Deubiquitinating enzymes and the proteasome regulate unique sets of ubiquitin substrates.

The ubiquitin-proteasome axis has been extensively explored at a system-wide level, but the impact of deubiquitinating enzymes (DUBs) on the ubiquitinome remains largely unknown. Using UbiSite technology and inhibitors, we have compared the contributions of the proteasome and DUBs on the ubiquitinome. We uncovered large differential dynamic Ub signalling networks with substrates and sites uniquely regulated by DUBs or by the proteasome, highlighting the role of DUBs in degradation-independent ubiquitin signalling. DUBs regulate substrates via nearly 40,000 unique sites. Moreover, we found that ubiquitin conjugated to SUMO2/3 forms a unidirectional proteasomal degradation signal with strikingly rapid kinetics compared to ubiquitin polymers only. We found that PARP1, is hyper ubiquitinated in response to DUB inhibition, increasing its enzymatic activity. Our findings highlight the key regulatory roles of DUBs on ubiquitin dynamics.