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Methods Mol Biol 2023 Jan 01;2591:219-236. doi: 10.1007/978-1-0716-2803-4_13.
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Identification of Deubiquitinase Substrates in Xenopus Egg Extract.

Rossio V , Paulo JA , King RW .

Deubiquitinases (DUBs) antagonize protein ubiquitination by removing ubiquitin from substrates. Identifying the physiological substrates of each DUB is critical for understanding DUB function and the principles that govern the specificity of this class of enzymes. Since multiple DUBs can act on the same substrate, it can be challenging to identify substrates using inactivating a single enzyme. Here, we outline a method that enables the identification of proteins whose stability depends on DUB activity and an approach to profile DUB specificity in Xenopus egg extract. By coupling broad DUB inhibition with quantitative proteomics, we circumvent DUB redundancy to identify DUB substrates. By adding back recombinant DUBs individually to the extract, we pinpoint DUBs sufficient to counteract proteasomal degradation of these newly identified substrates. We apply this method to Xenopus egg extract but suggest that it can also be adapted to other cell lysates.

PubMed ID: 36350551
PMC ID: PMC9662951
Article link: Methods Mol Biol
Grant support: [+]

Species referenced: Xenopus laevis
GO keywords: regulation of protein deubiquitination

Article Images: [+] show captions
References [+] :
Beckley, A Degenerate Cohort of Yeast Membrane Trafficking DUBs Mediates Cell Polarity and Survival. 2015, Pubmed