E. coli-mediated expedient biosynthesis of biotin-labeled ubiquitin probe enables activity-based profiling of deubiquitinases in cytoplasmic and nuclear compartments

Deubiquitinases (DUBs) that remove conjugated ubiquitin from substrates are critical regulators of protein homeostasis and cellular signaling. Understanding the spatial distribution of DUBs in subcellular compartments is essential for uncovering their roles in stress responses and disease pathogenesis. Herein, we developed an Escherichia coli (E. coli)-based biosynthetic strategy for the expedient generation of biotin-labeled ubiquitin probe (Biotin-Ub-PA) through co-expression of Avi-Ub and BirA in E. coli, thus bypassing tedious chemical synthesis. Combined with affinity purification and proteomics, we utilized the biosynthetic probe to covalently capture active DUBs in subcellular compartments and revealed the distribution of DUBs in the cytoplasm and nucleus. As a proof of concept, hydrogen peroxide-induced activity changes of DUBs in the cytoplasm and nucleus were analyzed by the same probe. The results suggest that some DUBs, such as ubiquitin-specific protease 36 (USP36) and USP16, may undergo subcellular translocation and activity alterations under oxidative stress. Our study presents a reliable workflow for the spatial distribution analysis of DUBs under different stimuli or disease conditions.