The Effect And Mechanisms Of E3 Ubiquitin Ligase SIAH2 In TRIF-Dependent TLR Singalling Pathway And The Biological Functions Of CG2974 In Drosophila

Background:Toll-like receptors（TLRs）are transmembrane proteins expressed by cells of the innate immune system,which recognize invading microbes and activate signaling pathways that launch immune and inflammatory responses to destroy the invaders.TLR signaling pathways are classified into My D88-and TRIF-dependent pathways,based on specific adaptor recruitment.These two pathways are collectively responsible for the activation of the MAPKs and IKKs,which in turn,activate a suite of transcription factors,such as Nuclear Factor kappa-light-chain-enhancer of activated B cells（NF-κB）and members of the interferon regulated factor（IRF）family.E3 ubiquitin ligase SIAH2 is composed of the Really Interesting New Gene（RING）catalytic domain,two zinc finger domains and a substrate binding domain.In mammals,SIAH2 mediates efficient ubiquitination and degradation of substrates that play an important role in diverse stress and cytokine-activated signaling pathways.Objective:To explore the mechanism of SIAH2 in TRIF-dependent TLR signaling pathways.Method:In our study,we used cell as models,overexpression and CRISPR/Cas9knockout as a research method.The localization and interaction of TRIF and SIAH2 were studied by Western blot and immunofluorescence confocal observation.The effects of SIAH2 on TLR pathway were investigated by co-transfection and dual-luciferase reporter gene assay.Results:The dual-luciferase reporter assay showed that overexpression of SIAH2inhibited TRIF-dependent TLR signaling pathway.The combination of SIAH2 and TRIF was found by immunoco-precipitation and GST-pull down experiment.The localization of TRIF in SIAH2^KO cells is different from wild-type.Conclusion:Here we report that SIAH2 negatively regulates TLR signaling pathway by targeting TRIF.In addition,SIAH2 and TRIF in the same complex in HEK 293T cells,and TRIF localization is changed in SIAH2^KO cells,suggesting that SIAH2 inhibits TRIF by affecting subcellular localization of TRIF.Background CG2974（NAD（P）H-hydrate epimerase,NAXE）,is highly conserved from fruit flies to mammals.However,the biological function of CG2974 in drosophila is not known yet.Sequence analysis showed that CG2974 in Drosophila had high homology with NAXE in human.Studies of NAXE protein in mammals have shown that it regulates the transport of cholesterol and repair of both epimers of NAD（P）HX to prevent excessive accumulation of toxic metabolites.In addition,according to clinical studies,the abnormal function of NAXE is closely related to human diseases.For example,biallelic mutations in NAXE were found in patients with an infantile,lethal,neurometabolic disease.In addition,NAXE was found mainly in the serum of urine,cerebrospinal fluid,and some sepsis patients,but not in the serum of healthy patients.These findings suggest that NAXE may play a key role in immune and metabolic processes.Objective To explore the biological functions of CG2974 in Drosophila.Method In this study,CG2974 mutant which was generated by CRISPR/Cas9 was used as model to explore the influence of CG2974 on survival rate in response to infection or stress stimulation.Results We found that the localization of NAXE cells in Drosophila was basically the same as that in mammals,and it was in the cytoplasm and nucleus.Howerer,the lifespan,immune response,stress,and metabolic response of the CG2974 mutant in drosophila had no significant different on its survival,and CG2974 is not located on mitochondria.Conclusion Due to homologue of Apolipoprotein A-I（apo A-I）,a protein which combined with NAXE was not in Drosophila.It suggests that CG2974 plays A similar function in mammals,also need to rely on the existence of apo A-I.Furthermore,CG2974 is highly conserved in evolution,and it is suggested that CG2974 has an indispensable and important function,so the redundancy of function may mask the phenotype that is missing in CG2974.All in all,Drosophila is a powerful model organism with multiple advantages,and its application in life science is of immeasurable value.Therefore,using Drosophila as a model organism to study the biological function of NAXE in Drosophila is of great significance to reveal the mechanism of the metabolite repair system affecting the immune response of the body and the fatal neurometabolic disorder in early childhood.