E3Ubiquitin-ligase ANKIB1Regulates TRAF6Mediated Nf-κB Signaling Pathway

Background ANKIB1contains rich domain and motif, such as RBR and TRAF6. RBR is closely related to E3ligase. Ubiquitin-ligase enzymes play an important role in the process of protein ubiquitin, and is closely related to cell apoptosis, cell cycle and tumorigenesis. TRAF6is a core joint protein and the important molecule of varieties of signal pathway (such as NF-κB, Smad), and is the main signal conduction of IL-1receptor, TNF receptor superfamily and toll-like receptor superfamily. ANKIB1contains three TRAF6motifs, which indicates ANKIB1probably regulates TRAF6mediated NF-κB Signaling pathway. The ubiquitin-ligase activity of ANKIB1and the influence of some signal pathway by studing the interaction between ANKIB1and TRAF6will be explored.Objectives1. To verify the ubiquitin-ligase enzyme activity of ANKIB1.2. To analyse the function of ANKIB1targeted the ubiquitination of TRAF6and NF-κB. Methods1. The research of the ubiquitin-ligase activity of ANKIB1His-Ub, GST-UbcH5b, GST-UbcH7, GST-UbcH8, GST-ANKIB1(1-480), His-ANKI B1(330-570)fusion proteins were expressed in E.coli BL21(DE3). The GST-fusion proteins were purified by Glutathione Sepharose4B affinity chromatography, and the His-fusion proteins were purified by Nickel agarose gel FF affinity chromatography. Screening ubiquitin-conjugation enzymes interacted with ANKIB1by GST pull-down, and verify the ubiquitin-ligase activity of GST-ANKIB1(1-480) and His-ANKIB1(330-570) by in vitro ubiquitination experiment.2. The research of ANKIB1targeted to the self-ubiquitination of TRAF6The interaction between ANKIB1and TRAF6were detected by immune coprecipitat- ion, and the influence of ANKIB1targeted to the self-ubiquitination of TRAF6:transfected or co-transfected ANKIB1, TRAF6, Ub recombinant plasmid, and used TRAF6antibodies for bait, then Western Blotting detected the changes of ubiquitin chain.3. The study of ANKIB1regulating TRAF6mediated NF-kB signal pathwayThe influence of ANKIB1toward NF-kB signal pathway were detected by dual-luciferase report assay. Firstly, transfected or co-transfected ANKIB1, TRAF6, ANKIB1(1-480), ANKIB1(481-780), ANKIB1(781-1089) and pNF-kappaB-Luc, pRL-SV40. Then tested the relative luciferase activity. Transfected or co-transfected ANKIB1, TRAF6, ANKIB1(1-480), ANKIB1(481-780), ANKIB1(781-1089) recombinant plasmids, and stimulated with IL-1, then we tested the changes of IKK, IkB by Western Blotting.Results1. His-Ub, GST-UbcH5b, GST-UbcH7, GST-UbcH8, GST-ANKIB1(1-48O), His-ANKIB1(330-570) fusion proteins were purified.2. ANKIB1could interact with ubiquitin-conjugation enzymes UbcH5b, UbcH7and UbcH8by GST pull-down, and ANKIB1has ubiquitin ligase activity preliminary.3. TRAF6could interact with ANKIB1, ANKIB1(1-480), ANKIB1(481-780) and ANKIB1(781-1089).4. ANKIB1reduced the self-ubiquitination of TRAF6, and then reduced the phosphorylation of IKK and decreased the degradation of IκB, thereby inhibited the NF-κB signaling pathway.Conclusions1. ANKIB1is an RBR type ubiquitin-ligase.2. ANKIB1reduces the ubiquitination of TRAF6and inhibits NF-κB signaling pathway, then influences the expression of downstream genes by interacting with TRAF6, which lays a foundation of the latter ANKIB1’s function research.