proteome. We predict that D. discoideum will be an important model to study the function of prion-like proteins and their mechanistic links to disease.KEYWORDS: amyloid, Hsp104, prion, molecular chaperone, phase separation, protein aggregate, protein misfolding, ubiquitin/proteasome system     

Pathogenic Polyglutamine Tracts Are Potent Inducers of Spontaneous Sup35 and Rnq1 Amyloidogenesis

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.     

A non-Q/N-rich prion domain of a foreign prion, [Het-s], can propagate as a prion in yeast

Prions are self-propagating, infectious aggregates of misfolded proteins. The mammalian prion, PrP(Sc), causes fatal neurodegenerative disorders. Fungi also have prions. While yeast prions depend upon glutamine/asparagine (Q/N)-rich regions, the Podospora anserina HET-s and PrP prion proteins lack such sequences. Nonetheless, we show that the HET-s prion domain fused to GFP propagates as a prion in yeast. Analogously to native yeast prions, transient overexpression of the HET-s fusion induces ring-like aggregates that propagate in daughter cells as cytoplasmically inherited, detergent-resistant dot aggregates. Efficient dot propagation, but not ring formation, is dependent upon the Hsp104 chaperone. The yeast prion [PIN(+)] enhances HET-s ring formation, suggesting that prions with and without Q/N-rich regions interact. Finally, HET-s aggregates propagated in yeast are infectious when introduced into Podospora. Taken together, these results demonstrate prion propagation in a truly foreign host. Since yeast can host non-Q/N-rich prions, such native yeast prions may exist.     

Prion-like proteins and their computational identification in proteomes

Introduction: The aberrant or misfolded forms of the prion protein have been described as the causative agents of rare transmissible spongiform encephalopathies. In addition, proteins associated with frequently occurring neurodegenerative disorders, such as Alzheimer’s and Parkinson’s, are shown to share prion-like properties and to spread the disease in the brain.

Areas covered: Interest in the prion phenomenon has crystallized in a series of computational methods aimed at uncovering prion-like proteins at the proteome level. These programs rely on the identification of sequence signatures similar to those of yeast prions, whose structural conversion is driven by specific domains enriched in glutamine/asparagine residues. A myriad of prion-like candidates, similar to those in yeast, are predicted to exist in organisms across all kingdoms of life. We review here the role of prions, prionoids and prion-like proteins in health and disease, with a special focus on the algorithms and databases developed for their prediction and classification.

Expert commentary: Computational approaches provide novel insights into prion-like protein functions, their regulation and their role in disease.     

Yeast prions assembly and propagation: Contributions of the prion and non-prion moieties and the nature of assemblies

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.