Discovery and mechanism of K63-linkage-directed deubiquitinase activity in USP53
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ABSTRACT: 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 progressive familial intrahepatic cholestasis. Here we report the discovery that USP53 and USP54 are 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 en bloc deubiquitinate substrate proteins 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 reagents and a mechanistic framework to investigate K63-polyubiquitin decoding, and establishes K63-linkage-directed deubiquitination as novel DUB activity.
INSTRUMENT(S): Orbitrap Fusion Lumos
ORGANISM(S): Homo Sapiens (human)
SUBMITTER: Jeroen Demmers
LAB HEAD: Jeroen Demmers
PROVIDER: PXD054748 | Pride | 2024-11-11
REPOSITORIES: pride
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