An Insight Into The Role Of Ubiquitination In Autophagic Regulation Of NLRP3 Inflammasome In Atherosclerosis

Objective:Atherosclerosis is the main cause for ischemic stroke and inflammation,especially involving NLRP3 inflammasome,is believed to play a primary role in the formation of atherosclerosis.Enhanced autophagy can inhibit the development of atherosclerosis.Recent studies have revealed that NLRP3 inflammasome can be degraded by autophagy in atherosclerosis and the process is accompanied by ubiquitination of the NLRP3 inflammasome constituent proteins.However,the relationship between NLRP3 inflammasome and autophagy in atherosclerosis needs further study and the role of ubiquitination in autophagic regulation of NLRP3 inflammasome remains obscure.We established a foam-cell model to investigate the impact of oxidized low-density lipoproteins(ox-LDLs)on autophagy and the inflammasome in atherosclerosis-related inflammation.We then explored the relationship between NLRP3 inflammasome and autophagy in the foam-cell model and the specific inflammatory proteins through which autophagy exerts its influence on NLRP3 inflammasome.Finally,the ubiquitin chains mediating the autophagic identification of these proteins are investigated.Methods:THP-1 derived macrophages(Mφ)were stimulated with ox-LDLs to establish the foam-cell model.Time interval series and concentration graded ox-LDLs were administered to foam-cells and the expression of NLRP3 inflammasome-related and autophagy-related proteins was detected via Western blot and ELISA.Rapamycin and 3-MA were employed to up-regulate or down-regulate autophagy in foam-cells.Detected was the expression of NLRP3 inflammasome-related proteins under both circumstances.P62-siRNA was transfected to foam-cells to investigate the influence of p62-knockdown on NLRP3-associated inflammation.Autophagy upregulated foam-cells were transfected with p62-siRNA and the expression of NLRP3 inflammasome-related proteins was detected by Western blot and ELISA to find out the role of p62 in the process of autophagic regulation of NLRP3 inflammasome.Immunoprecipitation was conducted to detect the inflammatory proteins bound to p62.Immunoprecipitation was employed to compare the ubiquitin modification of the inflammatory proteins in p62-knockdown foam-cells with that in control foam-cells and determine the ubiquitin chains recognized by p62.P62 and inflammatory proteins complex was pretreated with specific deubiquitination protease.Then,the amount of inflammatory protein bound to p62 was detected to confirm the role of the ubiquitin chains in connecting p62 and inflammatory proteins.Results:After stimulated with ox-LDLs,red oil O stained obvious droplets in THP-1 derived macrophages.Ox-LDLs promoted the expression of activated caspase-1(p20)and IL-1β in a dose-and time-dependent manner,during which expression of NLRP3 and pro-IL-1β were up-regulated while ASC and pro-caspase-1 remained uninfluenced.Meanwhile,OxLDLs decreased LC3II/LC3 I ratio and increased p62 expression in a dose-and timedependent manner.Up-regulating or down-regulating autophagy with rapamycin or 3-MA could inhibit or elevate the expression of caspase-1(p20)and IL-1β respectively and the processes were accompanied by changes in NLRP3 and ASC expression,whereas,procaspase-1 and pro-IL-1β exempted.Knocking-down p62 with p62-siRNA led to an increase in the expression of p20,IL-1β,NLRP3,and ASC.However,when rapamycin-treated foam-cells were transfected with p62-siRNA,the expression of p20,IL-1β,NLRP3,and ASC were not decreased by enhanced autophagy.Immunoprecipitation revealed that NLRP3 and ASC combined with p62,but NLRP3 and ASC formed a stable complex.Compared to performed with NLRP3 antibodies,more p62 detached from the NLRP3-ASC complex when immunoprecipitation was performed with ASC antibodies.We further observed that both NLRP3 and ASC undergone K48 and K63 ubiquitination.When we knocked-down p62 expression with p62-siRNA,K63 ubiquitin chains attached to NLRP3 increased significantly and K48 ubiquitin chains increased slightly,while both ubiquitin chains attached to ASC remained unchanged.Finally,pretreating p62 immunoprecipitates with K63-specific deubiquitination protease-AMSH resulted in less NLRP3 combined to p62 when compared to the control group.Conclusions:The present study suggests that:1.Ox-LDLs activate NLRP3 inflammasome and inhibit autophagy in foam-cells in a time-and dose-dependent manner.2.Enhanced autophagy can inhibit NLRP3 inflammasome activation and the proteins degraded in the process are NLRP3 and ASC,but not pro-caspase-1 and pro-IL-1β.3.Autophagy regulates NLRP3 inflammasome via the adaptor protein p62,which identifies K63 ubiquitin chains attached to NLRP3 to bind NLRP3 inflammasome and directs it to autophagosome for degradation.