Prion-like domains (PLDs) are low complexity sequences found in RNA binding proteins associated with the neurodegenerative disorder amyotrophic lateral sclerosis. Recently, PLDs have been implicated in mediating gene regulation via liquid-phase transitions that drive ribonucleoprotein granule assembly. In this paper, we report many PLDs in proteins associated with paraspeckles, subnuclear bodies that form around long noncoding RNA. We mapped the interactome network of paraspeckle proteins, finding enrichment of PLDs. We show that one protein, RBM14, connects key paraspeckle subcomplexes via interactions mediated by its PLD. We further show that the RBM14 PLD, as well as the PLD of another essential paraspeckle protein, FUS, is required to rescue paraspeckle formation in cells in which their endogenous counterpart has been knocked down. Similar to FUS, the RBM14 PLD also forms hydrogels with amyloid-like properties. These results suggest a role for PLD-mediated liquid-phase transitions in paraspeckle formation, highlighting this nuclear body as an excellent model system for understanding the perturbation of such processes in neurodegeneration.

Chaperones with aggregase activity promote and organize the aggregation of misfolded proteins and their deposition at specific intracellular sites. This activity represents a novel cytoprotective strategy of protein quality control systems; however, little is known about its mechanism. In yeast, the small heat shock protein Hsp42 orchestrates the stress-induced sequestration of misfolded proteins into cytosolic aggregates (CytoQ). In this study, we show that Hsp42 harbors a prion-like domain (PrLD) and a canonical intrinsically disordered domain (IDD) that act coordinately to promote and control protein aggregation. Hsp42 PrLD is essential for CytoQ formation and is bifunctional, mediating self-association as well as binding to misfolded proteins. Hsp42 IDD confines chaperone and aggregase activity and affects CytoQ numbers and stability in vivo. Hsp42 PrLD and IDD are both crucial for cellular fitness during heat stress, demonstrating the need for sequestering misfolded proteins in a regulated manner.

The conversion of cellular prion protein (PrPC) to disease-provoking conformer (PrPSc) is crucial in the pathogenesis of prion diseases. Heparin has been shown to enhance mammalian prion protein misfolding. As spontaneous prion disease has not been reported in non-mammalian species, such as chicken, it is interesting to explore the influence of heparin on the conversion of chicken prion protein (ChPrP). Herein, we investigated the influences of heparin on biochemical properties of full-length recombinant ChPrP, with murine prion protein (MoPrP) as control. The results showed that at low heparin concentration (10 μg/mL), a great loss of solubility was observed for both MoPrP and ChPrP using solubility assays. In contrast, when the concentration of heparin was high (30 μg/mL), the solubility of MoPrP and ChPrP both decreased slightly. Using circular dichroism, PK digestion and transmission electron microscopy, significantly increased β-sheet content, PK resistance and size of aggregates were observed for MoPrP interacted with 30 μg/mL heparin, whereas 30 μg/mL heparin-treated ChPrP showed less PK resistance and slight increase of β-sheet structure. Therefore, heparin can induce conformational changes in both MoPrP and ChPrP and the biochemical properties of the aggregates induced by heparin could be modified by heparin concentration. These results highlight the importance of concentration of cofactors affecting PrP misfolding.

Prions are proteinaceous particles that propagate alternative protein conformations/states to further copies of the same proteins, and are transmitted from cell-to-cell, and organism-to-organism. Prions are usually made of the beta-sheet rich assemblies termed amyloid. The original prion protein PrP causes devastating neurodegenerative disorders in humans and other mammals. In the yeast Saccharomyces cerevisiae, many prion-forming proteins have been observed; a prominent feature of these proteins is an intrinsically disordered domain rich in glutamine (Q) and asparagine (N) residues. Several human proteins that are yeast-prion-like, in particular those with poly-glutamine (poly-Q) expansions, have been experimentally implicated in human neurodegenerative diseases.

A hallmark of prion diseases is the accumulation of disease-associated isoforms (PrP(Sc)) which results from the conversion of host-encoded cellular prion proteins (PrP(C)). Using molecular biochemistry, several disease variants of the human Creutzfeldt-Jakob disease have been identified showing several PrP(Sc) variants in individuals and selective accumulation in specific brain regions. As PrP(C) is differentially expressed and post-translationally modified, certain distinct protein compositions may have the ability to convert more efficiently than others. The PrP(C) glycoprotein moiety represents a single yet divers mixture, but little is known about its exact composition. In this study, we separated and characterized PrP(C) derived from six defined human brain regions in regard to their solubility in several detergent solutions and glycoprotein profile formation. We identified four different but regionally distinct protein compositions. PrP(C) found in the neocortex exhibited dominant diglycosylated bands in the high as well as in the low soluble fractions. The proteins in the nucleus lentiformis, thalamus and hippocampus were more soluble with deoxycholic acid as the N-octyl-β-d-glucopyranoside and the diglycosylated bands displayed strong signals in the supernatants and weaker signals in the sediments. Two different protein profiles were established with PrP(C) derived from the medulla oblongata and the solubility of PrP(C) in the cerebellum clearly differed by the choice of detergent. Our findings indicate the existence of several distinct PrP(C) compositions localized in distinct brain regions. Protein variations may be induced by specific modifications to specific regional biological functions.

In molting animals, a cuticular extracellular matrix forms the first barrier to infection and other environmental insults. In the nematode Caenorhabditis elegans there are two types of cuticle: a well-studied collagenous cuticle lines the body, and a poorly-understood chitinous cuticle lines the pharynx. In the posterior end of the pharynx is the grinder, a tooth-like cuticular specialization that crushes food prior to transport to the intestine for digestion. We here show that the grinder increases in size only during the molt. To gain molecular insight into the structure of the grinder and pharyngeal cuticle, we performed a microarray analysis to identify mRNAs increased during the molt. We found strong transcriptional induction during the molt of 12 of 15 previously identified abu genes encoding Prion-like (P) glutamine (Q) and asparagine (N) rich PQN proteins, as well as 15 additional genes encoding closely related PQN proteins. abu/pqn genes, which we name the abu/pqn paralog group (APPG) genes, were expressed in pharyngeal cells and the proteins encoded by two APPG genes we tested localized to the pharyngeal cuticle. Deleting the APPG gene abu-14 caused abnormal pharyngeal cuticular structures and knocking down other APPG genes resulted in abnormal cuticular function. We propose that APPG proteins promote the assembly and function of a unique cuticular structure. The strong developmental regulation of the APPG genes raises the possibility that such genes would be identified in transcriptional profiling experiments in which the animals' developmental stage is not precisely staged.

Paralogs that arise from gene duplications during genome evolution enable genetic redundancy and phenotypic robustness. Variation in the coding or regulatory sequence of paralogous transcriptional regulators diversifies their functions and relationships, which provides developmental robustness against genetic or environmental perturbation. The fate transition of plant shoot stem cells for flowering and reproductive success requires a robust transcriptional control. However, how paralogs function and interact to achieve such robustness is unknown.

Recent cases of prion transmission in humans following transfusions using blood donated by patients with asymptomatic variant Creutzfeldt-Jakob disease (CJD) implicate the presence of prion infectivity in peripheral blood. In this study, we examined the levels of the normal, cellular prion protein (PrPC), and the disease-causing isoform (PrPSc) in subpopulations of circulating white blood cells (WBCs) from patients with sporadic (s) CJD, age-matched neurological controls and healthy donors. Though widely distributed, the highest levels of PrPC were found in a subpopulation of T lymphocytes: approximately 12,000 PrPC molecules were found per CD4+CD45RA-CD62L- effector memory T helper cell. Although platelets expressed low levels of PrPC on their surface, their high abundance in circulation resulted in the majority of PrPC being platelet associated. Using quantitative fluorescence-activated cell sorting analysis, we found that neither WBC composition nor the amount of cell-surface PrPC molecules was altered in patients with sCJD. Eight different WBC fraction types from the peripheral blood of patients with sCJD were assessed for PrPSc. We were unable to find any evidence for PrPSc in purified granulocytes, monocytes, B cells, CD4+ T cells, CD8+ T cells, natural killer cells, nonclassical gamma delta T cells, or platelets. If human WBCs harbor prion infectivity in patients with sCJD, then the levels are likely to be low.

Proteomics changes of brain tissues have been described in different neurodegenerative diseases including Alzheimer's disease and Parkinson's disease. However, the brain proteomics of human prion disease remains less understood. In the study, the proteomics patterns of cortex and cerebellum of brain tissues of sporadic Creutzfeldt-Jakob disease, fatal familial insomnia, and G114V genetic CJD were analyzed with isobaric tags for relative and absolute quantitation combined with multidimensional liquid chromatography and MS analysis, with the brains from three normal individuals as controls. Global protein profiling, significant pathway, and functional categories were analyzed. In total, 2287 proteins were identified with quantitative information both in cortex and cerebellum regions. Cerebellum tissues appeared to contain more up- and down-regulated proteins (727 proteins) than cortex regions (312 proteins) of Creutzfeldt-Jakob disease, fatal familial insomnia, and G114V genetic CJD. Viral myocarditis, Parkinson's disease, Alzheimer's disease, lysosome, oxidative phosphorylation, protein export, and drug metabolism-cytochrome P450 were the most commonly affected pathways of the three kinds of diseases. Almost coincident biological functions were identified in the brain tissues of the three diseases. In all, data here demonstrate that the brain tissues of Creutzfeldt-Jakob disease, fatal familial insomnia, and G114V genetic CJD have obvious proteomics changes at their terminal stages, which show the similarities not only among human prion diseases but also with other neurodegeneration diseases. This is the first study to provide a reference proteome map for human prion diseases and will be helpful for future studies focused on potential biomarkers for the diagnosis and therapy of human prion diseases.

RNA-binding proteins (RBPs) with prion-like domains (PrLDs) phase transition to functional liquids, which can mature into aberrant hydrogels composed of pathological fibrils that underpin fatal neurodegenerative disorders. Several nuclear RBPs with PrLDs, including TDP-43, FUS, hnRNPA1, and hnRNPA2, mislocalize to cytoplasmic inclusions in neurodegenerative disorders, and mutations in their PrLDs can accelerate fibrillization and cause disease. Here, we establish that nuclear-import receptors (NIRs) specifically chaperone and potently disaggregate wild-type and disease-linked RBPs bearing a NLS. Karyopherin-β2 (also called Transportin-1) engages PY-NLSs to inhibit and reverse FUS, TAF15, EWSR1, hnRNPA1, and hnRNPA2 fibrillization, whereas Importin-α plus Karyopherin-β1 prevent and reverse TDP-43 fibrillization. Remarkably, Karyopherin-β2 dissolves phase-separated liquids and aberrant fibrillar hydrogels formed by FUS and hnRNPA1. In vivo, Karyopherin-β2 prevents RBPs with PY-NLSs accumulating in stress granules, restores nuclear RBP localization and function, and rescues degeneration caused by disease-linked FUS and hnRNPA2. Thus, NIRs therapeutically restore RBP homeostasis and mitigate neurodegeneration.

The key event in prion pathogenesis is the structural conversion of the normal cellular protein, PrP(C), into an aberrant and partially proteinase K resistant isoform, PrP(Sc). Since the minimum requirement for a prion disease phenotype is the expression of endogenous PrP in the host, species carrying orthologue prion genes, such as fish, could in theory support prion pathogenesis. Our previous work has demonstrated the development of abnormal protein deposition in sea bream brain, following oral challenge of the fish with natural prion infectious material. In this study, we used a prion-infected mouse neuroblastoma cell line for the expression of three different mature fish PrP proteins and the evaluation of the resistance of the exogenously expressed proteins to proteinase K treatment (PK), as an indicator of a possible prion conversion. No evidence of resistance to PK was detected for any of the studied recombinant proteins. Although not indicative of an absolute inability of the fish PrPs to structurally convert to pathogenic isoforms, the absence of PK-resistance may be due to supramolecular and conformational differences between the mammalian and piscine PrPs.

Despite having therapeutic potential, anti-PrP antibodies caused a major controversy due to their neurotoxic effects. For instance, treating mice with ICSM antibodies delayed prion disease onset, but both were found to be either toxic or innocuous to neurons by researchers following cross-linking PrPC. In order to elucidate and understand the reasons that led to these contradictory outcomes, we conducted a comprehensive in silico study to assess the antibody-specific toxicity. Since most therapeutic anti-PrP antibodies were generated against human truncated recombinant PrP91-231 or full-length mouse PrP23-231, we reasoned that host specificity (human vs murine) of PrPC might influence the nature of the specific epitopes recognized by these antibodies at the structural level possibly explaining the 'toxicity' discrepancies reported previously. Initially, molecular dynamics simulation and pro-motif analysis of full-length human (hu)PrP and mouse (mo)PrP 3D structure displayed conspicuous structural differences between huPrP and moPrP. We identified 10 huPrP and 6 moPrP linear B-cell epitopes from the prion protein 3D structure where 5 out of 10 huPrP and 3 out of 6 moPrP B-cell epitopes were predicted to be potentially toxic in immunoinformatics approaches. Herein, we demonstrate that some of the predicted potentially 'toxic' epitopes identified by the in silico analysis were similar to the epitopes recognized by the toxic antibodies such as ICSM18 (146-159), POM1 (138-147), D18 (133-157), ICSM35 (91-110), D13 (95-103) and POM3 (95-100). This in silico study reveals the role of host specificity of PrPC in epitope-specific anti-PrP antibody toxicity.

Prions are often considered as molecular memory devices, generating reproducible memory of a conformational change. Prion-like proteins (PrLPs) have been widely demonstrated to be present in plants, but their role in plant stress and memory remains unexplored. In this work, we report the widespread presence of PrLPs in plants through a comprehensive meta-analysis of 39 genomes representing major taxonomic groups. We find diverse functional roles associated with these proteins in various species and term the full complement of PrLPs in a genome as its "prionome." In particular, we found the rice prionome being significantly enriched in transposons/retrotransposons (Ts/RTRs) and identified over 60 rice PrLPs that were differentially regulated in stress and developmental responses. This prompted us to explore whether and to what extent PrLPs may build stress memory. By integrating the available rice interactome, transcriptome, and regulome data sets, we could find links between stress and memory pathways that would not have otherwise been discernible. Regulatory inferences derived from the superimposition of these data sets revealed a complex network and cross talk between PrLPs, transcription factors (TFs), and the genes involved in stress priming. This integrative meta-analysis connects transient and transgenerational memory mechanisms in plants with PrLPs, suggesting that plant memory may rely upon protein-based signals in addition to chromatin-based epigenetic signals. Taken together, our work provides important insights into the anticipated role of prion-like candidates in stress and memory, paving the way for more focused studies for validating the role of the identified PrLPs in memory acclimation.

Proteins such as FUS phase separate to form liquid-like condensates that can harden into less dynamic structures. However, how these properties emerge from the collective interactions of many amino acids remains largely unknown. Here, we use extensive mutagenesis to identify a sequence-encoded molecular grammar underlying the driving forces of phase separation of proteins in the FUS family and test aspects of this grammar in cells. Phase separation is primarily governed by multivalent interactions among tyrosine residues from prion-like domains and arginine residues from RNA-binding domains, which are modulated by negatively charged residues. Glycine residues enhance the fluidity, whereas glutamine and serine residues promote hardening. We develop a model to show that the measured saturation concentrations of phase separation are inversely proportional to the product of the numbers of arginine and tyrosine residues. These results suggest it is possible to predict phase-separation properties based on amino acid sequences.

Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized by the accumulation of amyloid-β (Aβ) and the loss of synapses. Aggregation of the cellular prion protein (PrPC) by Aβ oligomers induced synapse damage in cultured neurons. PrPC is attached to membranes via a glycosylphosphatidylinositol (GPI) anchor, the composition of which affects protein targeting and cell signaling. Monoacylated PrPC incorporated into neurons bound "natural Aβ", sequestering Aβ outside lipid rafts and preventing its accumulation at synapses. The presence of monoacylated PrPC reduced the Aβ-induced activation of cytoplasmic phospholipase A2 (cPLA2) and Aβ-induced synapse damage. This protective effect was stimulus specific, as treated neurons remained sensitive to α-synuclein, a protein associated with synapse damage in Parkinson's disease. In synaptosomes, the aggregation of PrPC by Aβ oligomers triggered the formation of a signaling complex containing the cPLA2.a process, disrupted by monoacylated PrPC. We propose that monoacylated PrPC acts as a molecular sponge, binding Aβ oligomers at the neuronal perikarya without activating cPLA2 or triggering synapse damage.

Prions are a particular type of amyloids with the ability to self-perpetuate and propagate in vivo. Prion-like conversion underlies important biological processes but is also connected to human disease. Yeast prions are the best understood transmissible amyloids. In these proteins, prion formation from an initially soluble state involves a structural conversion, driven, in many cases, by specific domains enriched in glutamine/asparagine (Q/N) residues. Importantly, domains sharing this compositional bias are also present in the proteomes of higher organisms, thus suggesting that prion-like conversion might be an evolutionary conserved mechanism. We have recently shown that the identification and evaluation of the potency of amyloid nucleating sequences in putative prion domains allows discrimination of genuine prions. PrionW is a web application that exploits this principle to scan sequences in order to identify proteins containing Q/N enriched prion-like domains (PrLDs) in large datasets. When used to scan the complete yeast proteome, PrionW identifies previously experimentally validated prions with high accuracy. Users can analyze up to 10 000 sequences at a time, PrLD-containing proteins are identified and their putative PrLDs and amyloid nucleating cores visualized and scored. The output files can be downloaded for further analysis. PrionW server can be accessed at http://bioinf.uab.cat/prionw/.

Plasmodium falciparum encodes approximately 5300 proteins of which approximately 35% have repeats of amino acids, significantly higher than in other fully sequenced eukaryotes. The proportion of proteins with amino acid homorepeats varies from 4 to 54% amongst different functional classes of proteins. These homorepeats are dominated by asparagines, which are selected over lysines despite equivalent AT codon content. Surprisingly, asparagine repeats are absent from the variant surface antigen protein families of PfEMP1s, Stevors and Rifins. The PfEMP1 protein family is instead rich in recurrences of glutamates, similar to human cell surface proteins. Structural mapping of homorepeats suggests that these segments are likely to form surface exposed structures that protrude from the main protein cores. We also found an abundance of asparagine-rich prion-like domains in P. falciparum, significantly larger than in any other eukaryote. Domains rich in glutamines and asparagines have an innate predisposition to form self-propagating amyloid fibers, which are involved both in prion-based inheritance and in human neurodegenerative disorders. Nearly 24% (1302 polypeptides) of P. falciparum proteins contain prion-forming or prion-inducing domains, in comparison to Drosophila (approximately 3.4%) which to date showed the highest number of prion-like proteins. The unexpected properties of P. falciparum revealed here open new avenues for investigating parasite biology.

The formation of β-sheet rich prion oligomers and fibrils from native prion protein (PrP) is thought to be a key step in the development of prion diseases. Many methods are available to convert recombinant prion protein into β-sheet rich fibrils using various chemical denaturants (urea, SDS, GdnHCl), high temperature, phospholipids, or mildly acidic conditions (pH 4). Many of these methods also require shaking or another form of agitation to complete the conversion process. We have identified that shaking alone causes the conversion of recombinant PrP to β-sheet rich oligomers and fibrils at near physiological pH (pH 5.5 to pH 6.2) and temperature. This conversion does not require any denaturant, detergent, or any other chemical cofactor. Interestingly, this conversion does not occur when the water-air interface is eliminated in the shaken sample. We have analyzed shaking-induced conversion using circular dichroism, resolution enhanced native acidic gel electrophoresis (RENAGE), electron microscopy, Fourier transform infrared spectroscopy, thioflavin T fluorescence and proteinase K resistance. Our results show that shaking causes the formation of β-sheet rich oligomers with a population distribution ranging from octamers to dodecamers and that further shaking causes a transition to β-sheet fibrils. In addition, we show that shaking-induced conversion occurs for a wide range of full-length and truncated constructs of mouse, hamster and cervid prion proteins. We propose that this method of conversion provides a robust, reproducible and easily accessible model for scrapie-like amyloid formation, allowing the generation of milligram quantities of physiologically stable β-sheet rich oligomers and fibrils. These results may also have interesting implications regarding our understanding of prion conversion and propagation both within the brain and via techniques such as protein misfolding cyclic amplification (PMCA) and quaking induced conversion (QuIC).

Amyloids are non-branching fibrils that are composed of stacked monomers stabilized by intermolecular β-sheets. Some amyloids are associated with incurable diseases, whereas others, functional amyloids, regulate different vital processes. The prevalence and significance of functional amyloids in wildlife are still poorly understood. In recent years, by applying new approach of large-scale proteome screening, a number of novel candidate amyloids were identified in the yeast Saccharomyces cerevisiae, many of which are localized in the yeast cell wall. In this work, we showed that one of these proteins, Toh1, possess amyloid properties. The Toh1-YFP hybrid protein forms detergent-resistant aggregates in the yeast cells while being expressed under its own PTOH1 or inducible PCUP1 promoter. Using bacterial system for generation of extracellular amyloid aggregates C-DAG, we demonstrated that the N-terminal Toh1 fragment, containing amyloidogenic regions predicted in silico, binds Congo Red dye, manifests 'apple-green' birefringence when examined between crossed polarizers, and forms amyloid-like fibrillar aggregates visualized by TEM. We have established that the Toh1(20-365)-YFP hybrid protein fluorescent aggregates are co-localized with a high frequency with Rnq1C-CFP and Sup35NM-CFP aggregates in the yeast cells containing [PIN+] and [PSI+] prions, and physical interaction of these aggregated proteins was confirmed by FRET. This is one of a few known cases of physical interaction of non-Q/N-rich amyloid-like protein and Q/N-rich amyloids, suggesting that interaction of different amyloid proteins may be determined not only by similarity of their primary structures but also by similarity of their secondary structures and of conformational folds.

Prion diseases are associated with the conformational conversion of the cellular prion protein (PrP(C)) into the pathological scrapie isoform (PrP(Sc)) in the brain. Both the in vivo and in vitro conversion of PrP(C) into PrP(Sc) is significantly inhibited by differences in amino acid sequence between the two molecules. Using protein misfolding cyclic amplification (PMCA), we now report that the recombinant full-length human PrP (rHuPrP23-231) (that is unglycosylated and lacks the glycophosphatidylinositol anchor) is a strong inhibitor of human prion propagation. Furthermore, rHuPrP23-231 also inhibits mouse prion propagation in a scrapie-infected mouse cell line. Notably, it binds to PrP(Sc), but not PrP(C), suggesting that the inhibitory effect of recombinant PrP results from blocking the interaction of brain PrP(C) with PrP(Sc). Our findings suggest a new avenue for treating prion diseases, in which a patient's own unglycosylated and anchorless PrP is used to inhibit PrP(Sc) propagation without inducing immune response side effects.