| Inflammasomes are multi-protein complexes in cells,which play important roles in the innate immune system.Inflammasome is composed of inflammasome sensor,Apoptosis-associated speck-like protein containing a CARD(ASC)and effector mole-cule pro-caspase-1,which can sense microbial infection and danger signals in cells.ASC is a common adaptor protein of various types of inflammasomes,and plays a central role in mediating the activation of inflammasomes.When the inflammasome sensor receptor is activated,ASC is recruited,and the activated ASC promotes pro-caspase-1 cleavage,and further leads to the secretion of pro-inflammatory cytokines IL-1β and IL-18.The activity of ASC is regulated by many types of protein post-translational modifications.Among them,the ubiquitination modification of ASC,as a modified form with important biological functions,has received extensive attention.It has been reported that ASC can be modified by various types of ubiquitin chains such as linear ubiquitination and K63 ubiquitination to regulate its protein stability and activity.Ubiquitination modification is a reversible process.The ubiquitin chain of the substrate protein can be removed by deubiquitinating enzymes(DUBs).However,there are few reports about whether DUBs are involved in the regulation of ASC.In this study,we investigated the effects of 75 eukaryotic deubiquitinating enzymes on ASC and found that deubiquitinase USP3 can directly bind to ASC and cleaves its K29,K33,and K48 polyubiquitin chains.In addition,USP3 can inhibit the degradation of the proteasome pathway of ASC by regulation the ubiquitination of ASC,and enhance its protein stability.Furthermore,this study found that knocking down USP3 in mouse peritoneal macrophages or stably knocking out USP3 in THP-1 cells promotes the degradation of ASC,reduces ASC speck aggregation,and reduces the secretion of related cytokines in the signaling pathway,thereby inhibiting activation of the infla-mmasome pathway.This study systematically revealed the biological functions and molecular mechanisms of USP3 targeting ASC ubiquitination and positively regulated the activation of inflammasomes,provided new insights for understanding the occur-rence of inflammation and a new target for the treatment of inflammasome-related diseases.Objectives:Screening and identifying deubiquitinating enzymes that directly target ASC;elucidating the molecular mechanism of deubiquitinating enzymes by regulating the ubiquitination of ASC.Methods:1.To screen and identify the DUBs which can deubiquitinate ASCHEK293T cells were transfected with GFP-ASC and Flag-tagged or Myc-tagged DUBs for 36 h.The number of spots was observed under a fluorescence microscope;HEK293T cells were transfected with Flag-tagged or Myc-tagged ASC,HA-ubiquitin(HA-Ub)and Myc-tagged DUBs or Flag-tagged for 36 h.The cell lysates were detected by western blot;Specific and effective siRNA targeting DUBs was used to suppress the expression of endogenous DUBs in mouse macrophages,secretion of IL-1β were measured by ELISA.2.Generation of USP3-knockout THP-1 using CRISPR-Cas9.Lentiviral particles that express three different guide RNAs(gRNAs)that target USP3 were prepared by transfecting HEK293T cells with gRNA-expressing construct,pPAX2 and pMD2 plasmids.Lentiviruses were collected 48 h after transfection and THP1 cells were infected with lentivirus.Then cells were subjected to puromycin selection(0.5 μg/mL)to isolate USP3-knockout immortalized THP-1 cells.3.To detect the effect of USP3 on the deubiquitination of ASC3.1 HEK293T cells were transfected with Myc-ASC,HA-Ub and Flag-USP3/enzyme inactive mutant Flag-USP3-C168S.The cell lysates were detected by weste-rn blot3.2 In vitro deubiquitination assay:HEK293T cells were transfected with Flag-ASC and HA-Ub,the cell lysates were collected 48 h after transfection,and the USP3 purified protein was added to the lysate of enriched ubiquitinated ASC,incubated at 37℃ for 30 minutes,and then detected by Western blot with anti-HA Ab.3.3 Mouse peritoneal macrophages or USP3-knockout immortalized THP-1 cells were stimulated with LPS and ATP,the cell lysates were detected by western blot4.To detect the interaction between ASC and USP34.1 HEK293T cells were transfected with Myc-ASC and Flag-USP3.The cell lysates were detected by western blot.4.2 In vitro binding assay:HEK293T cells were transfected with Flag-ASC and Flag-USP3,the cell lysates were collected 48 h after transfection and the protein is purified and incubated in vitro,and then were detected by western blot4.3 Endogenous binding assay:Mouse peritoneal macrophages or USP3-knockout immortalized THP-1 cells were stimulated with LPS and ATP for indicated time points.The cell lysates were detected by western blot.4.4 Co-localization assay:HEK293T cells were transfected with GFP-ASC and Flag-USP3,and the co-localization of ASC and USP3 was detected by immune-fluorescence experiment.4.5 Domain binding detection:HEK293T cells were transfected with Myc-ASC-CARD,Myc-ASC-PYD,Flag-USP3-UCH,Flag-USP3-ZnF,the cell lysates were collected 48 h after transfection and the cell lysates were detected by western blot4.6 Detection of the specificity of USP3 and ASC binding:HEK293T cells were transfected with Myc-ASC,Flag-USP3,Myc-Caspase-1 and Myc-NLRP3.The cell lysates were detected by western blot5.To investigate the function of USP3 in the ASC stability5.1 To explore the ubiquination form of ASC regulated by USP3:HEK293T cells were co-transfected with Myc-ASC,Flag-USP3 and HA-Ub or its mutants for 48 h.The cell lysates were detected by western blot.5.2 To detect the influence of USP3 on the stability of ASC:HEEK293T cells were co-transfected with Myc-ASC,Flag-USP3/Flag-USP3-C168S and HA-Ub-K48 for 48 h.The cell lysates were detected by western blot with anti-Myc Ab;Mouse peritoneal macrophages or USP3-knockout immortalized THP-1 cells were stimulated with LPS and ATP for indicated time points,the cell lysates were detected by Western blot analysis with anti-ASC Ab.5.3 To detect the pathway of ASC degradation:USP3-knockout immortalized THP-1 cells were pretreated with proteasome degradation inhibitor MG132 and autophagy lysosome inhibitors CQ and 3-MA for 4h,and stimulated with LPS for 6 hours.The protein was collected,and the level of ASC protein was detected by Western blot.5.4 To detect the ASC aggregation:USP3-knockout immortalized THP-1 cells were stimulated with LPS and ATP for indicated time points,the cell lysates were treated with DSS cross-linking agent,and the changes in ASC aggregation were detected by Western blot.6.To detect the effect of USP3 on the inflammasome pathway6.1 To investigate the role of USP3 on the activation of inflammasome in cells:Mouse peritoneal macrophages or USP3-knockout immortalized THP-1 cells were stim ulated with LPS and ATP/Nigericin for indicated time points,the the supernatant were concentrated and then detected by Western blot analysis with anti-IL-1β p17 Ab and caspase-1 p20 Ab.6.2 To investigate the role of USP3 on the production of cytokines in cells:USP3-knockout immortalized THP-1 were stimulated with LPS and ATP for indicated time points,the secretion of IL-1 β,IL6 and TNF-α were detected by ELISA and qPCR.Results:1.USP3 directly regulates the deubiquitination of ASC via its deubiquitinating enzyme activityTo identify the DUBs involved in the regulation of ASC ubiquitination,We searched for DUBs by screening a library of DUBs expression vectors.We first determined the ability of DUBs to affect ASC specks formation in HEK293T cells,in which 8 DUBs had been identified.We then determined if overexpression of these DUBs might reduce the levels of ASC ubiquitination,and finally we determined if knock down of any of candidate DUBs might suppress IL-1β secretion triggered by ATP in LPS-primed macrophages.Based on the above step-by-step screening,we finally determined that USP3 can be involved in regulating deubiquitination of ASC.Furthermore,we transfected USP3-WT or enzyme-inactivated mutants USP3-C168S,Ub and ASC in HEK293T cells,and the ubiquitination of ASC was analyzed through IP and Western blotting,we found that USP3 can regulate the ubiquitination of ASC in an enzyme-dependent manner.In order to exclude the false positive results caused by exogenous overexpression,we conducted endogenous ubiquitination experiments in the USP3-knockout immortalized THP-1 cells and mouse peritoneal macrophages to detect the changes in the endogenous ubiquitination level of ASC.We found that knocking down or knocking out USP3 can significantly increase the ubiquitination level of ASC.At the same time,in order to detect whether USP3 has a direct deubiquiti-nation effect on ASC,we conducted an in vitro deubiquitination experiment and proved that USP3 can directly deubiquitinate ASC in an in vitro enzyme system.The results indicated that USP3 directly regulates the ubiquitination of ASC depending on its deubiquitinating enzyme activity.2.USP3 binds to ASCIn order to further determine whether USP3 regulates ASC ubiquitination by directly binding to ASC,we transfected USP3 and ASC in HEK293T cells,and analyzed through IP and Western blotting,we found that USP3 interacted with ASC.Next,we performed in vitro binding experiments,we analyzed through IP and Western blotting and found that USP3 interacted with ASC directly.In order to eliminate the false positive results caused by overexpression in vitro,we performed endogenous binding experiments in mouse peritoneal macrophages and THP-1 cells respectively.The cells were stimulated with LPS and ATP,then analyzed through IP and Western blotting,the result showed that USP3 can associate with ASC.At the same time,we overexpressed GFP-ASC and Flag-USP3 in the HEK293 cells,and found that USP3 and ASC co-localizated in the pernucleus through immunofluorescence experiments.To further determine the binding domains for USP3 and ASC,we overexpressed Flag-USP3,Flag-USP3-UCH,Flag-USP3-ZnF and Myc-ASC,Myc-ASC-CARD,Myc-ASC-PYD in HEK293T cells,then analyzed through IP and Western blotting,the results showed that UCH domain of USP3 interacts with the CARD domain of ASC.Since the inflammasome is a multi-molecule complex,it is necessary to further determine whether USP3 binds to other components.We overexpressed Flag-USP3 with Myc-ASC,Myc-Caspase-1 and Myc-NLRP3 in HEK293T cells,then analyzed through IP and Western blotting,the results showed that that USP3 specifically binds to ASC,but not to NLRP3 and Caspase-1.3.USP3 stabilizes ASCSince different forms of ubiquitination modification produces different functions,it is necessary to further clarify the ubiquitination form that USP3 mainly regulates ASC.We first overexpressed Flag-USP3,Myc-ASC and different types of ubiquitin in HEK293T cells,including wild-type(WT),K6,K11,K23,K27,K29,K33,K48,K63,and analyzed through IP and Western blotting,and found that USP3 mainly removed K29-,K33-and K48-linked polyubiquitination of ASC.Since K48-linked polyubi-quitination is often related to protein degradation,we overexpressed Flag-USP3 in HEK293T cells,and found that the ASC protein was downregulated in a dose-dependent manners,but the enzyme-inactivated mutants USP3-C168S had no effects.The results demonstrated that USP3 regulates the stability of ASC protein through its enzymatic activity.Protein degradation mainly includes proteasome degradation and autophagy lysosomal degradation.We further tested the degradation form of ASC mediated by USP3.We stimulated USP3-knockout immortalized THP-1 cells with LPS and ATP,and then added MG132,CQ and 3-MA to inhibit proteasome degradation and autophagy lysosomal degradation respectively,and we analyzed the ASC levels through Western blotting,the results showed that the addition of MG132 rescued ASC protein levels,but CQ and 3-MA failed to do so.These data indicated that USP3 promotes the stability of ASC protein mainly by enhancing the proteasome degradation of ASC.4.USP3 regulates the activation of inflammasome signaling pathwaysASC plays significant role in regulation of inflammation,so we further investigate whether USP3 affects the activation of inflammasome signaling pathways.We interfered USP3 in mouse peritoneal macrophages,after treatment with LPS and ATP at different time points,the protein was collected,and the expression and secretion of caspase-1 and IL-1β were detected by Western blot.The results showed the secretion of IL-1β cleavage fragment(p17)and caspase-1 cleavage fragment(p20)was significantly reduced after knocking down USP3.We also obtained similar results in USP3-knockout immortalized THP-1 cells.To gether these data suggest that USP3 positively regulates the inflammasome signaling pathway.5.USP3 regulates the secretion of inflammatory cytokinesThe assembly and activation of inflammasomes promote self-cleavage and activation of pro-caspase-1,which is responsible for the maturation of proinflammatory cytokines,such as interleukin 1β(IL-1 β)and IL-18.We interfered in mouse peritoneal macrophages and stimulated with LPS and ATP,then detected the secretion of IL-1β in the supernatant by ELISA.We found that a significant decrease in IL-1βsecretion,but no significant change in IL-6 and TNF-α secretion in supernatants of USP3-knockout cells as compared with control cells.Moreover,we treated the cells with Nigericin(Nig),another activator of inflammasome,and obtained the similar result.We repeated the above experiment in the USP3-knockout immortalized THP-1 cells,and also got consistent results.The results suggest that USP3 can positively regulate the activation of inflammasomes.Conclusion:1.USP3 directly binds to ASC and directly deubiquitinate ASC K48-linked ubiquitin chains and enhances the stability of ASC.2.USP3 suppresses ASC proteasome degradation.3.USP3 can positively regulate the activation of inflammasomes... |
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