Yeast prions form infectious amyloid inclusion bodies in bacteria

ABSTRACT: BACKGROUND: Prions were first identified as infectious proteins associated with fatal brain diseases in mammals. However, fungal prions behave as epigenetic regulators that can alter a range of cellular processes. These proteins propagate as self-perpetuating amyloid aggregates being an example of structural inheritance. The best-characterized examples are the Sup35 and Ure2 yeast proteins, corresponding to [PSI+] and [URE3] phenotypes, respectively. RESULTS: Here we show that both the prion domain of Sup35 (Sup35-NM) and the Ure2 protein (Ure2p) form inclusion bodies (IBs) displaying amyloid-like properties when expressed in bacteria. These intracellular aggregates template the conformational change and promote the aggregation of homologous, but not heterologous, soluble prionogenic molecules. Moreover, in the case of Sup35-NM, purified IBs are able to induce different [PSI+] phenotypes in yeast, indicating that at least a fraction of the protein embedded in these deposits adopts an infectious prion fold. CONCLUSIONS: An important feature of prion inheritance is the existence of strains, which are phenotypic variants encoded by different conformations of the same polypeptide. We show here that the proportion of infected yeast cells displaying strong and weak [PSI+] phenotypes depends on the conditions under which the prionogenic aggregates are formed in E. coli, suggesting that bacterial systems might become useful tools to generate prion strain diversity.     

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.     

Diagnosing prion diseases: needs, challenges and hopes

Prion diseases are among the most intriguing infectious diseases and are associated with unconventional proteinaceous infectious agents known as prions. Prions seem to lack nucleic acid and propagate by transmission of protein misfolding. The nature of prions and their unique mode of transmission present challenges for early diagnosis of prion diseases. In this article, state-of-the-art prion diagnostic techniques, together with the new strategies that are being used to develop sensitive, early and non-invasive diagnoses for these diseases are reviewed.     

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.     

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.     

Propagating prions in fungi and mammals

Prions constitute a rare class of protein, which can switch to a robust amyloid form and then propagate that form in the absence of a nucleic acid determinant, thereby creating a unique, protein-only infectious agent. Details of the mechanism that drives conversion to the prion form and then subsequent propagation of that form are beginning to emerge using a range of in vivo and in vitro approaches. Recent studies on both mammalian and fungal prions are providing a greater understanding of the structural features that distinguish prions from non-transmissible amyloids.     

The Hunt for Ancient Prions: Archaeal Prion-Like Domains Form Amyloid-Based Epigenetic Elements

Prions, proteins that can convert between structurally and functionally distinct states and serve as non-Mendelian mechanisms of inheritance, were initially discovered and only known in eukaryotes, and consequently considered to likely be a relatively late evolutionary acquisition. However, the recent discovery of prions in bacteria and viruses has intimated a potentially more ancient evolutionary origin. Here, we provide evidence that prion-forming domains exist in the domain archaea, the last domain of life left unexplored with regard to prions. We searched for archaeal candidate prion-forming protein sequences computationally, described their taxonomic distribution and phylogeny, and analyzed their associated functional annotations. Using biophysical in vitro assays, cell-based and microscopic approaches, and dye-binding analyses, we tested select candidate prion-forming domains for prionogenic characteristics. Out of the 16 tested, eight formed amyloids, and six acted as protein-based elements of information transfer driving non-Mendelian patterns of inheritance. We also identified short peptides from our archaeal prion candidates that can form amyloid fibrils independently. Lastly, candidates that tested positively in our assays had significantly higher tyrosine and phenylalanine content than candidates that tested negatively, an observation that may help future archaeal prion predictions. Taken together, our discovery of functional prion-forming domains in archaea provides evidence that multiple archaeal proteins are capable of acting as prions—thus expanding our knowledge of this epigenetic phenomenon to the third and final domain of life and bolstering the possibility that they were present at the time of the last universal common ancestor.     

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.     

Prions affect the appearance of other prions: the story of [PIN(+)

Prions are self-propagating protein conformations. Recent research brought insight into prion propagation, but how they first appear is unknown. We previously established that the yeast non-Mendelian trait [PIN(+)] is required for the de novo appearance of the [PSI(+)] prion. Here, we show that the presence of prions formed by Rnq1 or Ure2 is sufficient to make cells [PIN(+)]. Thus, [PIN(+)] can be caused by more than one prion. Furthermore, an unbiased functional screen for [PIN(+)] prions uncovered the known prion gene, URE2, the proposed prion gene, NEW1, and nine novel candidate prion genes all carrying prion domains. Importantly, the de novo appearance of Rnq1::GFP prion aggregates also requires the presence of other prions, suggesting the existence of a general mechanism by which the appearance of prions is enhanced by heterologous prion aggregates.     

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.     

Direct assessment in bacteria of prionoid propagation and phenotype selection by Hsp70 chaperone

Protein amyloid aggregates epigenetically determine either advantageous or proteinopathic phenotypes. Prions are infectious amyloidogenic proteins, whereas prionoids lack infectivity but spread from mother to daughter cells. While prion amyloidosis has been studied in yeast and mammalian cells models, the dynamics of transmission of an amyloid proteinopathy has not been addressed yet in bacteria. Using time‐lapse microscopy and a microfluidic set‐up, we have assessed in Escherichia coli the vertical transmission of the amyloidosis caused by the synthetic bacterial model prionoid RepA‐WH1 at single cell resolution within their lineage context. We identify in vivo the coexistence of two strain‐like types of amyloid aggregates within a genetically identical population and a controlled homogeneous environment. The amyloids are either toxic globular particles or single comet‐shaped aggregates that split during cytokinesis and exhibit milder toxicity. Both segregate and propagate in sublineages, yet show interconversion. ClpB (Hsp104) chaperone, key for spreading of yeast prions, has no effect on the dynamics of the two RepA‐WH1 aggregates. However, the propagation of the comet‐like species is DnaK (Hsp70)‐dependent. The bacterial RepA‐WH1 prionoid thus provides key qualitative and quantitative clues on the biology of intracellular amyloid proteinopathies.     

De novo generation of prion strains

Prions are self-replicating proteins that can cause neurodegenerative disorders such as bovine spongiform encephalopathy (also known as mad cow disease). Aberrant conformations of prion proteins accumulate in the central nervous system, causing spongiform changes in the brain and eventually death. Since the inception of the prion hypothesis - which states that misfolded proteins are the infectious agents that cause these diseases - researchers have sought to generate infectious proteins from defined components in the laboratory with varying degrees of success. Here, we discuss several recent studies that have produced an array of novel prion strains in vitro that exhibit increasingly high titres of infectivity. These advances promise unprecedented insight into the structure of prions and the mechanisms by which they originate and propagate.     

Nanoimaging for prion related diseases

Misfolding and aggregation of prion proteins is linked to a number of neurodegenerative disorders such as Creutzfeldt-Jacob disease (CJD) and its variants: Kuru, Gerstmann-Straussler-Scheinker syndrome and fatal familial insomnia. In prion diseases, infectious particles are proteins that propagate by transmitting a misfolded state of a protein, leading to the formation of aggregates and ultimately to neurodegeneration. Prion phenomenon is not restricted to humans. There are a number of prion-related diseases in a variety of mammals, including bovine spongiform encephalopathy (BSE, also known as “mad cow disease”) in cattle. All known prion diseases, collectively called transmissible spongiform encephalopathies (TSEs), are untreatable and fatal. Prion proteins were also found in some fungi where they are responsible for heritable traits. Prion proteins in fungi are easily accessible and provide a powerful model for understanding the general principles of prion phenomenon and molecular mechanisms of mammalian prion diseases. Presently, several fundamental questions related to prions remain unanswered. For example, it is not clear how prions cause the disease. Other unknowns include the nature and structure of infectious agent and how prions replicate. Generally, the phenomenon of misfolding of the prion protein into infectious conformations that have the ability to propagate their properties via aggregation is of significant interest. Despite the crucial importance of misfolding and aggregation, very little is currently known about the molecular mechanisms of these processes. While there is an apparent critical need to study molecular mechanisms underlying misfolding and aggregation, the detailed characterization of these single molecule processes is hindered by the limitation of conventional methods. Although some issues remain unresolved, much progress has been recently made primarily due to the application of nanoimaging tools. The use of nanoimaging methods shows great promise for understanding the molecular mechanisms of prion phenomenon, possibly leading toward early diagnosis and effective treatment of these devastating diseases. This review article summarizes recent reports which advanced our understanding of the prion phenomenon through the use of nanoimaging methods.Key words: protein misfolding, prion, atomic force microscopy, nanomedicine, force spectroscopy     

Estimating Prion Adsorption Capacity of Soil by BioAssay of Subtracted Infectivity from Complex Solutions (BASICS)

Prions, the infectious agent of scrapie, chronic wasting disease and other transmissible spongiform encephalopathies, are misfolded proteins that are highly stable and resistant to degradation. Prions are known to associate with clay and other soil components, enhancing their persistence and surprisingly, transmissibility. Currently, few detection and quantification methods exist for prions in soil, hindering an understanding of prion persistence and infectivity in the environment. Variability in apparent infectious titers of prions when bound to soil has complicated attempts to quantify the binding capacity of soil for prion infectivity. Here, we quantify the prion adsorption capacity of whole, sandy loam soil (SLS) typically found in CWD endemic areas in Colorado; and purified montmorillonite clay (Mte), previously shown to bind prions, by BioAssay of Subtracted Infectivity in Complex Solutions (BASICS). We incubated prion positive 10% brain homogenate from terminally sick mice infected with the Rocky Mountain Lab strain of mouse-adapted prions (RML) with 10% SLS or Mte. After 24 hours samples were centrifuged five minutes at 200×g and soil-free supernatant was intracerebrally inoculated into prion susceptible indicator mice. We used the number of days post inoculation to clinical disease to calculate the infectious titer remaining in the supernatant, which we subtracted from the starting titer to determine the infectious prion binding capacity of SLS and Mte. BASICS indicated SLS bound and removed ≥ 95% of infectivity. Mte bound and removed lethal doses (99.98%) of prions from inocula, effectively preventing disease in the mice. Our data reveal significant prion-binding capacity of soil and the utility of BASICS to estimate prion loads and investigate persistence and decomposition in the environment. Additionally, since Mte successfully rescued the mice from prion disease, Mte might be used for remediation and decontamination protocols.     

Bank Vole Prion Protein As an Apparently Universal Substrate for RT-QuIC-Based Detection and Discrimination of Prion Strains

Prions propagate as multiple strains in a wide variety of mammalian species. The detection of all such strains by a single ultrasensitive assay such as Real Time Quaking-induced Conversion (RT-QuIC) would facilitate prion disease diagnosis, surveillance and research. Previous studies have shown that bank voles, and transgenic mice expressing bank vole prion protein, are susceptible to most, if not all, types of prions. Here we show that bacterially expressed recombinant bank vole prion protein (residues 23-230) is an effective substrate for the sensitive RT-QuIC detection of all of the different prion types that we have tested so far – a total of 28 from humans, cattle, sheep, cervids and rodents, including several that have previously been undetectable by RT-QuIC or Protein Misfolding Cyclic Amplification. Furthermore, comparison of the relative abilities of different prions to seed positive RT-QuIC reactions with bank vole and not other recombinant prion proteins allowed discrimination of prion strains such as classical and atypical L-type bovine spongiform encephalopathy, classical and atypical Nor98 scrapie in sheep, and sporadic and variant Creutzfeldt-Jakob disease in humans. Comparison of protease-resistant RT-QuIC conversion products also aided strain discrimination and suggested the existence of several distinct classes of prion templates among the many strains tested.     

Nanoimaging for prion related diseases

Misfolding and aggregation of prion proteins is linked to a number of neurodegenerative disorders such as Creutzfeldt-Jacob disease (CJD) and its variants, kuru, Gerstmann-Straussler-Scheinker syndrome and fatal familial insomnia. In prion diseases, infectious particles are proteins that propagate by transmitting a misfolded state of a protein, leading to the formation of aggregates and ultimately to neurodegeneration. Prion phenomenon is not restricted to humans. There is a number of prion-related diseases in a variety of mammals, including bovine spongiform encephalopathy (BSE, also known as "mad cow disease") in cattle. All known prion diseases, collectively called transmissible spongiform encephalopathies (TSEs), are untreatable and fatal. Prion proteins were also found in some fungi where they are responsible for heritable traits. Prion proteins in fungi are easily accessible and provide a powerful model for understanding the general principles of prion phenomenon and molecular mechanisms of mammalian prion diseases. Presently, several fundamental questions related to prions remain unanswered. For example, it is not clear how prions cause the disease. Other unknowns include the nature and structure of infectious agent and how prions replicate? Generally, the phenomenon of misfolding of prion protein into infectious conformations that have the ability to propagate their properties via aggregation is of significant interest. Despite the crucial importance of misfolding and aggregation, very little is currently known about the molecular mechanisms of these processes. While there is an apparent critical need to study molecular mechanisms underlying misfolding and aggregation, the detailed characterization of these single molecule processes is hindered by the limitation of conventional methods. Although some issues remain unresolved, much progress has been recently made primarily due to the application of nanoimaging tools. The use of nanoimaging methods shows great promise for understanding the molecular mechanisms of prion phenomenon, possibly leading toward early diagnosis and effective treatment of these devastating diseases. This review article summarizes recent reports which advanced our understanding of the prion phenomenon through the use of nanoimaging methods.     

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.     

Protein misfolding cyclic amplification of infectious prions

Prions are proteinaceous infectious agents responsible for the transmission of prion diseases. The lack of a procedure for cultivating prions in the laboratory has been a major limitation to the study of the unorthodox nature of this infectious agent and the molecular mechanism by which the normal prion protein (PrP(C)) is converted into the abnormal isoform (PrP(Sc)). Protein misfolding cyclic amplification (PMCA), described in detail in this protocol, is a simple, fast and efficient methodology to mimic prion replication in the test tube. PMCA involves incubating materials containing minute amounts of infectious prions with an excess of PrP(C) and boosting the conversion by cycles of sonication to fragment the converting units, thereby leading to accelerated prion replication. PMCA is able to detect the equivalent of a single molecule of infectious PrP(Sc) and propagate prions that maintain high infectivity, strain properties and species specificity. A single PMCA assay takes little more than 3 d to replicate a large amount of prions, which could take years in an in vivo situation. Since its invention 10 years ago, PMCA has helped to answer fundamental questions about this intriguing infectious agent and has been broadly applied in research areas that include the food industry, blood bank safety and human and veterinary disease diagnosis.     

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.     

Prions on the move

Prions consist mainly, if not entirely, of PrP(Sc), an aggregated conformer of the host protein PrP(C). Prions come in different strains, all based on the same PrP(C) sequence, but differing in their conformations. The efficiency of prion transmission between species is usually low, but increases after serial transmission in the new host, suggesting a process involving mutation and selection. Even within the same species, the transfer of prions between cell types entails a selection of favoured 'substrains', and propagation of prions in the presence of an inhibitory drug can result in the appearance of drug-resistant prion populations. We propose that prion populations are comprised of a variety of conformers, constituting 'quasi-species', from which the one replicating most efficiently in a particular environment is selected.