Deubiquitinase USP13 Promotes The Activation Of NLRP3 Inflammasome By Enhancing The Stability Of NLRP3 Protein

Objective: The NLRP3 inflammasome is an important component of natural immunity,which consists of the nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3(NLRP3),apoptosis-associated speck-like protein(ASC)and Pro-Caspase-1.The stability of NLRP3 protein is essential for NLRP3 inflammasome activation and is tightly regulated by multiple post-translational modifications,but the specific molecular mechanisms have not been fully revealed.Ubiquitin-specific protease13(USP13)is an important member of the ubiquitin-specific protease family and is involved in a variety of physiopathological processes including tumor,inflammation,autophagy and endoplasmic reticulum-associated degradation by regulating the deubiquitination of substrates.In recent years,it has been suggested that USP13 is involved in the regulation of NLRP3 inflammasome activation,but there is a lack of evidence at the animal level and the molecular mechanism is unknown.The aim of this thesis is to clarify the regulatory role of USP13 on NLRP3 inflammasome activation using USP13 knockout mice and to elucidate its molecular mechanism.Methods: Isolation and culture of wild type(WT)and USP13 knockout mouse bone marrow-derived macrophages(BMDMs)and peritoneal resident macrophages(PRMs);establishment of human THP-1 USP13 knockdown cells using RNAi technology was used to establish human THP-1 USP13 knockdown cells;the activation status of NLRP3 inflammasome in these cells was measured by Cytometric bead array(CBA)for IL-1βsecretion in supernatant,the activation of Caspase-1 and inflammatory pathways were detected by Western blot,and NLRP3 inflammasome assembly was detected by immunofluorescence.NLRP3 protein stability was detected by CHX-chase assay.NLRP3 ubiquitination level was detected by Western blot.We compared the responses of WT mice and USP13 knockout mice to Monosodium urate(MSU)induced peritonitis,Lipopolysaccharides(LPS)induced sepsis,and Methionine-choline deficient(MCD)diet induced non-alcoholic steatohepatitis(NASH).ELISA was performed to detect the release of transaminases in serum,H&E staining and oil red staining were performed to observe the accumulation of liver lipids,Masson staining to observe the degree of liver fibrosis,Real-time PCR to detect the expression of transcript levels of fibrosis-related genes,and CBA and Western blot to detect the activation of NLRP3 inflammasome in the MCD model.Results: In BMDMs,PRMs and THP-1-derived macrophages,knockout of USP13 inhibited NLRP3 inflammasome activation-mediated IL-1β secretion.In PRMs,knockout of USP13 inhibited Caspase-1 activation and NLRP3 inflammasome assembly,but did not affect the responsiveness of PRMs to LPS.The above results demonstrate that deletion of USP13 inhibits NLRP3 inflammasome activation.The CHX-chase assay showed that deletion of USP13 decreased NLRP3 half-life.Both overexpression of WT USP13 and its catalytically inactive mutant promoted NLRP3 deubiquitination,and the USP13 inhibitor Spautin-1 did not reverse the effect of USP13 on NLRP3 ubiquitination,suggesting that the regulation of NLRP3 ubiquitination by USP13 may be independent on its enzymatic activity.USP13 inhibits E3 ubiquitin ligase TRIM31-mediated ubiquitination and degradation of NLRP3.Using multiple animal models of NLRP3 inflammasome-driven inflammatory diseases,we found that USP13 deficiency attenuated MSU-induced peritonitis,LPS-induced sepsis and MCD-induced liver fibrosis.Conclusion: USP13 deficiency inhibits NLRP3 inflammasome activation and attenuates MSU-induced peritonitis,LPS-induced sepsis and MCD-induced liver fibrosis.Mechanistically,USP13 may enhance NLRP3 protein stability by inhibiting ubiquitinated degradation of NLRP3 by TRIM31.