A cell-permeable Ub-Dha probe for profiling E1-E2-E3 enzymes in live cells.

Activity-based ubiquitin probes (Ub-ABPs) have recently been developed as effective tools for studying the capabilities of E1-E2-E3 enzymes, but most of them can only be used in cell lysates. Here, we report the first cell-penetrating Ub-Dha probes based on thiazolidine-protected cysteines, which enable successful delivery into cells confirmed by a fluorophore at the N-terminus of Ub and live-cell fluorescence microscopy. A total of 18 E1-E2-E3 enzymes in live cells were labelled and enriched in combination with label-free quantification (LFQ) mass spectrometry. This work provided a new cell-penetrating Ub tool for studying the activity and function of Ub-related enzymes.

Cell-Permeable Stimuli-Responsive Ubiquitin Probe for Time-Resolved Monitoring of Substrate Ubiquitination in Live Cells.

Dynamic monitoring of intracellular ubiquitin (Ub) conjugates is instrumental to understanding the Ub regulatory machinery. Although many biochemical approaches have been developed to characterize protein ubiquitination, chemical tools capable of temporal resolution probing of ubiquitination events remain to be developed. Here, we report the development of the first cell-permeable and stimuli-responsive Ub probe and its application for the temporal resolution profiling of ubiquitinated substrates in live cells. The probe carrying the photolabile group N-(2-nitrobenzyl)-Gly (Nbg) on the amide bond between Ub Gly75 and Gly76 is readily prepared through chemical synthesis and can be delivered to live cells by conjugation via a disulfide bond with the cyclic cell-penetrating peptide cR10D (i.e., 4-((4-(dimethylamino)phenyl)-azo)-benzoic acid-modified cyclic deca-arginine). Both in vitro and in vivo experiments showed that Ub-modifying enzymes (E1, E2s, and E3s) could not install the Ub probe onto substrate proteins prior to removal of the nitrobenzyl group, which was easily accomplished via photoirradiation. The utility and practicality of this probe were exemplified by the time-resolved biochemical and proteomic investigation of ubiquitination events in live cells during a H2O2-mediated oxidative stress response. This work shows a conceptually new family of chemical Ub tools for the time-resolved studies on dynamic protein ubiquitination in different biological processes and highlights the utility of modern chemical protein synthesis in obtaining custom-designed tools for biological studies.

Chemical synthesis of on demand-activated SUMO-based probe by a photocaged glycine-assisted strategy.

The transiently-activated SUMO probes are conducive to understand the dynamic control of SENPs activity. Here, we developed a photocaged glycine-assisted strategy for the construction of on demand-activated SUMO-ABPs. The light-sensitive groups installed at G92 and G64 backbone of SUMO-2 can temporarily block probes activity and hamper aspartimide formation, respectively, which enabled the efficient synthesis of inert SUMO-2 propargylamide (PA). The probe could be activated to capture SENPs upon photo-irradiation not only in vitro but also in intact cells, providing opportunities to further perform intracellular time-resolved proteome-wide profiling of SUMO-related enzymes.

Synthesis of Disulfide Surrogate Peptides through a Serine/Threonine Ligation-Assisted Diaminodiacid Strategy.

We report a new serine/threonine ligation (STL)-assisted diaminodiacid (DADA) strategy for the flexible construction of disulfide surrogates by the option of more abundant -Aa-Ser/Thr- ligation sites. The practicality of this strategy was evidenced by the synthesis of the intrachain disulfide surrogate of C-type natriuretic peptide and the interchain disulfide surrogate of insulin.