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.     

Molecular dynamics studies on the structural stability of wild-type dog prion protein

Prion diseases such as Creutzfeldt-Jakob disease, variant Creutzfeldt-Jakob diseases, Gerstmann-Str?ussler-Scheinker syndrome, Fatal Familial Insomnia, Kuru in humans, scrapie in sheep, bovine spongiform encephalopathy (or 'mad-cow' disease) and chronic wasting disease in cattle are invariably fatal and highly infectious neurodegenerative diseases affecting humans and animals. However, by now there have not been some effective therapeutic approaches to treat all these prion diseases. In 2008, canine mammals including dogs (canis familials) were the first time academically reported to be resistant to prion diseases (Vaccine 26: 2601-2614 (2008)). Thus, it is very worth studying the molecular structures of dog prion protein to obtain insights into the immunity of dogs to prion diseases. This paper studies the molecular structural dynamics of wild-type dog prion protein. The comparison analyses with rabbit prion protein show that the dog prion protein has stable molecular structures whether under neutral or low pH environments. We also find that the salt bridges such as D177-R163 contribute to the structural stability of wild-type rabbit prion protein under neutral pH environment.     

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.     

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.     

Molecular basis for transmission barrier and interference between closely related prion proteins in yeast

Replicating amyloids, called prions, are responsible for transmissible neurodegenerative diseases in mammals and some heritable phenotypes in fungi. The transmission of prions between species is usually inhibited, being highly sensitive to small differences in amino acid sequence of the prion-forming proteins. To understand the molecular basis of this prion interspecies barrier, we studied the transmission of the [PSI(+)] prion state from Sup35 of Saccharomyces cerevisiae to hybrid Sup35 proteins with prion-forming domains from four other closely related Saccharomyces species. Whereas all the hybrid Sup35 proteins could adopt a prion form in S. cerevisiae, they could not readily acquire the prion form from the [PSI(+)] prion of S. cerevisiae. Expression of the hybrid Sup35 proteins in S. cerevisiae [PSI(+)] cells often resulted in frequent loss of the native [PSI(+)] prion. Furthermore, all hybrid Sup35 proteins showed different patterns of interaction with the native [PSI(+)] prion in terms of co-polymerization, acquisition of the prion state, and induced prion loss, all of which were also dependent on the [PSI(+)] variant. The observed loss of S. cerevisiae [PSI(+)] can be related to inhibition of prion polymerization of S. cerevisiae Sup35 and formation of a non-heritable form of amyloid. We have therefore identified two distinct molecular origins of prion transmission barriers between closely sequence-related prion proteins: first, the inability of heterologous proteins to co-aggregate with host prion polymers, and second, acquisition by these proteins of a non-heritable amyloid fold.     

Sequence-dependent Prion Protein Misfolding and Neurotoxicity

Prion diseases are neurodegenerative disorders caused by misfolding of the normal prion protein (PrP) into a pathogenic “scrapie” conformation. To better understand the cellular and molecular mechanisms that govern the conformational changes (conversion) of PrP, we compared the dynamics of PrP from mammals susceptible (hamster and mouse) and resistant (rabbit) to prion diseases in transgenic flies. We recently showed that hamster PrP induces spongiform degeneration and accumulates into highly aggregated, scrapie-like conformers in transgenic flies. We show now that rabbit PrP does not induce spongiform degeneration and does not convert into scrapie-like conformers. Surprisingly, mouse PrP induces weak neurodegeneration and accumulates small amounts of scrapie-like conformers. Thus, the expression of three highly conserved mammalian prion proteins in transgenic flies uncovered prominent differences in their conformational dynamics. How these properties are encoded in the amino acid sequence remains to be elucidated.     

Transition of the prion protein from a structured cellular form (PrPC) to the infectious scrapie agent (PrPSc)

Prion diseases in mammals are caused by a conformational transition of the cellular prion protein from its native conformation (PrP^C) to a pathological isoform called “prion protein scrapie” (PrP^Sc). A molecular level of understanding of this conformational transition will be helpful in unveiling the disease etiology. Experimental structural biological techniques (NMR and X‐ray crystallography) have been used to unravel the atomic level structural information for the prion and its binding partners. More than one hundred three‐dimensional structures of the mammalian prions have been deposited in the protein databank. Structural studies on the prion protein and its structural transitions will deepen our understanding of the molecular basis of prion pathogenesis and will provide valuable guidance for future structure‐based drug discovery endeavors.     

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.     

Prion protein-related proteins from zebrafish are complex glycosylated and contain a glycosylphosphatidylinositol anchor

A hallmark of prion diseases in mammals is a conformational transition of the cellular prion protein (PrP(C)) into a pathogenic isoform termed PrP(Sc). PrP(C) is highly conserved in mammals, moreover, genes of PrP-related proteins have been recently identified in fish. While there is only little sequence homology to mammalian PrP, PrP-related fish proteins were predicted to be modified with N-linked glycans and a C-terminal glycosylphosphatidylinositol (GPI) anchor. We biochemically characterized two PrP-related proteins from zebrafish in cultured cells and show that both zePrP1 and zeSho2 are imported into the endoplasmic reticulum and are post-translationally modified with complex glycans and a C-terminal GPI anchor.     

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.     

Spatial sequestration and oligomer remodeling during de novo [PSI+] formation

Prions are misfolded, aggregated, infectious proteins found in a range of organisms from mammals to bacteria. In mammals, prion formation is difficult to study because misfolding and aggregation take place prior to symptom presentation. The study of the yeast prion [PSI⁺], which is the misfolded infectious form of Sup35p, provides a tractable system to monitor prion formation in real time. Recently, we showed that the de novo formation of prion aggregates begins with the appearance of highly mobile cytoplasmic foci, called early foci, which assemble into larger ring or dot structures. We also observed SDS-resistant oligomers during formation, and lysates containing newly formed oligomers can convert [psi^?] cells to the [PSI⁺] state, suggesting that these oligomers have infectious potential. Here, we further characterize two aspects of prion formation: spatial sequestration of early foci and oligomerization of endogenous Sup35p. Our data provides important insights into the process of prion formation and explores the minimal oligomer requirement for infectivity.     

Oxidative stress conditions increase the frequency of de novo formation of the yeast [PSI+] prion

Prions are self‐perpetuating amyloid protein aggregates which underlie various neurodegenerative diseases in mammals and heritable traits in yeast. The molecular basis of how yeast and mammalian prions form spontaneously into infectious amyloid‐like structures is poorly understood. We have explored the hypothesis that oxidative stress is a general trigger for prion formation using the yeast [PSI⁺] prion, which is the altered conformation of the Sup35 translation termination factor. We show that the frequency of [PSI⁺] prion formation is elevated under conditions of oxidative stress and in mutants lacking key antioxidants. We detect increased oxidation of Sup35 methionine residues in antioxidant mutants and show that overexpression of methionine sulphoxide reductase abrogates both the oxidation of Sup35 and its conversion to the [PSI⁺] prion. [PSI⁺] prion formation is particularly elevated in a mutant lacking the Sod1 Cu,Zn‐superoxide dismutase. We have used fluorescence microscopy to show that the de novo appearance of [PSI⁺] is both rapid and increased in frequency in this mutant. Finally, electron microscopy analysis of native Sup35 reveals that similar fibrillar structures are formed in both the wild‐type and antioxidant mutants. Together, our data indicate that oxidative stress is a general trigger of [PSI⁺] formation, which can be alleviated by antioxidant defenses.     

Fish models in prion biology: Underwater issues

Transmissible spongiform encephalopathies (TSEs), otherwise known as prion disorders, are fatal diseases causing neurodegeneration in a wide range of mammalian hosts, including humans. The causative agents - prions - are thought to be composed of a rogue isoform of the endogenous prion protein (PrP). Beyond these and other basic concepts, fundamental questions in prion biology remain unanswered, such as the physiological function of PrP, the molecular mechanisms underlying prion pathogenesis, and the origin of prions. To date, the occurrence of TSEs in lower vertebrates like fish and birds has received only limited attention, despite the fact that these animals possess bona fide PrPs. Recent findings, however, have brought fish before the footlights of prion research. Fish models are beginning to provide useful insights into the roles of PrP in health and disease, as well as the potential risk of prion transmission between fish and mammals. Although still in its infancy, the use of fish models in TSE research could significantly improve our basic understanding of prion diseases, and also help anticipate risks to public health. This article is part of a Special Issue entitled Zebrafish Models of Neurological 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.     

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     

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.     

Comparative proteomic analysis of cell lines and scrapings of the human intestinal epithelium

Background

The homologues of human disease genes are expected to contribute to better understanding of physiological and pathogenic processes. We made use of the present availability of vertebrate genomic sequences, and we have conducted the most comprehensive comparative genomic analysis of the prion protein gene PRNP and its homologues, shadow of prion protein gene SPRN and doppel gene PRND, and prion testis-specific gene PRNT so far.

Results

While the SPRN and PRNP homologues are present in all vertebrates, PRND is known in tetrapods, and PRNT is present in primates. PRNT could be viewed as a TE-associated gene. Using human as the base sequence for genomic sequence comparisons (VISTA), we annotated numerous potential cis-elements. The conserved regions in SPRNs harbour the potential Sp1 sites in promoters (mammals, birds), C-rich intron splicing enhancers and PTB intron splicing silencers in introns (mammals, birds), and hsa-miR-34a sites in 3'-UTRs (eutherians). We showed the conserved PRNP upstream regions, which may be potential enhancers or silencers (primates, dog). In the PRNP 3'-UTRs, there are conserved cytoplasmic polyadenylation element sites (mammals, birds). The PRND core promoters include highly conserved CCAAT, CArG and TATA boxes (mammals). We deduced 42 new protein primary structures, and performed the first phylogenetic analysis of all vertebrate prion genes. Using the protein alignment which included 122 sequences, we constructed the neighbour-joining tree which showed four major clusters, including shadoos, shadoo2s and prion protein-likes (cluster 1), fish prion proteins (cluster 2), tetrapode prion proteins (cluster 3) and doppels (cluster 4). We showed that the entire prion protein conformationally plastic region is well conserved between eutherian prion proteins and shadoos (18–25% identity and 28–34% similarity), and there could be a potential structural compatibility between shadoos and the left-handed parallel beta-helical fold.

Conclusion

It is likely that the conserved genomic elements identified in this analysis represent bona fide cis-elements. However, this idea needs to be confirmed by functional assays in transgenic systems.     

Processing of high-titer prions for mass spectrometry inactivates prion infectivity

Prions represent a class of universally fatal and transmissible neurodegenerative disorders that affect humans and other mammals. The prion agent contains a pathologically aggregated form of the host prion protein that can transmit infectivity without any bacterial or viral component and is thus difficult to inactivate using disinfection protocols designed for infectious microorganisms. Methods for prion inactivation include treatment with acids, bases, detergents, bleach, prolonged autoclaving and incineration. During these procedures, the sample is often either destroyed or damaged such that further analysis for research purposes is compromised. In this study we show that a straightforward denaturation and in-gel protease digestion protocol used to prepare prion-infected samples for mass spectroscopy leads to the loss of at least 7 logs of prion infectivity, yielding a final product that fails to transmit prion disease in vivo. We further show that the resultant sample remains suitable for mass spectrometry-based protein identifications. Thus, the procedure described can be used to prepare prion-infected samples for mass spectrometry analysis with greatly reduced biosafety concerns.     

Biological functions of amyloids: Facts and hypotheses

Amyloids are fibrous protein aggregates that arise via polymerization of proteins with their concurrent conformational rearrangement and the formation of a specific cross-β structure. Amyloids are of particular interest as a cause of a vast group of human and animal diseases called amyloidoses. Some of these diseases are caused by prions, a specific type of amyloids, and are transmissible. Apart from mammals, prion amyloids are described in lower eukaryotes, where they act as nonchromosomal genetic determinants. Although amyloids are usually associated with pathologies in humans and animals, the increasing number of findings suggests that the acquisition of an amyloid or prion form by a protein is of biological significance in some cases. The review summarizes the data on the biological significance of prion and nonprion amyloids in a wide range of species from bacteria to mammals.