Discovery and mechanism of K63-linkage-directed deubiquitinase activity in USP53

TY - UNPB

T1 - Discovery and mechanism of K63-linkage-directed deubiquitinase activity in USP53

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

PY - 2024/7/7

Y1 - 2024/7/7

N2 - 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.

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.

U2 - 10.1101/2024.07.07.602376

DO - 10.1101/2024.07.07.602376

M3 - Preprint

BT - Discovery and mechanism of K63-linkage-directed deubiquitinase activity in USP53

PB - BioRxiv

ER -