DUB-Resistant Ubiquitin to Survey Ubiquitination Switches in Mammalian Cells

The ubiquitin-modification status of proteins in cells is highly dynamic and maintained by specific ligation machineries (E3 ligases) that tag proteins with ubiquitin or by deubiquitinating enzymes (DUBs) that remove the ubiquitin tag. The development of tools that offset this balance is critical in characterizing signaling pathways that utilize such ubiquitination switches. Herein, we generated a DUB-resistant ubiquitin mutant that is recalcitrant to cleavage by various families of DUBs both in vitro and in mammalian cells. As a proof-of-principle experiment, ectopic expression of the uncleavable ubiquitin stabilized monoubiquitinated PCNA in the absence of DNA damage and also revealed a defect in the clearance of the DNA damage response at unprotected telomeres. Importantly, a proteomic survey using the uncleavable ubiquitin identified ubiquitinated substrates, validating the DUB-resistant ubiquitin expression system as a valuable tool for interrogating cell signaling pathways. [Display omitted] •UbL73P ubiquitin conjugates are not deubiquitinated by various DUB family members•Expression of DUB-resistant ubiquitin in cells stabilizes monoubiquitinated PCNA•Expression of UbL73P reduces the clearance of 53BP1 foci at unprotected telomeres•Proteomic survey of UbL73P-expressing cells identifies ubiquitinated substrates It has been postulated that the majority of proteins in a cell are regulated and modified by ubiquitin at some point; however, it has proven difficult to monitor the ubiquitination status of these proteins, because many of the modifications only exist transiently, owing largely to the multitude of deubiquitinases (DUBs) present in cells. Here, Huang and colleagues stabilize the ubiquitin proteome by expressing a ubiquitin mutant that is resistant to DUB cleavage. This allows them to identify ubiquitinated substrates and uncover a function for ubiquitination in the clearance of the DNA damage response at unprotected telomeres.