Proteomics

Dataset Information

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Unmasking functional redundancy of deubiquitylases enables specificity profiling and discovery of novel proteasome substrates


ABSTRACT: Deubiquitylating enzymes (DUBs) counteract ubiquitylation to control the stability or activity of their substrates. Identifying DUB substrates is challenging and genetic approaches can be thwarted by redundant action of DUBs. Here, we circumvented redundancy by broadly inhibiting DUBs in Xenopus egg extract and used quantitative mass spectrometry to identify over thirty proteins that undergo proteasomal degradation, the majority of which have not been reported as DUB substrates. These results were confirmed with recombinant human proteins, demonstrating the conservation of their DUB-dependent stability. We used these substrates to profile the ability of a panel of DUBs to rescue degradation. This approach revealed that USP7, uniquely among the 14 DUBs tested, has a broad ability to rescue degradation. USP21, which is used widely to nonspecifically deubiquitylate proteins in vitro, was unable to rescue degradation, highlighting the importance of profiling enzyme activity in a physiological system. Together, we identify new DUB substrates and present a system to characterize physiological DUB specificity, overcoming the challenges posed by DUB redundancy.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Xenopus Laevis (african Clawed Frog)

SUBMITTER: Joao Paulo  

LAB HEAD: Joao A. Paulo

PROVIDER: PXD016164 | Pride | 2022-09-02

REPOSITORIES: Pride

Dataset's files

Source:
Action DRS
333373_m16266_Val_ContiGG.mzIdentML Mzid
333374_m16267_Val_ContiGG.mzIdentML Mzid
333375_m16268_Val_ContiGG.mzIdentML Mzid
333376_m16269_Val_ContiGG.mzIdentML Mzid
333377_m16270_Val_ContiGG.mzIdentML Mzid
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Publications

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

Rossio Valentina V   Paulo Joao A JA   Chick Joel J   Brasher Bradley B   Gygi Steven P SP   King Randall W RW  

Cell chemical biology 20210107 4


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-  ...[more]

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