Mechanistic insights into the enhancement or inhibition of phase separation by polyubiquitin chains of different lengths or linkages

Abstract

SummaryUbiquitin-binding shuttle UBQLN2 mediates crosstalk between proteasomal degradation and autophagy, likely via interactions with K48- and K63-linked polyubiquitin chains, respectively. UBQLN2 is recruited to stress granules in cells and undergoes liquid-liquid phase separation (LLPS) in vitro. However, interactions with ubiquitin or multivalent K48-linked chains eliminate LLPS. Here, we found that, although some polyubiquitin chain types (K11-Ub4 and K48-Ub4) did generally inhibit UBQLN2 LLPS, others (K63-Ub4, M1-Ub4 and a designed tetrameric ubiquitin construct) significantly enhanced LLPS. Using nuclear magnetic resonance (NMR) spectroscopy and complementary biophysical techniques, we demonstrated that these opposing effects stem from differences in chain conformations, but not in affinities between chains and UBQLN2. Chains with extended conformations and increased accessibility to the ubiquitin binding surface significantly promoted UBQLN2 LLPS by enabling a switch between homotypically to partially heterotypically-driven phase separation. Our study provides mechanistic insights into how the structural and conformational properties of polyubiquitin chains contribute to heterotypic phase separation with ubiquitin-binding shuttles and adaptors.HighlightsUbiquitin or short polyubiquitin chains bind to phase separation-driving stickers on UBQLN2 and inhibit its phase separation whereas longer chains provide the multivalency needed to enhance UBQLN2 phase separation.Compact K11- and K48-linked Ub4 chains destabilized UBQLN2 phase separation, while extended M1- and K63-linked Ub4 promoted UBQLN2 phase separation.Chain conformation and accessibility of the Ub interacting surface is a driving factor of UBQLN2/polyUb co-phase separation.UBQLN2 condensates assemble during in vitro enzymatic assembly of K63-linked polyUb chains as free ubiquitin is depleted.

Uncovering the molecular mechanism for dual effect of ATP on phase separation in FUS solution

“…Notably, they were the first to show that monovalent ligands destabilize phase separation driven by homotypic interactions, and the nonmonotonic behavior of ligands as a function of the ligand concentration was also anticipated and demonstrated (60,61). Very recently, Dao et al (62) found that the type of the polyubiquitin chain could also play distinct roles in the phase separation of UBQLN2, which further bolsters the general conceptual foundations of the findings from Ruff et al These results share some similarity with the dual effect of ATP (on the phase separation) reported here, which provides a key conceptual lynchpin for our current work. In general, this work well explains the nonmonotonic effect of ATP on the phase separation.…”

Adsorption of RNA to interfaces of biomolecular condensates enables wetting transitions

“…The mechanisms of adsorption and wetting are distinct from the site-specific effects of ligands that are known to modulate driving forces for phase separation through preferential binding across phase boundaries 31, 32, 33, 34 . While preferential binding of ligands 35 can either compete against or add to extant interactions, thereby destabilizing or stabilizing condensates, adsorption of a ternary component has little effect on the interactions within a condensate 36 .…”