Pyroptosis,a form of programmed cell death primarily mediated by gasdermin D(GSD-MD),plays a crucial role in infections and inflammatory responses.Although the OTU deubiquiti-nase 4(OTUD4)has shown its importance in various cellular processes,its specific function in py-roptosis remains unclear,which necessitates a deeper understanding of its role.This study aims to explore the role of OTUD4 in regulating GSDMD-mediated pyroptosis.Through experiments inclu-ding cell transfection,lactate dehydrogenase(LDH)release assays,co-immunoprecipitation(Co-IP),and Western blot(WB)analysis,we investigated the interaction between OTUD4 and GSD-MD and their influence on GSDMD-mediated pyroptosis.Our results indicated that OTUD4 could bind to GSDMD,significantly reduce Caspase-1-mediated cleavage of GSDMD,and lower protein levels of GSDMD-p30,thereby inhibiting pyroptosis.Conversely,the enzymatically inactive mutant of OTUD4(C45A)exhibited a markedly diminished inhibitory effect on pyroptosis.Furthermore,OTUD4 also demonstrated a significant inhibitory effect on pyroptosis in porcine cells.In conclu-sion,this study reveals that OTUD4 can inhibit GSDMD-p30-mediated pyroptosis through its deu-biquitinating activity,highlighting the critical function of OTUD4 in the regulation of pyroptosis and suggesting a potential target for the development of new therapeutic strategies.

Pyroptosis,a form of programmed cell death primarily mediated by gasdermin D(GSD-MD),plays a crucial role in infections and inflammatory responses.Although the OTU deubiquiti-nase 4(OTUD4)has shown its importance in various cellular processes,its specific function in py-roptosis remains unclear,which necessitates a deeper understanding of its role.This study aims to explore the role of OTUD4 in regulating GSDMD-mediated pyroptosis.Through experiments inclu-ding cell transfection,lactate dehydrogenase(LDH)release assays,co-immunoprecipitation(Co-IP),and Western blot(WB)analysis,we investigated the interaction between OTUD4 and GSD-MD and their influence on GSDMD-mediated pyroptosis.Our results indicated that OTUD4 could bind to GSDMD,significantly reduce Caspase-1-mediated cleavage of GSDMD,and lower protein levels of GSDMD-p30,thereby inhibiting pyroptosis.Conversely,the enzymatically inactive mutant of OTUD4(C45A)exhibited a markedly diminished inhibitory effect on pyroptosis.Furthermore,OTUD4 also demonstrated a significant inhibitory effect on pyroptosis in porcine cells.In conclu-sion,this study reveals that OTUD4 can inhibit GSDMD-p30-mediated pyroptosis through its deu-biquitinating activity,highlighting the critical function of OTUD4 in the regulation of pyroptosis and suggesting a potential target for the development of new therapeutic strategies.