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.     

Infectious Fold and Amyloid Propagation in Podospora anserina

Aggregation of amyloid proteins is involved in serious neurodegenerative disorders such as Alzheimer disease and transmissible encephalopathies. The concept of an infectious protein (prion) proposed as the scrapie agent was successfully validated for several proteins of yeast and fungi. Ure2, Sup35 and Rnq1 in Saccharomyces cerevisiae and HET-s in Podospora anserina have been genetically, then biochemically identified as prion proteins. Studies on these proteins have brought critical informations on the mechanisms of prions appearance and propagation. The prion phenotype correlates with the aggregation state of these particular proteins. In vitro, the recombinant prion proteins form amyloid fibers characterized by a rich β-sheet content. In a previous work on the HET-s prion protein of Podospora we have demonstrated the infectivity of HET-s recombinant amyloid aggregates. More recently, the structural analysis of the prion domain of HET-s associated with in vivo mutagenesis allowed us to propose a model for the infectious fold of the HET-s prion domain. Further investigations to complete this model are discussed in this review as well as relevant questions about the [Het-s] system of Podospora anserina.     

Biological Roles of Prion Domains

In vivo amyloid formation is a widespread phenomenon in eukaryotes. Self-perpetuating amyloids provide a basis for the infectious or heritable protein isoforms (prions). At least for some proteins, amyloid-forming potential is conserved in evolution despite divergence of the amino acid (aa) sequences. In some cases, prion formation certainly represents a pathological process leading to a disease. However, there are several scenarios in which prions and other amyloids or amyloid-like aggregates are either shown or suspected to perform positive biological functions. Proven examples include self/nonself recognition, stress defense and scaffolding of other (functional) polymers. The role of prion-like phenomena in memory has been hypothesized. As an additional mechanism of heritable change, prion formation may in principle contribute to heritable variability at the population level. Moreover, it is possible that amyloid-based prions represent by-products of the transient feedback regulatory circuits, as normal cellular function of at least some prion proteins is decreased in the prion state.     

Biological Roles of Prion Domains

In vivo amyloid formation is a widespread phenomenon in eukaryotes. Self-perpetuating amyloids provide a basis for the infectious or heritable protein isoforms (prions). At least for some proteins, amyloid-forming potential is conserved in evolution despite divergence of the amino acid (aa) sequences. In some cases, prion formation certainly represents a pathological process leading to a disease. However, there are several scenarios in which prions and other amyloids or amyloid-like aggregates are either shown or suspected to perform positive biological functions. Proven examples include self/nonself recognition, stress defense and scaffolding of other (functional) polymers. The role of prion-like phenomena in memory has been hypothesized. As an additional mechanism of heritable change, prion formation may in principle contribute to heritable variability at the population level. Moreover, it is possible that amyloid-based prions represent by-products of the transient feedback regulatory circuits, as normal cellular function of at least some prion proteins is decreased in the prion state.Key Words: amyloid, amyloidosis, epigenetic, evolution, inheritance, mammals, misfolding, protein, stress, yeast     

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.     

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

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

Prion proteins as genetic material in fungi

Prions are infectious proteins. Several prions have been identified in fungi where they behave as non-Mendelian cytoplasmic genetic elements. Most of these prions propagate as self-perpetuating amyloid aggregates thus providing an example of structural heredity. In yeast, prion propagation requires the Hsp104 disaggregase presumably to sheer amyloid assemblies and generate more fiber ends. Recent work in yeast shows that amyloid structure polymorphism underlies the prion strain phenomenon and influences species barriers. Structural models for the amyloid form of several fungal prion proteins are now available. All propose a cross beta-organization with parallel beta-sheets. Whether or not some of the fungal prions might be beneficial to their host is still a debated issue.     

Infectious Fold and Amyloid Propagation in Podospora anserina

Amyloid protein aggregation is involved in serious neurodegenerative disorders such as Alzheimer''s disease and transmissible encephalopathies. The concept of an infectious protein (prion) being the scrapie agent was successfully validated for several yeast and fungi proteins. Ure2, Sup35 and Rnq1 in Saccharomyces cerevisiae and HET-s in Podospora anserina have been genetically and biochemically identified as prion proteins. Studies on these proteins have revealed critical information on the mechanisms of prions appearance and propagation. The prion phenotype correlates with the aggregation state of these particular proteins. In vitro, the recombinant prion proteins form amyloid fibers characterized by rich β sheet content. In a previous work on the HET-s prion protein Podospora, we demonstrated the infectivity of HET-s recombinant amyloid aggregates. More recently, the structural analysis of the HET-s prion domain associated with in vivo mutagenesis allowed us to propose a model for the infectious fold of the HET-s prion domain. Further investigations to complete this model are discussed in this review, as are relevant questions about the [Het-s] system of Podospora anserina.Key Words: prion, HET-s, Podospora, amyloid, infectious, β sheet, mutagenesis, fold, propagation     

Prions are novel infectious pathogens causing scrapie and Creutzfeldt-Jakob disease

Scrapie and Creutzfeldt–Jakob disease (CJD) are caused by prions, which appear to be different from both viruses and viroids. Prions contain protein which is required for infectivity, but no nucleic acid has been found within them. Prion proteins are encoded by a cellular gene and not by a nucleic acid within the infectious prion particle. A cellular homologue of the prion protein has been IDentified. The role of this homologue in metabolism is unknown. Prion proteins, but not the cellular homologue, aggregate into rod-shaped particles that are histo-chemically and ultrastructurally IDentical to amyloid. Extracellular collections of prion proteins form amyloid plaques in scrapie- and CJD-infected rodent brains as well as CJD-infected human brains. Within the plaques, prion proteins assemble to form amyloid filaments. Elucidating the molecular differences between the prion protein and its cellular homologue may be important in understanding the chemical structure and replication of prions.     

The expanding realm of prion phenomena in neurodegenerative disease

The aggregation of a soluble protein into insoluble, β-sheet rich amyloid fibrils is a defining characteristic of many neurodegenerative diseases, including prion disorders. The prion protein has so far been considered unique because of its infectious nature. Recent investigations, however, suggest that other amyloid-forming proteins associated with much more common diseases, such as tau, α-synuclein, amyloid β, and polyglutamine proteins, while not infectious in the classical sense, share certain essential properties with prions that may explain phenotypic diversity, and patterns of spread within the nervous system. We suggest a common mechanism of pathogenesis of myriad sporadic and inherited neurodegenerative diseases based on templated conformational change.     

The ultrastructure of infectious L-type bovine spongiform encephalopathy prions constrains molecular models

Bovine spongiform encephalopathy (BSE) is a prion disease of cattle that is caused by the misfolding of the cellular prion protein (PrP^C) into an infectious conformation (PrP^Sc). PrP^C is a predominantly α-helical membrane protein that misfolds into a β-sheet rich, infectious state, which has a high propensity to self-assemble into amyloid fibrils. Three strains of BSE prions can cause prion disease in cattle, including classical BSE (C-type) and two atypical strains, named L-type and H-type BSE. To date, there is no detailed information available about the structure of any of the infectious BSE prion strains. In this study, we purified L-type BSE prions from transgenic mouse brains and investigated their biochemical and ultrastructural characteristics using electron microscopy, image processing, and immunogold labeling techniques. By using phosphotungstate anions (PTA) to precipitate PrP^Sc combined with sucrose gradient centrifugation, a high yield of proteinase K-resistant BSE amyloid fibrils was obtained. A morphological examination using electron microscopy, two-dimensional class averages, and three-dimensional reconstructions revealed two structural classes of L-type BSE amyloid fibrils; fibrils that consisted of two protofilaments with a central gap and an average width of 22.5 nm and one-protofilament fibrils that were 10.6 nm wide. The one-protofilament fibrils were found to be more abundant compared to the thicker two-protofilament fibrils. Both fibrillar assemblies were successfully decorated with monoclonal antibodies against N- and C-terminal epitopes of PrP using immunogold-labeling techniques, confirming the presence of polypeptides that span residues 100–110 to 227–237. The fact that the one-protofilament fibrils contain both N- and C-terminal PrP epitopes constrains molecular models for the structure of the infectious conformer in favour of a compact four-rung β-solenoid fold.     

Lack of Prion Infectivity in Fixed Heart Tissue from Patients with Creutzfeldt-Jakob Disease or Amyloid Heart Disease

In most forms of prion disease, infectivity is present primarily in the central nervous system or immune system organs such as spleen and lymph node. However, a transgenic mouse model of prion disease has demonstrated that prion infectivity can also be present as amyloid deposits in heart tissue. Deposition of infectious prions as amyloid in human heart tissue would be a significant public health concern. Although abnormal disease-associated prion protein (PrP^Sc) has not been detected in heart tissue from several amyloid heart disease patients, it has been observed in the heart tissue of a patient with sporadic Creutzfeldt-Jakob Disease (sCJD), the most common form of human prion disease. In order to determine whether prion infectivity can be found in heart tissue, we have inoculated formaldehyde fixed brain and heart tissue from two sCJD patients, as well as prion protein positive fixed heart tissue from two amyloid heart disease patients, into transgenic mice overexpressing the human prion protein. Although the sCJD brain samples led to clinical or subclinical prion infection and deposition of PrP^Sc in the brain, none of the inoculated heart samples resulted in disease or the accumulation of PrP^Sc. Thus, our results suggest that prion infectivity is not likely present in cardiac tissue from sCJD or amyloid heart disease patients.     

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 many shades of prion strain adaptation

In several recent studies transmissible prion disease was induced in animals by inoculation with recombinant prion protein amyloid fibrils produced in vitro. Serial transmission of amyloid fibrils gave rise to a new class of prion strains of synthetic origin. Gradual transformation of disease phenotypes and PrP^Sc properties was observed during serial transmission of synthetic prions, a process that resembled the phenomenon of prion strain adaptation. The current article discusses the remarkable parallels between phenomena of prion strain adaptation that accompanies cross-species transmission and the evolution of synthetic prions occurring within the same host. Two alternative mechanisms underlying prion strain adaptation and synthetic strain evolution are discussed. The current article highlights the complexity of the prion transmission barrier and strain adaptation and proposes that the phenomenon of prion adaptation is more common than previously thought.     

The expanding realm of prion phenomena in neurodegenerative disease

The aggregation of a soluble protein into insoluble, β-sheet rich amyloid fibrils is a defining characteristic of many neurodegenerative diseases, including prion disorders. The prion protein has so far been considered unique because of its infectious nature. Recent investigations, however, suggest that other amyloidforming proteins associated with much more common diseases, such as tau, α-synuclein, amyloid β and polyglutamine proteins, while not infectious in the classical sense, share certain essential properties with prions that may explain phenotypic diversity, and patterns of spread within the nervous system. We suggest a common mechanism of pathogenesis of myriad sporadic and inherited neurodegenerative diseases based on templated conformational change.Key words: tau, prion, amyloid beta, α-synuclein, polyglutamine, neurodegeneration, fibril, propagation     

Tissue-specific biochemical differences between chronic wasting disease prions isolated from free-ranging white-tailed deer (Odocoileus virginianus)

Chronic wasting disease (CWD) is an invariably fatal prion disease affecting cervid species worldwide. Prions can manifest as distinct strains that can influence disease pathology and transmission. CWD is profoundly lymphotropic, and most infected cervids likely shed peripheral prions replicated in lymphoid organs. However, CWD is a neurodegenerative disease, and most research on prion strains has focused on neurogenic prions. Thus, a knowledge gap exists comparing neurogenic prions to lymphogenic prions. In this study, we compared prions from the obex and lymph nodes of naturally exposed white-tailed deer to identify potential biochemical strain differences. Here, we report biochemical evidence of strain differences between the brain and lymph node from these animals. Conformational stability assays, glycoform ratio analyses, and immunoreactivity scanning across the structured domain of the prion protein that refolds into the amyloid aggregate of the infectious prion reveal significantly more structural and glycoform variation in lymphogenic prions than neurogenic prions. Surprisingly, we observed greater biochemical differences among neurogenic prions than lymphogenic prions across individuals. We propose that the lymphoreticular system propagates a diverse array of prions from which the brain selects a more restricted pool of prions that may be quite different than those from another individual of the same species. Future work should examine the biological and zoonotic impact of these biochemical differences and examine more cervids from multiple locations to determine if these differences are conserved across species and locations.     

Prion domains: sequences, structures and interactions

Mammalian and most fungal infectious proteins (also known as prions) are self-propagating amyloid, a filamentous beta-sheet structure. A prion domain determines the infectious properties of a protein by forming the core of the amyloid. We compare the properties of known prion domains and their interactions with the remainder of the protein and with chaperones. Ure2p and Sup35p, two yeast prion proteins, can still form prions when the prion domains are shuffled, indicating a parallel in-register beta-sheet structure.     

Subclinical prion disease induced by oral inoculation

Natural transmission of prion disease is believed to occur by peripheral infection such as oral inoculation. Following this route of inoculation, both the peripheral nervous system and the lymphoreticular system may be involved in the subsequent neuroinvasion of the central nervous system by prions, which may not necessarily result in clinical signs of terminal disease. Subclinical prion disease, characterized by the presence of infectivity and PrP(Sc) in the absence of overt clinical signs, may occur. It is not known which host factors contribute to whether infection with prions culminates in a terminal or subclinical disease state. We have investigated whether the level of host PrP(c) protein expression is a factor in the development of subclinical prion disease. When RML prion inoculum was inoculated by either the i.c. or intraperitoneal route, wild-type and tga20 mice both succumbed to terminal prion disease. In contrast, orally inoculated tga20 mice succumbed to terminal prion disease, whereas wild-type mice showed no clinical signs. However, wild-type mice sacrificed 375 or 525 days after oral inoculation harbored significant levels of brain PrP(Sc) and infectivity. These data show that same-species transmission of prions by the oral route in animals that express normal levels of PrP(c) can result in subclinical prion disease. This indicates that the level of host PrP(c) protein expression is a contributing factor to the regulation of development of terminal prion disease. Events that increase PrP(c) expression may predispose a prion-infected animal to the more deleterious effects of prion pathology.     

Genetics of prion infections

Although the infectious prions causing scrapie and several human transmissible neurodegenerative diseases resemble viruses in many respects, molecular and genetic analyses indicate that prions are fundamentally different from viruses in their structure and the mechanisms by which they cause disease. The only macromolecule that has been identified in infectious prion preparations is a disease-specific isoform of the prion protein, which is encoded by a host gene. A growing body of data supports the contention that prion infections represent a novel host-pathogen interaction.     

Sequence specificity and fidelity of prion transmission in yeast

Amyloid formation is a widespread feature of various proteins. It is associated with both important diseases (including infectious mammalian prions) and biologically positive functions, and provides a basis for structural "templating" and protein-based epigenetic inheritance (for example, in the case of yeast prions). Amyloid templating is characterized by a high level of sequence specificity and conformational fidelity. Even slight variations in sequence may produce a strong barrier for prion transmission. Yeast models provide useful insight into a mechanism of amyloid specificity and fidelity. Accumulating evidence indicates that cross-species prion transmission is controlled by the identity of short sequences (specificity stretches) rather than by the overall level of sequence identity. Location of the specificity stretches determines the location and/or size of the cross-β amyloid region that controls patterns of prion variants. In some cases of cross-species prion transmission, fidelity of variant reproduction is impaired, leading to the formation of new structural variants. We propose that such a variant switch may occur due to choice of the alternatively located secondary specificity stretches, when interaction between the primary stretches is impaired due to sequence divergence.