| Fungal infection diseases are serious human health threats,especially HIV patients,solid and hematopoietic stem cell transplantation(HSCT),immune-compromised people with chemotherapy,or primary immune deficiencies.During fungal infection,C-type lectin receptors(CLRs)against fungal pathogens by sensing various fungal surface components to elicit a strong immune response.Upon agonist binding,CLR signaling triggers SYK translocation and activation,which then activates CLR pathway,leading to the production of proinflammatory cytokines and chemokines.Moreover,these cytokines and chemokines promote T cell differentiation,to elicit adaptive immune responses.As an essential adaptor and kinase of the CLR signaling complex,the translocation and activation of SYK is critical during fungal infection,but there has been poorly reported.Previous study suggested E3 ubiquitin ligase Cbl-b promotes the degradation of phosphorylated SYK.However,whether SYK activation is being modified by other E3 ubiquitin ligases is completely unknown.Here we screened which E3 ubiquitin ligase(s)may catalyze SYK ubiquitination and identified the E3 ligase TRIM31 as a crucial regulator of SYK activation.TRIM31 catalyzes K27-linked polyubiquitination at K375 and K517 of SYK.This K27-linked polyubiquitination of SYK promotes its plasma membrane translocation and binding with Dectin-1 or FcRy,and also prevents the interaction with the phosphatase SHP-1,thus promoting SYK kinase activity.Consistantly,deficiency of Trim31 in bone marrow-derived dendritic cells(BMDCs)and macrophages(BMDMs)dampened SYK-mediated signaling and inhibited the production of pro-inflammatory cytokines and chemokines against the fungal pathogen Candida albicans infection.Moreover,TRIM31 dificient BMDCs and BMDMs displayed reduced ability of phagocytosis of Candida albicans,and produced fewer ROS and NO for killing fungi.More importantly,Trim31-deficient mice were more susceptible to C.albicans systemic infection than wild-type mice,and exhibited reduced Thl and Thl7 responses.This study highlighted the significance of TRIM31 in anti-fungal immunity,enriched the activity regulation mechanism of SYK,and provided a new idea for treatment fungal diseases.Objectives1.To screen which E3 ubiquitin ligase(s)may catalyze SYK ubiquitination;2.To clarify the role of TRIM31 in antifungal immunity and explore its mechanism;3.To clarify the physiological significance of TRIM31 through mouse model infected with C.albicans.Methods1.To screen which E3 ubiquitin ligase(s)may catalyze SYK ubiquitinationIn order to screen which E3 ubiquitin ligase(s)may catalyze SYK ubiquitination.We over-expressed some TRIM family proteins,Ubiquitin(Ub)and SYK in HEK293T cells,IP was used to enrich SYK,and WB to detect the ubiquitination of SYK.2.To detect the interaction between TRIM31 and SYK2.1 Exogenous interactionGFP-TRIM31 and SYK,SHP-2,PKC?,CARD9,PLC?2,BCL10 were cotransfected into HEK293T cells,IP and WB to detect which molecules interact with TRIM31.2.2 Endogenous interactionBMDCs were stimulated with Zymd or a-mannan for indicated time points,antiTRIM31 was used to IP,and WB to detect the interaction of endogenous TRIM31 and SYK.2.3 In vitroThe recombinant proteins TRIM31 and SYK were co-incubated in vitro,the mixture was detected by WB with indicated antibodies.2.4 Deletion mutantsThe deletion mutants of Myc-SYK were constructed and co-transfected into HEK293T with GFP-TRIM31,anti-Myc was used to IP,and WB to detect which domains of SYK interact with TRIM31.2.5 ColocalizationmCherry-SYK and GFP-TRIM31 were transfected into HEK293T,after 24h,the co-localization was directly observed using a confocal microscope.3.To detect TRIM31 catalyzes polyubiquitination of SYK3.1 Exogenous ubiquitinationCo-IP analysed the ubiquitination of SYK in HEK293T cells transfected with Myc-SYK or Myc-MAVS,HA-Ub(WT),Flag-TRIM31 or Flag-TRIM31(C53A,C56A).Co-IP analysed the ubiquitination of the plasmids(CARD9,BCL10,PLCy2,SYK,SHP-2 and PKC?)in HEK293T cells transfected various combinations with HAUb(WT)and Flag-TRIM31 or Myc-TRIM31.Co-IP analysed the ubiquitination of the receptors(Dectin-1,Dectin-2,Dectin-3,Mincle)and the adaptor FcR? in HEK293T cells transfected with HA-Ub(WT)and Flag-TRIM31.3.2 The type of ubiquitinationCo-IP analysed the ubiquitination of SYK in HEK293T cells transfected with Flag-SYK,Myc-TRIM31 and HA-Ub or its mutants(K6,K11,K27,K29,K33,K48 and K63).3.3 Endogenous ubiquitinationIP analysed the ubiquitination of endogenous SYK in Trim31+/+and Trim31-/BMDCs stimulated with Zymd or ?-mannan for indicated time points.3.4 The site of ubiquitinationTo detect the lysine residues on SYK mediated by TRIM31,which were identified by mass spectrometry.Co-IP analysed the ubiquitination of SYK and its mutants in HEK293T cells transfected with Myc-TRIM31,Flag-SYK(WT or mutants)along with HA-Ub(K27).4.To investigate the role of TRIM31 on antifungal immunity responseTo clarify the physiological significance of TRIM31,Trim31+/+mice and Trim31-/-mice(8 week)were infected with a lethal dose of C.albicans(2×105 fungal cells per mouse)by intravenous injection,weight loss and survival were observed daily;To assess C.albicans colony forming units(CFU)in the kidney,spleen and liver of Trim31+/+and Trim31-/-mice infected with C.albicans for 5 days;Kidney sections of Trim31+/+and Trim31-/-mice were stained with HE,PAS and Ly-6G+to obtain inflammatory score;ELISA analysed the secretion of IL-6 and TNF-? from the supernatant of the organ of the Trim31+/+ and Trim31-/-mice infected with C.albicans for 5 days;ELISA analysed the levels of IgG in serum from Trim31+1+ and Trim31-/mice infected with C.albicans for 5 days.To determine whether adaptive immune reponse was involved in TRIM31 mediated antifungal immunity,ELISA analysed the concentration of IFN-y and IL-17A in the supernatant of splenic cells obtained from Trim31+/+and Trim31-/-mice infected with C.albicans(2×105 fungal cells per mouse)for 5 days,followed by stimulation with HKCA-Y for 2 days;Intracellular staining of IFN-y(Th1)and IL-17(Th17)were determined with FCM;qRT-PCR analysed of Ifng,Il17a,and Il17f from kidneys of Trim31+/+and Trim31-/-mice infected with C.albicans(2×105 fungal cells per mouse)for 5 days.Pro-inflammatory cytokines are crucial in host denfense against fungal infection,we analysed the levels of IL-6,TNF-?,IL-12,CXCL1 and CXCL2 in serum from Trim31+?+and Trim31-/-mice infected with C.albicans(1×106 fungal cells per mouse)for 24h via ELISA.To investigate the cellular basis of the TRIM31-related antifungal effect,we generated bone marrow(BM)-chimeric mice by reconstituting lethally irradiated Trim31+/+mice with syngeneic Trim31-/-or Trim31+/+BM,and infected with a lethal dose of C.albicans(2×105 fungal cells per mouse)by intravenous injection,weight loss and survival were observed daily.5.To detect the ability of TRIM31 in antifungal activity in cells5.1 To detect the role of TRIM31 in the CLRs-induced production of cytokinesBMDCs or BMDMs were obtained from Trim31+/+and Trim31-/-mice,followed by stimulation with Zymd,?-mannan,TDB,HKCA-Y and HKCA-H.ELISA analysis of proinflammatory cytokines including IL-6,TNF-?,IL-1?,IL-12,IL-23 and chemokines CXCL1 and CXCL2.5.2 To detect the role of TRIM31 in the CLRs-induced production of cytokines through gene restoration experimentWe reconstructed TRIM31 or catalytically inactive mutant mTRIM31(C52A,C55A)in Trim31-/-BMDCs and BMDMs through lentiviral infection,followed by stimulation with Zymd or ?-mannan.WB was used to detect the expression of TRIM31 or mTRIM31(C52A,C55A),ELISA analysis of the production of IL-6 and TNF-?.5.3 To detect the role of TRIM31 on the phagocytosisBMDCs or BMDMs were obtained from Trim31+/+and Trim31-/-mice,and cocultured with FITC-labeled C.albicans for 30 min or 60 min,followed by incubation with trypan blue.The rate of phagocytosis was assessed by flow cytometry.5.4 To detect the production of ROS induced by C.albicansBMDCs or BMDMs were obtained from Trim31+/+and Trim31-/-mice,followed by stimulation with Zymd,?-mannan,TDB,HKCA-Y and HKCA-H,the production of ROS was measured by flow cytometry.5.5 To detect the production of NO induced by C.albicansBMDCs or BMDMs were obtained from Trim31+/+and Trim31-/-mice,followed by stimulation with HKCA-Y or HKCA-H,the production of NO was detected.6.To detect the effect of TRIM31 on SYK kinase activity6.1 To detect the protein phosphorylation of CLR signaling pathwayBMDCs or BMDMs were obtained from Trim31+/+and Trim31-/-mice,followed by stimulation with HKCA-Y,HKCA-H,Zymd or ?-mannan.WB analysis the phosphorylation levels and total levels of SYK,PLCy2,PKC?,p65,ERK,JNK and p38.6.2 To detect the p-SYK by overexpressionOver-expressed Myc-Dectin-1,V5-SYK,Flag-TRIM31 or Flag-TRIM31(C53A,C56A)in HEK293T,followed by stimulation with HKCA-Y,WB analysis with p-SYK.Over-expressed Myc-Dectin-1,Flag-TRIM31,V5-SYK or V5-SYK(K375R,K517R)in HEK293T,followed by stimulation with HKCA-Y,WB analysis with pSYK.Over-expressed Myc-Dectin-2,HA-FcR?,V5-SYK,Flag-TRIM31 or FlagTRIM31(C53A,C56A)in HEK293T,followed by stimulation with HKCA-H,WB analysis with p-SYK.Over-expressed Myc-Dectin-2,HA-FcR?,Flag-TRIM31,V5-SYK or V5-SYK(K375R,K517R)in HEK293T,followed by stimulation with HKCA-H,WB analysis with p-SYK.6.3 Gene restoration experimentWe reconstructed SYK,SYK and TRIM31,SYK(K375R,K517R),SYK(K375R,K517R)and TRIM31 in Syk-/-BMDCs through lentiviral infection,followed by stimulation with Zymd or ?-mannan,WB to detect the expression of these plasmids,ELISA analysis of the production of IL-6 and TNF-?.7.To detect TRIM31 regulates SYK translocate to cell membrane and interact with Dectin-1 or FcR?7.1 Exogenous interactionOver-expressed Myc-Dectin-1 or Myc-Dectin-2,HA-FcR?,V5-SYK,FlagTRIM31 or Flag-TRIM31(C53A,C56A)in HEK293T,followed by stimulation with HKCA-Y or HKCA-H.Anti-Myc or anti-HA was used to IP,and WB to detect V5-SYK interact with Myc-Dectin-1 or HA-FcR?.Over-expressed Myc-Dectin-1 or Myc-Dectin-2,HA-FcR?,Flag-TRIM31,V5SYK or V5-SYK(K375R,K517R)in HEK293T,followed by stimulation with HKCAY or HKCA-H.Anti-Myc or anti-HA was used to IP,and WB to detect V5-SYK(K375R,K517R)interact with Myc-Dectin-1 or HA-FcR?.7.2 Endogenous interactionBMDCs were obtained from Trim31+/+and Trim31-/-mice,followed by stimulation with Zymd or ?-mannan,WB to detect the interaction between endogenous SYK and Dectin-1 or FcR?.7.3 To detect SYK translocate to cell membraneBMDCs were obtained from Trim31+/+and Trim31-/-mice,followed by stimulation with Zymd and ?-mannan,then prepared cell membrane and cytosolic fractions,WB to detect SYK.Meanwhile,Confocal microscopy was used to observe SYK translocated to cell membrane.8.To detect TRIM31 inhibits the interaction between SHP-1 and SYK8.1 To detect the p-SYK by overexpressionOver-expressed Myc-Dectin-1,Myc-SYK,V5-SHP-1,Flag-TRIM31 or FlagTRIM31(C53A,C56A)in HEK293T,followed by stimulation with HKCA-Y,WB analysis with p-SYK.Over-expressed Myc-Dectin-1,V5-SHP-1,Flag-TRIM31,Flag-SYK or FlagSYK(K375R,K517R)in HEK293T,followed by stimulation with HKCA-Y,WB analysis with p-SYK.8.2 To detect Endogenous interaction between SHP-1 and SYKBMDCs were obtained from Trim31+/+and Trim31-/-mice,followed by stimulation with Zymd or ?-mannan,WB to detect the interaction between endogenous SYK and SHP-1.8.3 To detect Exogenous interaction between SHP-1 and SYKOver-expressed Myc-SYK,V5-SHP-1,Flag-TRIM31 or Flag-TRIM31(C53A,C56A)in HEK293T,anti-V5 was used to IP,WB to detect the interaction between SYK and SHP-1.Results1.TRIM31 catalyzes polyubiquitination of SYKIt was found that TRIM25,TRIM31 and TRIM39 increased SYK polyubiquitination.Notably,TRIM31 had the highest efficiency to catalyze the ubiquitination of SYK.2.TRIM31 interacts with SYKGiven that TRIM31 catalyzes the polyubiquitination of SYK,we proposed that TRIM31 might interact with SYK.We over-expressed TRIM31-GFP and other molecules in the CLR signaling pathway in HEK293T cells,and found that TRIM31 interacted with SYK,and mainly depend on the KD domain,but not with other molecules.Further,the interaction between endogenous TRIM31 and SYK was increased in BMDCs upon stimulation with Zymd or ?-mannan.We also confirmed the direct interaction between TRIM31 and SYK in vitro using recombinant proteins.3.TRIM31 catalyzes the K27-linked polyubiquitination of SYK at K375 and K5173.1 TRIM31-mediated SYK polyubiquitination by its enzymatic activityTo further confirm that TRIM31 is involved in SYK ubiquitination,HEK293T cells were co-transfected with Myc-SYK,HA-Ub and Flag-TRIM31 WT or TRIM31(C53A,C56A)which lacks its enzymatic activity.We found that the polyubiquitination of SYK was readily detected in the presence of WT TRIM31.While,TRIM31(C53A,C56A)failed to do so.As a positive control,TRIM31 ubiquitinated MAVS as previously reported in our lab.And TRIM31 failed to catalyze the polyubiquitination of CARD9,BCL10,PLC-?2,SHP2,PKC-?,Dectin-1,Dectin-2,Dectin-3,Mincle and the adptor FcR?.3.2 TRIM31 catalyzes K27-linked polyubiquitination of SYKTo investigate the type of TRIM31-mediated SYK polyubiquitination,we used HA-Ub mutants K6,K11,K27,K29,K33,K48 and K63,which contain the one lysine residue at indicated position and other lysine residues were substituted by arginine.We found TRIM31 substantially increased SYK polyubiquitination in the presence of K27,but not with other ubiquitin mutants.We measured the polyubiquitination of SYK in BMDCs prepared from Trim31+/+and Trim31-/-mice upon stimulation with Zymd or ?-mannan.The endogenous SYK polyubiquitination was significantly increased followed by stimulations.And the endogenous SYK polyubiquitination was significantly decreased in Trim31-/-BMDCs as compared with Trim31+/+BMDCs after stimulation with Zymd or ?-mannan.Importantly,K27-linked SYK polyubiquitination was decreased,while K48-linked and K63-linked polyubiquitination remained unchanged.3.3 TRIM31 catalyzes the polyubiquitination of SYK at K375 and K517SYK has 49 lysine residues,we next investigated which lysine residues of SYK might be targeted by TRIM31 for the K27-linked polyubiquitination.We performed mass spectrometry(MS)analysis and systemically investigated the potential ubiquitinated lysine residues of SYK.A total of seven ubiquitinated lysine residues(K124,K165,K261,K368,K375,K386,K517)were identified in the MS analysis.We then constructed several SYK mutants including K124R,K165R,K261R,K368R,K375R,K386R and K517R,in which the potential ubiquitinated lysine residues were replaced with arginine.We transfected these SYK mutants into HEK293T cells together with Myc-TRIM31,and found that TRIM31-mediated K27-linked ubiquitination were decreased in K375R and K517R mutants.Taken together,these data indicate that TRIM31 directly interacts with and catalyzes the K27-linked polyubiquitination of SYK at K375 and K517.4.TRIM31 deficiency impairs anti-fungal immune responses in vivoTo confirm the in vivo function of TRIM31 anti-fungal immune responses,we intravenously infected Trim31-/-mice and Trim31+/+mice with a lethal dose of C.albicans.Trim31-/-mice manifested a massive weight loss after infection with C.albicans and eventually died of infection;we assessed the fungal loads in mice at 5 days after infection.We found Trim31-/-mice exhibited more C.albicans colony forming units(CFU)in the kidney,spleen and liver,compared to that in Trim31+/+mice;Histopathologic analysis of kidneys demonstrated that Trim31-/-mice exhibited increased renal inflammation and numbers of C.albicans yeast cells and hyphae.Additionally,the neutrophils infiltrated into the kidneys of infected Trim31-/-mice were significantly higher than Trim31+/+mice;Trim31-/-mice also had lower levels of IL-6 and TNF-? in the homogenates of kidney and liver after 5 days infection with C.albicans;Trim31-/-mice reduced production of IgG in their serum at 5 d post-infection as compared with Trim31+/+mice;After 5 days infection with C.albicans,Further,we isolated spleen cells from Trim31-/? and Trim31+/+mice infected with or without C.albicans.We found restimulation of the spleen cells with HKCA-Y from Trim31+/+mice infected with C.albicans greatly increased the secretion of IL-17A and IFN-y.While,the secretion of IL-17A and IFN-y was greatly attenuated in the spleen cells from infected Trim31-/mice upon re-stimulation with HKCA-Y.We also investigated the T-cell differentiation in the spleen at day 5 after C.albicans infection.The percentage of Thl and Th17 cells in the spleen was greatly decreased in Trim31-/-mice compared to that in the spleen from Trim31+/+mice.We found that expression of the genes encoding Ifng,Il17a and Il17f was also attenuated in the kidneys from mice compared to Trim31+/+mice.Further,at early time points of C.albicans infection,Trim31-/-mice showed significantly reduced production of IL-6,TNF-?,IL-12,CXCL1 and CXCL2 in their serum at 24 h post-infection as compared with Trim31+/+mice;To investigate the cellular basis of TRIM31 in anti-fungal protective function,we generated bone marrow(BM)-chimeric mice by reconstituting lethally irradiated Trim31+/+mice with syngeneic Trim31-/-or Trim31+/+BM.Trim31+/+mice reconstituted with Trim31-/-BM showed a phenotype similar to that of mice with total Trim31 deficiency after C.albicans infection,indicating the radiosensitive hematopoietic cells were involved in the anti-fungal immune response.Taken together,these results suggest that TRIM31 positively regulates the anti-fungal immune response in vtvo.5.TRIM31 positively regulates the antifungal activity in cellsWe next sought to determine the role of TRIM31 in CLR-induced cytokine and chemokine production.We obtained BMDCs from Trim31+/+and Trim31-/-mice,followed by stimulation with various CLR ligands Zymd,?-mannan,TDB,heat-killed C.albicans yeast and heat-killed C.albicans hyphae.We found that the production of proinflammatory cytokines including IL-6,TNF-?,IL-1?,IL-12,IL-23 and chemokines such as CXCL1 and CXCL2 were markedly reduced in Trim31-/-BMDCs compared to that in Trim31+/+BMDCs.Furthermore,we restored TRIM31 protein expression in Trim31-/-BMDCs or BMDMs through lentiviral infection.WB analysis showed successful restoration of TRIM31 protein expression.We found reconstitution of WT TRIM31,rather than the catalytically inactive mutant mTRIM31(C52A,C55A),was able to restore the induction of IL-6 and TNF-? upon stimulation with Zymd or ?-mannan stimulation,indicating TRIM31 E3 ligase activity was important for CLR-mediated proinflammatory cytokines production.Thus,our results indicate that TRIM31 is a positive regulator for cytokine and chemokine production in response to C.albicans infection.Macrophages are professional phagocytes and are essential in controlling fungal infections by phagocytosis and killing mechanisms.We found Trim31-/-BMDCs and BMDMs exhibited impaired phagocytosis of C.albicans yeast.To investigate whether TRIM31 affects the production of ROS and NO in innate immune cells,we isolated BMDCs or BMDMs from Trim31+/+and Trim31-/-mice and stimulated these cells with various CLR ligands.We found Trim31-/-BMDCs or BMDMs produced lower amounts of ROS after stimulation with Zymd,TDB,?-mannan,HKCA-Y and HKCAH respectively.Similarly,production of NO after stimulation with HKCA-Y and HKCA-H was also decreased in Trim31-/-BMDCs or BMDMs.These data suggest that TRIM31 controls the fungicidal activity of BMDCs or BMDMs by regulating ROS and NO production.Thus,our results indicate that TRIM31 is a positive regulator of antifungal activity in cells.6.TRIM31 promotes activation of SYKBased on the results abovementioned,we hypothesized that TRIM31 may affect the CLR pathways by directly regulating SYK kinase activation.Thus,we prepared BMDCs or BMDMs from Trim31+/+and Trim31-/-mice,followed by stimulation with HKCA-Y,HKCA-H,Zymd or ?-mannan and measured the phosphorylation(active form)of the CLR-induced signaling.We found that the phosphorylation of SYK,PLC?2,PKC?,p65,ERK,JNK and p38 in Trim31-/-BMDCs or BMDMs was significantly attenuated in response to stimulation compared to that in Trim31+/+BMDCs or BMDMs.Overexpression of WT TRIM31,but not TRIM31(C53A,C56A),greatly enhanced SYK phosphorylation at basal level,and fungal stimulation further elevated SYK phosphorylation.TRIM31 significantly enhanced the phosphorylation of WT SYK,but not SYK(K375R,K517R)mutant before and after HKCA-Y stimulation or HKCA-H.These data indicate that the K375/517-linked polyubiquitin chains mediated by TRIM31 is critical for SYK activation.To investigate whether TRIM31-mediated SYK ubiquitination is involved in antifungal immune responses,we further reconstituted Syk-/-BMDCs with WT SYK or SYK(K375R,K517R)mutant through lentiviral infection.WB analysis showed the successful reconstitution of SYK protein in Syk-/-BMDCs.As expected,SYK deficiency attenuated Zymd-or ?-mannan-induced IL-6 and TNF-? production.Reintroduction of SYK or SYK(K375R,K5 17R)efficiently restored the induction of IL-6 and TNF-? by Zymd or ?-mannan in Syk-/-BMDCs.Notably,TRIM31 greatly increased the production of IL-6 and TNF-? in Syk-/-BMDCs reconstituted with WT SYK,whereas SYK(K375R,K517R)mutant-mediated induction of IL-6 and TNF-?was not further increased in the presence of TRIM31.Taken together,these data demonstrate that SYK polyubiquitination at K375 and K517 mediated by TRIM31 is essential for SYK activation and the subsequent pro-inflammatory cytokines production.7.TRIM31 promotes recruitment SYK to Dectin-1 or FcR? and translocate to cell membrane7.1 TRIM31 promotes recruitment SYK to Dectin-1 or FcR?Because binding of SYK to CLRs promotes its phosphorylation/activity,we hypothesized that K27-linked polyubiquitination might affect SYK recruitment to CLRs.To confirm this,HEK293T cells were transfected with Dectin-1,SYK and TRIM31 or TRIM31(C53A,C56A),then the cells were treated with HKCA-Y.Overexpression of WTTRIM31,but not TRIM31(C53A,C56A),greatly increased the interaction of Dectin-1 and SYK,and fungal stimulation further elevated association between Dectin-1 and SYK.Similarly,the interaction between SYK and FcRy was also increased in the presence of WT TRIM31,but not TRIM31(C53A,C56A)in HEK293T cells before or after stimulation with HKCA-H.We then transfected Dectin-1,TRIM31,WT SYK or SYK(K375R,K517R)into HEK293T cells,followed by fungal stimulation.We found that TRIM31 greatly increased the interaction between WT SYK and Dectin-1.However,this increased interaction is not observed when SYK was mutated at K375R and K517R.Similarly,the interaction between WT SYK and FcR? was increased in the presence,while the interaction between SYK(K375R,K517R)and FcR? was unchanged in the presence of TRIM31.Further,we showed that Zymd and ?-mannan stimulation in BMDCs increased the interaction between SYK and Dectin-1 or FcR?.However,Zymd-and ?-mannaninduced interaction between SYK and Dectin-1/FcR? was decreased in Trim31-/BMDCs compared to that in Trim31+/+BMDCs.Thus,our results highlight the important role of TRIM31 in modulating the interaction between SYK and CLR receptors.7.2 TRIM31 promotes SYK translocate to cell membraneTo independently verify whether TRIM31 promotes SYK recruitment to cell membrane followed stimulation,we prepared cell membrane and cytosolic fractions from BMDCs after stimulation with Zymd or ?-mannan.We found these stimulations could induce SYK translocation to the membrane,while SYK membrane translocation was greatly decreased in Trim31-/-BMDCs.To directly visualize the translocation of SYK to the cell membrane,confocal microscopy was performed.In resting cells,SYK was found to be diffusely distributed in the cell cytosol of BMDCs,but Zymd or ?-mannan-induced recruitment of SYK to the cell membrane and colocalization with Dectin-1 or FcR?.However,the colocalization were decreased in Trim31-/-BMDCs.Previous study had shown that Dectin-1 agonists could induce a rearrangement of the cell membrane and trigger cellular phagocytosis.We also found Zymd induced less phagosomes in Trim31-/BMDCs.Taken together,these results suggest that TRIM31-mediated polyubiquitination promotes SYK recruitment to Dectin-1 or Dectin-2-FcR? and translocation to the cell membrane.8.TRIM31 inhibits SHP-1 mediated SYK desphosphorylation8.1 TRIM31-mediated polyubiquitination inhibits SHP-1-mediated SYK desphosphorylationSHP-1 has been shown to desphosphorylated SYK,we further explore whether TRIM31 influences the dephosphorylation of SYK by SHP-1,we then transfected Dectin-1,SYK,SHP-1,TRIM31 or TRIM31(C53A,C56A)into HEK293T cells,followed by HKCA-Y stimulation.As expected,transfection of SHP-1 greatly attenuated SYK phosphorylation,while,transfection of WT TRIM31,but not TRIM31(C53A,C56A),greatly inhibited SHP-1-mediated SYK dephosphorylation.To study whether polyubiquitination of SYK at K375 and K517 sites is required for SHP-1mediated dephosphorylation.We also co-expressed Dectin-1,SYK,SYK(K375R,K517R),SHP-1 and TRIM31 in HEK293T cells.SYK and SYK(K375R,K517R)mutant were dephosphorylated by SHP-1 as expected.Overexpression of TRIM31 attenuated SHP-1-mediated WT SYK dephosphorylation,while,TRIM31 did not prevent SHP-1-mediated dephosphorylation of SYK(K375R,K517R).Together,these data highlight that TRIM31-mediated polyubiquitination inhibits SHP-1-mediated SYK desphosphorylation.8.2 TRIM31 inhibits the interaction between SHP-1 and SYKTo investigate whether TRIM31-mediated SYK ubiquitination affected the binding between SYK and SHP-1,we prepared BMDCs from Trim31+/+and Trim31-/mice and stimulated with Zymd or ?-mannan.We found the interaction between SYK and SHP-1 was increased in Trim31-/-BMDCs compared to that in Trim31+/+BMDCs.Meanwhile,we co-expressed SYK,SHP-1 and WTTRIM31 or TRIM31(C53A,C56A)in HEK293T cells.WT TRIM31,but not TRIM31(C53A,C56A)attenuated the interaction between SYK and SHP-1.In summary,TRIM31 specifically catalyzed SYK at K375 and K517 for K27linked polyubiquitination,facilitated SYK translocation and activation,and decreased the association with SHP-1,thus positively regulated CLR signaling pathway,highlighted the significance of TRIM31 in anti-fungal immunity.Innovative topics1.Whether SYK is being modified by ubiquitination and the role of SYK ubiquitination is rarely reported.We found the E3 ubiquitin ligase TRIM31 specifically catalyzed SYK at K375 and K517 for K27-linked polyubiquitination for the first time.We fill up this knowledge gap.2.The kinase activity of SYK was regulated by many molecules,during the activation process,SYK changes its structure and translocates to the cell membrane.We found TRIM31 promotes its kinase activity and polyubiquitination might facilitate the shift of SYK conformation from an inactive status to the active status with the kinase domain exposed.Then,SYK binding with the CLRs,and TRIM31 prevented the interaction with SHP-1.We firstly demonstrate that ubiquitination promotes the activation and translocation of SYK.3.TRIM31 as a member of TRIM family proteins,has been reported play role in cancer,anti-virus and inflammation.We found TRIM31 can positively regulate the CLR pathway and regulate anti-fungal immunity for the first time,clarified the physiological significance in antifungal immunity. |
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