Marzahn, M. R. | Search bioRxiv

bioRxiv2025

Viscoelasticity and interface properties of multi-component condensates govern protein sequestration and suppression of amyloid formation

Stress granules (SGs) are multi-component biomolecular condensates widely implicated as sites of protein aggregation by virtue of the high concentrations of amyloidogenic RNA-binding proteins they contain. This model, in which SGs are viewed as crucibles for amyloid formation, has not been rigorously tested. Here, we employed twelve multi-component protein-nucleic acid condensate systems as SG-mimics with diverse physicochemical features. Utilizing three fibril-forming proteins, hnRNPA1, Tau, and FUS, which are concentrated more than 50-fold in condensates, we report that multi-component biomolecular condensates robustly suppress, rather than promote, amyloid formation. Multiscale experimental analyses, including quantitative kinetic measurements, rheology, and microscopy, combined with computational modelling, reveal that condensates serve as sinks for soluble protein, and fibrils form in the dilute phase, although interfaces can promote nucleation. Three key physicochemical properties of condensates govern suppression of fibril formation: First, condensate-mediated sequestration lowers the concentration of fibril-forming proteins in the dilute phase. Second, condensate viscoelasticity constrains efflux-driven fibril growth in the dilute phase. And third, dilution of fibril-forming proteins at condensate interfaces mitigates fibril nucleation. The sink potential of SG-mimics is recapitulated in G3BP1-RNA condensates and SGs reconstituted in mammalian cell lysate, suggesting that SGs may have evolved to suppress stress-induced protein aggregation.

biophysics↗

bioRxiv2023

Reduction of oligomer size modulates the competition between cluster formation and phase separation of the tumor suppressor SPOP

Phase separation is a ubiquitous process that compartmentalizes many cellular pathways. Given that the same interactions that drive phase separation mediate the formation of complexes below the saturation concentration, the contribution of condensates vs complexes to function is not always clear. Here, we characterized several new cancer-associated mutations of the tumor suppressor Speckle-type POZ protein (SPOP), a substrate recognition subunit of the Cullin3-RING ubiquitin ligase (CRL3), which pointed to a strategy for generating separation-of-function mutations. SPOP self-associates into linear oligomers and interacts with multivalent substrates, and this mediates the formation of condensates. These condensates bear the hallmarks of enzymatic ubiquitination activity. We characterized the effect of mutations in the dimerization domains of SPOP on its linear oligomerization, binding to the substrate DAXX, and phase separation with DAXX. We showed that the mutations reduce SPOP oligomerization and shift the size distribution of SPOP oligomers to smaller sizes. The mutations therefore reduce the binding affinity to DAXX, but enhance the poly-ubiquitination activity of SPOP towards DAXX. This unexpectedly enhanced activity may be explained by enhanced phase separation of DAXX with the SPOP mutants. Our results provide a comparative assessment of the functional role of clusters versus condensates and support a model in which phase separation is an important factor in SPOP function. Our findings also suggest that tuning of linear SPOP self-association could be used by the cell to modulate its activity, and provide insights into the mechanisms underlying hypermorphic SPOP mutations. The characteristics of these cancer-associated SPOP mutations suggest a route for designing separation-of-function mutations in other phase-separating systems.

biophysics↗