Advancing our Understanding of Ubiquitination Using the Ub-Toolkit.

Post-translational protein modification by ubiquitin (Ub) and Ub-like modifiers is orchestrated by the sequential action of Ub-activating, -conjugating, and -ligating enzymes to regulate a vast array of fundamental biological processes. Unsurprisingly, the dysregulation of the intricate interplay between ubiquitination and deubiquitination gives rise to numerous pathologies, most notably cancer and neurodegenerative diseases. While activity-based probes (ABPs) and assay reagents have been extensively developed and applied for deubiquitinating enzymes, similar tools for the Ub cascade have only recently emerged. Given the recent efforts to develop inhibitors for the Ub system, the urgency for developing ABPs and assay reagents is imminent. In this light, we comprehensively discuss the currently available ABPs with a focus on the newly developed reagents targeting the Ub cascade while illustrating their potential applications.