NAE1/UBA3-UBE2M are E1 and E2 enzymes for the URM1 modification.

Ubiquitin-related modifier 1 (URM1) is an evolutionarily conserved ubiquitin-like protein. In eukaryotes, it serves dual roles as a sulfur donor for tRNA modification and a posttranslational protein modifier. URM1 is proposed to be a primitive protein modifier and a potential precursor to the more complex ubiquitin system. However, no specific activating enzyme (E1), conjugating enzyme (E2), or ligase (E3) has been reported for the URM1 modification cascade in human cells. In this study, we design an activity-based URM1 probe to covalently capture cysteine enzymes functioning in the URM1 signaling pathway. Through proteomic characterization and cell-based validation, we identify NAE1/UBA3 and UBE2M as E1 and E2 enzymes, respectively, for the urmylation pathway under both normal and oxidative stress conditions. Pharmacologic perturbation of the UBE2M-DCN1 module suggests DCN1 may contribute to URM1 conjugation. Bioinformatic analysis further reveals that genetic knockdown of NAE1, UBE2M, and URM1 affects overlapping genes associated with pathways controlling cellular response to stress conditions or with implications in liver diseases. URM1 serves a protective role against oxidative stress. Pevonedistat, a potent NAE1 inhibitor that blocks protein urmylation in human cells, exhibits strong synergy with cisplatin, an agent known to induce oxidative stress, in killing liver cancer cells effectively.