USP24 is an ISG15 cross-reactive deubiquitinase that mediates IFN-I production by de-ISGylating the RNA helicase MOV10

The interferon-stimulated gene 15 (ISG15) is a ubiquitin-like modifier induced by type I Interferon (IFN-I) and plays a crucial role in the innate immune response against viral infections. ISG15 is conjugated to target proteins by an enzymatic cascade through a process called ISGylation. While ubiquitin-specific protease 18 (USP18) is a well-defined deISGylase counteracting ISG15 conjugation, ISG15 cross-reactive deubiquitylating enzymes (DUBs) have also been reported. Our study reports USP24 as a novel ISG15 cross-reactive DUB identified through activity-based protein profiling (ABPP). We demonstrate that recombinant USP24 processed pro-ISG15 and ISG15-linked synthetic substrates in vitro . Moreover, the depletion of USP24 significantly increased the accumulation of ISG15 conjugates upon IFN-β stimulation. An extensive proteomic analysis of the USP24-dependent ISGylome, integrating total proteome, GG-peptidome, and ISG15 interactome data, identified the helicase Moloney leukemia virus 10 (MOV10) as a specific target of USP24 for deISGylation. Further validation in cells revealed that ISGylated MOV10 enhances IFN-β production/secretion, whereas USP24 deISGylates MOV10 to negatively regulate the innate immune response. This study showcases USP24’s novel roles in modulating ISGylation and modulation of the IFN-I-dependent immune responses, with potential therapeutic implications in infectious diseases, cancer, autoimmunity, and neuroinflammation.