The Effect Of TRIM50 On NLRP3 Inflammasome Activation And Its Molecular Machanism

PurposesInflammation is a defense response triggered by microbial infection and tissue damage.The inflammatory response is like a double-edged sword,on one hand,it cleans up pathogens and damaged cells in the body;and on the other hand,an imbalanced inflammatory response can induce disruption of tissues and cells,cause inflammatory diseases or even lead to the death of the host.NOD-like receptor family protein 3(NLRP3)inflammasome is involved in various inflammatory responses in vivo by recognizing different pathogen-associated or tissue damageassociated molecular patterns,and plays an important role in a variety of diseases such as pathogenic infections,auto-inflammatory diseases,neurodegenerative diseases and type Ⅱdiabetes.However,the molecular mechanisms involved in the regulation of NLRP3 inflammasome activation have not been fully clarified,and the effect of the post-translational modifications of NLRP3 on the NLRP3 inflammasome activation needs to be further investigated.Therefore,it is important to clarify the molecular mechanism of NLRP3 inflammasome to development of new therapeutic strategy for NLRP3 inflammasome-related diseases.The tripartite motif-containing(TRIM)family members are a series of proteins with a wide range of biological activities.Most of the TRIM family members are E3 ubiquitin ligases,and they exert their biological effect by inducing the ubiquitination modification of target proteins.TRIM50 is a newly identified member of the TRIM family,and current studies have shown that it is a typical E3 ubiquitin ligase.However,the biological function of TRIM50 in the innate immune response remains unknown.The data of this study demonstrate that TRIM50 directly interacts with NLRP3 via its RING domain,inducing the abrogation of its RING-type ubiquitination ligase activity,and further leading to the NLRP3 oligomerization and NLRP3 inflammasome activation.This study provides new experimental evidences to clarify the regulatory mechanisms of NLRP3 inflammasome,and it also suggests the potential therapeutic strategy by targeting TRIM50 in a variety of NLRP3 inflammasome involved diseases.MethodsThe interaction between TRIM50 and NLRP3 was detected by co-immunoprecipitation(co-IP)and immunofluorescence(IF).Then we constructed a series of domain truncated mutants of TRIM50 and NLRP3 to define the domains that mediate the intereaction between TRIM50 and NLRP3.We further constructed a Trim50 knockdown cellular model by using Trim50 specific small interference RNA,and we constructed a Trim50 knockout mouse model to detect the activation of NLRP3 inflammasome by western blot and enzyme linked immunosorbent assay(ELISA).The molecular mechanisms of TRIM50-mediated regulation of NLRP3 inflammasome activation were investigated in HEK293T cells and macrophages by coIP and western blot.The molecular mechanisms involved in the positive regulation of NLRP3 inflammasome activation by TRIM50 were verified in Trim50 knockout mouse embryonic fibroblasts(MEFs).Then NLRP3-associated inflammation models were constructed by Trim50 knockout mouse to validate the regulatory role of TRIM50 in the NLRP3 inflammasomeassociated disease.Results1.TRIM50 directly interacts with NLRP3.The interaction and co-localization between TRIM50 and NLRP3 were verified using coimmunoprecipitation(Co-IP)and immunofluorescence(IF)in HEK293T cells and mouse peritoneal macrophages.In vitro protein expression system further demonstrated a direct interaction between TRIM50 and NLRP3 in vitro.Further co-IP and IF data indicated that the binding ability of TRIM50 with NLRP3 was significantly enhanced after NLRP3 inflammasome was activated.Then we constructed a series of domain truncated mutants of TRIM50 and NLRP3,and the interaction domains of TRIM50 and NLRP3 was detected by coIP assay.These data showed that the RING domain of TRIM50 interacted directly with the NACHT741 region of NLRP3.2.TRIM50 positively regulates NLRP3 inflammasome activation by increasing the expression of NLRP3 protein.We constructed a Trim50 knockdown cellular model,and further investigation showed that both the NLRP3 protein levels and the NLRP3 inflammasome activation were significantly decreased after Trim50 was interfered.Then we isolated peritoneal macrophages and bone marrow-deribed macrophages(BMDMs)from the Trim50 knockout mouse,and the western blot and ELISA assay revealed that TRIM50 positively regulated NLRP3 inflammasome activation.3.TRIM50 reduces NLRP3 ubiquitination and promotes the stability of NLRP3 protein.The ubiquitination of NLRP3 in HEK293T cells and mouse BMDM cells was detected by IP assay,and the data showed that TRIM50 significantly reduced the ubiquitination of NLRP3.Then we inhibited the de novo protein synthesis by cycloheximide(CHX),and western blot data showed that TRIM50 significantly enhanced the stability of NLRP3 protein.Further IP data revealed that the RING domain of TRIM50 was directly involved in the interaction between TRIM50 and NLRP3 as well as the regulation of NLRP3 inflammasome activation.4.TRIM50 directly induces NLRP3 oligomerization and promotes NLRP3 inflammasome activation.The oligomerization of NLRP3 in HEK293T cells,mouse peritoneal macrophages and BMDM cells were detected by western blot,and the data showed that TRIM50 significantly enhanced NLRP3 oligomerization.Western blot assay revealed that TRIM50 significantly enhanced the oligomerization of ASC during NLRP3 inflammasome activation.We further constructed an in vitro oligomerization system and an in vitro deubiquitination system,and the data showed that TRIM50 can directly induced NLRP3 oligomerization in vitro.The in vitro biochemical data indicated that the inhibition of ubiquitination was a subsequent indirect consequence following NLRP3 oligomerization.The direct regulatory effect of TRIM50 on NLRP3 was further validated by the application of NLRP3-specific inhibitors.5.TRIM50 induces NLRP3 oligomerization and promotes NLRP3 inflammasome activation via its coiled-coil domain.The NLRP3 ubiquitination assay showed that the coiled-coil domain deleted mutants of TRIM50 significantly rescued the inhibition of NLRP3 ubiquitination induced by TRIM50.NLRP3 oligomerization and protein stability assays further demonstrated that the coiled-coil domain of TRIM50 induced the oligomerization of NLRP3 and enhanced the stability of NLRP3 protein.We further transfected TRIM50 as well as TRIM50 mutants constructs back into MEFs from Trim50 knockout mice,and the data revealed that the coiled-coil domain of TRIM50 regulated NLRP3 inflammasome activation.6.TRIM50 exacebated NLRP3 inflammasome associated diseases.We constructed an LPS-induced endotoxic shock model by intraperitoneal injecting LPS in wild-type mice and Trim50-/-mice.Further investigation showed that the overall survival time of the LPS challenged Trim50-/-mice was significantly prolonged compared with the wild type mice.The analysis of inflammatory factors in sera and histopathological analysis of mice lungs indicated that knockout of Trim50 significantly alleviate the endotoxic shock induced by LPS.Then we constructed another alum-induced acute peritonitis model,and the data showed that TRIM50 promoted the recruitment of neutrophils and monocytes.We further constructed an acute tubular necrosis model in mice by folic acid(FA),and our data showed that knockout of Trim50 significantly inhibited the activation of NLRP3 inflammasome as well as the pathological kidney injury.Conclusion and Innovation1.TRIM50 directly interacts with NLRP3 via the RING domain and induces NLRP3 oligomerization through its coiled-coil domains,leading to the NLRP3 inflammasome activation.2.TRIM50 directly induces NLRP3 oligomerization,decreases NLRP3 ubiquitination and enhances NLRP3 protein stability.3.TRIM50 is involved in NLRP3 associated diseases,and provides a new therapeutic strategy for NLRP3 inflammasome associated diseases.