An official website of the United States government
Some proteins, including yeast translation termination factor Sup35 (eRF3) are capable of both stress-induced liquid-liquid phase separation (LLPS) and formation of solid fibrous aggregates (amyloids). Fragmentation and propagation of amyloid fibrils generates transmissible (in yeast, heritable) self-perpetuating protein agents, termed prions. Relationships between these processes are still poorly understood. Previous literature data suggested that the ability of Sup35 orthologs to form a prion is sporadically distributed in fungal evolution, and depends on amino acid composition of Sup35 prion domain (PrD), rather than on a evolutionarily variable specific sequence. We have studied two groups of proteins: 1) fungal Sup35 PrDs of various evolutionary origins, and 2) artificially synthesized “scrambled” variants of Saccharomyces cerevisiae Sup35 PrD, that possess identical amino acid composition but different sequences. These proteins were fused to fluorophores and expressed in S. cerevisiae cells. LLPS and amyloid/prion formation were assessed by fluorescence microscopy and biochemical approaches. Amino acid sequences were analyzed by various computational algorithms. Our data indicates that propagation of prion state strongly depends on the evolutionary distance from the host. In contrast, majority of proteins studied are capable of both LLPS and ability to form amyloid fibrils. These capabilities are associated with specific patterns of PrD amino acid distribution, that are broadly conserved among fungi. Notably, PrDs of different sequences differ from each other by their ability to convert from liquid condensates to amyloids, and relationship between these processes is apparently optimized in evolution. Moreover, heterotypic PrDs are can colocalize with each other within liquid condensates and influence amyloid conversion by each other. To conclude, LLPS and amyloid properties depend on specific evolutionarily conserved sequence patterns, indicating possible important biological roles for these processes. These patterns could potentially be used to predict LLPS and prion potential in other sequence contexts. This work was supported by NSF grant 2345660.
more »
« less
This content will become publicly available on November 5, 2026
From sequence to prion: comparison and evolutionary analysis of patterns controlling liquid-liquid phase separation and prion formation.
Introduction: Some proteins, including yeast prion protein Sup35 (eRF3) are capable of both stress-induced liquid-liquid phase separation (LLPS) and formation of prion state, propagated via solid fibrous aggregates (amyloids). Relationships between these processes are still poorly understood. Previous literature data suggested that prion formation by Sup35 is sporadically distributed in fungal evolution and depends on amino acid composition of its prion domain (PrD), rather than on a specific sequence which is highly variable. Objectives: Identify sequence patterns that control LLPS and amyloid formation by Sup35 PrD, and trace their conservation in fungal evolution. Methods: Fungal Sup35 PrDs of various evolutionary origins, as well as artificially synthesized “scrambled” variants of Saccharomyces cerevisiae Sup35 PrD, having identical amino acid composition but different sequences, were fused to fluorophores and expressed in S. cerevisiae cells. LLPS and amyloid/prion formation were assessed by fluorescence microscopy and biochemical approaches. Amino acid sequences were analyzed by various computational algorithms. Results/Discussion: While propagation of prion state depends on evolutionary distance from the host, both LLPS and ability to form an amyloid are associated with specific patterns of PrD amino acid distribution, that are broadly conserved among fungi. PrDs of different origins are capable of colocalizing within liquid condensates and influencing amyloid conversion by each other. Conclusion: LLPS and amyloid properties depend on specific evolutionarily conserved sequence patterns, indicating possible important biological roles for these processes. These patterns could potentially be used to predict LLPS and prion potential in other sequence contexts. Funding: NSF grant 2345660
more »
« less
- Award ID(s):
- 2345660
- PAR ID:
- 10652241
- Publisher / Repository:
- Proceedings of the Prion 2025 - Advancing the understanding and treatment of prion diseases. Anais eletrônicos..., Galoá
- Date Published:
- Volume:
- 1
- ISBN:
- 978-65-80968-48-0
- Page Range / eLocation ID:
- 328355
- Subject(s) / Keyword(s):
- Amyloid Evolution Phase separation Prion Yeast
- Format(s):
- Medium: X
- Location:
- Rio de Janeiro, Brazil
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
null (Ed.)Yeast prions provide self-templating protein-based mechanisms of inheritance whose conformational changes lead to the acquisition of diverse new phenotypes. The best studied of these is the prion domain (NM) of Sup35, which forms an amyloid that can adopt several distinct conformations (strains) that confer distinct phenotypes when introduced into cells that do not carry the prion. Classic dyes, such as thioflavin T and Congo red, exhibit large increases in fluorescence when bound to amyloids, but these dyes are not sensitive to local structural differences that distinguish amyloid strains. Here we describe the use of Michler’s hydrol blue (MHB) to investigate fibrils formed by the weak and strong prion fibrils of Sup35NM and find that MHB differentiates between these two polymorphs. Quantum mechanical time-dependent density functional theory (TDDFT) calculations indicate that the fluorescence properties of amyloid-bound MHB can be correlated to the change of binding site polarity and that a tyrosine to phenylalanine substitution at a binding site could be detected. Through the use of site-specific mutants, we demonstrate that MHB is a site-specific environmentally sensitive probe that can provide structural details about amyloid fibrils and their polymorphs.more » « less
-
na (Ed.)T-Cell Intracellular Antigen-1 (TIA1) is a 43 kDa multi-domain RNA-binding protein involved in stress granule formation during eukaryotic stress response, and has been implicated in neurodegenerative diseases including Welander distal myopathy and amyotrophic lateral sclerosis. TIA1 contains three RNA recognition motifs (RRMs), which are capable of binding nucleic acids and a C-terminal Q/N-rich prion-related domain (PRD) which has been variously described as intrinsically disordered or prion inducing and is believed to play a role in promoting liquid-liquid phase separation connected with the assembly of stress granule formation. Motivated by the fact that our prior work shows RRMs 2 and 3 are well-ordered in an oligomeric full-length form, while RRM1 and the PRD appear to phase separate, the present work addresses whether the oligomeric form is functional and competent for binding, and probes the consequences of nucleic acid binding for oligomerization and protein conformation change. New SSNMR data show that ssDNA binds to full-length oligomeric TIA1 primarily at the RRM2 domain, but also weakly at the RRM3 domain, and Zn2+ binds primarily to RRM3. Binding of Zn2+ and DNA was reversible for the full-length wild type oligomeric form, and did not lead to formation of amyloid fibrils, despite the presence of the C-terminal prion-related domain. While TIA1:DNA complexes appear as long “daisy chained” structures, the addition of Zn2+ caused the structures to collapse. We surmise that this points to a regulatory role for Zn2+. By occupying various “half” binding sites on RRM3 Zn2+ may shift the nucleic acid binding off RRM3 and onto RRM2. More importantly, the use of different half sites on different monomers may introduce a mesh of crosslinks in the supramolecular complex rendering it compact and markedly reducing the access to the nucleic acids (including transcripts) from solution.more » « less
-
Amyloids are self-perpetuating protein aggregates causing neurodegenerative diseases in mammals. Prions are transmissible protein isoforms (usually of amyloid nature). Prion features were recently reported for various proteins involved in amyloid and neural inclusion disorders. Heritable yeast prions share molecular properties (and in the case of polyglutamines, amino acid composition) with human disease-related amyloids. Fundamental protein quality control pathways, including chaperones, the ubiquitin proteasome system and autophagy are highly conserved between yeast and human cells. Crucial cellular proteins and conditions influencing amyloids and prions were uncovered in the yeast model. The treatments available for neurodegenerative amyloid-associated diseases are few and their efficiency is limited. Yeast models of amyloid-related neurodegenerative diseases have become powerful tools for high-throughput screening for chemical compounds and FDA-approved drugs that reduce aggregation and toxicity of amyloids. Although some environmental agents have been linked to certain amyloid diseases, the molecular basis of their action remains unclear. Environmental stresses trigger amyloid formation and loss, acting either via influencing intracellular concentrations of the amyloidogenic proteins or via heterologous inducers of prions. Studies of environmental and physiological regulation of yeast prions open new possibilities for pharmacological intervention and/or prophylactic procedures aiming on common cellular systems rather than the properties of specific amyloids.more » « less
-
Self-perpetuating transmissible protein aggregates, termed prions, are implicated in mammalian diseases and control phenotypically detectable traits in Saccharomyces cerevisiae . Yeast stress-inducible chaperone proteins, including Hsp104 and Hsp70-Ssa that counteract cytotoxic protein aggregation, also control prion propagation. Stress-damaged proteins that are not disaggregated by chaperones are cleared from daughter cells via mother-specific asymmetric segregation in cell divisions following heat shock. Short-term mild heat stress destabilizes [ PSI + ], a prion isoform of the yeast translation termination factor Sup35 . This destabilization is linked to the induction of the Hsp104 chaperone. Here, we show that the region of Hsp104 known to be required for curing by artificially overproduced Hsp104 is also required for heat-shock-mediated [ PSI + ] destabilization. Moreover, deletion of the SIR2 gene, coding for a deacetylase crucial for asymmetric segregation of heat-damaged proteins, also counteracts heat-shock-mediated destabilization of [ PSI + ], and Sup35 aggregates are colocalized with aggregates of heat-damaged proteins marked by Hsp104 -GFP. These results support the role of asymmetric segregation in prion destabilization. Finally, we show that depletion of the heat-shock noninducible ribosome-associated chaperone Hsp70-Ssb decreases heat-shock-mediated destabilization of [ PSI + ], while disruption of a cochaperone complex mediating the binding of Hsp70-Ssb to the ribosome increases prion loss. Our data indicate that Hsp70-Ssb relocates from the ribosome to the cytosol during heat stress. Cytosolic Hsp70-Ssb has been shown to antagonize the function of Hsp70-Ssa in prion propagation, which explains the Hsp70-Ssb effect on prion destabilization by heat shock. This result uncovers the stress-related role of a stress noninducible chaperone.more » « less
