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 foci ¹ , and proposed that such transient aggregate-associated droplets (TAADs) may facilitate aggregate removal ² . 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 agreement with our observation of confined diffusion ³ . We demonstrate that TAADs co-localize with cytosolic alpha-synuclein aggregates to facilitate their clearance. Inhibition of proteasome- or ubiquitination activity abolishes 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 reveals impact on key mitochondrial-associated processes even after TAAD-aggregate disengagement. Similar TAAD-aggregate co-localizations are found 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, should pathological aggregates persist, TAADs may remain engaged and conceivably sequester proteasomes from physiological activities, thus contributing to neurodegenerative disorders.