Photocaged Cell-Permeable Ubiquitin Probe for Temporal Profiling of Deubiquitinating Enzymes

Abstract

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.

Rational Development and Characterization of a Ubiquitin Variant with Selectivity for Ubiquitin C-Terminal Hydrolase L3

“…Therefore, for selective UbV-ABPs to find more use in DUB research strategies to make the ABPs cell permeable must be employed. However, the ability to deliver Ub-based probes to the cellular cytosol has previously been demonstrated using cell-penetrating peptides [ 38 , 39 , 40 ], which and as a result this may give rise to broader use of UbV-ABPs.…”

Lights on 2,5-diaryl tetrazoles: applications and limits of a versatile photoclick reaction

“…In this case, the authors did not employ a 2,5-diaryl tetrazole, but a mono-aromatic derivative; however, this type of scaffold showed activation properties comparable to previously described compounds and represents an interesting example of a genetically encoded probe that can be exploited to study protein-protein interactions in their native environment. More recently, an analogous of compound 29, has been conjugated to ubiquitin and used to monitor and clarify the activities of deubiquitinating enzymes in live cells, an important target against several diseases, upon activation at 365 nm [104]. In the same scenario, Hacker et al, exploited the high reactivity of 2,5-diaryl tetrazoles against carboxylates to identify new potential binding sites for inhibitors to treat bacterial infections.…”

Chemical methods for protein site-specific ubiquitination

“…Recently, several cell-permeable Ub probes have been reported allowing for intracellular profiling of DUBs. 111,[131][132][133] Achieving controlled polyubiquitination of a target protein in cells using bioorthogonal methods is still a challenge. To this end, new cell-compatible chemical reactions needs to be adopted for achieving chemical ubiquitination in live cells.…”