Linked-domain inhibitors designed to block UBE2D induce the unfolded protein response

Ubiquitin (Ub) is a protein post-translational modifier that controls proteostasis through mechanisms spanning transcription, translation, and protein degradation. Ub conjugation occurs through a cascade of three enzyme classes (E1, E2, and E3s) involving >1,000 proteins that regulate the ubiquitination of cellular proteins. The E2 Ub-conjugating enzymes are the midpoint, yet their cellular roles remain under-characterized. Here, we develop highly selective and potent pan-UBE2D/UBCH5 inhibitors by targeting the RING- and backside-binding sites with engineered linked-domain proteins. In HeLa cells, these inhibitors phenocopy the knockdown of UBE2D by enhancing chemosensitivity to cisplatin. Whole-cell proteomics reveals that ∼20% of the identified proteins are more abundant, and most do not have altered mRNA levels, suggesting that their protein turnover is regulated by UBE2D. Enrichment analysis of the altered mRNAs indicates that the linked-domain proteins trigger the unfolded protein response. These precision tools will enable new studies probing UBE2D's cellular roles and help to deconvolute complex Ub regulatory networks.