TRIM26 Positively Regulates The Inflammatory Immune Response Through K11-linked Ubiquitination Of TAB1

TGF-?-activated kinase 1(TAK1)belongs to the MAPK kinase kinase(MAPKKK)family,which is involved in a broad range of biological and pathological processes,especially TLRs-medicated signaling.The activity of TAK1 is critically regulated by its binding partners,TAK1-binding proteins(TAB1-3).It is well established that TAK1 and TAB 1-3 can form a complex,which are called TAK1TABs.Previous studies have demonstrated that the assembly and activation of TAK1-TABs is critically regulated by ubiquitination.Yet,the molecular mechanisms modulating TAK1-TABs activation are still unclear.Here,we demonstrated that Tripartite motif 26(TRIM26)as an essential regulator of TAB 1.Knockout of Trim26 impaired the expression and production of inflammatory cytokines following LPS,TNF-? and IL-1? stimulation.Consequently,Trim26 deficiency protects mice from LPS-induced septic shock in vivo.Moreover,Trim26 deficiency attenuates the severity of DSS-induced colitis.Mechanistically,TRIM26 positively regulates TAK1 activation and downstream NF-?B and MAPK signaling through the K11-linked ubiquitination of TAB1.This study indicated that TRIM26 functions as a positive modulator of TLRs-mediated inflammatory response and provided a novel insight into how TAK1 activation is regulated through TRIM26-mediated ubiquitination of TAB1.ObjectivesOur laboratory has previously reported that TRIM26 regulates the antiviral response by targeting IRF3 in the nucleus,but the role of TRIM26 in NF-?B and MAPK singnaling pathway remains unknown.As an E3 ubiquitin ligase,it is worth exploring whether existing a substrate of TRIM26 in the inflammatory response.Methods1.To identify the effect of TLR ligands,TNF-? and IL-1? on TRIM26 expression1.1 Expression of TRIM26 in PMs stimulated with various TLR ligandsPeritoneal macrophages(PMs)were stimulated with LPS(TLR4),Pam3CSK4(TLR2),Poly(I:C)(TLR3)or R848(TLR7/8),the lysates were followed by western blot analysis with anti-TRIM26 Ab.1.2 Expression of TRIM26 stimulated with TNF-? and IL-1?PMs were stimulated with TNF-? or IL-1?,the protein level of TRIM26 was detected by western blotting.2.To investigate the role of TRIM26 on TLRs-induced inflammatory response2.1 The role of TRIM26 in TLRs-induced production of inflammatory cytokinesWe designed mouse Trim26-specific small interfering(siRNA)and transfected into PMs to knockdown endogenous Trim26 expression.PMs stimulated with LPS,Poly(I:C),Pam3CSK4 or R848 for indicated times,mRNA of Tnfa,116 and Il12b was measured by qRT-PCR.We also designed human TRIM26-specific siRNA and transfected into THP-1 cells(human monocyte cell line).Secretion and mRNA level of Inflammatory cytokines were measured.PMs,BMDMs and MEFs from Trim26+/+mice and Trim26-/-mice,followed stimulation with various TLR ligands.mRNA level and secretion of TNF-?,IL-6 and IL-12p40 were measured by qRT-PCR and ELISA,respectively.2.2 To investigate the role of TRIM26 on the activation of TLRs signaling pathwayPMs from Trim26+/+and Trim26-/-mice were treated with LPS,Pam3CSK4 or Poly(I:C),the phosphorylation levels of IKK?/?,p65,JNK,ERK and p38 were detected by western blot analysis.2.3 Rescue experiments to determine the regulatory effect of TRIM26 on TLRs-induced inflammatory responsePMs from Trim26-/-mice were treated with mTRIM26 or its mutant lentivirus before stimulation with LPS for indicated times,mRNA level of Tnf?,116 and Il12b was measured by qRT-PCR.PMs from Trim26-/-mice were treated with mTRIM26 or its mutant lentivirus before stimulation with LPS for indicated times,the phosphorylation levels of IKK?/?,p65,JNK,ERK and p38 were detected by western blot analysis.3.To screen and identify the substrate of TRIM263.1 To idenfity the adaptors which ubiquitinated by TRIM26 in NF-?B and MAPK pathwayWe first transfected MyD88,IRAK1,IRAK4,TRAF6,TAK1-TABs and IKK complex,which are important molecules involved in NF-?B and MAPK activation in the TLRs pathway,together with HA-Ub and Flag-tagged or Myc-tagged TRIM26 into HEK293T cells.The ubiquitination of MyD88,IRAK1,IRAK4,TRAF6,TAK1-TABs or IKK complex was detected with anti-HA Ab.3.2 To identify the interaction between TRIM26 and TAB1Flag-TAB1 and Myc-TRIM26 expression plasmids were co-transfected into HEK293T cells,Co-IP analysis the interaction between TAB 1 and TRIM26;PMs were stimulated with LPS for indicated time points,the cell lysates were IP with anti-TAB1 Ab followed by western blot analysis with anti-TRIM26 or anti-TAB1 Ab;Recombinant TRIM26 and TAB1 protein were prepared in an in vitro transcription and translation system,IP analysis was performed using anti-TAB 1 Ab,immunoblot analysis was followed with anti-TRIM26 Ab.3.3 To confirm the co-localization of TRIM26 and TAB1 in cellsHela cells were transfected with GFP-TRIM26 and Myc-TAB1 expression plasmids for 24 h,Confocal microscopy was followed by labeling of TAB 1 with a Myc-specific primary antibody and fluorescent secondary antibody(red).The in vivo co-localization between TAB1 and TRIM26 in MEFs was examined by confocal microscopy stimulated with LPS for 1 h.4.To detect the ubiquitination linkage of TAB1 mediated by TRIM264.1 To identify the linkage type of TRIM26-induced TAB1 polyubiquitin chainIn order to explore which ubiquitination form of TAB 1 is mainly regulated by TRIM26,we overexpressed Myc-TAB1 and Flag-TRIM26 into HEK293T cells together with HA-Ub or its mutants with different lysine residue sites(K6?K11?K27?K29?K33?K48?K63).IP was performed by anti-Myc Ab.4.2 To confirm the polyubiquitin of TAB1 regulated by TRIM26 depends on its enzymatic activityHEK293T cells were transfected with Ub,TAB1 and TRIM26 or TRIM26 C16A or TRIM26 ?R.IP was performed by anti-Myc Ab and TAB1 ubiquitination was detected by western blot analysis.4.3 In vivo and in vitro ubiquitination assayPMs from Trim26+/+and Trim26-/-mice were prepared followed stimulation with LPS for indicated times.IP was performed by anti-TAB1 Ab and TAB1 ubiquitination was detected by immunoblot analysis with anti-Ub Ab.Recombinant TRIM26 and TAB1 were co-incubated together with E1,UbcH5c and Ub or K11-Ub.The ubiquitination of TAB 1 was examined by western blotting.5.To clarify the function of TRIM26 in LPS-induced septic shock in vivoTrim26+/+and Trim26-/-mice were injected intraperitoneally with LPS,the survival and weight loss of the mice were examined;TNF-?,IL-6 and IL-12p40 production in serum obtained from Trim26+/+and Trim26-/-mice after stimulation with LPS for 4 h were detected by ELISA.Lung tissues from Trim26+/+ and Trim26-/-mice after stimulation with LPS for 4 h were stained with H?E.6.To clarify the function of TRIM26 in DSS-induced colitis in vivoTrim26+/+(n=6)and Trim26-/-(n=6)mice were challenged with 3%DSS in the drinking water,the weight loss and colitis scores were examined every day;The length of the colons was measured on day 6.The pathological damage of the colon tissues was also examined.Results1.TRIM26 protein expression was increased upon TLR ligands,TNF-? and IL-1? stimulationTo investigate the possible function of TRIM26 in inflammatory responses,we first examined TRIM26 protein expression in PMs stimulated with various TLR ligands using immunoblot analysis.The expression of TRIM26 protein was increased upon stimulation with LPS.Similarly,Pam3CSK4,Poly(I:C)or R848 stimulation also enhanced TRIM26 protein expression in PMs.Notably,TNF-? and IL-1?,two inflammatory cytokines induced upon TLR ligands stimulation,also increased TRIM26 protein expression in PMs.2.TRIM26 positively regulates TLRs-induced inflammatory response2.1 TRIM26 positively regulates TLRs-induced production of inflammatory cytokinessiRNA-mediated knockdown of mouse Trim26 expression decreased mRNA level of Tnf?,116 and Il12b after LPS stimulation,which is recognized by TLR4.Further,we found Pam3CSK4-,Poly(I:C)-or R848-induced Tnf?,116 and Il12b mRNA expression was much lower in Trim26 knockdown PMs.We also designed human TRIM26-specific siRNA and transfected into THP-1 cells.Consistent with the data obtained from mice macrophages,we found LPS-induced expression of TNF-? and IL-6 was decreased in THP-1 cells transfected with TRIM26-specific siRNA.We prepared PMs from Trim26+/+ mice and Trim26-/-mice,followed stimulation with various TLR ligands.qRT-PCR and ELISA analysis unveiled that induction of TNF-?,IL-6 and IL-12p40 were decreased in Trim26-/-compared with Trim26+/+ cells following LPS stimulation.Consistently,Trim26 deficiency also attenuated the production of inflammatory cytokines induced by Pam3CSK4,Poly(I:C)or R848 in PMs.Similar results were observed in BMDMs and MEFs prepared from Trim26+/+and Trim26-/-mice stimulated with LPS,Pam3CSK4,Poly(I:C)or R848.Taken together,these data suggested that TRIM26 positively regulates TLRs-mediated production of cytokines.2.4 TRIM26 enhances TLRs-induced NF-?B and MAPK signalingWe prepared PMs from Trim26+/+and Trim26-/-mice,followed by stimulation with LPS,Poly(I:C),Pam3CSK4 or TNF-?.We analyzed the phosphorylation status of the main kinases in the TLRs-induced signaling.Knockout of Trim26 in PMs decreased LPS-induced phosphorylation of IKK?/? and p65,which are hallmarks of NF-?B activation.Moreover,p-JNK,p-ERK and p-p38 in the MAPK signaling was also attenuated in Trim26-/-PMs.Similarly,Poly(I:C)-,Pam3CSK4-or TNF-?-mediated NF-?B and MAPK signaling was also decreased in PMs from Trim26-/-mice.3.TRIM26 targets TAB1 and mediates TAB1 polyubiquitination3.1 TRIM26 mediates polyubiquitination of TAB1Co-IP and immunoblot analysis showed that the polyubiquitination of MyD88,IRAK1,IRAK4 and TRAF6 was not affected in the presence of TRIM26.We found polyubiquitination of TAB 1 but not TAK1 or TAB2 was increased in the presence of TRIM26.As a control,TRIM26-mediated polyubiquitination of IRF3 was readily detected,as previously reported.Co-IP and immunoblot analysis showed that none of these IKK complex subunits were polyubiquitinated by TRIM26.These data indicated that TRIM26 may target TAB1 for polyubiquitination.3.2 TRIM26 interacts with TAB1HEK293T cells were transfected with Myc-TRIM26 and Flag-TAB1,IP analysis was performed using anti-Flag Ab,immunoblot analysis was followed with anti-Myc Ab.We found TAB1 indeed could associate with TRIM26.To verify that TRIM26 associated with TAB1 directly,TRIM26 and TAB1 recombinant proteins were prepared and in vitro pull-down assay was performed.Recombinant TRIM26 was found to interact with TAB1.We also detected the interaction between endogenous TRIM26 and TAB1 in PMs.Notably,the interaction was increased upon stimulation with LPS.3.3 Identification of the co-localization of TRIM26 and TAB1Confocal microscope imaging demonstrated that TRIM26 co-localized with TAB1 in Hela.Considering that overexpressed proteins might affect their natural distributions in the cells,we also used MEFs to explore the co-localization.Consistently,confocal analysis showed that TRIM26 co-localized with TAB1 upon LPS stimulation.4.TRIM26 conjugates the K11-linked polyubiquitin of TAB1 depends on its enzymatic activity4.1 TRIM26 promotes K11-linked polyubiquitination of TAB1HEK293T cells were transfected with Myc-TAB1,Flag-TRIM26 and HA-Ub or ubiquitin mutants containing only one single lysine residue,IP analysis was performed using anti-Myc Ab,immunoblot analysis was followed with anti-HA Ab.The Co-IP result showed TRIM26 mainly promotes K11-linked polyubiquitination of TAB1.4.2 TRIM26 mediates polyubiquitination of TAB1 depends on its enzymatic activityHEK293T cells were transfected with Myc-TAB1,HA-Ub and Flag-TRIM26 or RING domain mutant Flag-TRIM26.TAB1 ubiquitination was easily detected in the presence of TRIM26.While,Flag-TRIM26 C16A or Flag-TRIM26 ?R abrogated TAB1 polyubiquitination.Similarly,TRIM26-mediated K11-linked polyubiquitination of TAB1 was attenuated in TRIM26 mutants C16A and ?R transfected HEK293T cells.4.3 TRIM26 directly ubiquitinates TAB1To exclude any artificial effect caused by overexpression and determine whether TRIM26-mediated TAB 1 ubiquitination occurs in physiological conditions,PMs from Trim26+/+and Trim26-/-mice were prepared followed stimulation with LPS.Co-IP experiments showed that the level of endogenous TAB1 ubiquitination was decreased in Trim26-/-PMs compared with that in Trim26+/+PMs upon LPS stimulation.In addition,Trim26 deficiency also inhibited TAB1 K11-linked Ub.Moreover,TAB1 was ubiquitinated by TRIM26 in the presence of WT and K11 Ub in vitro.Taken together,these data suggested that TRIM26 can directly ubiquitinate TAB1 in physiological conditions.5.TRIM26 promotes TAK1 phosphorylation depends on its enzymatic activity5.1 TRIM26 promotes TAK1 phosphorylationPMs from Trim26+/+and Trim26-/-mice were prepared followed stimulation with LPS,Poly(I:C)or Pam3CSK4 for indicated times.Western blot revealed that TAK1 phosphorylation was lower in PMs from Trim26-/-mice than in those from Trim26+/+mice.5.2 Rescue experiments to determine the regulatory effect of TRIM26 on the NF-?B and MAPK pathwayOverexpression of mTRIM26 but not the enzymatic mutant C16A could restore LPS-induced phosphorylation of TAK1,IKK?/?,p65,ERK,JNK and p38.Consistently,mTRIM26 instead of mTRIM26 C16A rescued the expression of Tnfa,Il6 and Il12b mRNA upon LPS stimulation.6.Trim26 deficiency protects mice from LPS-induced septic shockTo gain insight into the functions of TRIM26 in the regulation of TLRs-mediated inflammation in vivo,we monitored inflammatory cytokines expression as well as septicemia-induced death in Trim26+/+and Trim26-/-mice after intraperitoneal(i.p.)administration of LPS.LPS-triggered serum levels of TNF-?,IL-6 and IL-12p40 were decreased in Trim26-/-mice than in WT mice.Consistent herewith,Trim26-/-mice experienced later death onset and exhibited a lower death rate and they showed less lung inflammation and lower pathological assessment of lung severity scores than their WT counterparts.Collectively,these data suggested that TRIM26 positively regulates LPS-induced septic shock in vivo.7.Trim26 deficiency attenuates DSS-induced colitisFirst,the clinical features of Trim26-/-and Trim26-/-mice was detected in DSS-induced colitis over a 6-day period.Compared with Trim26+/+ mice,Trim26-/mice displayed attenuated colitis,as indicated by less weight loss,rectal bleeding scores and stool consistency scores;The colonic lengths of Trim26-/-mice were longer than those of Trim26+/+ mice;The colonic mucosa of Trim26-/-mice had less infiltrating leukocytes and was more intact;Trim26-/-mice showed less inflammatory cell infiltration in colonic tissues than their WT littermates after DSS treatment.The pathological assessment of colitis severity scores also reflected the same phenomenon.All together,these results suggested that Trim26 deficiency attenuates the severity of DSS-induced colitis.Innovation1.TAB1,a new substrate of TRIM26In recent years,various research of PTMs of TAKl-TABs have been reported,which foucs on the TAK1 and TAB2/3.TAB1,as a key component of this complex,which results in the phosphorylation and activation of TAK1 and subsequent NF-?B signaling.However,whether TAB1 could be regulated through nonclassical protein ubiquitination remained unknown.Here,we identified TRIM26 as a novel E3 ligase to induce a nonclassical polyubiquitination,K11-linked polyubiquitination of TAB 1.2.Identification of the role of TRIM26 in inflammatory responsePrevious research revealed that TRIM26 is related to antiviral innate immune response and tumor development,but the role of TRIM26 in inflammatory response remains unclear.Here,we discovered that TRIM26 acts as a positive regulator of TLRs-induced inflammatory responses.3.Our results revealed a novel regulatory mechanism of TAK1 activationTAK1 involved in a wide range of biological processes,its activation must be tightly controlled.Our research demonstrated that TRIM26 targets TAB1,the binding protein of TAK1,positively regulates TAK1 phosphorylation through its E3 ligase activity.This discovery can provide a new idea for the regulatory mechanism ofTAK1 activation.