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

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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			Action	DRS
	2547_CaCo_GG_Spectronaut_data.xlsx	Xlsx
	2547_X_KaiGallant_DIA_13dec23_GG_CaCo_2_3.raw	Raw
	2547_X_KaiGallant_DIA_13dec23_GG_CaCo_2_4.raw	Raw
	2547_X_KaiGallant_DIA_13dec23_GG_CaCo_2_Dox_3.raw	Raw
	2547_X_KaiGallant_DIA_13dec23_GG_CaCo_2_Dox_4.raw	Raw

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