TNFAIP8L2/TIPE2 Regulates Autolysosome Reformation Via RAC1-MTOR Axis

ObjectiveTNFAIP8L2,the tumor necrosis factor-induced protein 8-like 2(TIPE2),is a known negative regulator of innate and adaptive immunity and plays an important role in maintaining immune homeostasis.TNFAIP8L2 can decrease the phagocytosis of innate immunity and oxidative burst strength by combining and blocking RAC1 GTPase activity.However,the mechanism of TNFAIP8L2 regulats the immune response is still not completely clear.Moreover,RAC1 can bind to mammal rapamycin target protein(MTOR)directly and raised its kinase activity.Many signaling pathway and effector proteins are invoved in MTOR complex activation.The mechanism of RAC 1 regulating MTOR signaling pathways is still not entirely illuminated.Autophagy,which is ubiquitous in eukaryotes,eliminates intracellular wrong structure proteins or damaged organelles by lysosome degradation pathway and reused rely on autophagy related protein(ATGs).Studies have shown that autophagy invoved many diseases such as alzheimer’s disease,tumor,inflammation,aging and so on.Autophagy also played an important role in metabolic diseases.Therefore,further research of the mechanism for autophagy will provide new theoretical basis for disease treatment targeting autophagy.MTORC1 activity in response to nutrient oscillations and other environmental changes stimulated by feeding or fasting and control.MTORC1 applies inhibitory phosphorylation marks to unc-51-like autophagy-activating kinasel(ULK1)and ATG13,two key early effectors in the induction of autophagy.Recently,it was found that MTORC1 not only control the initiation of autophagy,also involved in the terminal stage of autophagy,the regeneration of autophagic lysosome refermation.It greatly improved the people understanding of autophagy.In our previous study found that TNFAIP8L2 can promote the tumor cell death induced by starvation and related to the autophagy pathway,so we try to clarify whether the mechanism of TNFAIP8L2 regulates tumor cell death is related to its negative regulation of immune function.In order to define the mechanism of TNFAIP8L2 promotes tumor cell death and impacts on autophagy,our research include following there parts:Part I.TNFAIP8L2 impaired autophagy flux and inhibited MTOR signaling pathway.Part II.The mechanism of TNFAIP8L2 regulated MTOR signaling pathway.Part Ⅲ.TNFAIP8L2 inhibited inflammation by regulation MTOR in LPS-induced endotoxemia model.Methods1.TNFAIP8L2 promotes cell death by inhibiting MTORC1 and blocking ALR.1.The expression of TNFAIP8L2 in different cell lines and knockout mice.Western blot was used to detect the expression of TNFAIP8L2 in HepG2 cell line,BEL-7402 hepatocellular carcinoma cell line,HEK-293T cell line,BMDMs,HeLa cell line and H1975 lung cancer cell line.2.TNFAIP8L2 regulated autophagic flux induced by starvation.In BMDMs of Tnfaip812 knockout mice or HepG2 cells overexpressed of TNFAIP8L2 plasmid,autophagy was induced by starving cells different time.And then autophagy marker LC3B and SQSTM1 were detected by western blot.Secondly,the dynamic changes of autophagy flow were monitored after HepG2 cells were infected by mCherry-GFP-LC3B in tandem with fluorescent adenovirus in accordance with the adenovirus operation manual.3.The effect of TNFAIP8L2 on initiation and fusion of autophagy.Treated the BMDMs of Tnfaip8l2 knockout mice or HepG2 cells overexpressed of TNFAIP8L2 plasmid with RAPA,BafAl for 6 hrs.LC3B and SQSTMlwere detected by western blot method and autophagosome LC3B markers were observed by immunofluorescence method.4.TNFAIP8L2 promoted starvation-linduced cell death by impairing autophagic lysosome reformation.After 24 hours of overexpression of TNFAIP8L2 plasmid in HepG2,autophagy was induced by starving them with glutamine-free DMEM.The formation of autophagosomes and autophagolysosomes were observed by immunofluorescence assay.The number and size of the autophagosomes and autophagolysosomes were statistical.5.TNFAIP8L2 inhibits normal activation and starvation-induced reactivation of MTORC1 signaling pathway.HepG2 with overexpression of TNFAIP8L2 plasmid or BMDMs from Tnfaip8l2 knockout mice were starved with glutamine-free DMEM to reactivate the MTORC1.The expression levels of p-MTOR,p-RPS6 and other molecules were detected by western blot.In the same system,the level of p-RPS6 was detected and analyzed by immunofluorescence.II.The mechanism of TNFAIP8L2 regulates MTORC1.1.TNFAIP8L2 interacts with RAC1 and enables the disassociation of RAC1-MTOR complex.a.In HEK-293T cells,the combination of RAC1 to endogenous MTOR was confermed by Co-IP.b.Co-IP detected whether MTOR could bind to the TNFAIP8L2-RAC1 complex.c.Co-IP detected the binding of RAC1 and MTOR in BMDMs from Tnfaip8l2 knockout mice.d.HEK-293T cells were transfected with MYC-tagged RAC1 in the presence of increasing Flag-tagged TNFAIP8L2 plasmids(0,1.5 and 3 μg).Cell lysate were performed Co-IP.e.The RAC1Q61L mutant plasmid and WT TNFAIP8L2 plasmid of different concentrations were transfected to HEK-293T cells.The binding of RAC1 and MTOR was detected by Co-IP.f.Both the TNFAIP8L2k15/16Q mutant or WT TNFAIP8L2 plasmid and the RAC1 plasmid were overexpressed in HEK-293T cells.The binding of RAC1 and MTOR was detected by immunoprecipitation.2.TNFAIP8L2 competitively inhibited RAC1 bind to MTOR on lysosome.a.Lysosomes were isolated from cells,and RAC1 was detected by western blot.b.Cells transfected with wild type,inactivation and activation of RAC1 mutant plasmids or treated with NSC23766-inhibitors of RAC1 GTPase were used to isolate lysosomes and then detected the RAC1 type by western blot.c.Lysosomes were isolated after the HEK-293T cells were transfected with MYC-tagged RAC1 active mutants in the presence of increasing Flag-tagged TNFAIP8L2,and p-MTORC1 in lysosome lysate was detected by western blot.3.TNFA1P8L2 inhibits normal and serum-induced reactivation of MTORC1.In the system of short-term starvation and serum-induced restimulation,the activation of MTORC1 was detected by western blot after TNFAIP8L2 was overexpressed or knocked out,respectively.To verify the above results,immunofluorescence was performed in the same system.4.TNFAIP8L2 negatively regulates MTORC1 signaling pathway by RAC1.a.Cells transfected with wild type,inactivation and activation of RAC1 mutant plasmids,and the level of MTORC1 signaling pathway under starvation and serum reactivation conditions were detected by western blot.b.Both wild TNFAIP8L2 plasmid and RAC1Q61L mutant plasmid or not were transfected to cells.The level of MTORC1 signaling pathway were detected by western blot.c.Cells were overexpressed the TNFAIP8L2K15/16Q or the RAC1Q61L mutant plasmid,the level of MTORC1 signaling pathway were detected by western blot.5.TNFAIP8L2 affects the autophagy pathway in a RAC1-dependent manner.Cells were overexpressed the WT TNFAIP8L2 and TNFAIP8L2K15/16Q mutants plasmid,and western blot were used to detected LC3B and SQSTM1.Ⅲ.Tnfai8l2 deficiency leads to overactivation of MTORC1 and inflammation.a.BMDMs from WT or Tnfai8l2 knockout mice were activated by LPS.RAPA were used to inhibit MTORC1 pathway.The expression of IL-6 and TNF were detected by ELISA.And the level2 of MTORC1 was detected by western blot.b.Mice endotoxemia model was constructed by intraperitoneal injection of LPS into the wild type and knockout mice with or without treated with rapamycin.The level of cytokine were detected by ELISA and p-RPS6 expression in lung tissue was detected by IHC.Inflammatory cell infiltration in lung tissue was observed by HE.Results1.TNFAIP8L2 promotes cell death by inhibiting MTORC1 and blocking ALR.1.The expression of TNFAIP8L2 in different cell lines and knockout mice.First of all,we tested the expression of TNFAIP8L2 in HepG2 cells,BEL-7402 cells,HEK-293T cells,HeLa cells,H1975 cells and BMDMs.The results showed that BMDMs,HeLa and H1975 with TNFAIP8L2 expression level in turn reduce.2.TNFAIP8L2 regulated autophagic flux induced by starvation.The BMDMs of Tnfai8l2-/-mice and TNFAIP8L2 overexpressed HepG2 were induced autophagy by starvation cells with glutamic-free medium,and LC3B and SQSTM1 were detected by western blot.The results showed that autophagy marker LC3B and SQSTM1 increased simultaneously after overexpression of TNFAIP8L2,while both LC3B and SQSTM1 decreased after knockout of Tnfai8l2.These results suggested that autophagy flow was blocked.3.The effect of TNFAIP8L2 on initiation and fusion of autophagy.We further examined which stage would likely be controlled by TNFAIP8L2.We,therefore,measured the changes in LC3B and SQSTM1 in response to the MTOR inhibitor,rapamycin and inhibitor of the autophagosome-lysosome fusion,bafilomycin A1,separately.Treatment with bafilomycin A1 had no obvious impact on the differences of LC3B-II and SQSTM1 induced by TNFAIP8L2;however,rapamycin treatments almost completely abolished these differences in both HepG2 and BMDMs cell lines.Further,the immunofluorescence analysis results were consistent with these previous findings4.TNFAIP8L2 promoted starvation-induced cell death by impairing autophagic lysosome reformation.a.Immunofluorescence were used to observe the ALR.When TNFAIP8L2 was overproduced,the numbers of autolysosomes indicated by LAMP 1+LC3B+(yellow)puncta were significantly decreased after 4 h of starvation,and its mean size was clearly much larger than wild type cells after 0 h,8 h,and 10 h of starvation.Further,much fewer lysosomes(LAMP+vesicles)were regenerated in TNFAIP8L2-overexpressing cells after prolonged starvation for 8 h and 10 h.These data showed that ALR was impaired,and the full complement of lysosomes restoration was blocked.b.Cells with overexpression of TNFAIP8L2 and starvation cells at different time induced autophagic cell death.The number of cell death was detected by flow cytometry.The results showed that the number of cell death of overexpressed TNFAIP8L2 increased significantly compared with the control group.5.TNFAIP8L2 inhibits normal activation and starvation-induced reactivation of MTORC1 signaling pathway.BMDMs of TNFAIP8L2 knockout mice and HepG2 of liver cancer cells with overexpression of TNFAIP8L2 were used to induce the reactivation of MTORC1 signaling pathway by starvation them with glutamine-free DMEM,and the expression levels of p-MTORand p-RPS6 were detected by western blot.The results showed that p-MTOR and p-RPS6 decreased in the basal and reactivated state after overexpression of TNFAIP8L2.It indicated that the MTORC1 signaling pathway was inhibited.While p-MTOR and p-RPS6 increased after TNFAIP8L2 deletion.Ⅱ.The mechanism of TNFAIP8L2 regulates MTORC1 signaling pathway.1.TNFAIP8L2 interacts with RAC1 and enables the disassociation of RAC1-MTOR complex.a.In HEK-293T cells,the RAC1 plasmid was overexpressed and the binding of RAC1 to endogenous MTOR was detected by immunoprecipitation.b.Immunoprecipitation was used to detected the exogenous TNFAIP8L2 and the immunoco-precipitation method verified that TNFAIP8L2 could bind to RAC1,and found that RAC1 could not bind to MTOR after binding to TNFAIP8L2.c.The binding of RAC1 to MTOR was enhanced in BMDMs with TNFAIP8L2 knockout.d.Both the RAC1 plasmid and the WT TNFAIP8L2 plasmid of different concentrations were overexpressed in HEK-293T cells.The binding of RAC1 and MTOR was detected by immunoco-precipitation.The results of immunoco-precipitation showed that TNFAIP8L2 could competitively inhibit the binding of RAC 1 and MTOR and was concentration-dependent.e.In HEK-293T cells,RAC1Q61L and inreased TNFAIP8L2 plasmids were overexpressed simultaneously.The results of immunoprecipitation showed that TNFAIP8L2 could inhibit the binding of RAC 1 and MTOR and was concentration-dependent.f.In HEK-293T cells,both the TNFAIP8L2 plasmid and the RAC1 plasmid with over-expression of mutations in the RAC1 binding region and the RAC1 binding region were overexpressed.g.The binding of RAC 1 and MTOR was detected by immunocoprecipitation,and the inhibitory effect of TNFAIP8L2 mutant on the binding of RAC1 and MTOR was found disappeared.h.Immunofluorescence was used to detect the combination of RAC1 and MTOR in BMDMs or HepG2 overexpressed TNFAIP8L2.2.TNFAIP8L2 competitively inhibited RAC1 bind to MTOR on lysosome.a.First of all,separating lysosomes to figure out whether RAC1 can locate on lysosome or not.b.Cells were overexpressed WT,inactivation and activation of RAC1 plasmid and then were separated lysosome.Results show that the active form of RAC1 easier to locate to the lysosomes.c.Lysosomes were isolated after RAC1Q61L and increased TNFAIP8L2 overexpressed,and the activation of MTORC1 was detected by western blot.3.TNFAIP8L2 inhibits normal and serum-induced reactivation of MTOR signaling pathway.Overexpression or knockout TNFAIP8L2,short time sarvation to inhibit MTOR,then cells treated by FBS for 20 min to induced MTOR reactivation.western blot and immunofluorescence method to detect MTORC1 activation.Results showed that overexpression TNFAIP8L2 inhibit basic and reactivated of MTOR signaling pathways.4.TNFAIP8L2 negatively regulates MTORC1 signaling pathway by RAC1.a.western blot were used to detect MTORC1 signaling pathway after overexpression of wild type,inactivated mutants and activated mutants of RAC1.It was found that the activity of MTORC1 signaling pathway increased with the increase of RAC1 activity.b.Overexpression of both WT TNFAIP8L2 and RAC1Q61L plasmid or not,western blot were used to detect the changes of MTORC1 signaling pathway under basic,starvation and serum reactivation conditions.The results showed that TNFAIP8L2 could block the activation of MTORC1 by blocking RAC1Q61L.c.Cells were treated same as(b),WT TNFAIP8L2 instead of TNFAIP8L2K15/16Q The results showed that TNFAIP8L2K15/16Q mutant plasmid inhibited the upregulation of MTORC1 activity by RAC1Q61L active mutants.5.TNFAIP8L2 affects the autophagy pathway in a RAC1-dependent manner.Overexpression of WT TNFAIP8L2 and TNFAIP8L2K15/16Q,LC3B and SQSTM1 were detected by western blot.The results showed that WT TNFAIP8L2 promoted the accumulation of LC3B and SQSTM1,while the mutant TNFAIP8L2K15/16Q had no effect on the accumulation of LC3B and substrate SQSTM1.Ⅲ.Tnfai8l2 deficiency leads to overactivation of MTORC1 and inflammation in BMDMs.1.Tnfai8l2 deficiency leads to the upregulation of inflammatory cytokines result from the overactivation of MTORC1 in BMDMs.To evaluate whether the anti-inflammatory role of TNFAIP8L2 depends on the MTORC1 pathway,we generated Tnfaip8l2 knockout mice using CRISPR-Cas9 as described in the Methods.In macrophages,Tnfaip8l2 deficiency exacerbated inflammatory responses by increasing pro-inflammatory cytokines IL6 and TNF upon the activation of the Toll-like receptor pathway by lipopolysaccharide(LPS)stimulation.At the same time,the increased IL6 and TNF were markedly attenuated by blocking MTOR activity with rapamycin.This result indicated that TNFAIP8L2 might control inflammation by modulating MTOR activity.2.Tnfai8l2 deficiency exacerbates inflammation by up-regulation MTOR activity in LPS-induced endotoxemia model.We further measured the MTOR activity by blotting for p-MTOR and its downstream signal,p-RPS6.Compared to wild type mice,Tnfaip8l2 deficiency led to higher levels of p-MTOR and p-RPS6 in macrophages in the absence and presence of LPS stimulation and the rapamycin treatment obviously abrogated the increase of p-MTOR and p-RPS6.Thus,it is conceivable for us to conclude that Tnfaip8l2 deficiency can promote inflammatory responses by upregulating MTOR activity.Furthermore,in the LPS-induced endotoxemia model,we found that circulating pro-inflammatory cytokines IL6 and TNF levels in plasma were also enhanced in Tnfaip8l2-deficient mice compared with wild type mice,and these increases were almost eliminated by rapamycin.The Tnfaip8l2-deficient mice showed the lowest body temperature while inhibiting MTOR activity could alleviate this effect.Conclusion1.TNFAIP8L2 impairs autophagy flux following starvation and induces cell death.2.TNFAIP8L2 inhibits autolysosome reformation via negatively modulating MTOR reactivation.3.TNFAIP8L2 interacts with RAC1 and enables the disassociation of the RAC1-MTOR complex.4.TNFAIP8L2 suppresses the colocalization of active RAC1 and MTOR on the lysosome membrane by competitively binding to active RAC1.5.Activation of MTORC1 is negatively regulated by TNFAIP8L2 through interacting with GTP binding RAC 1.6.Tnfaip8l2 deficiency exacerbates inflammation by the upregulation of MTOR activity in an LPS-induced endotoxemia model.Innovation and significance1.We identified a novel mechanism of autolysosome reformation by TIPE2 mediated the inhibition of mTOR reactivation.2.TNFAIP8L2 suppresses mTOR activation via interacting with RAC1 and disassociating it from RAC 1-mTOR complex.3.TNFAIP8L2 controls the innate immunity by negatively regulating mTOR activation.Limitations1.The role of TNFAIP8L2 in other immune-related diseases needs further exploration.2.The regulation of autophagy by TNFAIP8L2 needs to more exploration.