A journey through the species barrier

The species barrier in prion infectivity is believed to reside in the degree of amino acid sequence heterology between the infectious prion protein, PrP(Sc), of the donor and the normal PrP of the host. bring new evidence that distinct PrP(Sc) species or prion strains may have different conformations even when they have identical amino acid sequence and that the conformation of the exogenous prion strain is a determinant of the species barrier in hosts that have identical PrP genotype.     

Mammalian prion proteins

The past two years have seen the extension of our knowledge on the cellular prion protein structure with new NMR data on both the hamster and human proteins. In addition, the folding dynamics of two cellular prion proteins have been elucidated. There are now several examples of recombinant prion proteins that are able to adopt different conformations in solution and recent work on the molecular basis of prion strains has done much to consolidate the protein-only hypothesis. Important advances in relating disease to structure have also been made through the identification of the minimal prion protein fragment that is capable of conferring susceptibility to and propagation of the scrapie agent.     

Prion diseases of yeast: amyloid structure and biology

Prion "variants" or "strains" are prions with the identical protein sequence, but different characteristics of the prion infection: e.g. different incubation periods for scrapie strains or different phenotype intensities for yeast prion variants. We have shown that infectious amyloids of the yeast prions [PSI+], [URE3] and [PIN+] each have an in-register parallel β-sheet architecture. Moreover, we have pointed out that this amyloid architecture can explain how one protein can faithfully transmit any of several conformations to new protein monomers. This explains how proteins can be genes.     

Mechanism of aggregation and membrane interactions of mammalian prion protein

The cellular prion protein (PrP^C), which is present ubiquitously in all mammalian neurons, is normally found to be linked to the cell membrane through a glycosylphosphatidylinositol (GPI) anchor. The conformational conversion of PrP^C into misfolded and aggregated forms is associated with transmissible neurodegenerative diseases known as prion diseases. The importance of different misfolded conformations in prion diseases, and the mechanism by which prion aggregates induce neurotoxicity remain poorly understood. Multiple studies have been shown that the toxicity of misfolded prion protein is directly correlated with its ability to interact with and perturb membranes. This review describes the current progress toward understanding prion protein misfolding and aggregation, as well as the interaction of prion protein aggregates with lipid membrane.     

Strain conformation controls the specificity of cross-species prion transmission in the yeast model

Transmissible self-assembled fibrous cross-β polymer infectious proteins (prions) cause neurodegenerative diseases in mammals and control non-Mendelian heritable traits in yeast. Cross-species prion transmission is frequently impaired, due to sequence differences in prion-forming proteins. Recent studies of prion species barrier on the model of closely related yeast species show that colocalization of divergent proteins is not sufficient for the cross-species prion transmission, and that an identity of specific amino acid sequences and a type of prion conformational variant (strain) play a major role in the control of transmission specificity. In contrast, chemical compounds primarily influence transmission specificity via favoring certain strain conformations, while the species origin of the host cell has only a relatively minor input. Strain alterations may occur during cross-species prion conversion in some combinations. The model is discussed which suggests that different recipient proteins can acquire different spectra of prion strain conformations, which could be either compatible or incompatible with a particular donor strain.     

The state of the prion

There is little doubt that the main component of the transmissible agent of spongiform encephalopathies - the prion - is a conformational variant of the ubiquitous host protein PrP(C), and that the differing properties of various prion strains are associated with different abnormal conformations of this protein. The precise structure of the prion is not yet known, nor are the mechanisms of infection, conformational conversion and pathogenesis understood.     

Prion Stability and Infectivity in the Environment

The biology of normal prion protein and the property of infectivity observed in abnormal folding conformations remain thinly characterized. However, enough is known to understand that prion proteins stretch traditional views of proteins in biological systems. Numerous investigators are resolving details of the novel mechanism of infectivity, which appears to feature a protein-only, homologous replication of misfolded isoforms. Many other features of prion biology are equally extraordinary. This review focuses on the status of infectious prions in various natural and man-made environments. The picture that emerges is that prion proteins are durable under extreme conditions of environmental exposure that are uncommon in biological phenomena, and this durability offers the potential for environmental reservoirs of persistent infectivity lasting for years. A recurrent theme in prion research is a propensity for these proteins to bind to mineral and metal surfaces, and several investigators have provided evidence that the normal cellular functions of prion protein may include metalloprotein interactions. This structural propensity for binding to mineral and metal ions offers the hypothesis that prion polypeptides are intrinsically predisposed to non-physiological folding conformations that would account for their environmental durability and persistent infectivity. Similarly, the avidity of binding and potency of prion infectivity from environmental sources also offers a recent hypothesis that prion polypeptides bound to soil minerals are actually more infectious than studies with purified polypeptides would predict. Since certain of the prion diseases have a history of epidemics in economically important animal species and have the potential to transmit to humans, urgency is attached to understanding the environmental transmission of prion diseases and the development of protocols for their containment and inactivation. Special issue article in honor of Dr. George DeVries.     

Prion-Prion Interactions

The term prion has been used to describe self-replicating protein conformations that can convert other protein molecules of the same primary structure into its prion conformation. Several different proteins have now been found to exist as prions in Saccharomyces cerevisiae. Surprisingly, these heterologous prion proteins have a strong influence on each others’ appearance and propagation, which may result from structural similarity between the prions. Both positive and negative effects of a prion on the de novo appearance of a heterologous prion have been observed in genetic studies. Other examples of reported interactions include mutual or unilateral inhibition and destabilization when two prions are present together in a single cell. In vitro work showing that one purified prion stimulates the conversion of a purified heterologous protein into a prion form, suggests that facilitation of de novo prion formation by heterologous prions in vivo is a result of a direct interaction between the prion proteins (a cross-seeding mechanism) and does not require other cellular components. However, other cellular structures, e.g., the cytoskeleton, may provide a scaffold for these interactions in vivo and chaperones can further facilitate or inhibit this process. Some negative prion-prion interactions may also occur via a direct interaction between the prion proteins. Another explanation is a competition between the prions for cellular factors involved in prion propagation or differential effects of chaperones stimulated by one prion on the heterologous prions.     

Did the prion protein become vulnerable to misfolding after an evolutionary divide and conquer event?

Despite high sequence identity among mammalian prion proteins (PrPs), mammals have varying rates of susceptibility to prion disease resulting in a so-called species barrier. The species barrier follows no clear pattern, with closely related species or similar sequences being no more likely to infect each other, and remains an unresolved enigma. Variation of the conformationally flexible regions may alter the thermodynamics of the conformational change, commonly referred to as the conformational conversion, which occurs in the pathogenic process of the mammalian prion protein. A conformational ensemble scenario is supported by the species barrier in prion disease and evidence that there are strains of pathogenic prion with different conformations within species. To study how conformational flexibility has evolved in the prion protein, an investigation was undertaken on the evolutionary dynamics of structurally disordered regions in the mammalian prion protein, non-mammalian prion protein that is not vulnerable to prion disease, and remote homologs Doppel and Shadoo. Structural disorder prediction analyzed in an evolutionary context revealed that the occurrence of increased or altered conformational flexibility in mammalian PrPs coincides with key events among PrP, Doppel, and Shadoo. Comparatively rapid evolutionary dynamics of conformational flexibility in the prion protein suggest that the species barrier is not a static phenomenon. A small number of amino acid substitutions can repopulate the conformational ensemble and have a disproportionately large effect on pathogenesis.     

Mechanism of cross-species prion transmission: an infectious conformation compatible with two highly divergent yeast prion proteins

Efficiency of interspecies prion transmission decreases as the primary structures of the infectious proteins diverge. Yet, a single prion protein can misfold into multiple infectious conformations, and such differences in "strain conformation" also alter infection specificity. Here, we explored the relationship between prion strains and species barriers by creating distinct synthetic prion forms of the yeast prion protein Sup35. We identified a strain conformation of Sup35 that allows transmission from the S. cerevisiae (Sc) Sup35 to the highly divergent C. albicans (Ca) Sup35 both in vivo and in vitro. Remarkably, cross-species transmission leads to a novel Ca strain that in turn can infect the Sc protein. Structural studies reveal strain-specific conformational differences in regions of the prion domain that are involved in intermolecular contacts. Our findings support a model whereby strain conformation is the critical determinant of cross-species prion transmission while primary structure affects transmission specificity by altering the spectrum of preferred amyloid conformations.     

Strain-specific prion-protein conformation determined by metal ions

In animals infected with a transmissible spongiform encephalopathy, or prion disease, conformational isomers (known as PrPSc proteins) of the wild-type, host-encoded cellular prion protein (PrPc) accumulate. The infectious agents, prions, are composed mainly of these conformational isomers, with distinct prion isolates or strains being associated with different PrPSc conformations and patterns of glycosylation. Here we show that two different human PrPSc types, seen in clinically distinct subtypes of classical Creutzfeldt-Jakob disease, can be interconverted in vitro by altering their metal-ion occupancy. The dependence of PrPSc conformation on the binding of copper and zinc represents a new mechanism for post-translational modification of PrP and for the generation of multiple prion strains, with widespread implications for both the molecular classification and the pathogenesis of prion diseases in humans and animals.     

Origins and kinetic consequences of diversity in Sup35 yeast prion fibers

A remarkable feature of prions is that infectious particles composed of the same prion protein can give rise to different phenotypes. This strain phenomenon suggests that a single prion protein can adopt multiple infectious conformations. Here we use a novel single fiber growth assay to examine the heterogeneity of amyloid fibers formed by the yeast Sup35 prion protein. Sup35 spontaneously forms multiple, distinct and faithfully propagating fiber types, which differ dramatically both in their degrees of polarity and overall growth rates. Both in terms of the number of distinct self-propagating fiber types, as well as the ability of these differences to dictate the rate of prion growth, this diversity is well suited to account for the range of prion strain phenotypes observed in vivo.     

Spontaneous Variants of the [RNQ+] Prion in Yeast Demonstrate the Extensive Conformational Diversity Possible with Prion Proteins

Prion strains (or variants) are structurally distinct amyloid conformations arising from a single polypeptide sequence. The existence of prion strains has been well documented in mammalian prion diseases. In many cases, prion strains manifest as variation in disease progression and pathology, and in some cases, these prion strains also show distinct biochemical properties. Yet, the underlying basis of prion propagation and the extent of conformational possibilities available to amyloidogenic proteins remain largely undefined. Prion proteins in yeast that are also capable of maintaining multiple self-propagating structures have provided much insight into prion biology. Here, we explore the vast structural diversity of the yeast prion [RNQ+] in Saccharomyces cerevisiae. We screened for the formation of [RNQ+] in vivo, allowing us to calculate the rate of spontaneous formation as ~2.96x10^-6, and successfully isolate several different [RNQ+] variants. Through a comprehensive set of biochemical and biological analyses, we show that these prion variants are indeed novel. No individual property or set of properties, including aggregate stability and size, was sufficient to explain the physical basis and range of prion variants and their resulting cellular phenotypes. Furthermore, all of the [RNQ+] variants that we isolated were able to facilitate the de novo formation of the yeast prion [PSI+], an epigenetic determinant of translation termination. This supports the hypothesis that [RNQ+] acts as a functional amyloid in regulating the formation of [PSI+] to produce phenotypic diversity within a yeast population and promote adaptation. Collectively, this work shows the broad spectrum of available amyloid conformations, and thereby expands the foundation for studying the complex factors that interact to regulate the propagation of distinct aggregate structures.     

Analysis of yeast prion aggregates with amyloid-staining compound in vivo

Yeast prions are protein-based genetic elements whose non-Mendelian patterns of inheritance are explained by their inheritance of altered conformations. Here we showed that aggregates made by overexpression of two different prion domains of Sup35 and Rnq1, were stained in yeast by thioflavin-S, an amyloid binding compound. These results suggested that yeast prion domains take the form of amyloid in vivo, and supported the idea that the self-propagating property of amyloids is responsible for the heritable traits of yeast prions.     

Mammalian prion amyloid formation in bacteria

Mammalian prion proteins (PrPs) that cause transmissible spongiform encephalopathies are misfolded conformations of the host cellular PrP. The misfolded form, the scrapie PrP (PrP^Sc), can aggregate into amyloid fibrils that progressively accumulate in the brain, evolving to a pathological phenotype. A particular characteristic of PrP^Sc is to be found as different strains, related to the diversity of conformational states it can adopt. Prion strains are responsible for the multiple phenotypes observed in prion diseases, presenting different incubation times and diverse deposition profiles in the brain. PrP biochemical properties are also strain-dependent, such as different digestion pattern after proteolysis and different stability. Although they have long been studied, strain formation is still a major unsolved issue in prion biology. The recreation of strain-specific conformational features is of fundamental importance to study this unique pathogenic phenomenon. In our recent paper, we described that murine PrP, when expressed in bacteria, forms amyloid inclusion bodies that possess different strain-like characteristics, depending on the PrP construct. Here, we present an extra-view of these data and propose that bacteria might become a successful model to generate preparative amounts of prion strain-specific assemblies for high-resolution structural analysis as well as for addressing the determinants of infectivity and transmissibility.     

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.     

The same primary structure of the prion protein yields two distinct self-propagating states

The question of whether distinct self-propagating structures could be formed within the same amino acid sequence in the absence of external cofactors or templates has important implications for a number of issues, including the origin of prion strains and the engineering of smart, self-assembling peptide-based biomaterials. In the current study, we showed that chemically identical prion protein can give rise to conformationally distinct, self-propagating amyloid structures in the absence of cellular cofactors, post-translational modification, or PrP(Sc)-specified templates. Even more surprising, two self-replicating states were produced under identical solvent conditions, but under different shaking modes. Individual prion conformations were inherited by daughter fibrils in seeding experiments conducted under alternative shaking modes, illustrating the high fidelity of fibrillation reactions. Our study showed that the ability to acquire conformationally different self-propagating structures is an intrinsic ability of protein fibrillation and strongly supports the hypothesis that conformational variation in self-propagating protein states underlies prion strain diversity.