| Background and ObjectiveNLRP3(nucleotide binding domain like receptor protein 3)inflammasome is a multimolecular complex,composed of NOD-like receptor NLRP3,an adapter protein apoptosis-associated speck-like protein(ASC),and an effector pro-caspase-1,and plays fundamental roles in inflammation.NLRP3 inflammasome recognizes pathogen associated molecular patterns(PAMPs)from invading microbes and endogenous danger signals(damage-associated molecular patterns,DAMPs)released from damaged or dying cells.NLRP3 inflammasome is the most thoroughly studied inflammasome at present.Aberrant NLRP3 inflammasome activation is closely related to the occurrence and development of many types of diseases,such as type II diabetes,gout,infectious diseases,atherosclerosis,Alzheimer’s disease,and cancers.Therefore,NLRP3 inflammasome activity should be tightly regulated to avoid such detrimental disorders.NLRP3 expression level is considered as a limiting step in inflammasome activation.Therefore,the regulation of NLRP3 expression level has become an important means to intervene NLRP3 inflammasome activation.At present,the regulation of NLRP3 post-translational modifications(PTMs),such as ubiquitination and phosphorylation,is the hottest means.The E3 ubiquitin ligase TRIM31(Tripartite motif-containing protein 31)attenuates NLRP3 inflammasome activation by promoting the K48-linked ubiquitination and proteasomal degradation of NLRP3.Tumor suppressor protein PTEN(phosphatase and tension homolog deleted on chromosome ten)could directly bind to NLRP3,dephosphorylate its Tyr32,and then promotes the interaction between NLRP3 and ASC as well as inflammasome assembly,thus positively regulating NLRP3 inflammasome activation.ABRO1(Abraxas brother protein 1)could promote the assembly and activation of NLRP3 inflammasomes by recruiting BRCC3(BRCA1/BRCA2-containing complex subunit 3)to remove the K63-linked ubiquitination of NLRP3.However,the role of NLRP3 K48-linked deubiquitination in the activation of NLRP3 inflammasome remains largely unclear.Ubiquitin specific peptidase 1(USP1)-associated factor 1(UAF1,also called WDR48 or p80)is a stoichiometric binding partner of three deubiquitinating enzymes and constitutes three deubiquitinating enzyme complexes,including UAF1/USP1,UAF1/USP12,and UAF1/USP46.UAF1 constitutively binds to USP1,USP12,and USP46,and this binding greatly enhances their deubiquitinase activity.The UAF1/USP1 complex deubiquitinates a wide range of substrates and has been implicated in the regulation of DNA repair processes,tumor pathogenesis,and antiviral innate immunity.USP1 could deubiquitinate and stabilize inhibitors of DNA binding proteins(IDs),including ID1,ID2 and ID3,suppress osteoblastic differentiation,and subsequently promote the maintenance of mesenchymal stem cells in osteosarcoma.USP12 and USP46 are also involved in tumor pathogenesis by deubiquitinating and stabilizing different targets,such as PH domain leucine-rich repeat protein phosphatase 1(PHLPP1),Tumor suppressor p53(TP53)and androgen receptor(AR).However,the potential roles of UAF1 deubiquitinase complexes in inflammation are unclear.In our previous study,we found that the deubiquitination enzyme USP1 could promote NLRP3 inflammasome activation(the relevant results have been summarized in my Master’s Thesis).However,whether UAF1 could promote NLRP3 inflammasome activation by enhancing USP1 deubiquitination enzyme activity,and the roles of UAF1/USP12 and UAF1/USP46 deubiquitinase complexes in inflammation are unknown.In the present study,we explored the roles of UAF1 deubiquitinase complexes(including UAF1/USP1,UAF1/USP12,and UAF1/USP46)in the activation of NLRP3 inflammasome and the molecular mechanisms.It provides new ideas for the study of the regulation mechanism of NLRP3 inflammasome activation and theoretical basis for immunomodulatory therapy for the prevention and treatment of NLRP3 inflammasome related diseases.Materials and Methods1.To explore the roles of UAF1 in the activation of NLRP3 inflammosome1.1 To detect the effects of Uaf1 knockout or knockdown on NLRP3 inflammosome activationDesigned and constructed the Uaf1 conditional knockout mice in myeloid cells using the CRISPR-Cas9 technology.The genotypes of the mice were identified and the homozygous knockout mice(called Uaf1CKO here)were screened out for the use of the following study.Macrophages were induced by intraperitoneal injection of 6%starch in wild-type(WT)mice and Uaf1CKO mice.ELISA analysis was used to detect the expressions of IL-1β,IL-6 and TNF induced by the activation of NLRP3 inflammasome;Western blot analysis was used to detect the expressions of caspase-1 p20,IL-1β P17,NLRP3,pro-caspase-1 and IL-1β.Designed and synthesized Uaf1 specific siRNAs,and transfected them in mouse primary peritoneal macrophages from WT mice by using transfection reagent Interferin and further cultured for 48h.Western blot analysis was used to detect the expression of UAF1 to screen out the most efficient siRNA for the use of the following study.Used Interferin to transfect the Uaf1-siRNA and Ctrl-siRNA in mouse primary peritoneal macrophages from WT mice.ELISA analysis was used to detect the expression of IL-1β by the activation of NLRP3 inflammasome;Western blot analysis was used to detect the expressions of caspase-1 p20,IL-1β P17,NLRP3,pro-caspase-1 and IL-1β.1.2 To detect the roles of UAF1 in LPS-induced EndotoxemiaWT mice and Uaf1CKO mice were intraperitoneally injected with LPS(10mg/kg).After 90 min,the mice were sacrificed and blood was collected for measurement of serum cytokines IL-1 β,TNF,and IL-6 by ELISA.2.To explore the effects of UAF1 on the expression of NLRP32.1 To detect the effects of Uaf1 knockout or knockdown on NLRP3 expressionMacrophages from Wild-type and Uaf1CKO mice were stimulated with LPS.Western blot analysis was used to investgate the expressions of NLRP3,NLRC4,and AIM2;RT-PCR analysis was used to detect the mRNA level of Nlrp3:ELISA analysis was used to detect the secretion of IL-1 β induced by the activation of NLRC4 and AIM2 inflammasome.Mouse primary peritoneal macrophages from WT mice were transfected with Uaf1-siRNA and Ctrl-siRNA by using Interferon.After 48 hours,Western blot analysis was used to investgate the expressions of NLRP3,NLRC4,and AIM2 proteins induced by LPS stimulation.2.2 To detect the effects of UAFl on the activation of NF-κBMacrophages from Wild-type and Uaf1CKO mice were stimulated with LPS or poly(I:C)for the different time periods.Western blot analysis was used to investgate the expressions of p-p65;RT-PCR analysis was used to detect the RNA level of Il1b,Tnf and 116.Flag-tagged UAF1 plasmid and Flag-tagged vector plasmid were transfected in HEK293T cells together with MyD88,TRIF plasmid and NF-κB reporter plasmid.Then cultured for 24h and lysed cells.The cell lysates were detected for NF-κB luciferase activity.3.To explore the effects of USP1,USP12,and USP46 on inflammasome activation and NLRP3 expression3.1 To detect the effects of USP1,USP12,and USP46 on inflammasome activationDesigned and synthesized Usp1,Usp12,and Usp46 specific siRNAs respectively,and transfected them by Interferin in mouse primary peritoneal macrophages from WT mice.After 48h,used Western blot to respectively investgate the expressions of USP1,USP12,and USP46,and screened out the most efficient siRNAs for the use of the following study.Mouse primary peritoneal macrophages from WT mice were respectively transfected with the screened Usp1,Usp12,and Usp46 siRNA.After 48h,the macrophages were primed with LPS for 8h and subsequent stimulated with ATP for 40min.ELISA analysis was used to detect the secretion of IL-1β induced by NLRP3 inflammasome activation;Western blot analysis was used to investgate the expressions of caspase-1 p20,IL-1β P17,NLRP3,pro-caspase-1 and IL-1β.3.2 To detect the effects of USP1,USP12,and USP46 on NLRP3 expressionMouse primary peritoneal macrophages from WT mice respectively transfected with Usp1,Usp12,and Usp46 siRNA,were stimulated with LPS for different time periods(0,4h,8h).Then used Western blot to investgate the expression of NLRP3 induced by LPS;RT-PCR analysis was used to detect the mRNA expression of Nlrp3.Mouse primary peritoneal macrophages from WT mice primed with increasing amount of ML323(a specific inhibitor of UAF1/USP1)for 4h,were stimulated with LPS for 8h.Western blot analysis was used to investgate the expressions of NLRP3 and caspase-1.4.To explore the molecular mechanisms of UAF1/USP12 and UAF1/USP46 regulating the NLRP3 inflammasome activation and NLRP3 expression4.1 To detect the effects of USP1,USP12,and USP46 on the NF-κB activationMouse primary peritoneal macrophages from WT mice were respectively transfected with Usp1,Uspl2,and Usp46 siRNA.RT-PCR analysis was used to detect the RNA expression of Il1b,Tnf and Il6 induced by LPS.Flag-tagged USP1/USP12/USP46 plasmid and Flag-tagged vector plasmid were transfected in HEK293T cells together with MyD88,TRAF6,TAK1,p65 plasmid and NF-κB reporter plasmid.Then cultured for 24h and lysed cells.The cell lysates were detected for NF-κB luciferase activity.Mouse primary peritoneal macrophages from WT mice were respectively transfected with Usp1,Usp12,and Usp46 siRNA.Western blot analysis was used to detect the expressions of p-p65 and p65 induced by LPS.Macrophages from Wild-type and Uaf1CKO mice were stimulated with LPS,and used Western blot to investgate p-p65 and p65 expressions.4.2 To detect the interactions of UAF1/USP1/USP12/USP46 and p65Mouse primary peritoneal macrophages were stimulated with LPS for different time periods(0,2h,4h).Used the special antibody of p65 for co-immunoprecipitation(co-IP),and used Western blot analysis to investgate the endogenous interactions of p65 and UAF1/USP1/USP12/USP46.Flag-tagged UAF1/USP1/USP12/USP46 plasmids and Flag-tagged vector plasmid were transfected by LipofectamineTM2000 in HEK293T cells together with Myc-tagged p65 plasmid.Used Myc antibody for co-IP,and detected the exogenous interactions of p65 and UAF1/USP1/USP12/USP46 by Western bolt.4.3 To detect the effects of UAF1/USP12/USP46 on the the ubiquitination of p65Myc-p65,HA-tagged Ub,and Flag-U AF1/USP12/U SP46 plasmids were together transfected by LipofectamineTM2000 in HEK293T cells.The ubiquitination of p65 was investigated by co-IP with anti-Myc and Western bolt.Macrophages from Wild-type and Uaf1CKO mice were stimulated with LPS for 6h.Used the special antibody of p65 for co-IP,and detected the ubiquitination of p65 by Western bolt.Myc-p65,HA-tagged K48-Ub,K63-Ub plasmid,Flag-UAF1/USP12/USP46 plasmid and Flag-tagged vector plasmids were together transfected by LipofectamineTM2000 in HEK293T cells.Used anti-Myc for co-IP,and detected the ubiquitination of p65 by Western bolt.5.To explore the interactions of UAF1/USP1 and NLRP3Myc-tagged NLRP3 and HA-tagged UAF1 or GFP-tagged USP1 plasmid were transfected in MEFs(mouse embryonic fibroblasts).After 24h,the cells were stimulated with LPS for 2h.Then detected the colocalizations of NLRP3 and UAF1 or USP1 by immunofluorescence(IF)and confocal microscopy analysis.Myc-NLRP3,Flag-UAF1 or Flag-USP1 and vector plasmids were transfected by LipofectamineTM2000 in HEK293T cells.After 24h,cells were lysed and the cell lysates were used for co-IP with anti-Flag.Then used Western blot to investgate the exogenous interactions of NLRP3 and UAF1 or USP1.Mouse primary peritoneal macrophages from WT mice were stimulated with LPS for different time periods(0,2h,4h).Used the specific UAF1 or USP1 antibody for co-IP,and detected the endogenous interactions of NLRP3 and UAF1 or USP1 by Western blot.Myc-NLRP3,pcmv6-USP 1,and HA-UAF1 plasmids were respectively transcribed and translated by the TNT(?)T7 Quick Coupled Transcription/Translation System.Then used the proteins for co-IP,and detected the interactions of NLRP3 and UAF1 or USP1 by Western blot.Designed the primers to construct the mutants of NLRP3 plasmid by KOD-plus point mutation kit.Myc-NLRP3 and its mutants,Flag-UAF1 or Flag-USP1 and vector plasmids were transfected by LipofectamineTM2000 into HEK293T cells.After 24h,cells were lysed,and the cell lysates were used for co-IP with anti-Flag.Then the interactions of NLRP3 or its mutants and UAF1 or USP1 were detected by Western blot.6.To detect the roles of UAF1/USP1 in NLRP3 ubiquitination6.1 To detect the effects of UAF1/USP1 on NLRP3 ubiquitinationIn HEK293T cells,Myc-NLRP3,HA-Ub,and Flag-UAF1 or USP1 and USP1 C90S(USP1 mutant plasmid losing its deubiquitination enzyme activity)plasmids were together transfected by LipofectamineTM2000.The ubiquitination of NLRP3 was detected by co-IP with anti-Myc and Western bolt.MEFs primed with ML323 for 4h,were stimulated by LPS for 6h.Then cells were lysed and the cell lysates were used for co-IP with anti-NLRP3 and detected the ubiquitination of NLRP3 by Western bolt.Macrophages from Wild-type and Uaf1CKO mice were stimulated with LPS for 6h.Used the special antibody of NLRP3 for co-IP,and detected the ubiquitination of NLRP3 by Western bolt.Myc-NLRP3,HA-tagged K48-Ub or K63-Ub,and Flag-UAF1 or USP1 plasmids and Flag-tagged vector plasmids were together transfected by LipofectamineTM2000 in HEK293T cells.Used anti-Myc for co-IP,and detected the ubiquitination of NLRP3 by Western bolt.6.2 To detect the effects of UAF1/USP1 on the degradation of NLRP3Mouse peritoneal macrophages from WT mice were pretreated with DMSO or ML323 for 4h and then stimulated with LPS for 4h,followed by treatment with cycloheximide(CHX)for different time periods(0,4h,8h,12h).Western blot was used to investgate the expression of NLRP3 and caspase-1.WT and Uaf1-deficient mouse peritoneal macrophages were pretreated with LPS for 8h,followed by treatment with MG132 for 4 h before harvesting the cells.Used Western blot to investgate the expression of NLRP3.7.To explore the roles of ML323 or Uaf1 deficiency in NLRP3-dependent diseases7.1 To detect the effects of ML323 on NLRP3 inflammasome activation in macrophagesMouse peritoneal macrophages were pretreated with DMSO or ML323 for 4h,followed by LPS stimulation for 8h and treatment with ATP or Nig for the last 40 min.ELISA analysis was used to detect the secretion of IL-1 β.Mouse peritoneal macrophages were pretreated with DMSO or ML323 for 4h,followed by LPS stimulation for different time periods(0,4h,8h)and treatment with ATP for the last 40 min.Western blot analysis was used to investgate the expressions of caspase-1 p20 and IL-1β P17 in the culture supernatants,and NLRP3,pro-caspase-1 and IL-1β expressions in the cell lysates.7.2 To detect the roles of ML323 in LPS-induced endotoxemia modelC57BL/6J mice were initially i.p.injected with DMSO or ML323(10mg/kg)for 4h and later with LPS(10mg/kg)for 90min.ELISA analysis was used to detect the serum levels of IL-1 β,TNF and IL-6.7.3 To detect the roles of ML323 in FA-induced acute tubular necrosis(ATN)modelC57BL/6J mice were initially i.p.injected with DMSO or ML323(10mg/kg)for 4h and then i.p.injected with folic acid(FA,250mg/kg):(1)After injected FA for 12h,ELISA analysis was used to detect the serum level of IL-1β.(2)After injected FA for 36h,used Western blot analysis to detect NLRP3,UAF1 and USP1 expressions in kidney samples;H&E staining was used to investgate the structural changes of kidney tissue sections;PAS staining was used to investgate the glycogen accumulation of the kidney tissue sections and renal inflammation was scored based upon PAS staining.(3)Observed the survival of mice at different time(0,12h,24h,36h,48h,60h,72h,84h).7.4 To detect the roles of Uafl deficiency in ATN modelWT or Uaf1CKO mice were i.p.injected with FA for 36h.Western blot analysis was used to detect IL-1β p17 and p31 expressions in kidney samples.H&E staining was used to detect the structural changes of kidney tissue sections;PAS staining was used to detect the glycogen accumulation of the kidney tissue sections and renal inflammation was scored based upon PAS staining.Results1.UAFl facilitates NLRP3 inflammasome activationBoth Uaf1 deficiency and knockdown could markedly attenuate the secretion of IL-1β induced by NLRP3 inflammasome activation.In addition,Uaf1 deficiency could also inhibit TNF and IL-6 secretions.Western blot data revealed Uaf1 deficiency and knockdown substantially suppressed the cleavage of caspase-1 and IL-1β.In vivo,the secretions of IL-1β,TNF and IL-6 induced by the LPS injection was much lower in the sera of Uaf1CKO mice than in the sera of WT mice.These results indicate that UAF1 facilitates NLRP3 inflammasome activation,and promotes TNF and IL-6 secretions.2.UAF1 enhances NLRP3 expressionUaf1 deficiency and knockdown could markedly inhibited NLRP3 protein expression,but Uaf1 deficiency had no effect on the expressions of AIM2 and NLRC4.RT-PCR data showed Nlrp3,Il1b,Tnf and Il6 mRNA expressions were also attenuated by Uaf1 deficiency.Furthermore,Western blot data revealed Uaf1 deficiency inhibited LPS-induced or poly(I:C)-induced phosphorylation of p65.In HEK293T cells,UAF1 overexpression enhanced NF-κB activation induced by MyD88 and TRIF.These results indicate that UAF1 enhances NLRP3 and pro-IL-1βexpression at both the protein and mRNA levels.3.USP1/12/46 enhance NLRP3 expression and inflammasome activation.Usp1,Usp12,or Usp46 knockdown considerably inhibited IL-1β secretion,caspase-1 and IL-1β cleavage induced by NLRP3 inflammasome activation in mouse peritoneal macrophages.Besides,Usp1,Usp12,or Usp46 knockdown also markedly attenuated LPS-induced NLRP3 expression,although Usp1 knockdown had no effect on NLRP3 expression in unstimulated macrophages.Furthermore,ML323,an allosteric and specific inhibitor of the UAF1/USP1 deubiquitinase complex,inhibited LPS-induced NLRP3 expression in a dose-dependent manner.RT-PCR data showed that Usp12,or Usp46 knockdown significantly inhibited Nlrp3 mRNA expression,while Usp1 knockdown had no effect on Nlrp3 mRNA expression.These data suggest that USP1,USP12,and USP46 enhance NLRP3 inflammasome activation and NLRP3 expression.4.UAF1/USP12 and UAF1/USP46 complexes promote NLRP3 transcriptionRT-PCR data showed Usp12 and Usp46 knockdown suppressed LPS-induced Il1b,Tnf,116 and Nlrp3 mRNA expressions in mouse peritoneal macrophages,suggesting that USP12 and USP46 may regulate the priming process of NLRP3 inflammasome activation.As the activation of NF-κB plays vital roles in NLRP3 inflammasome activation and the mRNA expressions of Il1b,Tnf,Il6 and Nlrp3.Then we examined the effects of USP12 and USP46 on NF-κB activation.In HEK293T cells,USP12 and USP46 overexpression enhanced MyD88-,TRAF6-,TAK1-,and p65-induced NF-κB luciferase reporter activation,while the effect of USP1 was not obvious.In addition,Usp12/Usp46 knockdown,and Uaf1 deficiency considerably attenuated LPS-induced phosphorylation of p65 and total p65 expression.Collectively,these results indicate that UAF1/USP12 and UAF1/USP46 complexes promote NF-κB activation by enhancing p65 expression.Co-immunoprecipitation(co-IP)and Western blot assays showed that p65 interacted with USP12,USP46 and UAF1 in both resting and LPS-stimulated macrophages,but exhibited no association with USP1.Consistently,p65 interacted with UAF1,USP12 and USP46 in HEK293T cells.As p65 can be inactivated and degraded by proteasomes,we then examined whether UAF1/USP12 and UAF1/USP46 could target p65 to remove its polyubiquitin chains.The co-IP and Western blot data showed that all the three could remove p65 polyubiquitination.Under physiological conditions,endogenous p65 was ubiquitinated upon LPS stimulation in macrophages,and Uaf1 deficiency markedly increased endogenous p65 ubiquitination.Furthermore,UAF1,USP12,and USP46 markedly inhibited K48-linked ubiquitination of p65,but had no effect on K63-linked ubiquitination of p65.Collectively,these results indicate that UAF1/USP12 and UAF1/uSP46 complexes remove K48-linked ubiquitination of p65,enhance its expression,and then promote NLRP3 transcription,thereby facilitating NLRP3 inflammasome activation.5.UAF1/USP1 interacts with NLRP3USP1 enhanced NLRP3 protein expression,with no effect on Nlrp3 mRNA expression,suggesting that the UAF1/USP1 complex regulated the posttranslational modifications(PTMs)of NLRP3.Next,we firstly examined the association between NLRP3 and UAF1/USP1.Confocal analysis suggested the colocalization between USP1/UAF1 and NLRP3 upon LPS stimulation.In HEK293T cells,NLRP3 could be immunoprecipitated with USP1 or UAF1.In vitro binding assays demonstrated that NLRP3 could directly interact with UAF1/USP1.Besides,co-IP and Western blot assays showed an association between NLRP3 and UAF1/USP1 in LPS-stimulated mouse peritoneal macrophages.Taken together,these data suggest that NLRP3 could directly interact with UAF1/USP1.To further determine the domains of NLRP3 responsible for the interaction with UAF1/USP1,a series of Myc-tagged NLRP3 truncated mutants were constructed,and co-IP assay indicated the LRR and NACHT domains of NLRP3 interact with UAF1/USP1.These results suggest UAF1/USP1 interacts with the LRR and NACHT domains of NLRP3.6.UAF1/USP1 removes NLRP3 ubiquitination and stabilizes NLRP3NLRP3 could be ubiquitinated with both K48 and K63 linkage.Next,we explored whether the deubiquitinase complex UAF1/USP1 could remove the ubiquitination of NLRP3.Co-IP and re-IP assay showed both USP1 and UAF1 considerably inhibited the polyubiquitination of NLRP3.Besides,the USP1 point mutation(C90S)with substitution of the cysteine residue with serine at position90,lost the ability to inhibit the polyubiquitination of NLRP3.Furthermore,USP1 and UAF1 both inhibited NLRP3 polyubiquitination with K48-linkage,but not with K63-linkage.In addition,polyubiquitination of NLRP3 was enhanced in ML323-treated MEFs and Uaf1-deficient macrophages.Collectively,these data indicate that UAF1/USP1 eliminates K48-linked polyubiquitination of NLRP3 through its deubiquitinase activity.As K48-linked protein ubiquitination leads to degradation of the corresponding proteins.We then investigated the effects of UAF1/USP1 complex on NLRP3 protein degradation by cycloheximide(CHX)chase experiment.Western blot data showed ML323 significantly promoted NLRP3 protein degradation,with no effect on caspase-1.In addition,Uaf1 deficiency resulted in a loss of the inhibitory effects on NLRP3 expression in MG132-treated macrophages.These data indicate that the USP1/UAF1 deubiquitinase complex removes the K48-linked ubiquitination of NLRP3 and inhibits its degradation,thus facilitating NLRP3 inflammasome activation.7.ML323 and Uaf1 deficiency ameliorate NLRP3-dependent inflammation7.1 ML323 relieves NLRP3-dependent inflammationIn macrophages,we detected the roles of ML323 in NLRP3 inflammasome activation.ELISA data showed ML323 attenuated NLRP3-dependent IL-1β secretion;Western blot data showed ML323 inhibited caspase-1 and IL-1β cleavage induced by NLRP3 inflammasome activation.ML323-treated mice produced significantly less IL-1β and TNF in sera after i.p.injection of LPS than DMSO-treated mice,while no difference in IL-6 secretion was observed.In FA-induced acute tubular necrosis(ATN)model,ML323-treated mice produced less IL-1β in sera and NLRP3 expression in the kidneys compared with DMSO-treated mice.Renal inflammation and edema were greatly ameliorated in the kidneys of ML323-treated mice.Concordantly,ML323-treated mice were found to be more resistant than DMSO-treated mice in survival assays upon FA injection.These results reveal ML323 inhibits IL-1β secretion and therefore ameliorates NLRP3-dependent inflammation both in vitro and in vivo.7.2 Uaf1 deficiency ameliorates the inflammations in FA-induced ATN modelIn FA-induced ATN,Uaf1 deficiency in myeloid cells markedly suppressed IL-1β secretion and pro-IL-1β expression in the kidneys.Furthermore,less severe renal inflammation and edema were observed in Uaf1CKO mice compared with WT mice.These data indicate that Uaf1 deficiency in myeloid cells ameliorates FA-induced ATN.Conclusions1.UAF1 promotes the activation of NLRP3 inflammasome,and palys cruial regulatory roles in NLRP3-dependent disease models,such as Endotoxemia and ATN.2.UAF1/USP12 and UAF1/USP46 complexes target p65,remove its K48-linked polyubiquitination,and promote NF-κB activation,thus facilating NLRP3 inflammasome activation.3.UAF1/USP1 complex promotes the activation of NLRP3 inflammasome by removing NLRP3 K48-linked polyubiquitination and stabilizing its expression.4.The UAF1/USP1 specific inhibitor ML323 could ameliorate NLRP3-related inflammations,such as Endotoxemia and ATN.Innovation and Significance1.Our study revealed that UAF1 deubiquitinase complexes could promote the activation of NLRP3 inflammasome via different ways,and elucidated their important regulatory roles in inflammation.2.Our study further revealed the vital roles of NLRP3 deubiquitination in stabilizing NLRP3 expression and promoting NLRP3 inflammasome activation.3.Our study reported that ML323,as a specific inhibitor of UAF1/USP1,could promote NLRP3 inflammasome activation and plays vital roles in NLRP3 inflammasome related diseases,thus providing new ideas for the prevention and treatment of related disorders.LimitationsAs the specific sites of NLRP3 ubiquitination have not been determined at present,we didn’t explore the specific sites of NLRP3 deubiquitination in this study. |
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