Designed, linked-domain proteins are pan-UBE2D inhibitors and induce proteostress and the unfolded protein response in HeLa cells

Ubiquitin (Ub) is a protein post-translational modification 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 >1000 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, multivalent, designed protein inhibitors against all UBE2D/UBCH5 isoforms by targeting the RING- and backside-binding sites. In HeLa cells, these inhibitors phenocopy knockdown of UBE2D by enhancing chemosensitivity to cisplatin. Whole-cell proteomics reveal ~20% of the identified proteins are more abundant and most do not have altered RNA levels, suggesting their protein turnover is regulated by UBE2D. Enrichment analysis of the altered mRNAs indicates the UBE2D inhibitors trigger the unfolded protein response. These precision tools will enable new studies about UBE2D’s cellular roles and help to deconvolute complex ubiquitin regulatory network.