Isolation of drugs active against mammalian prions using a yeast-based screening assay

We have developed a rapid, yeast-based, two-step assay to screen for antiprion drugs. The method allowed us to identify several compounds effective against budding yeast prions responsible for the [PSI+] and [URE3] phenotypes. These inhibitors include the kastellpaolitines, a new class of compounds, and two previously known molecules, phenanthridine and 6-aminophenanthridine. Two potent promoters of mammalian prion clearance in vitro, quinacrine and chlorpromazine, which share structural similarities with the kastellpaolitines, were also active in the assay. The compounds isolated here were also active in promoting mammalian prion clearance. These results validate the present method as an efficient high-throughput screening approach to identify new prion inhibitors and furthermore suggest that biochemical pathways controlling prion formation and/or maintenance are conserved from yeast to humans.     

Using budding yeast to screen for anti-prion drugs

Prions are misfolded proteins capable of propagating their altered conformation which are commonly considered as the causative agent of transmissible spongiform encephalopathies, a class of fatal neurodegenerative diseases. Currently, no treatment for prion-based diseases is available. Recently we have developed a rapid, yeast-based, two-step assay to screen for anti-prion drugs [1]. This new method allowed us to identify several compounds that are effective in vivo against budding yeast [PSI+] and [URE3] prions but also able to promote mammalian prion clearance in three different cell culture-based assays. Taken together, these results validate our method as an economic and efficient high-throughput screening approach to identify novel prion inhibitors or to carry on comprehensive structure-activity studies for already isolated anti-mammalian prion drugs. These results suggest furthermore that biochemical pathways controlling prion formation and/or maintenance are conserved from yeast to human and thus amenable to pharmacological and genetic analysis. Finally, it would be very interesting to test active drugs isolated using the yeast-based assay in models for other diseases (neurodegenerative or not) involving amyloid fibers like Huntington's, Parkinson's or Alzheimer's diseases.     

The Toll-Like Receptor Agonist Imiquimod Is Active against Prions

Using a yeast-based assay, a previously unsuspected antiprion activity was found for imiquimod (IQ), a potent Toll-like receptor 7 (TLR7) agonist already used for clinical applications. The antiprion activity of IQ was first detected against yeast prions [PSI ⁺] and [URE3], and then against mammalian prion both ex vivo in a cell-based assay and in vivo in a transgenic mouse model for prion diseases. In order to facilitate structure-activity relationship studies, we conducted a new synthetic pathway which provides a more efficient means of producing new IQ chemical derivatives, the activity of which was tested against both yeast and mammalian prions. The comparable antiprion activity of IQ and its chemical derivatives in the above life forms further emphasizes the conservation of prion controlling mechanisms throughout evolution. Interestingly, this study also demonstrated that the antiprion activity of IQ and IQ-derived compounds is independent from their ability to stimulate TLRs. Furthermore, we found that IQ and its active chemical derivatives inhibit the protein folding activity of the ribosome (PFAR) in vitro.     

Recent advances of research on the [PSI+] prion in Saccharomyces cerevisiae

Prion diseases such as bovine spongiform encephalopathy or Creutzfeldt-Jakob disease have been extensively studied in recent years. Research in this field is being done in highly secured laboratories because of potential transmission of prions to humans. Emerging similarities between mammalian and yeast prions allow using yeast-based assays to examine the activity of anti-prion drugs. Besides the intensively studied clinical aspects of prion diseases, the evolutionary aspects of prion proteins present in the yeast Saccharomyces cerevisiae are also extensively investigated. One of the key feature of prions, the ability to be stable in two alternative conformations, seems to play an important role in the evolution of this fungi, although some authors point out the negative influence of these particles upon yeast physiology. In this review, the most intensively studied fields of the research carried out on [PSI+] prion in yeast are summarized.     

Yeast as a model for studying the prion and amyloid occurrence

Recent data on the use of yeast as a model for studying the molecular basis of prion infection are summarized. In contrast to mammalian prions, which are related to incurable neurodegenerative diseases, yeast prions determine the appearance of non-chromosomally inherited phenotypic traits. Prions of yeast are structurally similar to amyloids of mammals and their replication involves not only growth, but also fragmentation of prion amyloid-like fibrils. In mammals the fragmentation should lead to an increase in infectious titer. The use of yeast for study of the mechanisms of human amyloidoses, development of new anti-prion drugs and search for new proteins with prion properties is described.     

New aspects of research upon the yeast Saccharomyces cerevisiae [PSI+] prion

One of the key feature of prions is the ability to be stable in two alternative conformations. Besides the intensively studied mammalian prions, there are also prion proteins present in the yeast Saccharomyces cerevisiae. Research in this field has lead to opposite hypotheses that explain the sense of presence of [PSI+] prion in yeast cells. Some authors postulate e of role of the prions in the evolution of S. cerevisiae, whereas other investigators point out the negative influence of these particles upon the yeast physiology. In recent years, yeast prions are used for anti-prion drug screening, because of common features with mammalian prions. This work presents the most intensively studied fields of the research carried out on [PSI+] prion in yeast.     

Newly identified prions in budding yeast, and their possible functions

Yeast prions are atypical genetic elements that are transmitted as heritable protein conformations. [PSI+], [URE3], and [PIN+] are three well-studied prions in the budding yeast, Saccharomyces cerevisiae. In the last three years, several additional prions have been reported in yeast, including [SWI+], [OCT+], [MCA], [GAR+], [MOT3+], [ISP+], and [NSI+]. The growing number of yeast prions suggests that protein-based inheritance might be a widespread biological phenomenon. In this review, we summarize the characteristics of each prion element, and discuss their potential functional roles in yeast biology.     

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.     

New anti-prion drugs make yeast blush

Prions are misfolded proteins capable of propagating their altered conformational state. They have been identified as the causative agents of a class of neurodegenerative diseases termed spongiform encephalopathies. No treatment for prion diseases is currently available. In a recent paper, Bach et al. describe a yeast-based approach for the development of anti-prion drugs. This approach could prove convenient for the identification and improvement of anti-prion pharmacologicals. From a fundamental point of view this study underscores the mechanistic similarities between mammalian and yeast prions.     

Prions in yeast

The concept of a prion as an infectious self-propagating protein isoform was initially proposed to explain certain mammalian diseases. It is now clear that yeast also has heritable elements transmitted via protein. Indeed, the "protein only" model of prion transmission was first proven using a yeast prion. Typically, known prions are ordered cross-β aggregates (amyloids). Recently, there has been an explosion in the number of recognized prions in yeast. Yeast continues to lead the way in understanding cellular control of prion propagation, prion structure, mechanisms of de novo prion formation, specificity of prion transmission, and the biological roles of prions. This review summarizes what has been learned from yeast prions.     

Antihypertensive drug guanabenz is active in vivo against both yeast and mammalian prions

Background

Prion-based diseases are incurable transmissible neurodegenerative disorders affecting animals and humans.

Methodology/Principal Findings

Here we report the discovery of the in vivo antiprion activity of Guanabenz (GA), an agonist of α2-adrenergic receptors routinely used in human medicine as an antihypertensive drug. We isolated GA in a screen for drugs active in vivo against two different yeast prions using a previously described yeast-based two steps assay. GA was then shown to promote ovine PrP^Sc clearance in a cell-based assay. These effects are very specific as evidenced by the lack of activity of some GA analogues that we generated. GA antiprion activity does not involve its agonist activity on α2-adrenergic receptors as other chemically close anti-hypertensive agents possessing related mechanism of action were found inactive against prions. Finally, GA showed activity in a transgenic mouse-based in vivo assay for ovine prion propagation, prolonging slightly but significantly the survival of treated animals.

Conclusion/Significance

GA thus adds to the short list of compounds active in vivo in animal models for the treatment of prion-based diseases. Because it has been administrated for many years to treat hypertension on a daily basis, without major side-effects, our results suggest that it could be evaluated in human as a potential treatment for prion-based diseases.     

The [PSI+] prion of yeast: a problem of inheritance

The [PSI(+)] prion of the yeast Saccharomyces cerevisiae was first identified by Brian Cox some 40 years ago as a non-Mendelian genetic element that modulated the efficiency of nonsense suppression. Following the suggestion by Reed Wickner in 1994 that such elements might be accounted for by invoking a prion-based model, it was subsequently established that the [PSI(+)] determinant was the prion form of the Sup35p protein. In this article, we review how a combination of classical genetic approaches and modern molecular and biochemical methods has provided conclusive evidence of the prion basis of the [PSI(+)] determinant. In so doing we have tried to provide a historical context, but also describe the results of more recent experiments aimed at elucidating the mechanism by which the [PSI(+)] (and other yeast prions) are efficiently propagated in dividing cells. While understanding of the [PSI(+)] prion and its mode of propagation has, and will continue to have, an impact on mammalian prion biology nevertheless the very existence of a protein-based mechanism that can have a beneficial impact on a cell's fitness provides equally sound justification to fully explore yeast prions.     

Cross-talk between RNA and prions

As concepts evolve in mammalian and yeast prion biology, rather preliminary research investigating the interplay between prion and RNA processes are gaining momentum. The yeast prion [PSI+] represents an aggregated state of the translation termination factor Sup35 resulting in the tendency of ribosomes to readthrough stop codons. This "nonsense suppression" activity is investigated for its possible physiological role to engender on Saccharomyces cerevisiae the ability to respond to stress or variable growth conditions and thereby act as a capacitor to evolve. The interaction between prion and RNA is a two way street--the cell may have adopted RNA processes in translation to govern the presence of prions and the [PSI+] prion's nonsense suppressor phenotype may exhibit different growth phenotypes by its control of translation termination. RNA processes in the mammalian cell also effect and are affected by prions.     

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.     

Modulation of prion-dependent polyglutamine aggregation and toxicity by chaperone proteins in the yeast model

In yeast, aggregation and toxicity of the expanded polyglutamine fragment of human huntingtin strictly depend on the presence of the endogenous self-perpetuating aggregated proteins (prions), which contain glutamine/asparagine-rich domains. Some chaperones of the Hsp100/70/40 complex, modulating propagation of yeast prions, were also reported to influence polyglutamine aggregation in yeast, but it was not clear whether they do it directly or via affecting prions. Our data show that although some chaperone alterations indeed act on polyglutamines via curing endogenous prions, other alterations decrease size and ameliorate toxicity of polyglutamine aggregates without affecting prion propagation. Therefore, the role of yeast chaperones in polyglutamine aggregation and toxicity is not restricted only to their effects on the endogenous prions. Moreover, chaperone interactions with prion and polyglutamine aggregates appear to be of a highly specific nature. One and the same chaperone alteration, substitution A503V in the middle region of the chaperone Hsp104, exhibited opposite effects on one of the endogenous prions ([PSI(+)], the prion form of Sup35) and on polyglutamines, increasing aggregate size and toxicity in the former case and decreasing them in the latter case. On the other hand, different members of a single chaperone family exhibited opposite effects on one and the same type of aggregates: excess of the Hsp40 chaperone Ydj1 increased polyglutamine aggregate size and toxicity, whereas excess of the other Hsp40 chaperone, Sis1, decreased them. As many stress-defense proteins are conserved between yeast and mammals, these data shed light on possible mechanisms modulating polyglutamine aggregation and toxicity in mammalian cells.     

Chaperoning prions: the cellular machinery for propagating an infectious protein?

Newly made polypeptide chains require the help of molecular chaperones not only to rapidly reach their final three-dimensional forms, but also to unfold and then correctly refold them back to their biologically active form should they misfold. Most prions are an unusual type of protein that can exist in one of two stable conformations, one of which leads to formation of an infectious alternatively folded form. Studies in Baker's yeast (Saccharomyces cerevisiae) have revealed that prions can exploit the molecular chaperone machinery in the cell in order to ensure stable propagation of the infectious, aggregation-prone form. The disaggregation of yeast prion aggregates by molecular chaperones generates forms of the prion protein that can seed the protein polymerisation that underlies the prion propagation cycle. In this article, we review what we have learnt about the role of molecular chaperones in yeast prion propagation, describe a model that can explain the role of various classes of molecular chaperones and their co-chaperones, and speculate on the possible involvement of chaperones in the propagation of mammalian prions.     

The [RNQ+] prion: A model of both functional and pathological amyloid

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.     

The yeast prions [PSI+] and [URE3] are molecular degenerative diseases

The yeast prions [URE3] and [PSI] are not found in wild strains, suggesting they are not an advantage. Prion-forming ability is not conserved, even within Saccharomyces, suggesting it is a disease. Prion domains have non-prion functions, explaining some conservation of sequence. However, in spite of the sequence being constrained in evolution by these non-prion functions, the prion domains vary more rapidly than the remainder of the molecule, and these changes produce a transmission barrier, suggesting that these changes were selected to block prion infection. Yeast prions [PSI] and [URE3] induce a cellular stress response (Hsp104 and Hsp70 induction), suggesting the cells are not happy about being infected. Recently, we showed that the array of [PSI] and [URE3] prions includes a majority of lethal or very toxic variants, a result not expected if either prion were an adaptive cellular response to stress.Key words: [URE3], [PSI⁺], prion, Sup35p, Ure2pfMammalian prions are uniformly fatal, but a lethal yeast prion would not be detected by the usual procedure, which requires growth of a colony under some selective condition. As a result, the prion variants commonly studied are quite mild in their effects. This circumstance has led to the suggestion that yeast prions actually benefit their host. Sup35p, the translation termination subunit whose amyloid becomes the [PSI⁺] prion, is essential for growth and Ure2p, the nitrogen regulation protein whose amyloid constitutes the [URE3] prion, is important for growth, with ure2 mutants showing noticeably slowed growth.When yeast prions were discovered,¹ we assumed they were diseases, by analogy with the mammalian diseases and the many non-prion amyloid diseases. Inactivating the essential Sup35p or the desireable Ure2p did not seem like a useful strategy. While control of either protein''s activity might be advantageous, and Ure2p activity control is the key to regulation of nitrogen catabolism, prion formation is a stochastic process, so it makes control of activity of these proteins random instead of appropriate to the circumstances. The [Het-s] prion changed that picture.² Here was a prion necessary for a normal function, heterokaryon incompatibility, and we suggested that it was the first beneficial prion.³     

An insight into the complex prion-prion interaction network in the budding yeast Saccharomyces cerevisiae

The budding yeast Saccharomyces cerevisiae is a valuable model system for studying prion-prion interactions as it contains multiple prion proteins. A recent study from our laboratory showed that the existence of Swi1 prion ([SWI⁺]) and overproduction of Swi1 can have strong impacts on the formation of 2 other extensively studied yeast prions, [PSI⁺] and [PIN⁺] ([RNQ⁺]) (Genetics, Vol. 197, 685–700). We showed that a single yeast cell is capable of harboring at least 3 heterologous prion elements and these prions can influence each other's appearance positively and/or negatively. We also showed that during the de novo [PSI⁺] formation process upon Sup35 overproduction, the aggregation patterns of a preexisting inducer ([RNQ⁺] or [SWI⁺]) can undergo significant remodeling from stably transmitted dot-shaped aggregates to aggregates that co-localize with the newly formed Sup35 aggregates that are ring/ribbon/rod- shaped. Such co-localization disappears once the newly formed [PSI⁺] prion stabilizes. Our finding provides strong evidence supporting the “cross-seeding” model for prion-prion interactions and confirms earlier reports that the interactions among different prions and their prion proteins mostly occur at the initiation stages of prionogenesis. Our results also highlight a complex prion interaction network in yeast. We believe that elucidating the mechanism underlying the yeast prion-prion interaction network will not only provide insight into the process of prion de novo generation and propagation in yeast but also shed light on the mechanisms that govern protein misfolding, aggregation, and amyloidogenesis in higher eukaryotes.     

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.