The targeting of non-fibrillar polyQ via distinct VCP-proteasome coupling

The aggregation of proteins containing expanded poly-glutamine (polyQ) repeat sequences is a cytopathological hallmark of several dominantly inherited neurodegenerative diseases (ND), including Huntingtons disease (HD). Previously, we observed that long polyQ repeats form a fibrillar core, surrounded by amorphous and soluble intermediates, which compared to the fibrils, are more readily taken up by autophagosomes 1. In this study, we investigated how the alternative major degradation pathway in cultured cells, ubiquitin proteasome system (UPS), interacts with the different pools of polyQ. We observed that the AAA+ ATPase, valosin-containing protein VCP/p97, in collaboration with proteasomes, plays a crucial role in degrading non-fibrillar polyQ. As both VCP and proteasomes were recruited to the polyQ intermediates peripheral to the fibrils, we imaged these regions by in situ cryo-ET for subtomogram averaging. VCP predominantly adopts an ATP-bound state, often in an active processing conformation with resolved NPLOC4-like cofactor density. The region was also enriched with the 26S proteasome (26S), 20S proteasome core (20S), and 19S regulatory particles (19S). Distance analysis of the macromolecules revealed a striking proximity between VCP and the 20S in situ. This is likely mediated by VCPs C-terminal hydrophobic-tyrosine-X (HbYX) motif, which binds the 20S via hydrophobic interactions, as confirmed by cryo-EM single-particle analysis (SPA) and in vitro assays. Our study highlights a distinct VCP-20S coupling mechanism, where VCP functionally overlaps with the 19S complex in substrate unfolding, facilitating the degradation of polyQ intermediates by the 20S catalytic core.