A yeast-based assay to isolate drugs active against mammalian prions

Recently, we have developed a yeast-based (Saccharomyces cerevisiae) assay to isolate drugs active against mammalian prions. The initial assumption was that mechanisms controlling prion appearance and/or propagation could be conserved from yeast to human, as it is the case for most of the major cell biology regulatory mechanisms. Indeed, the vast majority of drugs we isolated as active against both [PSI(+)] and [URE3] budding yeast prions turned out to be also active against mammalian prion in three different mammalian cell-based assays. These results strongly argue in favor of common prion controlling mechanisms conserved in eukaryotes, thus validating our yeast-based assay and also the use of budding yeast to identify antiprion compounds and to study the prion world.     

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.     

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.     

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.     

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.     

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.     

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.     

Yeast prions, mammalian amyloidoses, and the problem of proteomic networks

Prion proteins are infective amyloids and cause several neurodegenerative diseases in humans and animals. In yeasts, prions are expressed as cytoplasmic heritable determinants of a protein nature. Yeast prion [PSI], which results from a conformational rearrangement and oligomerization of translation termination factor eRF3, is used as an example to consider the structural--functional relationships in a potentially prion molecule, specifics of its evolution, and interactions with other prions, which form so-called prion networks. In addition, the review considers the results of modeling mammalian prion diseases and other amyloidoses in yeast cells. A hypothesis of proteomic networks is proposed by analogy with prion networks, involving interactions of different amyloids in mammals.     

Neurodegenerative amyloidoses: the yeast model

More than 20 human diseases are related to protein misfolding which causes formation of amyloids, fibrillar aggregates of normally soluble proteins. Such diseases are called amyloid diseases or amyloidoses. Of them only prion diseases are transmissible. Amyloids of the prion type are described in lower eukaryotes. However, in contrast to mammalian prions, which cause incurable neurodegenerative diseases, prions of lower eukaryotes are related to some non-chromosomally inherited phenotypic traits. Here we summarize the results of studies of prions of the yeast Saccharomyces cerevisiae and of the use of yeast model for investigation of some human amyloidoses, such as prion diseases, Alzheimer's, Parkinson's, and Huntington's diseases.     

Yeast prions, mammalian amyloidoses, and the problem of proteomic networks

Prion proteins are infective amyloids and cause several neurodegenerative diseases in humans and animals. In yeasts, prions are detected as the cytoplasmic heritable determinants of a protein nature. Yeast prion [PSI], which results from a conformational rearrangement and oligomerization of translation termination factor eRF3, is used as an example to consider the structural-functional relationships in a potentially prion molecule, specifics of its evolution, and interactions with other prions, which form so-called prion networks. In addition, the review considers the results of modeling mammalian prion diseases and other amyloidoses in yeast cells. A hypothesis of proteomic networks is proposed by analogy with prion networks, involving interactions of different amyloids in mammals.     

Prion Domain of Yeast Ure2 Protein Adopts a Completely Disordered Structure: A Solid-Support EPR Study

Amyloid fibril formation is associated with a range of neurodegenerative diseases in humans, including Alzheimer’s, Parkinson’s, and prion diseases. In yeast, amyloid underlies several non-Mendelian phenotypes referred to as yeast prions. Mechanism of amyloid formation is critical for a complete understanding of the yeast prion phenomenon and human amyloid-related diseases. Ure2 protein is the basis of yeast prion [URE3]. The Ure2p prion domain is largely disordered. Residual structures, if any, in the disordered region may play an important role in the aggregation process. Studies of Ure2p prion domain are complicated by its high aggregation propensity, which results in a mixture of monomer and aggregates in solution. Previously we have developed a solid-support electron paramagnetic resonance (EPR) approach to address this problem and have identified a structured state for the Alzheimer’s amyloid-β monomer. Here we use solid-support EPR to study the structure of Ure2p prion domain. EPR spectra of Ure2p prion domain with spin labels at every fifth residue from position 10 to position 75 show similar residue mobility profile for denaturing and native buffers after accounting for the effect of solution viscosity. These results suggest that Ure2p prion domain adopts a completely disordered structure in the native buffer. A completely disordered Ure2p prion domain implies that the amyloid formation of Ure2p, and likely other Q/N-rich yeast prion proteins, is primarily driven by inter-molecular interactions.     

Prion protein interconversions

The transmissible spongiform encephalopathies (TSEs), or prion diseases, remain mysterious neurodegenerative diseases that involve perturbations in prion protein (PrP) structure. This article summarizes our use of in vitro models to describe how PrP is converted to the disease-associated, protease-resistant form. These models reflect many important biological parameters of TSE diseases and have been used to identify inhibitors of the PrP conversion as lead compounds in the development of anti-TSE drugs.     

朊病毒病治疗方法的研究进展

朊病毒是一种由体内正常朊蛋白转化形成的传染性蛋白质,朊病毒病是由朊病毒引发的致命性神经退行性疾病。目前临床虽然尚无治疗朊病毒病的方法,但是大量的研究者已从多个角度进行研究,并取得了一定进展。对近期有关传统化学药物、基因治疗方法、免疫学治疗方法和同源朊蛋白的朊病毒病治疗方法进行了综述,并重点分析了新型靶向细胞内信号通路药物以及有潜在利用价值的线粒体相关朊病毒胞内作用信号通路,旨在为朊病毒新的研究方向提供理论依据,从而促进朊病毒病治疗方法应用于临床。     

Use of yeast as a system to study amyloid toxicity

The formation of amyloid-like fibrils is a hallmark of several neurodegenerative diseases. How the assembly of amyloid-like fibrils contributes to cell death is a major unresolved question in the field. The budding yeast Saccharomyces cerevisiae is a powerful model organism to study basic mechanisms for how cellular pathways regulate amyloid assembly and proteotoxicity. For example, studies of the amyloidogenic yeast prion [RNQ(+)] have revealed novel roles by which molecular chaperones protect cells from the accumulation of cytotoxic protein species. In budding yeast there are a variety of cellular assays that can be employed to analyze the assembly of amyloid-like aggregates and mechanistically dissect how cellular pathways influence proteotoxicity. In this review, we describe several assays that are routinely used to investigate aggregation and toxicity of the [RNQ(+)] prion in yeast.     

Neurodegenerative amyloidoses: Yeast model

More than 20 human diseases are associated with protein misfolding, which results in the appearance of amyloids, fibrillar aggregates of normally soluble proteins. Such diseases are termed amyloid diseases, or amyloidoses. Of these, only prion diseases are transmissible. Amyloids of the prion type are known for lower eukaryotes. While mammalian prions cause neurodegenerative diseases, prions of lower eukaryotes are associated with some nonchromosomally inherited phenotypic traits. The review summarizes the results of studying the prions of yeast Saccharomyces cerevisiae and data obtained using S. cerevisiae as a model to investigate some human amyloidoses such as Alzheimer’s, Parkinson’s, Huntington’s, and prion diseases.     

A panel of monoclonal antibodies against the prion protein proves that there is no prion protein in human pancreatic ductal epithelial cells

Prion diseases are a group of neurodegenerative diseases that are fatal. The study of these unique diseases in China is hampered by a lack of resources. Amongst the most important resources for biological study are monoclonal antibodies. Here, we characterize a panel of monoclonal antibodies specific for cellular prion protein by enzyme-linked immunosorbent assay（ELISA）, immunofluorescent staining, flow cytometry, and western blotting. We identify several antibodies that can be used for specific applications and we demonstrate that there is no prion protein expression in human pancreatic ductal epithelial cells（HPDC）.     

Countering amyloid polymorphism and drug resistance with minimal drug cocktails

Several fatal, progressive neurodegenerative diseases, including various prion and prion-like disorders, are connected with the misfolding of specific proteins. These proteins misfold into toxic oligomeric species and a spectrum of distinct self-templating amyloid structures, termed strains. Hence, small molecules that prevent or reverse these protein-misfolding events might have therapeutic utility. Yet it is unclear whether a single small molecule can antagonize the complete repertoire of misfolded forms encompassing diverse amyloid polymorphs and soluble oligomers. We have begun to investigate this issue using the yeast prion protein Sup35 as an experimental paradigm. We have discovered that a polyphenol, (−)epigallocatechin-3-gallate (EGCG), effectively inhibited the formation of infectious amyloid forms (prions) of Sup35 and even remodeled preassembled prions. Surprisingly, EGCG selectively modulated specific prion strains and even selected for EGCG-resistant prion strains with novel structural and biological characteristics. Thus, treatment with a single small molecule antagonist of amyloidogenesis can select for novel, drug-resistant amyloid polymorphs. Importantly, combining EGCG with another small molecule, 4,5-bis-(4-methoxyanilino)phthalimide, synergistically antagonized and remodeled a wide array of Sup35 prion strains without producing any drug-resistant prions. We suggest that minimal drug cocktails, small collections of drugs that collectively antagonize all amyloid polymorphs, should be identified to besiege various neurodegenerative disorders.Key words: amyloid, yeast prion, Sup35, prion strains, EGCG, DAPH-12     

Heterologous cross-seeding mimics cross-species prion conversion in a yeast model

Background  

Prions are self-perpetuating, infectious, aggregated proteins that are associated with several neurodegenerative diseases in mammals and heritable traits in yeast. Sup35p, the protein determinant of the yeast prion [PSI ⁺], has a conserved C terminal domain that performs the Sup35p function and a prion domain that is highly divergent. Prions formed by chimeras of the prion domain of various species fused to the C domain of Saccharomyces cerevisiae exhibit a 'species barrier', a phenomenon first observed in mammals, and often fail to transmit the prion state to chimeras with prion domains of other species.     

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.