The Regulation And Mechanism Of E3 SUMO Ligase TRIM28 In NLRP3 Inflammasome Activation

NLRP3 inflammasome,which can recognize pathogen infection and intracellular danger signals,is a large cytoplasmic protein complex composed of NLRP3,ASC,and pro-caspase-1.NLRP3 inflammasome activation induces the maturation and secretion of cytokines IL-1β and IL-18,and thus further induces pyroptosis.NLRP3 inflammasome plays an important role in various physiological and pathological processes such as antiviral,inflammation,infectious diseases,Alzheimer’s disease,and cancer.The cellular NLRP3 protein level is crucial for the assembly and activation of NLRP3 inflammasome.Various post-translational modifications(PTMs),including phosphorylation and ubiquitination,control NLRP3 protein degradation,and NLRP3 inflammasome activation.To further reveal the role of PTMs in NLRP3 expression and NLRP3 inflammasome activation,we first used immunoprecipitation combined with liquid mass spectrometry to screen for specific NLRP3 binding molecules in mouse peritoneal macrophages.It was found that E3 SUMO ligase TRIM28 could bind to NLRP3.TRIM28,a member of the VI subfamily of TRIM family,has E3 SUMO ligase activity.SUMOylation,a small ubiquitin-like modifier(SUMO)protein conjugated modification which is similar to ubiquitination,is mediated by El-activating enzymes,E2-conjugating enzyme,and E3 SUMO ligases.At present,three SUMO proteins,including SUMO1,SUMO2,and SUMO3,can be covalently conjugated to proteins as a single moiety(SUMO1)or as polymeric SUMO chains(SUMO2 and SUMO3).SUMOylation is a dynamic and reversible process in which the SUMO-deconjugating enzymes can excise the SUMO molecule from the target protein,resulting in the deSUMOylation of the target protein.SUMOylation contributes significantly to mediating protein interaction,regulating protein localization in cells,regulating the activity of transcription factors,maintaining genomic stability,and transcriptional regulation.Recently,SUMOylation was identified in NLRP3 during inflammasome activation.The E3 SUMO protein ligase MUL1 catalyzes SUMO2/3 modification of NLRP3 and restrains NLRP3 inflammasome activation,whereas SUMO1-catalyzed SUMOylation of NLRP3 facilitates inflammasome activation.Although SUMO1 modification of NLRP3 is required for inflammasome activation,both the mechanism details and the E3 SUMO ligase mediates SUMO1-catalyzed SUMOylation of NLRP3 are unclear.The purpose of this study was to investigate the regulatory role of E3 SUMO ligase TRIM28 in NLRP3 inflammasome activation and to clarify the molecular mechanism of its action.To elucidate the interaction between SUMOylation and ubiquitination of NLRP3 during NLRP3 inflammasome activation,and further reveal the mechanism of NLRP3 inflammasome activation.First,we used mouse peritoneal macrophages,HEK293T cells,and mouse embryonic fibroblasts for immunoprecipitation,Western blotting,and immunofluorescence experiments to further confirm that TRIM28 can bind specifically to NLRP3 but not to other components of NLRP3 inflammation.Next,mouse PMs from Trim28CKO mice or transfected with Trim28 siRNA,were used to confirm that Trim28 did not affect the priming of NLRP3 inflammation activation by Western blotting and RT-PCR experiments.Subsequently,through ELISA and Western blot experiments,I found that deficiency attenuates NLRP3 inflammasome activation in vivo and in vitro.We used mouse PMs from Trim28CKO mice,transfected with Trim28 siRNA and HEK293T cells,as well as protein synthesis inhibitor CHX,ubiquitin-proteasome pathway inhibitor MG132,autophagy inhibitor Chloroquine,and lysosomal pathway inhibitor 3-MA.Western blots showed that TRIM28 could inhibit the proteasomal degradation of NLRP3 and thus stabilize NLRP3protein expression.We used mouse PMs from Trim28CKO mice and HEK293T cells and found that TRIM28 could inhibit the K48-linked ubiquitination of NLRP3 without affecting the K63-linked ubiquitination through transfection plasmid and Western blot experiments.A point mutant plasmid TRIM28-C651A that inactivated the TRIM28 E3 SUMO ligase is constructed.After transfection of the plasmid into HEK293T cells and immunoprecipitation and Western blot experiments,it was found that the TRIM28-C651A mutant plasmid lost the ability to bind to E2 SUMO binding enzyme UBC9,further confirming that the E3 SUMO ligase activity was inactivated after TRIM28 C651A point mutant.Moreover,TRIM28-C651A point mutant caused it to lose the ability to inhibit NLRP3 ubiquitination and degradation,and it was clear that TRIM28 specifically inhibited NLRP3 K48-linked ubiquitination and NLRP3 degradation through its E3 SUMO ligase activity.Finally,we used mouse PMs from Trim28CKO mice,transfected with Trim28 siRNA and HEK293T cells to do immunoprecipitation,transfection plasmid,and Western blot.It was found that TRIM28 can promote SUMO1,SUMO2,and SUMO3-catalyzed SUMOylation of NLRP3 in mouse PMs and HEK293T cells,and the TRIM28 C651A point mutant has lost the ability to mediate NLRP3 SUMOylation.In summary,this study clarified the role of E3 SUMO ligase TRIM28 in NLRP3 inflammasome activation,clarified the specific mechanism of TRIM28 regulating NLRP3 inflammasome,interpreted the protein level of NLRP3 in SUMO-regulated cells and the mechanism of inflammasome activation.The interaction between the two PTMs(SUMOylation and ubiquitination)of NLRP3 and the mechanism of fine-regulation of inflammasome activation was revealed.It also provides a new potential target for the prevention and control of diseases related to abnormal activation of the inflammasome.