Ubiquitin Azapeptide Esters as Next-Generation Activity-Based Probes for Cysteine Enzymes in the Ubiquitin Signal Pathway

Ubiquitination is a pivotal cellular process that controls protein homeostasis and regulates numerous biological functions. Its pathway operates through a cascade of enzyme reactions involving ubiquitin-activating (E1), ubiquitin-conjugating (E2), and ubiquitin-ligating (E3) enzymes and deubiquitinases (DUBs), many of which are cysteine enzymes. Activity-based ubiquitin probes were previously developed for profiling these enzymes. However, most conventional probes do not mimic natural enzyme-substrate interactions and involve chemical mechanisms different from enzyme catalysis. Their uses potentially affect the comprehensiveness of enzyme profiling results. The current study introduces a novel class of activity-based ubiquitin probes, ubiquitin azapeptide esters, designed to overcome these limitations. These probes incorporate an azaglycine ester at the ubiquitin <i>C</i>-terminus. They structurally mimic a ubiquitinated protein substrate and react with a cysteine enzyme via a mechanism like the enzyme catalysis. It was demonstrated that ubiquitin azapeptide esters are reactive toward a large variety of DUBs and several tested E1, E2, and E3 enzymes as well. Compared to a conventional probe, ubiquitin propargylamine, ubiquitin azapeptide esters generally provide superior labeling and profiling of active cysteine enzymes in the ubiquitination/deubiquitination cascade in both HEK293T cells and mouse tissue lysates. Activity-based protein profiling using these probes in mouse tissue lysates also revealed distinct patterns of labeled enzymes, confirming their potential in understanding the unique roles of these enzymes in different tissues.