Aug 2026· Proteins: Structure, Function, and Bioinformatics· 0 citations· 75 references
Medicine
TL;DR
This study enhances the understanding of the E. coli-genome nature and suggests the existence of specific and experimentally testable novel prions in this organism, and moves a step forward towards the identification of new prion proteins in bacteria.
Abstract
Prions are infectious proteins that bear misfolded conformations capable of converting folded states into misfolded aggregates under physiologically relevant conditions. In mammals, prions cause deadly maladies including Creutzfeldt-Jakob and chronic wasting disease. To date, several prion proteins have been identified in eukaryotes, primarily vertebrates and fungi. There are, however, very few established prions in bacteria. Interestingly, the sequence of most vertebrate and yeast prions has bimodal characteristics. Namely, it comprises one intrinsically disordered and one folded region of comparable size. Here, we took advantage of this property to develop the PUFF algorithm, which can computationally identify Protein Unfolding/Folding Frameworks based on amino-acid sequence alone. After extensive validations with known prions from different organisms, we employed PUFF to predict juxtaposed intrinsically disordered and folded large regions across the E. coli proteome. Based on this criterion, PUFF predicted the presence of 102 novel 1st-generation prions. Additional analysis, taking sub-cellular compartmentalization and tryptophan distribution into account, led to establishing a refined group of 6 cytoplasmic 2nd-generation prion candidates. Some of these have unknown functions, while others are experimentally well-characterized proteins that are primarily involved in gene expression but were not previously flagged as prions. This study enhances our understanding of the E. coli-genome nature and suggests the existence of specific and experimentally testable novel prions in this organism. In all, our work moves a step forward towards the identification of new prions in bacteria.
Prion diseases are neurodegenerative disorders associated with the structural conversion of the cellular prion protein (PrPc) into its misfolded infectious isoform (PrPSc). Despite substantial efforts, no disease-modifying therapy or cure is currently available. Here, we present an integrated computational-experimental pipeline for the rational design of cyclic peptides targeting PrPc to inhibit its pathogenic conversion. Starting from crystal structures of antibody-bound mouse PrPc, we develop a rational design strategy combined with iterative molecular dynamics simulations and sequence optimization to generate peptides with enhanced binding and structural impact. Three candidates were selected for experimental validation. Our results show that (49YGPDPSDSYT58, antibody numbering) that binds stably to the α2–α3 interface most effectively reduced PrPSc levels in GT1-7 cells, essentially by inducing allosteric re-arrangements that reinforce the intramolecular helical bundle. (89GQSNTKPYT97) and (89RQSNTWPYT97) binding the β1-α1/α3 junction exerted more modest effects due to the potential competition of the flexible tail to bind at this site. These results establish a mechanistic link between peptide-induced stabilization of PrPc and inhibition of prion propagation and provide a generalizable framework for designing conformational stabilizers of aggregation-prone proteins.
Elpiniki Paspali, C. Morales, Daria De Raffele et al.· bioRxiv· 0 citations
Prions are misfolded, self-propagating versions of cellular proteins. In humans and animals, misfolding of the cellular prion protein (PrPC) causes invariably fatal transmissible neurodegenerative diseases. Little is known about how mammalian prions replicate in the brain, including whether other proteins participate in prion replication in vivo. Several members of the protein disulfide isomerase family have been shown to reside in close spatial proximity to PrPC in cells and mice, implying that they could be involved in prion biogenesis. Here, we show that stable knock-down of the protein disulfide isomerase P4HB (also called PDIA1) in prion-susceptible CAD5 cells reduces PrPC levels and hinders the generation of protease-resistant PrP (PrPres) following infection with two different prion strains. Moreover, transient knock-down of P4HB decreases PrPres levels in cells with established prion infection. Partial reduction of P4HB activity using the P4HB-selective inhibitor KSC-34 also decreases PrPC levels in uninfected CAD5 cells whereas treatment of prion-infected CAD5 cells with KSC-34 results in higher levels of PrPres. A proportion of P4HB reaches the cell surface where PrPC is located, and a secreted P4HB variant increases PrPres levels in cells. Collectively, these results suggest that P4HB influences PrPC homeostasis and modulates the conversion of PrPC into misfolded species. Thus, targeting P4HB during prion disease may have therapeutic benefit.
Genki Amano, H. Arshad, Zeel Patel et al.· PLoS Pathogens· 0 citations
Protein intrinsically disordered regions (IDRs) play important biological roles despite lacking stable structures. IDRs drive the formation of both biomolecular condensates via liquid–liquid phase separation (LLPS) and solid fibrous amyloid aggregates. Amyloids can be pathogenic and may exhibit self-perpetuating (prion) properties. Relationships between LLPS and the amyloid-forming and prion-propagating abilities of IDRs remain poorly understood. The N-proximal IDR of the yeast translation termination factor eRF3 (Sup35) can form both liquid condensates and heritable amyloid-based prions and serves as a powerful model for investigating these phenomena due to the availability of simple phenotypic, cytological and biochemical assays. Deletion analysis demonstrates that the N-proximal prion domain (Sup35N) of Sup35 is sufficient for chaperone-dependent prion propagation and that various regions of this domain show differential impacts on LLPS, amyloid aggregation, and prion inheritance. Specifically, the N-terminal NQ-rich stretch and the region of oligopeptide repeats are the most important contributors to the LLPS and formation of amyloid fibrils, while oligopeptide repeats and the C-terminal region of Sup35N are crucial for prion inheritance. Contrary to previous reports, the NQ-rich stretch is not required for prion formation and inheritance in yeast. Our data indicate that, in addition to amino acid composition, specific sequence motifs control reversible and heritable assemblies of Sup35.
A. Grizel, Natalia A. Gorsheneva, Ismat Jahan Anee et al.· International Journal of Mol...· 0 citations
Cryo-electron microscopy (cryo-EM) studies of amyloid fibrils have revealed endpoint structures of disease-relevant filaments and polymorphic intermediates formed during in vitro assembly of prion-like proteins. However, how transmissible prion or prion-like amyloids evolve during de novo formation and maturation in living cells remains unknown. Here, using the yeast prion [PSI+] as a model, we isolated Sup35NM amyloid fibrils from successive stages of [PSI+] maturation in Saccharomyces cerevisiae and characterised their near-atomic structures and population-level structural diversity by combining cryo-EM and atomic force microscopy. We show that intermediate and mature states differ in predominant fibril structure and the regions of the Sup35 sequence incorporated into the core, and that structural diversity decreases during maturation. Curing of [PSI+] at the mature state by guanidine hydrochloride (GdnHCl), which selectively inhibits ATPase activity of the chaperone Hsp104, restored both the predominant intermediate amyloid structure and the broader structural diversity characteristic of the intermediate state. In addition, Hsp104, Ssa1 (Hsp70) and Sis1 (Hsp40) associate differently with fibrils from the two states. Together, these findings provide direct structural evidence for amyloid evolution in vivo and support a chaperone-mediated mechanism of conformer selection within a polymorphic amyloid population.
Ziang Wang, Samantha L. Weetman, Barbara Altenhuber et al.· bioRxiv· 0 citations
Many components of human innate immunity are conserved in prokaryotes 1,2. While pathogens are known to evade host defenses 3, whether mechanisms of immune evasion share a similarly deep evolutionary or functional conservation across the tree of life remains largely unresolved. Here, we systematically explore this question by establishing The Viral Compendium (TVC), a database of over 350,000 proteins and 790,000 domains from eukaryotic, bacterial, and archaeal viruses. We find that protein structure alignments identify pan-viral clusters of proteins and domains, vastly increasing viral protein annotation rates compared to sequence-based methods. Domain co-association analysis revealed 1,351 combinations of domains that are conserved across archaeal, eukaryotic, and bacterial viruses, including fusion proteins that reconstitute the nuclease-ATPase core of the Mre11-Rad50 multiprotein complex involved in cellular DNA repair 4. Leveraging structural comparisons, we identify widely shared structural folds that mediate immune suppression: conserved phosphodiesterase folds encoded by both viral and bacterial pathogens that degrade nucleotide messengers, and double-stranded RNA binding domains employed across eukaryotic and prokaryotic viruses to suppress cellular sensing. Together, our results demonstrate that pathogen immune evasion is built upon conserved structural building blocks, revealing unified mechanisms and effectors of immune antagonism spanning all domains of life.
L. Profeta, Erin E. Doherty, Johanna M. Ebner et al.· bioRxiv· 0 citations
Many proteins have slow folding times in vitro that are physiologically untenable. To combat this challenge, ATP-dependent chaperonins are thought to possess the unique ability to catalyze protein folding. Performing quantitative model selection using protein folding and unfolding data, we here show that short nucleic acids containing Gquadruplex (G4) structure can also catalyze protein folding. Performing the experiments as a function of temperature demonstrates that the G4 reshapes the underlying driving forces of protein folding. To understand the structural basis of this catalytic activity, we introduce NMR method to solve the structures, at base-level resolution, of a multiconformer G4 with chaperone activity without chemical shift assignments. We then perform structure-function studies via mutation and chaperone assays to test the G4 properties important for chaperoning protein aggregation and protein folding. Together, our finding uncovers a previously underappreciated role for nucleic acid in proteostasis and offer a new strategy for studying nucleic acid structure-function relationship at residue level.
Zijue Huang· 0 citations
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