Deubiquitinase dynamics: methodologies for understanding substrate interactions

Abstract

Deubiquitinases (DUBs) are essential regulators of proteinhomeostasis that influence cellular signaling, protein stability,and degradation by removing ubiquitin chains from substrateproteins. Understanding DUB–substrate interactions is criticalto elucidate their functional roles and therapeutic potential. This review highlights key methodologies to investigate DUBactivity and substrate interactions, including biochemical assays,fluorescence-based approaches, and in vitro deubiquitinationassays. Biochemical methods, such as those measuring proteindegradation rates, ubiquitination dynamics, and protein–proteininteractions, provide valuable insights into DUB function andspecificity. Fluorescence-based techniques that include photoconvertiblereporters, fluorescent timers, and FRET enable the realtimemonitoring of DUB dynamics and substrate turnover in livecells. Furthermore, in vitro deubiquitination assays provide directmechanistic insights into DUB activity on target substrates. Whileeach method provides unique insights, they also present challenges,like limited specificity or sensitivity, technical difficulties,or insufficient physiological relevance. Integrating complementaryapproaches can enhance accuracy and providedeeper insights into DUB–substrate interactions, facilitating thedevelopment of DUB-targeted therapeutic strategies.