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.     

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 ⁺].     

Ure2p function is enhanced by its prion domain in Saccharomyces cerevisiae

The Ure2 protein of Saccharomyces cerevisiae can become a prion (infectious protein). At very low frequencies Ure2p forms an insoluble, infectious amyloid known as [URE3], which is efficiently transmitted to progeny cells or mating partners that consequently lose the normal Ure2p nitrogen regulatory function. The [URE3] prion causes yeast cells to grow slowly, has never been identified in the wild, and confers no obvious phenotypic advantage. An N-terminal asparagine-rich domain determines Ure2p prion-forming ability. Since ure2Delta strains are complemented by plasmids that overexpress truncated forms of Ure2p lacking the prion domain, the existence of the [URE3] prion and the evolutionary conservation of an N-terminal extension have remained mysteries. We find that Ure2p function is actually compromised in vivo by truncation of the prion domain. Moreover, Ure2p stability is diminished without the full-length prion domain. Mca1p, like Ure2p, has an N-terminal Q/N-rich domain whose deletion reduces its steady-state levels. Finally, we demonstrate that the prion domain may affect the interaction of Ure2p with other components of the nitrogen regulation system, specifically the negative regulator of nitrogen catabolic genes, Gzf3p.     

Controlling the prion propensity of glutamine/asparagine-rich proteins

ABSTRACT

The yeast Saccharomyces cerevisiae can harbor a number of distinct prions. Most of the yeast prion proteins contain a glutamine/asparagine (Q/N) rich region that drives prion formation. Prion-like domains, defined as regions with high compositional similarity to yeast prion domains, are common in eukaryotic proteomes, and mutations in various human proteins containing prion-like domains have been linked to degenerative diseases, including amyotrophic lateral sclerosis. Here, we discuss a recent study in which we utilized two strategies to generate prion activity in non-prion Q/N-rich domains. First, we made targeted mutations in four non-prion Q/N-rich domains, replacing predicted prion-inhibiting amino acids with prion-promoting amino acids. All four mutants formed foci when expressed in yeast, and two acquired bona fide prion activity. Prion activity could be generated with as few as two mutations, suggesting that many non-prion Q/N-rich proteins may be just a small number of mutations from acquiring aggregation or prion activity. Second, we created tandem repeats of short prion-prone segments, and observed length-dependent prion activity. These studies demonstrate the considerable progress that has been made in understanding the sequence basis for aggregation of prion and prion-like domains, and suggest possible mechanisms by which new prion domains could evolve.     

Mapping the Fibril Core of the Prion Subdomain of the Mammalian CPEB3 that is Involved in Long Term Memory Retention

Long-term memory storage is modulated by the prion nature of CPEB3 forming the molecular basis for the maintenance of synaptic facilitation. Here we report that the first prion sub-domain PRD1 of mouse CPEB3 can autonomously form amyloid fibrils in vitro and punctate-like structures in vivo. A ninety-four amino acid sequence within the PRD1 domain, PRD1-core, displays high propensity towards aggregation and associated amyloid characteristics. PRD1-core is characterized using electron microscopy, X-ray diffraction, and solution-state NMR deuterium exchange experiments. Secondary structure elements deduced from solid-state NMR reveal a β-rich core comprising of forty amino acids at the N-terminus of PRD1-core. The synthesized twenty-three amino acid long peptide containing the longest rigid segment (E124-H145) of the PRD1-core rapidly self-aggregates and forms fibrils, indicating a limited aggregation-prone region that could potentially activate the aggregation of the full-length protein. This study provides the first step in identifying the structural trigger for the CPEB3 aggregation process.     

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.     

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.     

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.     

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.     

Stress-dependent Proteolytic Processing of the Actin Assembly Protein Lsb1 Modulates a Yeast Prion

Yeast prions are self-propagating amyloid-like aggregates of Q/N-rich protein that confer heritable traits and provide a model of mammalian amyloidoses. [PSI⁺] is a prion isoform of the translation termination factor Sup35. Propagation of [PSI⁺] during cell division under normal conditions and during the recovery from damaging environmental stress depends on cellular chaperones and is influenced by ubiquitin proteolysis and the actin cytoskeleton. The paralogous yeast proteins Lsb1 and Lsb2 bind the actin assembly protein Las17 (a yeast homolog of human Wiskott-Aldrich syndrome protein) and participate in the endocytic pathway. Lsb2 was shown to modulate maintenance of [PSI⁺] during and after heat shock. Here, we demonstrate that Lsb1 also regulates maintenance of the Sup35 prion during and after heat shock. These data point to the involvement of Lsb proteins in the partitioning of protein aggregates in stressed cells. Lsb1 abundance and cycling between actin patches, endoplasmic reticulum, and cytosol is regulated by the Guided Entry of Tail-anchored proteins pathway and Rsp5-dependent ubiquitination. Heat shock-induced proteolytic processing of Lsb1 is crucial for prion maintenance during stress. Our findings identify Lsb1 as another component of a tightly regulated pathway controlling protein aggregation in changing environments.     

Prion Variants and Species Barriers Among Saccharomyces Ure2 Proteins

As hamster scrapie cannot infect mice, due to sequence differences in their PrP proteins, we find “species barriers” to transmission of the [URE3] prion in Saccharomyces cerevisiae among Ure2 proteins of S. cerevisiae, paradoxus, bayanus, cariocanus, and mikatae on the basis of differences among their Ure2p prion domain sequences. The rapid variation of the N-terminal Ure2p prion domains results in protection against the detrimental effects of infection by a prion, just as the PrP residue 129 Met/Val polymorphism may have arisen to protect humans from the effects of cannibalism. Just as spread of bovine spongiform encephalopathy prion variant is less impaired by species barriers than is sheep scrapie, we find that some [URE3] prion variants are infectious to another yeast species while other variants (with the identical amino acid sequence) are not. The species barrier is thus prion variant dependent as in mammals. [URE3] prion variant characteristics are maintained even on passage through the Ure2p of another species. Ure2p of Saccharomyces castelli has an N-terminal Q/N-rich “prion domain” but does not form prions (in S. cerevisiae) and is not infected with [URE3] from Ure2p of other Saccharomyces. This implies that conservation of its prion domain is not for the purpose of forming prions. Indeed the Ure2p prion domain has been shown to be important, though not essential, for the nitrogen catabolism regulatory role of the protein.     

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.     

Smad3 contributes to positioning of proliferating cells in colonic crypts by inducing EphB receptor protein expression

In prion diseases cellular prion protein (PrP^C) undergoes conformational transition into the β-sheet-rich form (PrP^Sc). PrP^C consists of the disordered N-terminal part and a C-terminal globular domain containing three α-helices (H1, H2, H3) and an antiparallel beta sheet (B1, B2). B2–H2 loop, which has a focal role in the species barrier, contains the highest density of asparagine (N) and glutamine (Q) residues in the whole sequence. Q/N-rich domains are essential for the conversion of yeast prions. We investigated the role of Q/N residues in the B2–H2 loop in PrP conversion. We prepared mouse PrP mutants with increasing number of consecutive Q/N residues in the B2–H2 loop. Stability of the mutants decreased with the increasing number of inserted glutamines. In vitro conversion of mutants yielded fibrils of similar morphology as the wild-type PrP. Q/N mutants accelerated fibrillization in comparison to the wild-type PrP, with mutant containing the most glutamines having the shortest lag phase. The effect of Q/N residues was specific for the B2–H2 loop and was not due to simple increase in flexibility as the introduction of Gly-Ser or Ala residues slowed the conversion despite their decreased stability. Our results thus suggest that Q/N residues in the B2–H2 loop of PrP promote protein conversion and may represent a link to conversion of Q/N-rich prions.     

A glutamine/asparagine-rich fragment of Gln3, but not the full-length protein,aggregates in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

The amino acid sequence of protein Gln3 in yeast Saccharomyces cerevisiae has a region enriched with Gln (Q) and Asn (N) residues. In this study, we analyzed the effects of overexpression of Gln3 and its Q/N-rich fragment fused with yellow fluorescent protein (YFP). Being overexpressed, full-length Gln3-YFP does not form aggregates, inhibits vegetative growth, and demonstrates nuclear localization, while the Q/N-rich fragment (Gln3QN) fused with YFP forms aggregates that do not colocalize with the nucleus and do not affect growth of the cells. Although detergent-resistant aggregates of Gln3QN are formed in the absence of yeast prions, the aggregation of Gln3QN significantly increases in the presence of [PIN⁺] prion, while in the presence of two prions, [PSI⁺] and [PIN⁺], the percentage of cells with Gln3QN aggregates is significantly lower than in the strain bearing only [PIN⁺]. Data on colocalization demonstrate that this effect is mediated by interaction between Gln3QN aggregates and [PSI⁺] and [PIN⁺] prions.     

Synthesis of pathological and nonpathological human exon 1 huntingtin

Huntington's disease (HD) is a neurodegenerative disorder that affects approximately 1 in 10 000 individuals. The underlying gene mutation was identified as a CAG‐triplet repeat expansion in the gene huntingtin. The CAG sequence codes for glutamine, and in HD, an expansion of the polyglutamine (poly‐Q) stretch above 35 glutamine residues results in pathogenicity. It has been demonstrated in various animal models that only the expression of exon 1 huntingtin, a 67‐amino acid‐long polypeptide plus a variable poly‐Q stretch, is sufficient to cause full HD‐like pathology. Therefore, a deeper understanding of exon 1 huntingtin, its structure, aggregation mechanism and interaction with other proteins is crucial for a better understanding of the disease. Here, we describe the synthesis of a 109‐amino acid‐long exon 1 huntingtin peptide including a poly‐Q stretch of 42 glutamines. This microwave‐assisted solid phase peptide synthesis resulted in milligram amounts of peptide with high purity. We also synthesized a nonpathogenic version of exon 1 huntingtin (90‐amino acid long including a poly‐Q stretch of 23 glutamine residues) using the same strategy. In circular dichroism spectroscopy, both polypeptides showed weak alpha‐helical properties with the longer peptide showing a higher helical degree. These model peptides have great potential for further biomedical analyses, e.g. for large‐scale pre‐screenings for aggregation inhibitors, further structural analyses as well as protein–protein interaction studies. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Structure and Assembly Properties of the N-Terminal Domain of the Prion Ure2p in Isolation and in Its Natural Context

Background

The aggregation of the baker''s yeast prion Ure2p is at the origin of the [URE3] trait. The Q- and N-rich N-terminal part of the protein is believed to drive Ure2p assembly into fibrils of amyloid nature and the fibrillar forms of full-length Ure2p and its N-terminal part generated in vitro have been shown to induce [URE3] occurrence when introduced into yeast cells. This has led to the view that the fibrillar form of the N-terminal part of the protein is sufficient for the recruitment of constitutive Ure2p and that it imprints its amyloid structure to full-length Ure2p.

Results

Here we generate a set of Ure2p N-terminal fragments, document their assembly and structural properties and compare them to that of full-length Ure2p. We identify the minimal region critical for the assembly of Ure2p N-terminal part into amyloids and show that such fibrils are unable to seed the assembly of full length Ure2p unlike fibrils made of intact Ure2p.

Conclusion

Our results clearly indicate that fibrillar Ure2p shares no structural similarities with the amyloid fibrils made of Ure2p N-terminal part. Our results further suggest that the induction of [URE3] by fibrils made of full-length Ure2p is likely the consequence of fibrils growth by depletion of cytosolic Ure2p while it is the consequence of de novo formation of prion particles following, for example, titration within the cells of a specific set of molecular chaperones when fibrils made of Ure2p N-terminal domain are introduced within the cytoplasm.     

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.     

Thermodynamic Description of Polymorphism in Q- and N-Rich Peptide Aggregates Revealed by Atomistic Simulation

Amyloid fibrils are long, helically symmetric protein aggregates that can display substantial variation (polymorphism), including alterations in twist and structure at the β-strand and protofilament levels, even when grown under the same experimental conditions. The structural and thermodynamic origins of this behavior are not yet understood. We performed molecular-dynamics simulations to determine the thermodynamic properties of different polymorphs of the peptide GNNQQNY, modeling fibrils containing different numbers of protofilaments based on the structure of amyloid-like cross-β crystals of this peptide. We also modeled fibrils with new orientations of the side chains, as well as a de novo designed structure based on antiparallel β-strands. The simulations show that these polymorphs are approximately isoenergetic under a range of conditions. Structural analysis reveals a dynamic reorganization of electrostatics and hydrogen bonding in the main and side chains of the Gln and Asn residues that characterize this peptide sequence. Q/N-rich stretches are found in several amyloidogenic proteins and peptides, including the yeast prions Sup35-N and Ure2p, as well as in the human poly-Q disease proteins, including the ataxins and huntingtin. Based on our results, we propose that these residues imbue a unique structural plasticity to the amyloid fibrils that they comprise, rationalizing the ability of proteins enriched in these amino acids to form prion strains with heritable and different phenotypic traits.     

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

Rationale-Based Engineering of a Potent Long-Acting FGF21 Analog for the Treatment of Type 2 Diabetes

Fibroblast growth factor 21 (FGF21) is a promising drug candidate for the treatment of type 2 diabetes. However, the use of wild type native FGF21 is challenging due to several limitations. Among these are its short half-life, its susceptibility to in vivo proteolytic degradation and its propensity to in vitro aggregation. We here describe a rationale-based protein engineering approach to generate a potent long-acting FGF21 analog with improved resistance to proteolysis and aggregation. A recombinant Fc-FGF21 fusion protein was constructed by fusing the Fc domain of human IgG1 to the N-terminus of human mature FGF21 via a linker peptide. The Fc positioned at the N-terminus was determined to be superior to the C-terminus as the N-terminal Fc fusion retained the βKlotho binding affinity and the in vitro and in vivo potency similar to native FGF21. Two specific point mutations were introduced into FGF21. The leucine to arginine substitution at position 98 (L98R) suppressed FGF21 aggregation at high concentrations and elevated temperatures. The proline to glycine replacement at position 171 (P171G) eliminated a site-specific proteolytic cleavage of FGF21 identified in mice and cynomolgus monkeys. The derived Fc-FGF21(RG) molecule demonstrated a significantly improved circulating half-life while maintaining the in vitro activity similar to that of wild type protein. The half-life of Fc-FGF21(RG) was 11 h in mice and 30 h in monkeys as compared to 1-2 h for native FGF21 or Fc-FGF21 wild type. A single administration of Fc-FGF21(RG) in diabetic mice resulted in a sustained reduction in blood glucose levels and body weight gains up to 5-7 days, whereas the efficacy of FGF21 or Fc-FGF21 lasted only for 1 day. In summary, we engineered a potent and efficacious long-acting FGF21 analog with a favorable pharmaceutical property for potential clinical development.