USP53 (ubiquitin-specific peptidase 53) is a deubiquitinase that specifically cleaves K63-linked polyubiquitin chains from substrate proteins through an en bloc mechanism, recognizing ubiquitin chains at the S2 position 1. Previously annotated as a catalytically inactive pseudoenzyme, USP53 was discovered to be an active DUB with high specificity for K63-linked polyubiquitin 1. Its primary physiological function involves mediating deubiquitination of tight junction proteins, particularly MARVELD2 and LSR, thereby modulating barrier properties and mechanical stability of cellular junctions 1. This deubiquitinase activity is essential for auditory hair cell survival and hearing function 2. Loss-of-function mutations in USP53 cause progressive familial intrahepatic cholestasis (PFIC), with increased K63-linked ubiquitination of tricellular junction components 1. Clinical data from 29 PFIC patients with USP53 mutations showed good outcomes, with 82.7% achieving native liver survival 3. Beyond cholestasis, USP53 exhibits pleiotropic functions: it regulates bone homeostasis by controlling RANKL expression 4, suppresses tumorigenesis in colorectal cancer via MORF4L1 deubiquitination 5, and modulates atherosclerosis-related foam cell formation through SR-A stabilization 6. USP53 mutations have also been associated with hearing loss and potential schizophrenia risk 7, establishing it as a multifunctional deubiquitinase with significant clinical relevance.