Discovery and mechanism of K63-linkage-directed deubiquitinase activity in USP53
Wendrich K, Gallant K, Recknagel S, Petroulia S, Kazi NH, Hane JA, Führer S, Bezstarosti K, O'Dea R, Demmers J, Gersch M (2024). Nat Chemical Biology
USP53 and USP54 belong to a family of human deubiquitinases (DUBs), but were annotated as catalytically inactive pseudoenzymes. Conspicuously, mutations within the DUB domain of USP53 cause progressive familial intrahepatic cholestasis. Here, work by Kim Wendrich, Kai Gallant and colleagues from the Gersch lab report that USP53 and USP54 are active DUBs with high specificity for K63-linked polyubiquitin. The work demonstrates how USP53 patients mutations abrogate catalytic activity, implicating loss of DUB activity in USP53-mediated pathology. The study further shows that depletion of USP53 increases K63-linked ubiquitination of tricellular junction components, suggesting a mechanism of pathogenicity. Assays with substrate-bound polyubiquitin reveal that USP54 cleaves within K63-linked chains, whereas - surprisingly - USP53 can en bloc deubiquitinate substrate proteins in a K63-linkage-dependent manner. Biochemical and structural analyses uncover K63-specific S2 ubiquitin-binding sites within their catalytic domains, which give rise to these activities. Collectively, this work revises the annotation of USP53 and USP54, provides reagents and a mechanistic framework to investigate K63-linked polyubiquitin decoding and establishes K63-linkage-directed deubiquitination as a novel DUB activity.