Systematic Identification of Candidate Substrates of the UCH Family of Deubiquitinases

Deubiquitinases (DUBs) of the ubiquitin C-terminal hydrolase (UCH) family play critical roles in regulating ubiquitin-dependent signaling, yet their substrate specificity and functional organization remain incompletely defined. Here, we systematically characterized the ubiquitination landscape associated with UCH family members (UCHL1, UCHL3, UCHL5, and BAP1) using a quantitative proteomics workflow combining siRNA-mediated DUB silencing, bioUb-based enrichment of ubiquitinated proteins, and label-free mass spectrometry. Proteins exhibiting increased ubiquitination following DUB depletion were identified as candidate substrates. Our analysis revealed that UCH family members regulate largely distinct subsets of ubiquitinated proteins, with limited overlap in their candidate profiles despite sharing conserved catalytic domains. In parallel, site-resolved ubiquitinomics identified 851 ubiquitination sites, including 106 not previously reported, expanding the current landscape of experimentally validated ubiquitin-modified lysines. Functionally, substrate networks link individual UCH enzymes to distinct yet partially overlapping cellular processes, including hypoxia signaling, RNA metabolism, vesicle trafficking, apoptosis, and chromatin regulation. Notably, known substrates such as HIF-1α validated the approach, while recurrent detection of key regulators, including MCL1, pointed to convergence at the level of apoptosis control. Together, these findings provide a resource for exploring the distinct and potentially overlapping roles of UCH deubiquitinases within cellular ubiquitin-regulatory networks.