Prion diseases such as Creutzfeldt-Jakob disease in humans, bovine spongiform encephalopathy in cattle, and scrapie in sheep are fatal neurodegenerative diseases for which there is no effective treatment. The pathology of these diseases involves the conversion of a protease sensitive form of the cellular prion protein (PrPC) into a protease resistant infectious form (PrPsc or PrPres). Both in vitro (cell culture and cell free conversion assays) and in vivo (animal) studies have demonstrated the strong dependence of this conversion process on protein sequence homology between the initial prion inoculum and the host's own cellular prion protein. The presence of non-homologous (heterologous) proteins is often inhibitory to this conversion process. We hypothesize that the presence of heterologous prion proteins from one species might therefore constitute an effective treatment for prion disease in another species. To test this hypothesis, we infected mice intracerebrally with murine adapted RML-Chandler scrapie and treated them with heterologous prion protein (purified bacterially expressed recombinant hamster prion protein) or vehicle alone. Treated animals demonstrated reduced disease associated pathology, decreased accumulation of protease-resistant disease-associated prion protein, with delayed onset of clinical symptoms and motor deficits. This was concomitant with significantly increased survival times relative to mock-treated animals. These results provide proof of principle that recombinant hamster prion proteins can effectively and safely inhibit prion disease in mice, and suggest that hamster or other non-human prion proteins may be a viable treatment for prion diseases in humans.

Prions are transmissible, propagating alternative states of proteins, and are usually made from the fibrillar, beta-sheet-rich assemblies termed amyloid. Prions in the budding yeast Saccharomyces cerevisiae propagate heritable phenotypes, uncover hidden genetic variation, function in large-scale gene regulation, and can act like diseases. Almost all these amyloid prions have asparagine/glutamine-rich (N/Q-rich) domains. Other proteins, that we term here 'prionogenic amyloid formers' (PAFs), have been shown to form amyloid in vivo, and to have N/Q-rich domains that can propagate heritable states in yeast cells. Also, there are >200 other S.cerevisiae proteins with prion-like N/Q-rich sequence composition. Furthermore, human proteins with such N/Q-rich composition have been linked to the pathomechanisms of neurodegenerative amyloid diseases.

Prion diseases are fatal infectious neurodegenerative disorders in humans and other animals and are caused by misfolding of the cellular prion protein (PrPC) into the pathological isoform PrPSc These diseases have the potential to transmit within or between species, including zoonotic transmission to humans. Elucidating the molecular and cellular mechanisms underlying prion propagation and transmission is therefore critical for developing molecular strategies for disease intervention. We have shown previously that impaired quality control mechanisms directly influence prion propagation. In this study, we manipulated cellular quality control pathways in vitro by stably and transiently overexpressing selected quality control folding (ERp57) and cargo (VIP36) proteins and investigated the effects of this overexpression on prion propagation. We found that ERp57 or VIP36 overexpression in persistently prion-infected neuroblastoma cells significantly reduces the amount of PrPSc in immunoblots and prion-seeding activity in the real-time quaking-induced conversion (RT-QuIC) assay. Using different cell lines infected with various prion strains confirmed that this effect is not cell type- or prion strain-specific. Moreover, de novo prion infection revealed that the overexpression significantly reduced newly formed PrPSc in acutely infected cells. ERp57-overexpressing cells significantly overcame endoplasmic reticulum stress, as revealed by expression of lower levels of the stress markers BiP and CHOP, accompanied by a decrease in PrP aggregates. Furthermore, application of ERp57-expressing lentiviruses prolonged the survival of prion-infected mice. Taken together, improved cellular quality control via ERp57 or VIP36 overexpression impairs prion propagation and could be utilized as a potential therapeutic strategy.

Prions in eukaryotes have been linked to diseases, evolutionary capacitance, large-scale genetic control, and long-term memory formation. Prion formation and propagation have been studied extensively in the budding yeast Saccharomyces cerevisiae. Here, we have analysed the conservation of sequence and of prion-like composition for prion-forming proteins and for other prion-like proteins from S. cerevisiae, across three evolutionary levels. We discover that prion-like status is well-conserved for about half the set of prion-formers at the Saccharomycetes level, and that prion-forming domains evolve more quickly as sequences than other prion-like domains do. Such increased mutation rates may be linked to the acquisition of functional roles for prion-forming domains during the evolutionary epoch of Saccharomycetes. Domain scores for prion-like composition in S. cerevisiae are strongly correlated with scores for such composition weighted evolutionarily over the dozens of fungal species examined, indicating conservation of such prion-like status. Examples of notable prion-like proteins that are highly conserved both in sequence and prion-like composition are discussed.

Prions in eukaryotes have been linked to diseases, evolutionary capacitance, large-scale genetic control and long-term memory formation. In bacteria, constructed prion-forming proteins have been described, such as the prion-forming protein recently described for Clostridium botulinum transcription terminator Rho. Here, I analyzed the evolution of the Rho prion-forming domain across bacteria, and discovered that its conservation is sporadic both in the Clostridium genus and in bacteria generally. Nonetheless, it has an apparent evolutionary reach into eight or more different bacterial phyla. Motivated by these results, I investigated whether this pattern of wide-ranging evolutionary sporadicity is typical of bacterial prion-like domains. A measure of coverage of a domain (C) within its evolutionary range was derived, which is effectively a weighted fraction of the number of species in which the domain is found. I observe that occurrence across multiple phyla is not uncommon for bacterial prion-like protein domain families, but that they tend to sample of a low fraction of species within their evolutionary range, like Rho. The Rho prion-like domain family is one of the top three most widely distributed prion-like protein domain families in terms of number of phyla. There are >60 prion-like protein domain families that have at least the evolutionary coverage of Rho, and are found in multiple phyla. The implications of these findings for evolution and for experimental investigations into prion-forming proteins are discussed.

The cellular prion protein (PrPC) is renowned for its infectious conformational isoform PrPSc, capable of templating subsequent conversions of healthy PrPCs and thus triggering the group of incurable diseases known as transmissible spongiform encephalopathies. Besides this mechanism not being fully uncovered, the protein's physiological role is also elusive. PrPC and its newest, less understood paralog Shadoo are glycosylphosphatidylinositol-anchored proteins highly expressed in the central nervous system. While they share some attributes and neuroprotective actions, opposing roles have also been reported for the two; however, the amount of data about their exact functions is lacking. Protein-protein interactions and membrane microdomain localizations are key determinants of protein function. Accurate identification of these functions for a membrane protein, however, can become biased due to interactions occurring during sample processing. To avoid such artifacts, we apply a non-detergent-based membrane-fractionation approach to study the prion protein and Shadoo. We show that the two proteins occupy similarly raft and non-raft membrane fractions when expressed in N2a cells and that both proteins pull down the chaperone calnexin in both rafts and non-rafts. These indicate their possible binding to calnexin in both types of membrane domains, which might be a necessary requisite to aid the inherently unstable native conformation during their lifetime.

Approximately 15 % of human prion disease is associated with autosomal-dominant pathogenic mutations in the prion protein (PrP) gene. Previous attempts to model these diseases in mice have expressed human PrP mutations in murine PrP, but this may have different structural consequences. Here, we describe transgenic mice expressing human PrP with P102L or E200K mutations and methionine (M) at the polymorphic residue 129. Although no spontaneous disease developed in aged animals, these mice were readily susceptible to prion infection from patients with the homotypic pathogenic mutation. However, while variant Creutzfeldt-Jakob disease (CJD) prions transmitted infection efficiently to both lines of mice, markedly different susceptibilities to classical (sporadic and iatrogenic) CJD prions were observed. Prions from E200K and classical CJD M129 homozygous patients, transmitted disease with equivalent efficiencies and short incubation periods in human PrP 200K, 129M transgenic mice. However, mismatch at residue 129 between inoculum and host dramatically increased the incubation period. In human PrP 102L, 129M transgenic mice, short disease incubation periods were only observed with transmissions of prions from P102L patients, whereas classical CJD prions showed prolonged and variable incubation periods irrespective of the codon 129 genotype. Analysis of disease-related PrP (PrP(Sc)) showed marked alteration in the PrP(Sc) glycoform ratio propagated after transmission of classical CJD prions, consistent with the PrP point mutations directly influencing PrP(Sc) assembly. These data indicate that P102L or E200K mutations of human PrP have differing effects on prion propagation that depend upon prion strain type and can be significantly influenced by mismatch at the polymorphic residue 129.

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.

Prions are self-perpetuating proteins able to switch between a soluble state and an aggregated-and-transmissible conformation. These proteinaceous entities have been widely studied in yeast, where they are involved in hereditable phenotypic adaptations. The notion that such proteins could play functional roles and be positively selected by evolution has triggered the development of computational tools to identify prion-like proteins in different kingdoms of life. These algorithms have succeeded in screening multiple proteomes, allowing the identification of prion-like proteins in a diversity of unrelated organisms, evidencing that the prion phenomenon is well conserved among species. Interestingly enough, prion-like proteins are not only connected with the formation of functional membraneless protein-nucleic acid coacervates, but are also linked to human diseases. This review addresses state-of-the-art computational approaches to identify prion-like proteins, describes proteome-wide analysis efforts, discusses these unique proteins' functional role, and illustrates recently validated examples in different domains of life.

Prions are transmissible, propagating alternative states of proteins. Prions in budding yeast propagate heritable phenotypes and can function in large-scale gene regulation, or in some cases occur as diseases of yeast. Other 'prionogenic' proteins are likely prions that have been determined experimentally to form amyloid in vivo, and to have prion-like domains that are able to propagate heritable states. Furthermore, there are over 300 additional 'prion-like' yeast proteins that have similar amino-acid composition to prions (primarily a bias for asparagines and glutamines). Here, we examine the protein functional and interaction networks that involve prion, prionogenic and prion-like proteins. Set against a marked overall preference for N/Q-rich prion-like proteins not to interact with each other, we observe a significant tendency of prion/prionogenic proteins to interact with other, N/Q-rich prion-like proteins. This tendency is mostly due to a small number of networks involving the proteins NUP100p, LSM4p and PUB1p. In general, different data analyses of functional and interaction networks converge to indicate a strong linkage of prionogenic and prion-like proteins, to stress-granule assembly and related biological processes. These results further elucidate how prions may impact gene regulation, and reveal a broader horizon for the functional relevance of N/Q-rich prion-like domains.

Enhanced protein aggregation and/or impaired clearance of aggregates can lead to neurodegenerative disorders such as Alzheimer's Disease, Huntington's Disease, and prion diseases. Therefore, many protein quality control factors specialize in recognizing and degrading aggregation-prone proteins. Prions, which generally result from self-propagating protein aggregates, must therefore evade or outcompete these quality control systems in order to form and propagate in a cellular context. We developed a genetic screen in yeast that allowed us to explore the sequence features that promote degradation versus aggregation of a model glutamine/asparagine (Q/N)-rich prion domain from the yeast prion protein, Sup35, and two model glycine (G)-rich prion-like domains from the human proteins hnRNPA1 and hnRNPA2. Unexpectedly, we found that aggregation propensity and degradation propensity could be uncoupled in multiple ways. First, only a subset of classically aggregation-promoting amino acids elicited a strong degradation response in the G-rich prion-like domains. Specifically, large aliphatic residues enhanced degradation of the prion-like domains, whereas aromatic residues promoted prion aggregation without enhancing degradation. Second, the degradation-promoting effect of aliphatic residues was suppressed in the context of the Q/N-rich prion domain, and instead led to a dose-dependent increase in the frequency of spontaneous prion formation. Degradation suppression correlated with Q/N content of the surrounding prion domain, potentially indicating an underappreciated activity for these residues in yeast prion domains. Collectively, these results provide key insights into how certain aggregation-prone proteins may evade protein quality control degradation systems.

Prion diseases are fatal neurodegenerative disorders caused by aberrant metabolism of the cellular prion protein (PrP(C)). In genetic forms of these diseases, mutations in the globular C-terminal domain are hypothesized to favor the spontaneous generation of misfolded PrP conformers (including the transmissible PrP(Sc) form) that trigger downstream pathways leading to neuronal death. A mechanistic understanding of these diseases therefore requires knowledge of the quality control pathways that recognize and degrade aberrant PrPs. Here, we present comparative analyses of the biosynthesis, trafficking, and metabolism of a panel of genetic disease-causing prion protein mutants in the C-terminal domain. Using quantitative imaging and biochemistry, we identify a misfolded subpopulation of each mutant PrP characterized by relative detergent insolubility, inaccessibility to the cell surface, and incomplete glycan modifications. The misfolded populations of mutant PrPs were neither recognized by ER quality control pathways nor routed to ER-associated degradation despite demonstrable misfolding in the ER. Instead, mutant PrPs trafficked to the Golgi, from where the misfolded subpopulation was selectively trafficked for degradation in acidic compartments. Surprisingly, selective re-routing was dependent not only on a mutant globular domain, but on an additional lysine-based motif in the highly conserved unstructured N-terminus. These results define a specific trafficking and degradation pathway shared by many disease-causing PrP mutants. As the acidic lysosomal environment has been implicated in facilitating the conversion of PrP(C) to PrP(Sc), our identification of a mutant-selective trafficking pathway to this compartment may provide a cell biological basis for spontaneous generation of PrP(Sc) in familial prion disease.

Prion-like behaviour is attracting much attention due to the growing evidences that amyloid-like self-assembly may reach beyond neurodegeneration and be a conserved functional mechanism. The best characterized functional prions correspond to a subset of yeast proteins involved in translation or transcription. Their conformational promiscuity is encoded in Prion Forming Domains (PFDs), usually long and intrinsically disordered protein segments of low complexity. The compositional bias of these regions seems to be important for the transition between soluble and amyloid-like states. We have proposed that the presence of cryptic soft amyloid cores embedded in yeast PFDs can also be important for their assembly and demonstrated their existence and self-propagating abilities. Here, we used an orthogonal approach in the search of human domains that share yeast PFDs compositional bias and exhibit a predicted nucleating core, identifying 535 prion-like candidates. We selected seven proteins involved in transcriptional or translational regulation and associated to disease to characterize the properties of their amyloid cores. All of them self-assemble spontaneously into amyloid-like structures able to propagate their polymeric state. This provides support for the presence of short sequences able to trigger conformational conversion in prion-like human proteins, potentially regulating their functionality.

The basic molecular underpinnings of the pathological changes that unfold in prion disease remain elusive. A key role of increased oxidative stress has been hypothesized. Given the transient nature of most intermediate molecules implicated, increased oxidative stress is better assessed by quantitating the damage it causes to macromolecules. We used mass spectrometry-based methods to measure specific products of protein oxidation, glycoxidation, and lipoxidation in brains from patients suffering from Creutzfeldt-Jakob disease and Syrian hamsters affected by scrapie. In both cases, increased amounts of glutamic and aminoadipic semialdehydes, products of metal-catalyzed oxidation, malondialdehydelysine (a product of lipoxidation), N-epsilon-carboxyethyllysine (a product of glycoxidation), and N-epsilon-carboxymethyllysine (generated by lipoxidation and glycoxidation) were measured. PrP(Sc), the infectious isoform of the prion protein that accumulates in prion disease, was itself shown to be a target of increased oxidative modification. These changes were accompanied by alterations in fatty acid composition and increased phosphorylation of ERK(1/2) and p38, protein kinases known to respond to increased flows of ROS. These data support an important role of oxidative damage in the pathology of prion disease.

Prion-like behavior has been in the spotlight since it was first associated with the onset of mammalian neurodegenerative diseases. However, a growing body of evidence suggests that this mechanism could be behind the regulation of processes such as transcription and translation in multiple species. Here, we perform a stringent computational survey to identify prion-like proteins in the human proteome. We detected 242 candidate polypeptides and computationally assessed their function, protein-protein interaction networks, tissular expression, and their link to disease. Human prion-like proteins constitute a subset of modular polypeptides broadly expressed across different cell types and tissues, significantly associated with disease, embedded in highly connected interaction networks, and involved in the flow of genetic information in the cell. Our analysis suggests that these proteins might play a relevant role not only in neurological disorders, but also in different types of cancer and viral infections.

Prion protein (PrP) mutations are linked to genetic prion diseases, a class of phenotypically heterogeneous neurodegenerative disorders with invariably fatal outcome. How mutant PrP triggers neurodegeneration is not known. Synaptic dysfunction precedes neuronal loss but it is not clear whether, and through which mechanisms, disruption of synaptic activity ultimately leads to neuronal death. Here we show that mutant PrP impairs the secretory trafficking of AMPA receptors (AMPARs). Specifically, intracellular retention of the GluA2 subunit results in synaptic exposure of GluA2-lacking, calcium-permeable AMPARs, leading to increased calcium permeability and enhanced sensitivity to excitotoxic cell death. Mutant PrPs linked to different genetic prion diseases affect AMPAR trafficking and function in different ways. Our findings identify AMPARs as pathogenic targets in genetic prion diseases, and support the involvement of excitotoxicity in neurodegeneration. They also suggest a mechanistic explanation for how different mutant PrPs may cause distinct disease phenotypes.

Prion proteins were initially associated with diseases such as Creutzfeldt Jakob and transmissible spongiform encephalopathies. However, deeper research revealed them as versatile tools, exploited by the cells to execute fascinating functions, acting as epigenetic elements or building membrane free compartments in eukaryotes. One of the most intriguing properties of prion proteins is their ability to propagate a conformational assembly, even across species. In this context, it has been observed that bacterial amyloids can trigger the formation of protein aggregates by interacting with host proteins. As our life is closely linked to bacteria, either through a parasitic or symbiotic relationship, prion-like proteins produced by bacterial cells might play a role in this association. Bioinformatics is helping us to understand the factors that determine conformational conversion and infectivity in prion-like proteins. We have used PrionScan to detect prion domains in 839 different bacteria proteomes, detecting 2200 putative prions in these organisms. We studied this set of proteins in order to try to understand their functional role and structural properties. Our results suggest that these bacterial polypeptides are associated to peripheral rearrangement, macromolecular assembly, cell adaptability, and invasion. Overall, these data could reveal new threats and therapeutic targets associated to infectious diseases.

Prion diseases are lethal, infectious diseases associated with prion protein (PrP) misfolding. A large number of mammals are susceptible to both sporadic and acquired prion diseases. Although PrP is highly conserved and ubiquitously expressed in all mammals, not all species exhibit prion disease. By employing full length recombinant PrP from five known prion susceptible species (human, cattle, cat, mouse and hamster) and two species considered to be prion resistant (pig and dog) the amyloidogenicity of these PrPs has been delineated. All the mammalian PrPs, even from resistant species, were swiftly converted from the native state to amyloid-like structure when subjected to a native condition conversion assay. The PrPs displayed amyloidotypic tinctorial and ultrastructural hallmarks. Self-seeded conversion of the PrPs displayed significantly decreased lag phases demonstrating that nucleation dependent polymerization is a dominating mechanism in the fibrillation process. Fibrils from Aβ1-40, Aβ1-42, Lysozyme, Insulin and Transthyretin did not accelerate conversion of HuPrP whereas fibrils from HuPrP90-231 and HuPrP121-231 as well as full length PrPs of all PrPs efficiently seeded conversion showing specificity of the assay requiring the C-terminal PrP sequence. Our findings have implications for PrP misfolding and could have ramifications in the context of prion resistant species and silent carriers.

Prions are a singular subset of proteins able to switch between a soluble conformation and a self-perpetuating amyloid state. Traditionally associated with neurodegenerative diseases, increasing evidence indicates that organisms exploit prion-like mechanisms for beneficial purposes. The ability to transit between conformations is encoded in the so-called prion domains, long disordered regions usually enriched in glutamine/asparagine residues. Interestingly, Plasmodium falciparum, the parasite that causes the most virulent form of malaria, is exceptionally rich in proteins bearing long Q/N-rich sequence stretches, accounting for roughly 30% of the proteome. This biased composition suggests that these protein regions might correspond to prion-like domains (PrLDs) and potentially form amyloid assemblies. To investigate this possibility, we performed a stringent computational survey for Q/N-rich PrLDs on P. falciparum. Our data indicate that ∼10% of P. falciparum protein sequences have prionic signatures, and that this subproteome is enriched in regulatory proteins, such as transcription factors and RNA-binding proteins. Furthermore, we experimentally demonstrate for several of the identified PrLDs that, despite their disordered nature, they contain inner short sequences able to spontaneously self-assemble into amyloid-like structures. Although the ability of these sequences to nucleate the conformational conversion of the respective full-length proteins should still be demonstrated, our analysis suggests that, as previously described for other organisms, prion-like proteins might also play a functional role in P. falciparum.

Membrane invagination and vesicle formation are key steps in endocytosis and cellular trafficking. Here, we show that endocytic coat proteins with prion-like domains (PLDs) form hemispherical puncta in the budding yeast, Saccharomyces cerevisiae These puncta have the hallmarks of biomolecular condensates and organize proteins at the membrane for actin-dependent endocytosis. They also enable membrane remodeling to drive actin-independent endocytosis. The puncta, which we refer to as endocytic condensates, form and dissolve reversibly in response to changes in temperature and solution conditions. We find that endocytic condensates are organized around dynamic protein-protein interaction networks, which involve interactions among PLDs with high glutamine contents. The endocytic coat protein Sla1 is at the hub of the protein-protein interaction network. Using active rheology, we inferred the material properties of endocytic condensates. These experiments show that endocytic condensates are akin to viscoelastic materials. We use these characterizations to estimate the interfacial tension between endocytic condensates and their surroundings. We then adapt the physics of contact mechanics, specifically modifications of Hertz theory, to develop a quantitative framework for describing how interfacial tensions among condensates, the membrane, and the cytosol can deform the plasma membrane to enable actin-independent endocytosis.