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

Abstract

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 timeresolved biochemical and proteomic investigation of ubiquitination events in live cells during a H 2 O 2 -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 customdesigned tools for biological studies.

Three Rounds of Stability-Guided Optimization and Systematical Evaluation of Oncolytic Peptide LTX-315

“…A robust synthetic method was established for the accelerated manual synthesis of LTX-315-derived peptides and hybrid peptides. Previous studies indicated that the DIC/Oxyma condensation system, which featured high condensation efficiency and low risk of explosion, exhibited significant potency to inhibit side reactions represented by the racemization. − Nevertheless, the DIC/Oxyma-based condensation system was mostly conducted at 70 °C or even higher temperatures, which may induce burns to operators and instrument damage. − Using our previously established DIC/Oxyma-based strategy under moderate condensation conditions, the peptides were efficiently synthesized in a normal constant-temperature shaker at 50 °C (Figure A). − Meanwhile, to attach the CPT, the CPT was first reacted with succinic anhydride, affording the CPT-COOH with the isolated yield of 73.4% and the RP-HPLC purity above 95% (Figures B, S26, and S27). The HATU/HOAt/DIEA system was applied for the condensation of N-terminal Abz, AEEA, CPT-COOH, or 7-(diethylamino)­coumarin-3-carboxylic acid at 28 °C.…”