| CD4+T cells are the most important immune cells of adaptive immune system.It can recognize the exogenous antigens which are presented by antigen-presenting cells(APC),then regulates the activation of macrophage,and promotes the activation of B cell to produce antibodies against all kinds of pathogenic microorganism by the secretion of cytokines.It can also identify autoantigens and induces immune tolerance,then prevents the generation of autoimmune diseases.Thus,dysregulation of T cell activation can lead to severe immune deficiency or autoimmune diseases.The activation of T cell requires two signals.The first signal is that the complex of MHC-antigen peptide of APC are presented to the TCR-CD3 complex on the surface of T cells,which recruits ZAP70 and activates the LAT signaling complex,then including PLC?1-PKC?-NF-?B pathway,RAS-RAF-MEK-ERK pathway,and the Ca2+pathway is activated.The main second signal is the CD28,which binds to the B7 molecule on the APC membrane to activate the AKT signaling pathway and promotes the proliferation of T cell and the production of cytokines.RAF-1,which plays an extremely important role in RAS-mediated T cell proliferation and activation.In the resting state,it exists a self-inhibitory structure and can be recruited to the membrane by RAS upon activation.The earlier studies on the regulation of RAF-1 mainly focuses on phosphorylation,while the ubiquitination and o-glycosylation are involved in the stability of RAF-1 protein.Whether other E3 ligases regulate the activation of RAF-1 in a non-proteolytic manner remains unknown.In this study,we found that E3 ubiquitin ligase TRIM31 catalyzed K33-linked polyubiquitination at K327 and K493 of RAF-1,inhibited its binding to RAS on the membrane of cells,and ultimately affected its activation.TRIM31 deficiency in CD4+T cells could significantly promote the phosphorylation of RAF-1 and ERK,and the production of growth factors IL-2,IFN-? and TNF-? were significantly increased.The expression of CD69 and CD25,which are markers of T cell activation,were also significantly increased,and the proliferation of T cells was also significantly accelerated in TRIM31 dificient CD4+T cells.TRIM31 deficiency promoted CD4+T cell activation and Th1 cell polarization in mice.Compared with wild-type mice,Trim31fl/flCd4Cre mice were more susceptible to EAE,but were also able to resistance to melanomas.Objectives1.To clarify the role of TRIM31 in CD4+T cell activation and explore the specific molecular mechanism;2.To determine the role of TRIM31 in regulating CD4+T cell activation and immune response though EAE and B16 melanoma model in vivo.Methods1.The Changes of TRIM31 protein in T cell proliferation and activationCD4+T cells were obtained from wild type mice and stimulated with anti-CD3(5?g/ml)plus anti-CD28(1?g/ml)to detect the protein expression of TRIM31 at different time points.2.TRIM31 regulates CD4+T cell proliferation and activation2.1 TRIM31 affects the proliferation of CD4+T cellsCD4+T cells were obtained from Trim31fl/fl or Trim31fl/flCd4Cre mice and incubated with CFSE for 5 min,then treated with anti-CD3(5 ?g/ml)and soluble anti-CD28(1?g/ml)for 0,48 or 72 h,the frequencies of CFSElo cells were acquired by flow cytometry.2.2 TRIM31 affects the expression of IL-2,IFN-y and TNF-?CD4+ T cells were obtained from Trim31fl/fl or Trim31fl/flCd4Cre mice.Enzymelinked immunosorbent assay(ELISA)assays the productin of IL-2 in supernatants of CD4+T cells(1×106 cells per ml)treated for 0,24,48,72 h with anti-CD3 plus antiCD28,or for 72 h with various concentrations(1,2,5 pg/ml)of anti-CD3 alone.qRTPCR analysis the mRNA expression of Il2,Ifng and Tnfa of CD4+T cells treated with anti-CD3 plus anti-CD28 for various time.2.3 TRIM31 affects the expression of CD69 and CD25CD4+T cells from Trim31fl/fl or Trim31fl/flCd4Cre mice were incubated with antiCD3 plus anti-CD28 antibodies for 24 h,48 h and 72 h.The expression of activation markers CD69 and CD25 was analyzed by flow cytometry.2.4 TRIM31 reconstitution in Trim31fl/flCd4Cre CD4+T cellsTo restore TRIM31 or mutant mTRIM31(C52A,C55A)in Trim31fl/flCd4Cre CD4+T cells through lentiviral infection,followed by stimulation with anti-CD3 puls antiCD28.ELISA analysis of the production of IL-2 and IFN-?.3.TRIM31 affects the TCR signal pathwayWestern blot analysis of the total proteins and phosphorylated proteins(p-)of TCR signal pathway in Trim31fl/fl or Trim31fl/flCd4Cre CD4+T cells treated for various times(above lanes)with anti-CD3(5 pg/ml)plus anti-CD28(1?g/ml).4.To screen the targeting moleculars of TRIM31 in TCR signalHEK293T cell were transfected with GFP-TRIM31 or Flag-TRIM31 together with expression plasmids for Myc-RAS,Myc-Raf-1,Myc-MAP2K1,Myc-MAP2K2,MycMAPK3,Myc-MAPK1,Myc-MAVS.The cell lysates were IP with anti-Myc or antiFlag antibody,then followed by western blot analysis with anti-GFP or anti-Myc antibody.5.To detect the interaction between TRIM31 and SYK5.1 ColocalizationHEK293T cells were transfected with plasmids encoding GFP-TRIM31 together with Myc-RAF-1,followed by incubated with a Myc-specific primary antibody and a secondary antibody conjugated to Alexa Fluor 568,the co-localization was directly observed using a confocal microscope.5.2 The binding assay in vitroRecombinant TRIM31 and RAF-1 proteins were incubated and subjected to IP with anti-TRIM31 antibody followed by western blot analysis with anti-RAF-1.5.3 Endogenous interationCD4+T cells were obtained from Trim31fl/fl or Trim31fl/flCd4Cre mice and treated for 0,5,15,30 min with anti-CD3 plus anti-CD28.The cell lysates were subjected to IP with anti-TRIM31,followed by western blot analysis with anti-RAF1 antibody.5.4 The domain required for the binding between TRIM31 and RAF-1The truncation mutants of Flag-TRIM31 or Myc-RAF-1 were constructed and cotransfected into HEK293T with Myc-RAF-1 or GFP-TRIM31.The cell lysates were IP with anti-Flag or anti-Myc antibody,then followed by western blot analysis with antiMyc or anti-GFP antibody.6.To detect TRIM31 catalyzes polyubiquitination of RAF-16.1 Exogenous ubiquitinationHEK293T cells were transfected with Myc-RAF-1,HA-Ub(WT),Flag-TRIM31 or Flag-TRIM31(C53A,C56A),and immunoprecipitation analysis of ubiquitination of Myc-RAF-1.Co-immunoprecipitation analysis the ubiquitination of CD3?,ZAP70,LAT,RAS,MAP2K1,MAP2K2,MAPK3,MAPK1,BRAF in the same way.Coimmunoprecipitation analysis of the ubiquitination of RAF-1 in HEK293T cells transfected with Flag-TRIM10,TRIM15,TRIM25,TRIM26,TRIM27,TRIM31,TRIM40,TRIM56 and TRIM62 or HA-TRIM14,TRIM30?,TRIM38,TRIM39,TRIM41,TRIM65 together with HA-Ubiquitin or V5-Ubiquitin.6.2 Endogenous ubiquitinationCD4+T cells were obtained from Trim31fl/fl or Trim31fl/flCd4Cre mice,and treated for 0,20 min with anti-CD3 and anti-CD28,immunoprecipitation with anti-RAF-1,western blot analysis with anti-P4D1 antibody.6.3 The type of ubiquitinationHEK293T cells were transfected with Myc-RAF-1,Flag-TRIM31 and HAubiquitin or its mutants(K6,K11,K27,K29,K33,K48 and K63),and coimmunoprecipitation analysis the ubiquitination of RAF-1.In-vitro-translated MycRAF-1 and Flag-TRIM31,were incubated with E1,UbcH5a,and ubiquitin(WT)or ubiquitin(K48)for 90 min.Western blot analysis of ubiquitination of Myc-RAF-1 in vitro.6.4 To search for the sites of ubiquitinationHEK293T cells were transfected with Myc-RAF-1,Flag-TRIM31 or FlagTRIM31(C53A,C56A)together with HA-Ubiquitin.Mass spectrometry analysis of ubiquitination of RAF-1 lysine residues.Co-immunoprecipitation analyzed the ubiquitination of RAF-1 mutants in HEK293T cells transfected with Flag-TRIM31,HA-Ub(K33)together with Myc-RAF-1(WT or mutants).7.TRIM31 affects the activation of RAF-17.1 To detect the combination of TRIM31 with RAS in CD4+T cellCD4+T cells from Trim31fl/fl or Trim31fl/flCd4Cre mice were treated with anti-CD3 plus anti-CD28 antibodies for 0,5,15 min,followed by IP with anti-RAF-1,western blot analyzed the interaction of RAF-lwith RAS.The co-localization was directly observed using a confocal microscope.7.2 The combination of TRIM31 with RAS in HEK293T cellHEK293T cells were transfected with Myc-RAS,HA-RAF-1,Flag-TRIM31 or Flag-TRIM31(C53A,C56A)by various combinations.Cell lysates were IP with antiMyc,then followed by western blot analysis with the interaction of HA-RAF-1 with Myc-RAS.Myc-RAS,Flag-TRIM31,HA-RAF-1 or HA-RAF-1(K327R,K493R)were transfected into HEK293T cells in the same way,and western blot analyzed the interaction of HA-RAF-1 or RAF-1(K327R,K493R)with Myc-RAS.8.To investigate the role of TRIM31 in EAE modelTrim31fl/fl Trim31fl/flCd4Cre female mice(6-7 week)were infected with MOG3555(200 ?l,1 ?g/ml)antigen by subcutaneous injection,and infected with pertussis toxin(200 ?l,1 ?g/ml)by intraperitoneal injection at day 0 and day 2.Clinical scores were observed daily.HE and LFB staining of spinal cord tissue and ELISA analyzed the production of IFN-y,IL-17A in serum obtained from Trim31fl/fl or Trim31fl/flCd4Cre mice infected for 20 days.Single cells were obtained from brain,spleen and lymph nodes of mice infected for 20 days,and the infiltrated CD4+T in the CNS,effector T cell and differentiation of T cell subsets were detected by flow cytometry.9.To investigate the role of TRIM31 in B16 melanoma modelTrim31fl/fl and Trim31fl/flCd4Cre mice(8 week)were injected with B16 luc(1×106 per mice)by subcutaneously.Then mice were injected with 200 ?l D-luciferase potassium salt(15 mg/ml)into the tail vein on day 7,11,15 and 19,respectively.Tumor size was recorded by IVIS spectrum.ELISA analyzed the production of IFN-? and IL17A in serum obtained from Trim31fl/fl and Trim31fl/flCd4Cre mice.Single cells were obtained from tumor,spleen and lymph nodes of mice,and the differentiation of T cell subsets was detected by flow cytometry.Results1.The protein expression of TRIM31 was elevated during T cell activationPrevious study has shown that TRIM31 expression was higher in CD4+T cells than CD8+T cells,and higher in activated T cells than naive T cells.To verify this conclusion,CD4+T cells were obtained from wild type mice and stimulated with antiCD3(5 ?g/ml)plus anti-CD28(1 ?g/ml).We found TRIM31 expression was elevated in response to these stimuli and maintained at high levels with treatment.2.TRIM31 deficiency promotes T cell proliferation and activationTo confirm TRIM31 regulates T cells activation,we obtained CD4+T cells from the spleen of Trim31fl/fl or Trim31fl/flCd4Cre mice.FCM analysis showed that the proliferation of CD4+T was significantly enhanced in T cells obtained from Trim31fl/flCd4Cre mice compared to that from Trim31fl/fl mice.Because IL-2 is the major growth factor for T-cell proliferation,we found Trim31fl/flCd4Cre CD4+T cells also produced significantly higher amounts of IL-2 compared to Trim31fl/fl CD4+T cells in response to anti-CD3 alone or together with anti-CD28 stimulation.The expression of Il2,Ifng and Tnfa mRNA were increased in CD4+T cell obtained from Trim31fl/flCd4Cre mice compared to that from Trim31fl/fl mice.Additionally,Trim31fl/flCd4Cre CD4+T cells displayed increased expression of the cytokine receptor CD25 and the activation markers CD69 when compared with T cells from Trim31fl/fl mice.Then,we restored TRIM31 protein expression in Trim31fl/flCd4Cre T cells through lentiviral infection,and found reconstitution of TRIM31(WT),rather than the mutant mTRIM31(C52A,C55A),was able to suppress the induction of IL-2 and IFN-?.Together,these data demonstrated that TCR engagement increases TRIM31 expression and TRIM31 suppresses the activation and proliferation of CD4+T cells.3.TRIM31 deficiency exaggerates TCR signalingTo assess the effect of TRIM31 deficiency on TCR signaling,we next investigated the activation of important signaling mediators in TCR signaling.We isolated spleen CD4+T cells from Trim31fl/fl or Trim31fl/flCd4Cre mice,then stimulated with anti-CD3 and anti-CD28 antibody.TRIM31 deficiency did not affect the phosphorylation of Za70,PLC?1 as well as several downstream signaling mediators including JNK,p65,p38.However,Trim31fl/flCd4Cre CD4+T cells showed enhanced phosphorylated RAF-1 and ERK.These data suggested that TRIM31 might be involved in the RAS-RAF-MEKERK pathway to inhibit TCR signaling.4.TRIM31 targets RAF-1To study the molecular mechanism of inhibition of TCR signaling by TRIM31,HEK293T were transfected expression plasmids for TRIM31 together with RAS,RAF1,MAP2K1,MAP2K2,MAPK3 or MAPK1.Co-Immunoprecipitation(Co-IP)experiments and western blot showed that TRIM31 associated with RAF-1 but not with RAS,MAP2K1,MAP2K2,MAPK3 and MAPK1.To further confirm that TRIM31 associated with RAF-1,we performed confocal microscopy.We found that GFPTRIM31 colocalized with Myc-RAF-1.We also confirmed the direct interaction between TRIM31 and RAF-1 in vitro by using recombinant proteins.Furthermore,CoIP experiments suggested that endogenous TRIM31 formed a complex with RAF-1 in CD4+T cells after treatment with plate-bound anti-CD3 plus anti-CD28 antibodies.We next assessed the domains involved in the interaction between TRIM31 and RAF-1.Co-immunoprecipitation assays of HEK293T cells transfected with wild-type or mutant TRIM31 plus RAF-1 demonstrated that the coiled-coil motif of TRIM31 is involved in the interaction with RAF-1.The same assays revealed that the CR2(conserved regions 2)of RAF-1 is required for the interaction with TRIM31.Taken together,these data demonstrated that the coilded-coil motif of TRIM31 interacts with the CR2 domain of RAF-1.5.TRIM31 promotes the K33-linked polyubiquitination of RAF-1 at K327 and K493Because TRIM31 is an E3 ubiquitin ligases,we then investigated whether TRIM31 ubiquitinates RAF-1.We co-transfected various combination of plasmids encoding Myc-tagged RAF-1,hemagglutinin(HA)-tagged ubiquitin and Flag-tagged TRIM31 into HEK293T cells.Immunoprecipitation assays indicated that TRIM31 promotes polyubiquitination of RAF-1.Importantly,the E3 ligase inactive mutant TRIM31(C53A,C56A),in which the cysteine residues at positions 53 and 56 were replaced with alanine in the ring domain,lost the ability to increase the polyubiquitination of RAF-1.These data suggested that the E3 ligase activity of TRIM31 is required for the polyubiquitination of RAF-1.Furthermore,we also found that the polyubiquitination of RAF-1 was significantly impaired in TRIM31-deficient CD4+T cells.Consistent with the data that TRIM31 interacts with RAF-1,TRIM31 did not promote the ubiquitination of CD3?,Zap70,RAS,MAP2K1,MAPK2K2,MAPK3,MAPK1 and BRAF.Besides,we screened some TRIM family members which play essential roles in innate immunity,and found that TRIM31 had the highest efficiency to promote the polyubiquitination of RAF-1.We next investigated which type of ubiquitin-chain linkage was catalyzed by TRIM31 on RAF-1.We co-transfected wild-type ubiquitin or various ubiquitin mutant plasmids,in which only one of each of the seven lysine residues remained,together with RAF-1 and TRIM31 into HEK293T cells.We found that RAF-1 was ubiquitinated by TRIM31 in WT and K33-transfected cells,indicating TRIM31 mainly catalyzed the conjugation of K33-linked polyubiquitination on RAF-1.In vitro ubiquitination assay,we also confirm TRIM31 promote K33-linked polyubiquitination of RAF-1 by using recombinant proteins,but not K48-lined polyubiquitination.To explore which ubiquitin-modification sites of RAF-1 might be modified by TRIM31,mass spectrometry(MS)was performed.We found that there are two different lysine residues of RAF-1(K327,K493)have ubiquitin modification.Thus,we constructed and transfected those RAF-1 mutants into HEK293T cells together with HA-ubiquitin(K33).Co-Immunoprecipitation experiments and Western blot showed that polyubiquitination of the RAF-1(K327R,K493R)mutant was significantly decreased,the ubiquitination of RAF-1(K327R)or RAF-1(K493R)also were impaired.These results indicated that TRIM31 mediates the K33-linked polyubiquitination of RAF-1 at K327 and K493.6.TRIM31 inhibits RAF-1 interact with RAS.RAF-1 is recruited to the RAS,is an essential step in its activation.We hypothesized that K33-linked polyubiquitination of RAF-1 might affect the function of recruitment to RAS.To investigate this possibility,we obtained the CD4+T cell from Trim31fl/fl or Trim3lfl/flCd4Cre mice.Co-immunoprecipitation assay showed that the interaction between RAF-1 and RAS was increased in Trim3lfl/flCd4Cre CD4+T cell compared to that in Trim31fl/fl mice,which treated with anti-CD3 plus anti-CD28.The similar results were also obtained by confocal microscope.To further confirm that TRIM31 inhibits RAF-1 interact with RAS,Myc-RAS,HA-RAF-1 and TRIM31 or TRIM31(C53A,C56A)were transfected into HEK293T cells.The assay showed that overexpression of WT TRIM31,but not TRIM31(C53A,C56A),greatly decreased the interaction between RAF-1 with RAS.We also found that TRIM31 failed to impair the interaction between RAS and RAF-1(K327R,K493R).These results suggested TRIM31 mediated ubiquitination of RAF-1 could inhibit the binding of RAF-1 to RAS,K327 and K493 of RAF-1 were vital for the recruitment of RAF-1 to RAS.7.TRIM31 deficiency accelerates EAE progression in vivoCD4+T cells play an important role in experimental autoimmune encephalitis(EAE)model.To verified the function of TRIM31 in vivo,we challenged Trim31fl/fl or Trim31fl/flCd4Cre mice with MOG35-55.We found that Trim31fl/flCd4Cre mice were more susceptible to EAE than Trim31fl/fl counterparts.Tissue sections of the spinal cord showed more inflammatory cell infiltration and demyelination in Trim31fl/flCd4Cre mice.Twenty days after infection,the production of IFN-? in serum obtained from Trim31fl/flCd4Cre was significantly higher than Trim31fl/fl mice.We obtained the brain,spleen and lymph node,and found more CD4+T cell in the CNS of Trim31fl/flCd4Cre mice,Trim31fl/flCd4Cre CD4+T cells produced more IFN-y than Trim31fl/fl counterparts,but not IL-4 and IL-17A.The frequency of Treg cells were similar in Trim31fl/fl mice and Trim31fl/flCd4Cre.These results suggest that TRIM31 deficiency leads to elevated T cell activaton,produces larger amounts of IFN-y,and accelerates the process of EAE disease.8.TRIM31 deficiency inhibits the development of B16 melanoma in vivoCD4+T cells are indispensable in tumor immunity.On the one hand,they can activate CD8+T cells to differentiate into cytotoxic T cells and kill tumor cells.On the other hand,the secretion of IFN-y can fulfill the anti-tumor ability.Therefore,we subcutaneously injected Trim31fl/fl and Trim31fl/flCd4Cre mice with B16 luc cells.The sizes of B16 melanoma in mice at 7 days,11 days,15 days,19 days after infection were detected by IVIS Spectrum.The results showed that tumor sizes were smaller and the luciferase activity was lower in Trim31fl/flCd4Cre mice.The production of IFN-y in serum obtained from Trim31fl/flCd4Cre was significantly higher than Trim31fl/fl mic.Subsequently,single cells were obtained from lymph nodes,spleen and tumors of mice.And flow cytometry results showed that the production of IFN-y was increased in CD4+T cells of Trim31fl/flCd4Cre mice.Based on the above results,it indicates that TRIM31 can inhibit the activation of CD4+T cells in physiological state.Once deficiency,the mice were susceptible to autoimmune disease,but enhanced anti-tumor ability.Conclusions and innovative topics1.TRIM31 plays an important role in the development of various tumors and innate antiviral and antifungal immunity.In this study,we found that TRIM31 also plays a role in adaptive immunity,which can inhibit CD4+T activation by promoting the ubiquitination of RAF-1.Our studies enrich the function of TRIM31 in the immune system.2.We found a novel negative regulatory mechanism of T cell activation that TRIM31 inhibited the activation of CD4+T cell and suppressed Th1 polarization in vivo.3.The ubiquitination of RAF-1 mostly focuses on degradation.However,we firstly found that TRIM31 can mediate the K33-linked ubiquitination of RAF-1 rely on non-proteolytic regulation,and identified the modification sites of K327 and K493. |
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