Destruction or potentiation of different prions catalyzed by similar Hsp104 remodeling activities

Yeast prions are protein-based genetic elements that self-perpetuate changes in protein conformation and function. A protein-remodeling factor, Hsp104, controls the inheritance of several yeast prions, including those formed by Sup35 and Ure2. Perplexingly, deletion of Hsp104 eliminates Sup35 and Ure2 prions, whereas overexpression of Hsp104 purges cells of Sup35 prions, but not Ure2 prions. Here, we used pure components to dissect how Hsp104 regulates prion formation, growth, and division. For both Sup35 and Ure2, Hsp104 catalyzes de novo prion nucleation from soluble, native protein. Using a distinct mechanism, Hsp104 fragments both prions to generate new prion assembly surfaces. For Sup35, the fragmentation endpoint is an ensemble of noninfectious, amyloid-like aggregates and soluble protein that cannot replicate conformation. In vivid distinction, the endpoint of Ure2 fragmentation is short prion fibers with enhanced infectivity and self-replicating ability. These advances explain the distinct effects of Hsp104 on the inheritance of the two prions.     

Interspecies transmission of prions

Mammalian prions are infectious agents of proteinaceous nature that cause several incurable neurodegenerative diseases. Interspecies transmission of prions is usually impeded or impossible. Barriers in prion transmission are caused by small interspecies differences in the primary structure of prion proteins. The barriers can also depend on the strain (variant) of a transmitted prion. Interspecies barriers were also shown for yeast prions, which define some heritable phenotypes. Yeast prions reproduce all the main traits of prion transmission barriers observed for mammals. This allowed to show that the barrier in prion transmission can be observed even upon copolymerization of two prionogenic proteins. Available data allow elucidation of the mechanisms that impede prion transmission or make it impossible.     

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.     

Prion-based memory of heat stress in yeast

Amyloids and amyloid-based prions are self-perpetuating protein aggregates which can spread by converting a normal protein of the same sequence into a prion form. They are associated with diseases in humans and mammals, and control heritable traits in yeast and other fungi. Some amyloids are implicated in biologically beneficial processes. As prion formation generates reproducible memory of a conformational change, prions can be considered as molecular memory devices. We have demonstrated that in yeast, stress-inducible cytoskeleton-associated protein Lsb2 forms a metastable prion in response to high temperature. This prion promotes conversion of other proteins into prions and can persist in a fraction of cells for a significant number of cell generations after stress, thus maintaining the memory of stress in a population of surviving cells. Acquisition of an amino acid substitution required for Lsb2 to form a prion coincides with acquisition of increased thermotolerance in the evolution of Saccharomyces yeast. Thus the ability to form an Lsb2 prion in response to stress coincides with yeast adaptation to growth at higher temperatures. These findings intimately connect prion formation to the cellular response to environmental stresses.     

Hsp70 targets Hsp100 chaperones to substrates for protein disaggregation and prion fragmentation

Hsp100 and Hsp70 chaperones in bacteria, yeast, and plants cooperate to reactivate aggregated proteins. Disaggregation relies on Hsp70 function and on ATP-dependent threading of aggregated polypeptides through the pore of the Hsp100 AAA(+) hexamer. In yeast, both chaperones also promote propagation of prions by fibril fragmentation, but their functional interplay is controversial. Here, we demonstrate that Hsp70 chaperones were essential for species-specific targeting of their Hsp100 partner chaperones ClpB and Hsp104, respectively, to heat-induced protein aggregates in vivo. Hsp70 inactivation in yeast also abrogated Hsp104 targeting to almost all prions tested and reduced fibril mobility, which indicates that fibril fragmentation by Hsp104 requires Hsp70. The Sup35 prion was unique in allowing Hsp70-independent association of Hsp104 via its N-terminal domain, which, however, was nonproductive. Hsp104 overproduction even outcompeted Hsp70 for Sup35 prion binding, which explains why this condition prevented Sup35 fragmentation and caused prion curing. Our findings indicate a conserved mechanism of Hsp70-Hsp100 cooperation at the surface of protein aggregates and prion fibrils.     

Life cycle of cytosolic prions

Prions are self-templating protein aggregates that were originally identified as the causative agent of prion diseases in mammals, but have since been discovered in other kingdoms. Mammalian prions represent a unique class of infectious agents that are composed of misfolded prion protein. Prion proteins usually exist as soluble proteins but can refold and assemble into highly ordered, self-propagating prion polymers. The prion concept is also applicable to a growing number of non-Mendelian elements of inheritance in lower eukaryotes. While prions identified in mammals are clearly pathogens, prions in lower eukaryotes can be either detrimental or beneficial to the host. Prion phenotypes in fungi are transmitted vertically from mother to daughter cells during cell division and horizontally during mating or abortive mating, but extracellular phases have not been reported. Recent findings now demonstrate that in a mammalian cell environment, protein aggregates derived from yeast prion domains exhibit a prion life cycle similar to mammalian prions propagated ex vivo. This life cycle includes a soluble state of the protein, an induction phase by exogenous prion fibrils, stable replication of prion entities, vertical transmission to progeny and natural horizontal transmission to neighboring cells. Our data reveal that mammalian cells contain all co-factors required for cytosolic prion propagation and dissemination. This has important implications for understanding prion-like properties of disease-related protein aggregates. In light of the growing number of identified functional amyloids, cell-to-cell propagation of cytosolic protein conformers might not only be relevant for the spreading of disease-associated proteins, but might also be of more general relevance under non-disease conditions.     

Prions

Prions were originally defined as infectious agents of protein nature, which caused neurodegenerative diseases in animals and humans. The prion concept implies that the infectious agent is a protein in special conformation that can be transmitted to the normal molecules of the same protein through protein-protein interactions. Until the 1990s, the prion phenomenon was associated with the single protein named PrP. Discovery of prions in lower eukaryotes, the yeast Saccharomyces cerevisiae and fungus Podospora anserina, suggests that prions have wider significance. Prions of lower eukaryotes are not related to diseases; their propagation caused by aggregation of prion-like proteins underlies the inheritance of phenotypic traits and most likely has adaptive significance. This review covers prions of mammals and lower eukaryotes, mechanisms of their appearance de novo and maintenance, structure of prion particles, and prospects for the treatment of prion diseases. Recent data concerning the search for new prion-like proteins is included. The paper focuses on the [PSI+] prion of S. cerevisiae, since at present it is the most investigated one. The biological significance of prions is discussed.     

The reconstitution of mammalian prion infectivity de novo

The discovery of prion disease transmission in mammals, as well as a non-Mendelian type of inheritance in yeast, has led to the establishment of a new concept in biology, the prion hypothesis. The prion hypothesis postulates that an abnormal protein conformation propagates itself in an autocatalytic manner using the normal isoform of the same protein as a substrate and thereby acts either as a transmissible agent of disease (in mammals), or as a heritable determinant of phenotype (in yeast and fungus). While the prion biology of yeast and fungus supports this idea strongly, the direct proof of the prion hypothesis in mammals, specifically the reconstitution of the disease-associated isoform of the prion protein (PrP(Sc)) in vitro de novo from noninfectious prion protein, has been difficult to achieve despite many years of effort. The present review summarizes our current knowledge about the biochemical nature of the prion infectious agent and structure of PrP(Sc), describes potential strategies for generating prion infectivity de novo and provides some insight on why the reconstitution of infectivity has been difficult to achieve in vitro. Several hypotheses are proposed to explain the apparently low infectivity of the first generation of recently reported synthetic mammalian prions.     

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.     

Distinct Amino Acid Compositional Requirements for Formation and Maintenance of the [PSI+] Prion in Yeast

Multiple yeast prions have been identified that result from the structural conversion of proteins into a self-propagating amyloid form. Amyloid-based prion activity in yeast requires a series of discrete steps. First, the prion protein must form an amyloid nucleus that can recruit and structurally convert additional soluble proteins. Subsequently, maintenance of the prion during cell division requires fragmentation of these aggregates to create new heritable propagons. For the Saccharomyces cerevisiae prion protein Sup35, these different activities are encoded by different regions of the Sup35 prion domain. An N-terminal glutamine/asparagine-rich nucleation domain is required for nucleation and fiber growth, while an adjacent oligopeptide repeat domain is largely dispensable for prion nucleation and fiber growth but is required for chaperone-dependent prion maintenance. Although prion activity of glutamine/asparagine-rich proteins is predominantly determined by amino acid composition, the nucleation and oligopeptide repeat domains of Sup35 have distinct compositional requirements. Here, we quantitatively define these compositional requirements in vivo. We show that aromatic residues strongly promote both prion formation and chaperone-dependent prion maintenance. In contrast, nonaromatic hydrophobic residues strongly promote prion formation but inhibit prion propagation. These results provide insight into why some aggregation-prone proteins are unable to propagate as prions.     

Insights into the mechanism of prion propagation

Proteins with prion properties have been identified in both mammals and fungi. The tractability of yeast as a genetic model has contributed significantly to our understanding of prion formation and propagation. A number of molecular chaperones have been found to modulate the ability of yeast prion proteins to propagate. The results of recent genetic and in vitro studies have shed light on the mechanism of prion propagation, the physical and structural basis of different prion strains and the species barrier, as well as the function and mechanism of the chaperones that interact with the prion proteins. Whether aspects of the mechanisms of formation, maintenance and clearance of prions are conserved between fungi and mammals remains to be seen.     

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.     

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.     

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.     

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.     

Converting the prion protein: what makes the protein infectious

The discovery of prion disease transmission in mammals, as well as a non-Mendelian type of inheritance in yeast, has led to the establishment of a new concept in biology, the prion hypothesis. The prion hypothesis postulates that an abnormal protein conformation propagates itself in an autocatalytic manner via recruitment of the normal isoform of the same protein as a substrate, and thereby acts either as a transmissible agent of disease (in mammals) or as a heritable determinant of phenotype (in yeast and fungus). Although reconstitution of fully infectious PrP(Sc)in vitro from synthetic components has not yet been achieved, numerous lines of evidence indicate that the prion protein is the major and essential component, if not the only one, of the prion infectious agent. This article summarizes our current knowledge about the chemical nature of the prion infectious agent, describes potential strategies and challenges related to the generation of prion infectivity de novo, proposes new hypotheses to explain the apparently low infectivity observed in the first synthetic mammalian prions, and describes plausible effects of chemical modifications on prion conversion.     

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.     

The utility of prions

Infectious, self-propagating protein aggregates (prions) as well as structurally related amyloid fibrils have traditionally been associated with neurodegenerative diseases in mammals. However, recent work in fungi indicates that prions are not simply aberrations of protein folding, but are in fact widespread, conserved, and in certain cases, apparently beneficial. Analysis of prion behavior in yeast has led to insights into the mechanisms of prion appearance and propagation as well as the effect of prions on cellular physiology and perhaps evolution. The prion-forming proteins of Saccharomyces cerevisiae are members of a larger class of Gln/Asn-rich proteins that is abundantly represented in the genomes of higher eukaryotes, raising the prospect of genetically programmed prion-like behavior in other organisms.     

Appearance and propagation of polyglutamine-based amyloids in yeast: tyrosine residues enable polymer fragmentation

In yeast, fragmentation of amyloid polymers by the Hsp104 chaperone allows them to propagate as prions. The prion-forming domain of the yeast Sup35 protein is rich in glutamine, asparagine, tyrosine, and glycine residues, which may define its prion properties. Long polyglutamine stretches can also drive amyloid polymerization in yeast, but these polymers are unable to propagate because of poor fragmentation and exist through constant seeding with the Rnq1 prion polymers. We proposed that fragmentation of polyglutamine amyloids may be improved by incorporation of hydrophobic amino acid residues into polyglutamine stretches. To investigate this, we constructed sets of polyglutamine with or without tyrosine stretches fused to the non-prion domains of Sup35. Polymerization of these chimeras started rapidly, and its efficiency increased with stretch size. Polymerization of proteins with polyglutamine stretches shorter than 70 residues required Rnq1 prion seeds. Proteins with longer stretches polymerized independently of Rnq1 and thus could propagate. The presence of tyrosines within polyglutamine stretches dramatically enhanced polymer fragmentation and allowed polymer propagation in the absence of Rnq1 and, in some cases, of Hsp104.     

Yeast Prions Compared to Functional Prions and Amyloids

Saccharomyces cerevisiae is an occasional host to an array of prions, most based on self-propagating, self-templating amyloid filaments of a normally soluble protein. [URE3] is a prion of Ure2p, a regulator of nitrogen catabolism, while [PSI +] is a prion of Sup35p, a subunit of the translation termination factor Sup35p. In contrast to the functional prions, [Het-s] of Podospora anserina and [BETA] of yeast, the amyloid-based yeast prions are rare in wild strains, arise sporadically, have an array of prion variants for a single prion protein sequence, have a folded in-register parallel β-sheet amyloid architecture, are detrimental to their hosts, arouse a stress response in the host, and are subject to curing by various host anti-prion systems. These characteristics allow a logical basis for distinction between functional amyloids/prions and prion diseases. These infectious yeast amyloidoses are outstanding models for the many common human amyloid-based diseases that are increasingly found to have some infectious characteristics.