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

Abstract

1Deubiquitylating enzymes (DUBs) counteract ubiquitylation to control the stability or activity of 2 their substrates. Identifying DUB substrates is challenging and genetic approaches can be 3 thwarted by redundant action of DUBs. Here, we circumvented redundancy by broadly inhibiting 4 DUBs in Xenopus egg extract and used quantitative mass spectrometry to identify over thirty 5 proteins that undergo proteasomal degradation, the majority of which have not been reported as 6 DUB substrates. These results were confirmed with recombinant human proteins, demonstrating 7 the conservation of their DUB-dependent stability. We used these substrates to profile the ability 8 of a panel of DUBs to rescue degradation. This approach revealed that USP7, uniquely among 9 the 14 DUBs tested, has a broad ability to rescue degradation. USP21, which is used widely to 10 nonspecifically deubiquitylate proteins in vitro, was unable to rescue degradation, highlighting 11 the importance of profiling enzyme activity in a physiological system. Together, we identify new 12 DUB substrates and present a system to characterize physiological DUB specificity, overcoming 13 the challenges posed by DUB redundancy. 14 2 Here, we first took an unbiased quantitative proteomic approach and discovered new 53 substrates protected from proteasomal degradation by DUBs, using the Xenopus egg extract 54 model system. By broadly inhibiting cysteine-protease DUBs, we circumvented the possible 55 effects of their redundancy on proteome stability. Because transcription and translation are not 56 active in this system, protein instability caused by DUB inhibition is not a consequence of 57 protein quality control ubiquitination activity. We recapitulated protein destabilization with 58 expression of recombinant human orthologs in extract, demonstrating that DUB-dependent 59 4 protein stability is evolutionarily conserved. Next, we took advantage of these newly identified 60 DUB substrates and tested the ability of a set of DUBs to rescue their degradation, uncovering a 61 broad ability of USP7 to rescue protein instability in extract. However, specific inhibition of 62 USP7 with a small molecule inhibitor was not sufficient to promote degradation of most of the 63 substrates we identified, suggesting that USP7 functions redundantly with other DUBs in this 64 system. Our work highlights the impact of DUB redundancy on proteome stability and reveals 65 the specificity and activity of DUBs whose function would otherwise be masked by redundancy. 66 67Results 68 UbVS treatment induces rapid depletion of available ubiquitin and labels a broad set of 69 cysteine-protease DUBs in Xenopus extract 70Our laboratory previously showed that the broad cysteine-protease DUB inhibitor UbVS 71 induces depletion of available ubiquitin in Xenopus extract 22 . In particular, UbVS-treatment 72 inhibited proteasome degradation of cyclin B, which could be efficiently rescued by addition of 73 50 µM exogenous ubiquitin 22 . We confirmed that 10 µM UbVS was sufficient to rapidly deplet...