Activity-based ubiquitin-protein probes revealtarget protein specificity of deubiquitinatingenzymes

摘要

Ubiquitination is an essential eukaryotic post-translational modification that regulates various cellularprocesses. The removal of ubiquitin from its target protein is catalyzed by deubiquitinating enzymes(DUBs). Although it was proposed that many DUBs specifically interact and recognize ubiquitinatedproteins as substrates, more direct evidence is needed to support this notion. Here we report proteintargeting activity-based DUB probes that allowed the identification of DUBs recognizingmonoubiquitinated proliferating cell nuclear antigen (PCNA) in Saccharomyces cerevisiae. This new classof DUB probes contain a Michael acceptor as a warhead between ubiquitin and the target protein PCNAthrough a linkage that mimics the native isopeptide bond. We selected two known and biologicallyrelevant ubiquitination sites on PCNA to generate the DUB probes. This allowed us to interrogate thesite-specific deubiquitination of a target protein by DUBs. DUBs were profiled in yeast cell lysates usingthe two Ub-PCNA DUB probes in conjunction with two control probes that contain a noncleavablelinkage but no warhead. We identified yeast DUBs through pulldown coupled with quantitative massspectrometry analysis of the pulled down proteins. Our results showed that specific yeast DUBsrecognize monoubiquitinated PCNA and corroborated previous genetic study. We also identified DUBsas potential new deubiquitinase of PCNA. Remarkably, identified DUBs clearly distinguish the differentmodification sites on PCNA, thus supporting a high level of DUB specificity beyond the target proteinidentity.