期刊
CELL CHEMICAL BIOLOGY
卷 28, 期 4, 页码 487-+出版社
CELL PRESS
DOI: 10.1016/j.chembiol.2020.12.007
关键词
-
资金
- NIH [R01 GM132129, R01 GM67945, R35 GM127032]
This study involves chemically inhibiting multiple DUBs to identify DUB substrates and characterize their specificity, overcoming challenges posed by DUB redundancy. USP7 is found to have a unique ability to broadly antagonize degradation.
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 profiled the ability of DUBs to rescue degradation of these substrates. We found that USP7 has a unique ability to broadly antagonize degradation. Together, we present an approach to identify DUB substrates and characterize DUB specificity that overcomes challenges posed by DUB redundancy.
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