Advancement in our knowledge of deubiquitinases (DUBs) and their biological functions requires biochemical tools permitting interrogation of DUB activities under physiologically relevant conditions. Activity-based DUB probes (DUB ABPs) have been widely used in investigating the function and activity of DUBs. However, most ubiquitin (Ub)-based DUB ABPs are not cell-permeable, limiting their utility to purified proteins and cell lysates. Lysis of cells usually leads to dilution of the cytoplasm and disruption of the normal cellular organization, which may alter the activity of many DUBs and DUB complexes. Here, we report a new class of cell-permeable DUB ABPs that enable intracellular DUB profiling. We used a semisynthetic approach to generate modular ubiquitin-based DUB probes containing a reactive warhead for covalent trapping of DUBs with a catalytic cysteine. We employed cell-penetrating peptides (CPPs), particualrly cyclic polyarginine (cR10), to deliver the DUB ABPs into cells, as confirmed using live-cell fluorescence microscopy and DUB ABPs containing a fluorophore at the C-terminus of Ub. In comparison to TAT, enhanced intacellular delivery was observed through conjugation of a cyclic polyarginine (cR10) to the N-terminus of ubiquitin via a disulfide linkage. Using the new cell-permeable DUB ABPs, we carried out DUB profiling in intact HeLa cells, and identified active DUBs using immunocapture and label-free quantitative mass spectrometry. Additionally, we demonstrated that the cell-permeable DUB ABPs can be used in assessing the inhibition of DUBs by small-molecule inhibitors in intact cells. Our results indicate that cell-permeable DUB ABPs hold great promise in providing a better understanding of the cellular functions of DUBs and advancing drug discovery efforts targeting human DUBs.

Activity-based Ub-PCNA probes identify deubiquitinating enzymes (DUBs) specific for PCNA and demonstrate site-specific deubiquitination by DUBs.

Photocaged cell-permeable ubiquitin probe holds promise in profiling the activity of cellular deubiquitinating enzymes (DUBs) with the much needed temporal control. Here we report a new photocaged cell-permeable ubiquitin probe that undergoes photoactivation upon 365 nm UV treatment and enables intracellular deubiquitinating enzyme profiling. We used a semisynthetic approach to generate modular ubiquitin-based probe containing a tetrazole-derived warhead at the C-terminus of ubiquitin and employed a cyclic polyarginine cell-penetrating peptide (cR10) conjugated to the N-terminus of ubiquitin via a disulfide linkage to deliver the probe into live cells. Upon 365 nm UV irradiation, the tetrazole group is converted to a nitrilimine intermediate in situ, which reacts with nearby nucleophilic cysteine residue from the DUB active site. The new photocaged cell-permeable probe showed good reactivity toward purified DUBs, including USP2, UCHL1, and UCHL3, upon photoirradiation. The Ub-tetrazole probe was also assessed in HeLa cell lysate and showed robust labeling only upon photoactivation. We further carried out protein profiling in intact HeLa cells using the new photocaged cell-permeable ubiquitin probe and identified DUBs captured by the probe using label-free quantitative (LFQ) mass spectrometry. Importantly, the photocaged cell-permeable ubiquitin probe captured DUBs specifically in respective G1/S and G2/M phases in synchronized HeLa cells. Moreover, using this probe DUBs were profiled at different time points following the release of HeLa cells from G1/S phase. Our results showed that photocaged cell-permeable probe represents a valuable new tool for achieving a better understanding of the cellular functions of DUBs.