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

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.