PT  - JOURNAL ARTICLE
AU  - Wendrich, Kim
AU  - Gallant, Kai
AU  - Recknagel, Sarah
AU  - Petroulia, Stavroula
AU  - Führer, Siska
AU  - Bezstarosti, Karel
AU  - O’Dea, Rachel
AU  - Demmers, Jeroen
AU  - Gersch, Malte
TI  - Discovery and mechanism of K63-linkage-directed deubiquitinase activity in USP53
AID  - 10.1101/2024.07.07.602376
DP  - 2024 Jan 01
TA  - bioRxiv
PG  - 2024.07.07.602376
4099  - http://biorxiv.org/content/early/2024/07/07/2024.07.07.602376.short
4100  - http://biorxiv.org/content/early/2024/07/07/2024.07.07.602376.full
AB  - Ubiquitin-specific proteases (USPs) are the largest class of human deubiquitinases (DUBs) and comprise its phylogenetically most distant members USP53 and USP54, which are annotated as catalytically inactive pseudo-enzymes. Conspicuously, mutations in the USP domain of USP53 cause familial intrahepatic cholestasis. Here we report the discovery that USP53 and USP54 are in fact active DUBs with high specificity for K63-linked polyubiquitin. We demonstrate how USP53 patient mutations abrogate catalytic activity, implicating loss of DUB activity in USP53-mediated pathology. Depletion of USP53 increases K63-linked ubiquitination of tricellular junction components. Assays with substrate-bound polyubiquitin reveal that USP54 cleaves within K63-linked chains, whereas USP53 can deubiquitinate a substrate in a K63-linkage-dependent manner. Biochemical and structural analyses uncover underlying K63-specific S2-ubiquitin-binding sites within their catalytic domains. Collectively, our work revises the annotation of USP53 and USP54, provides chemical reagents and a mechanistic framework to broadly investigate K63-polyubiquitin chain length decoding, and establishes K63-linkage-directed deubiquitination as novel DUB activity.Competing Interest StatementThe authors have declared no competing interest.