RT Journal Article
SR Electronic
T1 Discovery and mechanism of K63-linkage-directed deubiquitinase activity in USP53
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2024.07.07.602376
DO 10.1101/2024.07.07.602376
A1 Wendrich, Kim
A1 Gallant, Kai
A1 Recknagel, Sarah
A1 Petroulia, Stavroula
A1 Führer, Siska
A1 Bezstarosti, Karel
A1 O’Dea, Rachel
A1 Demmers, Jeroen
A1 Gersch, Malte
YR 2024
UL http://biorxiv.org/content/early/2024/07/07/2024.07.07.602376.abstract
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.