The E3 Ligase WAZX2 Positively Regulates TBK1-mediated Antiviral Response Through Ubiquitination Of TBK1

ObjectivePart 1:TBK1 is the essential factor for the production of IFN-?and anti-viral innate immunity response.The modification of TBK1 can be an influence factor.Here we conformed an E3 ligase WAZX2,catalyzed the polyubiquitination of TBK1,and investigated the function of WAZX2 in anti-viral innate immunity response.Part 2:Sézary syndrome,a leukemic variant of cutaneous T-cell lymphoma,is characterized by its resistance to apoptosis.Recent evidence has revealed the importance of SATB1 in the apoptosis resistance of Sézary syndrome.In this study,we showed that SNF5 is an upstream regulator of SATB1in several conditions and that both are deficient in Sézary cells.We have confirmed the effect of SNF5 and SATB1 in apoptosis resistance.Thus,targeting SNF/SWI complex may represent a promising approach for Sézary syndrome therapy.Methods Part 1:1.VSV-?51 GFP infected 293T cells,then observed the GFP cells that are infected cases by inverted microscope and analyzed by flow cytometry.2.After 24h infection of SeV in 293T cells,RNA from the cells was extracted,and the expression of IFN-?was detected by real-time PCR.In addition,the luciferase reporter was detected by the instrument.3.Used LPS to stimulate or VSV virus infect iBMDM cells,the RNA of cells was collected at 4h,8h and 12h respectively,and the expression of the WAZX2 in mRNA level was detected.4.The effect of WAZX2 on the activity of the promoter of IFN-?was detected by using the fluorescence reporting gene technique with RIG-I N,MAVS,TBK1,IKKi and IRF3,5D stimulated signaling.And the interaction was detected by Co-immunoprecipitation.5.Used label immune coprecipitation method to detect the ubiquitin level changes of TBK1.And the modification is whether K48-linked or K63-linked ubiquitin.Part 2:1.Genome wide gene expression profile analysis between SATB1 and SNF5.2.qRT-PCR and Western Blot was used to find out the expression of SATB1 in snf^-/-MEF cells,thymocyte cells,T cells and lymphocyte.3.We performed Co-IP assay to investigate the association between SATB1 and SNF5 in cellular extract from 293Tcells stably expressing Flag-hSNF5 or Jurkat cells.4.qRT-PCR were used to find out the expression of SATB1 and SNF5 in Sézary cells.Flow cytometry analysis and Western Blot was performed to analyze the apoptosis in Sézary cells with repressing SNF5 and SATB1 or sh hSATB1.Results:Part 1:1.The cells were infected with VSV-?51 GFP,we observed the GFP cells were less than control in WAZX2 group.2.The cell with WAZX2 expression after 24h infection of SeV,mRNA level of IFN-?was elevated.And the result of fluorescence reporter assay was elevated,too.3.The cells were stimulated with LPS,the expression of the WAZX2 in mRNA level was elevated.The same result was shown in the VSV virus-infected iBMDM cells.4.The effect of WAZX2 on the activity of the promoter of IFN-?was positive in RIG-I N,MAVS,TBK1 induced signaling.The interaction of WAZX2 and adaptor was detected by Co-immunoprecipitation.5.WAZX2 increase TBK1 ubiquitination.WAZX2 promotes K63-linked ubiquitination of TBK1.Part 2:1.Genome wide gene expression profile analysis shows that the loss expression of SNF5 with low expression of SATB1.2.qRT-PCR and Western Blot assay shows that the expression of SATB1 is low in snf^-/-MEF cells,thymocyte cells,T cells and lymphocyte.3.The similarity of STAB1 from human and mice reached 95%,and the homology of hSATB1 and scSNF5 reached 26%.The homology of hSNF5 and scSNF5 reached36%.Co-IP assay shows that SATB1 and SNF5 interacts with each other in Hut78cells.4.Expression of SATB1 is reduced in and SNF^-/-CD8⁺T-cells lymphoma,and the same situation observed in CD4⁺T-cells and Sézary cells.Hut78 cells with repressing SATB1 and SNF5 show the poor apoptosis resistance.And the same situation in the expression of cleaved Caspase 8 and cleaved Caspase 3.Conclusion:Part 1:1.WAZX2 positively regulates anti-viral innate immunity response2.WAZX2 positively regulates the produce of IFN-?and the signaling.3.VSV infection and LPS stimulation induce WAZX2 expression in macrophage cells4.The effect of WAZX2 on the activity of the promoter of IFN-?was positive in RIG-I N,MAVS,TBK1 induced signaling.The interaction between WAZX2 and TBK1 is detected by Co-immunoprecipitation.5.WAZX2 promotes ubiquitination of TBK1.The ubiquitination of TBK1 is attenuated with WAZX2 loss.WAZX2 promotes K63-linked ubiquitination of TBK1to enhance anti-viral innate immunity response.Part 2:1.Microarray reveals that SATB1 expression level is reduced upon SNF5 loss.2.qRT-PCR and Western blot confirm that SATB1 expression level is reduced upon SNF5 loss.3.SATB1 physically associates with SWI/SNF complex.4.hSNF5 deficiency induces apoptosis resistance by repression hSATB1 expression in Sézary syndrome.