| The innate immunity of lower vertebrates,as the first line of defense of the body,plays a key role in the fight against viral infection and invasion of pathogenic microorganisms.The research on TDP43(TAR DNA binding protein 43)in mammals mainly focuses on its role in regulating neurorelated diseases,and there are few reports on its role in the innate immune response of fish.In this study,the model organism zebrafish was used to explore the relationship between zebrafish TDP43 protein and protein kinase Z(PKZ),and their regulation on the expression of type I interferon.Firstly,obtain the gene sequence of Dr TDP43(Danio rerio TDP43),predict its structural domain,and construct its phylogenetic tree.It was found that Dr TDP43 has three conserved domains,namely NTD,RRM1,and RRM2.In addition to this,TDP43 also has a C-terminal low complexity tail.The protein shares over 80% homology with mammalian TDP43.There are two subtype genes in zebrafish that are homologous to human TDP43,namely tardbp(CDS: 1236bp)and tardbpl(CDS: 912 bp).In this article,Dr TDP43 is a variant of the tardbpl gene,CDS 1191 bp.At the same time,we detected the expression level of Dr TDP43 m RNA at the individual zebrafish and grass carp cell levels.After infection with GCRV or viral analogue B-DNA,the expression of Dr TDP43 m RNA in different tissues(brain,gills,liver,skin,eye,heart,kidney)of zebrafish or grass carp kidney cells was studied during the stimulation gradient time period(0 h,6 h,12-96 h).The results showed that after virus stimulation,the m RNA expression level of Dr TDP43 was upregulated compared to the control group.By using subcellular localization to observe the distribution of zebrafish TDP43 in CIK cells,we found that zebrafish TDP43 is mainly distributed in the nucleus,with only a small portion located in the cytoplasm,while PKZ only locates the cytoplasm.Subsequently,experimental methods using Co-IP and WB were used to confirm that TDP43 can bind to PKZ.Subsequently,the expression regulation of IFN Ⅰ by TDP43 was explored.Overexpression of TDP43 alone inhibits the expression of IFN Ⅰ,and knocking down Dr TDP43 upregulates the expression of IFN Ⅰ.Co-transfection of TDP43 and PKZ downregulated the expression of IFN Ⅰ compared to single transfection of PKZ.We also explored the regulation of PKZ activity by TDP43.Overexpression of TDP43 resulted in a downregulation of PKZ expression and inhibition of PKZ and IRF3 phosphorylation;After interfering with TDP43,the expression of PKZ was upregulated,and the phosphorylation of PKZ and IRF3 was enhanced.In short,zebrafish TDP43 responds to viral stimulate and binds to PKZ,inhibiting PKZ and IRF3 phosphorylation and PKZ dimerization,and ultimately downregulating IFN Ⅰ expression. |
