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1. amyloid-predict and LLPS-predict: Predicting phase separation propensities in the intrinsically disordered proteome

   https://doi.org/10.1073/pnas.2531932123  

   Lobo, Samuel; Griem, Leif; Shell, M Scott; Shea, Joan-Emma (June 2026, Proceedings of the National Academy of Sciences)

   Amyloid formation and liquid–liquid phase separation (LLPS) are two important phenomena in cellular biology, linked to both normal physiological functions and various pathologies. Here, we present a computational framework that scores amyloid propensities (amyloid-predict) or LLPS propensities (LLPS-predict) from protein language model embeddings, enabling rapid proteome-wide annotation of peptides and residues. amyloid-predict achieves classification performance that exceeds existing AI and physics-based tools on a hexapeptide benchmark while enabling substantially faster high-throughput screening; notably, amyloid-predict is sensitive to subtle mutational effects and is influenced by sequence patterning and context rather than amino acid composition alone. We apply these protein language model classifiers to all the IDRs in the human proteome and uncover several protein categories with significant enhancement in amyloid and/or LLPS propensity, suggesting insights into the biological roles of these protein categories. For example, signaling receptors, carbohydrate-binding proteins, and Ca²⁺binding proteins are enriched in aggregation propensity, while mRNA-binding proteins, ribonucleoprotein complex, and nuclear matrix proteins are enriched in LLPS propensity. Interestingly, we observe patterns of both high amyloid and LLPS propensity in several amyloid-forming and prionic proteins. Together, these results provide side-by-side landscapes of LLPS and amyloid potential across the disordered human proteome while offering a rapid screening tool for basic biology, disease-mechanism studies, and rational design of peptide therapeutics. 

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2. Hydrophobicity of arginine leads to reentrant liquid-liquid phase separation behaviors of arginine-rich proteins

   https://doi.org/10.1038/s41467-022-35001-1  

   Hong, Yuri; Najafi, Saeed; Casey, Thomas; Shea, Joan-Emma; Han, Song-I; Hwang, Dong Soo (December 2022, Nature Communications)

   Abstract Intrinsically disordered proteins rich in cationic amino acid groups can undergo Liquid-Liquid Phase Separation (LLPS) in the presence of charge-balancing anionic counterparts. Arginine and Lysine are the two most prevalent cationic amino acids in proteins that undergo LLPS, with arginine-rich proteins observed to undergo LLPS more readily than lysine-rich proteins, a feature commonly attributed to arginine’s ability to form stronger cation-π interactions with aromatic groups. Here, we show that arginine’s ability to promote LLPS is independent of the presence of aromatic partners, and that arginine-rich peptides, but not lysine-rich peptides, display re-entrant phase behavior at high salt concentrations. We further demonstrate that the hydrophobicity of arginine is the determining factor giving rise to the reentrant phase behavior and tunable viscoelastic properties of the dense LLPS phase. Controlling arginine-induced reentrant LLPS behavior using temperature and salt concentration opens avenues for the bioengineering of stress-triggered biological phenomena and drug delivery systems. 

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3. From sequence to prion: comparison and evolutionary analysis of patterns controlling liquid-liquid phase separation and prion formation.

   Chernoff, Yury; Grizel, Anastasia; Stevenson, Jonathan; Anee, Ismat J; Paulius, Kristupas (November 2025, Proceedings of the Prion 2025 - Advancing the understanding and treatment of prion diseases. Anais eletrônicos..., Galoá)

   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 

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4. Previously uncharacterized interactions between the folded and intrinsically disordered domains impart asymmetric effects on UBQLN2 phase separation

   https://doi.org/10.1002/pro.4128  

   Zheng, Tongyin; Galagedera, Sarasi K.; Castañeda, Carlos A. (July 2021, Protein Science)

   null (Ed.)

   Shuttle protein UBQLN2 functions in protein quality control (PQC) by binding to proteasomal receptors and ubiquitinated substrates via its N-terminal ubiquitin-like (UBL) and C-terminal ubiquitin-associated (UBA) domains, respectively. Between these two folded domains are low complexity STI1-I and STI1-II regions, connected by disordered linkers. The STI1 regions bind other components, such as HSP70, that are important to the PQC functions of UBQLN2. We recently determined that the STI1-II region enables UBQLN2 to undergo liquid-liquid phase separation (LLPS) to form liquid droplets in vitro and biomolecular condensates in cells. However, how the interplay between the folded (UBL/UBA) domains and the intrinsically-disordered regions mediates phase separation is largely unknown. Using engineered domain deletion constructs, we found that removing the UBA domain inhibits UBQLN2 LLPS while removing the UBL domain enhances LLPS, suggesting that UBA and UBL domains contribute asymmetrically in modulating UBQLN2 LLPS. To explain these differential effects, we interrogated the interactions that involve the UBA and UBL domains across the entire UBQLN2 molecule using NMR spectroscopy. To our surprise, aside from well-studied canonical UBL:UBA interactions, there also exist moderate interactions between the UBL and several disordered regions, including STI1-I and residues 555-570, the latter of which is a known contributor to UBQLN2 LLPS. Our findings are essential for the understanding of both the molecular driving forces of UBQLN2 LLPS and the effects of ligand binding to UBL, UBA, or disordered regions on the phase behavior and physiological functions of UBQLN2. 

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5. Microstructural Organization in α-Synuclein Solutions

   https://doi.org/10.1021/acs.macromol.1c02550  

   Das, Shibananda; Muthukumar, Murugappan (June 2022, Macromolecules)

   We have investigated the structural evolution in solutions of the intrinsically disordered protein, α-synuclein, as a function of protein concentration and added salt concentration. Accounting for electrostatic and excluded volume interactions based on the protein sequence, our Langevin dynamics simulations reveal that α-synuclein molecules assemble into aggregates and percolated structures with a spontaneous selection of a dominant structure characteristic of microphase separation. This microphase assembly is mainly driven by electrostatic interactions between the residues in N-terminal and C-terminal of the protein molecules, and presence of salt loosens the compactness of the microstructures. We have quantified the features of the spontaneously formed microstructures using interchain radial distribution functions, and experimentally measurable inter-residue contact maps and static structure factors. Our results are in contrast to the commonly hypothesized mechanism of liquid–liquid phase separation (LLPS) for the formation of droplets in solutions of intrinsically disordered proteins, opening a new paradigm to understand the birth and structure of membraneless organelles. In general, construction of phase diagrams of intrinsically disordered proteins and other biomacromolecular systems needs to incorporate features of microphase separation into other mechanisms of macrophase separation and percolation. 

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