Proteomics of broad deubiquitylase inhibition unmasks redundant enzyme function to reveal substrates and assess enzyme specificity

Deubiquitylating enzymes (DUBs) counteract ubiquitylation to control stability or activity of substrates. Identification of DUB substrates is challenging because multiple DUBs can act on the same substrate, thwarting genetic approaches. Here, we circumvent redundancy by chemically inhibiting multiple DUBs simultaneously in Xenopus egg extract. We discovered a set of proteins that depends on DUBs for their stability and we confirmed their DUB-dependent regulation with human orthologs, demonstrating evolutionary conservation. We next extended this approach, developing a new method to profile DUB specificity. By adding recombinant DUBs to extract where DUB activity was broadly inhibited, but ubiquitylation and degradation were active at physiological rates, we profiled the ability of DUBs to rescue degradation of these new substrates. We found that USP7 has a unique ability to broadly antagonize their degradation. Together, we identify novel DUB substrates and present an approach to characterize DUB specificity that overcomes challenges posed by DUB redundancy.