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

ABSTRACT: 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 used quantitative mass spectrometry to identify proteins whose ubiquitylation or stability is altered by broad DUB inhibition, and confirmed their DUB-dependent regulation with human orthologs, demonstrating evolutionary conservation. We next extended this 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 profi

SUBMITTER: Rossio V 

PROVIDER: S-EPMC8052291 | biostudies-literature | 2021 Apr

REPOSITORIES: biostudies-literature