Aug 2026· International Journal of Molecular Sciences· Vol 27, pp. 7516· 0 citations· 77 references
TL;DR
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.
Abstract
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.
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
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.
Katherine Shreeve, Jinoh Jang, Mr. S Srivathsan et al.· Proteins: Structure, Functio...· 0 citations
Understanding the link between phase separation (PS) of disease‐linked proteins to form liquid‐like condensates, and their aggregation, requires insights into the conformational changes that the proteins undergo inside the condensates as they age and become solid‐like. In this study, the structural changes undergone by the mouse prion protein (moPrP) inside condensate induced by PS have been characterized. Hydrogen‐deuterium exchange in conjunction with mass spectrometry reveals that the N‐terminal region (NTR), which is unstructured in monomeric native moPrP, gains significant stable structure as the condensate ages. The structured C‐terminal domain remains native‐like, albeit with higher stability, but subtle changes are seen. Conformational change initiates in the native monomer inside the condensate, with different regions undergoing rapid, slow, or no conformational change as it ages. The β1‐α1 loop undergoes rapid conformational change to lose stability, while the NTR gains structure slowly concomitantly with conformational change at the C‐terminal end of α3. Infrared (IR) spectroscopy shows that β‐structure forms, IR and circular dichroism spectroscopy indicate that secondary structure becomes heterogeneous, and dynamic light scattering measurements reveal that the protein forms oligomeric nanoscale assemblies as the condensate ages. The formation of the nanoscale assemblies inside the condensate is responsible for the fraction of protein present as mobile monomer decreasing with time of aging, when fluorescence recovery after photobleaching is quantified. Such assembly and the resultant conformational change in the protein appear to be responsible for a change in its material properties of the condensate, which manifests itself as a liquid‐like to solid‐like transition.
Suman Pal, J. Udgaonkar· Protein Science· 0 citations
The Parkinson’s disease-related protein α-synuclein can form solid amyloid fibrils through liquid–liquid phase separation (LLPS) and liquid-to-solid phase transition. The most deleterious familial mutation E46K has recently been shown to enhance α-synuclein LLPS and subsequent solidification; yet, the precise mechanisms remain largely unknown. Here, using molecular dynamics simulations at different spatiotemporal scales combined with biochemical experiments, we show that the E46K mutation acts as an electrostatic switch to remodel interactions between the oppositely charged N-terminal domain (NTD) and C-terminal domain (CTD) of α-synuclein. This remodeling shifts the interaction site of NTD with CTD from its N-terminus to the mutation region. Such reorganization increases the hydrophobic solvent exposure of key LLPS-promoting motifs, potentially facilitating intermolecular interactions that initiate phase separation. Phase coexistence simulations further support this hypothesis, showing that the E46K mutant exhibits enhanced LLPS and solidification propensities. This enhancement is primarily driven by intermolecular electrostatic interactions between the mutation region and the CTD, followed by the hydrophobic NAC–NAC interactions mediated by the LLPS-promoting motifs. Collectively, these results reveal that NTD–CTD electrostatic crosstalk acts as the key modulator of α-synuclein phase separation, while NAC–NAC interactions play an auxiliary role, both of which synergistically govern α-synuclein phase separation. This study offers a complete and detailed mechanistic framework for understanding α-synuclein phase separation and its enhancement induced by the E46K mutation.
The yeast prion [PSI+] is a self-propagating amyloid of the translation termination factor Sup35p (eRF3). Among 79 ribosomal proteins in Saccharomyces cerevisiae, through a genetic screen for novel anti-prion factors, we identified RPL24B, a 60S ribosomal subunit protein, as a key determinant of prion generation. Our findings revealed that the functional distinction between the ribosomal paralogs RPL24A and RPL24B is the primary driver of ribosomal heterogeneity-based anti-prion generation. Heterogeneity establishes a critical genetic checkpoint that strictly regulates the emergence and heritable diversity of prions and their variants. We demonstrate that deleting RPL24B (rpl24bΔ) significantly increased the frequency of [PSI+] generation compared to the wild-type strain. Crucially, the resulting [PSI+] prions in the rpl24bΔ mutants exhibited distinct heritable diversity, characterized by high thermostability and exceptionally high propagon numbers. This anti-prion activity is uniquely specific to the RPL24B paralog, because deletion of its highly homologous counterpart, RPL24A, does not reproduce the effects observed in rpl24bΔ. Mechanistically, we identified valine 138 (V138) in Rpl24b as the major molecular determinant of its anti-prion generation activity. Loss of Rpl24b perturbs the cellular protein quality control landscape, leading to high accumulation of misfolded protein aggregates. This work suggests that RPL24B functions as a specialized retainer of genetic robustness, providing a translational barrier against protein misfolding diseases by filtering out robust amyloid variants.
Min Suk Jang, Sia Han, Eunsil Choi et al.· Genetics· 0 citations
Peptide self-assembly and liquid-liquid phase separation (LLPS), often mediated by intrinsically disordered regions (IDRs), are natural mechanisms that translate protein molecular features into complex nano- and mesoscale architectures. Although the thermodynamics and kinetics of these processes are well understood, synthetic materials integrating both functionalities remain rare. Inspired by the conserved IDR-assembly domain (AD) architecture of amyloidogenic proteins, we hypothesized that modular recombinant constructs combining LLPS-capable IDRs with β-sheet-forming ADs could generate materials with tunable structural properties. To test this, we engineered a library of elastin-like polypeptides (ELPs) fused to amphiphilic anionic or cationic amyloidogenic peptides, enabling systematic investigation of how sequence parameters─including ELP length, AD charge, and hydrophilicity─and environmental conditions, including temperature, pH, and salt concentration, influence material behavior. Our results reveal links between molecular design and emergent multiscale structures, including micelles and vesicles embedded within coacervates. This work provides a framework for designing hybrid proteins coupling LLPS and self-assembly.
Yulia Shmidov, Li-Xin Fan, Max R. Ney et al.· Biomacromolecules· 0 citations
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