UBE3C retrofits the proteasome to enforce degradation of ultra-stable folds

Abstract Neurodegeneration and proteinopathies arise from mutated or misfolded proteins that form ultra-stable, proteotoxic aggregates that evade proteasomal degradation despite ubiquitylation 1–8 . These conditions are exacerbated by the deubiquitylase USP14 that can prematurely rescue substrates 9–11 . To counter these obstacles to effective proteolysis, eukaryotes evolved the highly conserved HECT-type E3 ligase UBE3C—dysregulated in neurodegeneration 12,13 and overexpressed in cancers 14–17 —to reprogram the 26S proteasome toward heightened activity via unknown mechanisms 18–25 . Here we visualized functional dynamics of the human UBE3C-retrofitted proteasome enforcing degradation of a re-engineered superfolder GFP that normally escapes proteolysis, using time-resolved cryo-electron microscopy. A continuum of non-equilibrium conformations, comprising fourteen proteasome conformers orthogonally combined with four UBE3C states or three USP14 states, reveals a cryptic UBE3C-receptor site in the proteasomal lid and captures key intermediates of ubiquitin-chain elongation and branching at four linkage-specific ubiquitin-binding sites. Remarkably, UBE3C creates an extreme shortcut for ubiquitin shuttling that bypasses USP14, promotes USP14 recycling and allosterically strengthens substrate-unfolding forces of the AAA-ATPase unfoldase to ultimately surmount the unfolding energy barrier of ultra-stable proteins. These findings define the complete functional cycle of the UBE3C-reprogrammed proteasome, illuminating how UBE3C simultaneously controls USP14 and the proteasome to enforce clearance of ultra-stable folds, and establish crucial mechanistic foundations for proteolysis-targeting therapeutic discovery.