Proteomics-Based Identification of DUB Substrates Using Selective Inhibitors.

Deubiquitinating enzymes (DUBs) catalyze the removal of ubiquitin, thereby reversing the activity of E3 ubiquitin ligases and are central to the control of protein abundance and function. Despite the growing interest in DUBs as therapeutic targets, cellular functions for DUBs remain largely unknown and technical challenges often preclude the identification of DUB substrates in a comprehensive manner. Here, we demonstrate that treatment with potent DUB inhibitors coupled to mass spectrometry-based proteomics can identify DUB substrates at a proteome-wide scale. We applied this approach to USP7, a DUB widely investigated as a therapeutic target and identified many known substrates and additional targets. We demonstrate that USP7 substrates are enriched for DNA repair enzymes and E3 ubiquitin ligases. This work provides not only a comprehensive annotation of USP7 substrates, but a general protocol widely applicable to other DUBs, which is critical for translational development of DUB targeted agents. • Commonly used DUB inhibitors show a wide range of selectivity and cellular activity • Cellular proteomics of USP7, USP47, USP25, USP28, USP30, UCHL1, and USP1 inhibitors • Use of multiple USP7 inhibitors to confirm known and new targets of USP7 • Practical insights for profiling DUB inhibitors earlier in the development pipeline Bushman et al. profile commonly used inhibitors of deubiquitinating enzymes to assess their biochemical selectivity and cellular activity. USP7 is used to demonstrate proof-of-concept for using potent and selective inhibitors of deubiquitinating enzymes to identify their substrates by mass spectrometry. [ABSTRACT FROM AUTHOR]