Research On The Role For BRISC In The Control Of Toll-like Receptors(TLRs)-mediated Hepatic Inflammatory Responses And Regulation Mechanism For BRCC3 Protein Stability

BRISC(BRCC3 isopeptidase complex)is a deubiquitinating enzyme(DUB)that specifically cleaves K63-linked ubiquitin chains,which consists of four subunits including ABRO1,BRCC3,MERIT40,and BRE.Previous studies have suggested that BRISC plays a critical role in inflammation,cell cycle regulation,hematopoietic stem cell expansion,as well as tumor diseases.As one of the most important DUB in the cytoplasm,BRISC has a variety of substrates,which indicates that BRISC may have more complicated biological functions.Moreover,in addition to transcriptional regulation,the expression of BRISC components is also regulated by post-translational modifications,but the mechanism is still unclear.In this study,we focused on the role of BRISC in the regulation of Toll-like receptors(TLRs)signaling pathway and the possible mechanisms.We also demonstrated the regulation mechanism of how ABRO1 controls the stability of BRCC3.Main achievements of this research work are listed as follows:1.Analyze the expression of BRISC components in inflammation related liver diseases.Clinical samples and databases were used to analyze the expression of BRISC components in human patients with hepatitis.Additionally,the expression of BRISC components in liver and Kupffer cells(KCs)was detected in mice with acute and chronic hepatitis.The results indicate that hepatic BRISC components are significantly upregulated in response to inflammation.2.Evaluate the role of BRISC in TLRs-mediated inflammatory liver diseases.BRISC-deficient and wild-type(WT)mice were intraperitoneally injected with Dgalactosamine(D-Gal N),combined with either lipopolysaccharide(LPS),poly(I:C),or Cp G-ODN,to induce acute liver injury,as well as fed with methionine-choline deficient(MCD)or high-fat diet(HFD)to induce steatohepatitis.Serum samples were collected to measure aminotransferases and proinflammatory cytokines;liver tissues were analyzed by histology,immunohistochemistry,real-time PCR,and cytometric bead array(CBA).Results show that mice lack of BRISC displays attenuated liver injury in D-Gal N/TLRs induced acute liver failure.BRISC ablation suppresses progression of diet-induced steatohepatitis and liver fibrosis by decreasing hepatic inflammation.3.Investigate the possible mechanism of BRISC regulating TLRs-mediated liver inflammation.We demonstrated that KCs are the key effector cells involved in the hepatoprotective efficacy of BRISC-deficient mice in the D-Gal N/LPS model by bone marrow transplantation experiments.Furthermore,KCs were isolated and stimulated with TLR ligands then analyzed by CBA,real-time PCR,and western blot.The results show that BRISC-deficient KCs have defects in the production of TNF-α and IL-6 after TLR2,TLR3,TLR4,and TLR9 stimulation.BRISC regulates TLRs-mediated NF-κB activation in a cell-specific manner and thereby inhibits KCs activation and ameliorates inflammation-associated liver disease.4.Investigate the regulation mechanism for BRCC3 protein stability.Our results showed that ABRO1 deficiency results in increased ubiquitination and diminished protein level of BRCC3.Co-immunoprecipitation assay showed that ABRO1 completes with WWP2 to bind to the MPN domain of BRCC3.Overexpression of ABRO1 profoundly weakened the interaction between WWP2 and BRCC3,thus prevented WWP2-mediated ubiquitination of BRCC3.In addition,overexpression of WWP2 results in inhibition of NLRP3 deubiquitination in macrophages,which may further prevent the subsequent activation of NLRP3 inflammasome.