A novel type of gel-like proteasome condensate induced by toxic protein aggregates

Proteasomes reversibly form foci bodies in a liquid-liquid phase separation (LLPS)-dependent manner upon stress. We previously reported that internalized protein aggregates were targeted by proteasome-dense foci1, and proposed that such transient aggregate-associated droplets (TAADs) may facilitate aggregate removal2. Here we use quantitative imaging to show that TAADs represent a novel type of gel-like proteasome condensate. TAADs are irregular in shape and slow to disperse, sequestering proteasomes in alignment with our observation of confined diffusion3. We demonstrate that TAADs co-localize with cytosolic alpha-synuclein aggregates to facilitate their clearance. Inhibition of proteasome or ubiquitination activity abolish this aggregate clearance. We identify RAD23B necessary for TAAD formation, amid other co-localizing chaperones and (co-)proteins of the ubiquitin-proteasomes system. TAAD formation is associated with higher proteasomal substrate turnover whilst retaining overall catalytic efficiency, suggestive of altered degradation mechanisms upon aggregate engagement. Proteomics analysis show impact on key mitochondrial-associated processes even after apparent clearance of aggregates. Similar TAAD-aggregate co-localizations can be observed in iPSC-differentiated neurons and in disease-relevant samples, with no detection of compromised proteasome activity. Together, our results indicate a model where TAADs concentrate local proteasome activity, which facilitates aggregate clearance in healthy ageing cells. Potentially with persisting pathological aggregates, TAADs may remain engaged and conceivably sequester proteasomes from physiological activities, thus contributing to neurodegenerative disorders.