Study On Molecular Regulation Mechanism Of MITA And TBK1 In Miichthys Miiuy

The host has evolved an immune system composed of innate immunity and adaptive immunity in the process of resisting pathogens.As the primary defense system of host,the innate immune system is the first line of defense against the invasion of exogenous pathogens.In the lower vertebrates(such as fish,etc.),the innate immune system is the most important defense system to exercise immune function.The pathogen associated molecular patterns(PAMPs)are recognized by a class of receptors called pattern recognition receptors(PRRs)in innate immunity.After recognizing pathogens,PRRs can activate the signaling pathways to produce cytokines to eliminate invading pathogens.As one of the most important PRRs,RIG-I receptors(RLRs)are the most important recognition receptors in the cytoplasm,capable of recognizing viral RNA and playing a crucial role in antiviral innate immunity.Mediator of IRF3 activation(MITA),an interferon stimulator,can activate transcription factor IRF3,so it is one of the most important bridging molecules in RLRs antiviral signaling pathway.MITA can mediate viral ds DNA and RNA-induced signaling pathways.When viral ds DNA is recognized by intracellular receptors,MITA is dimerized and phosphorylated to activate downstream signaling pathways and promote transcriptional expression of interferon.RLRs can recognize viral RNA and activate the downstream signaling pathway,while MITA can undertake the signals of upstream molecules and further amplify and transmit the signals.Tank-binding kinase 1(TBK1)can interact with TANK and TRAF2 to regulate NF-κB signaling pathway.As a member of the non-classical IκB kinase family,TBK1 can also target the activation of IRF3 and IRF7 to promote their dimerization into the nucleus,thus activating the transcriptional expression of interferon.TBK1 plays an important role in the innate immune system by inducing activation of the transcription factors NF-κB and IRF3.At present,studies on the RLRs signaling pathway are mostly focused on higher vertebrates,while studies on the regulatory mode and mechanism of the RLRs signaling pathway in fish are few.In this study,we found molecules that can regulate MITA and TBK1 in the RLRs signaling pathway,and discussed the regulatory mechanism.It is expected to add some new ideas to the molecular regulation and regulation mechanism of the antiviral immune signaling pathway mediated by RLRs in bony fish.The main results of this study are as follows:1.IRF4 b and IRF8 inhibit NF-κB signaling pathway by promoting the degradation of MITA.Firstly,bioinformatics was used to analyze the evolutionary conservation of IRF4 b and IRF8 genes from fish to mammals.The results were found that IRF4 b and IRF8 were relatively conserved in evolution.Then q RT-PCR detection showed that the expression levels of IRF4 b and IRF8 in liver tissue of Miichthys miiuy increased with the increase of infection time under poly(I:C)stimulation and SCRV infection.It was determined that IRF4 b and IRF8 could participate in the antiviral immune response of the host.In addition,this study demonstrated that MITA can activate the NF-κB signaling pathway.However,IRF4 b and IRF8 could inhibit the activation of NF-κB signaling pathway by inhibiting MITA.At the protein level,IRF4 b and IRF8 could promote the degradation of MITA.By constructing sh RNA knockdown plasmids of IRF4 b and IRF8,this study proved that MITA could not be degraded after knockdown of IRF4 b and IRF8.When studying the structure of IRF4 b and IRF8 proteins,it is found that they have two same domains: the IRF domain and the IRF3 domain.In the mutant experiment,it was found that IRF4 b and IRF8 could not promote the degradation of MITA at the protein level after the IRF domains were mutated.When studying the degradation mechanism,the experimental results showed that IRF4 b and IRF8 could promote the degradation of MITA,but this process could be blocked by proteasome inhibitors.Therefore,it was determined that IRF4 b and IRF8 promote the degradation of MITA through ubiquitin-proteasome pathway.Subsequently,the effects of IRF4 b and IRF8 on MITA ubiquitination were tested,and it was found that they could promote the polyubiquitination of MITA.In conclusion,IRF4 b and IRF8 can inhibit the activation of NF-κB signaling pathway by promoting the degradation of MITA.2.Crlf3 inhibits the antiviral immune pathway by promoting the degradation of TBK1.In this study,Crlf3 was screened as a possible molecule involved in innate immune regulation.Bioinformatics was used to analyze the evolutionary conserved nature of Crlf3 gene from fish to mammals,and it was found that Crlf3 was relatively conserved.When the expression level of Crlf3 was analyzed,it was found that the expression level of Crlf3 in kidney and spleen of Miichthys miiuy was relatively higher in both m RNA level and protein level.Subsequent studies found that not only the expression level of Crlf3 increased with SCRV infection,but also Crlf3 promoted SCRV replication.Therefore,Crlf3 can be determined to be involved in the antiviral immune response and possibly in the escape immune mechanism of the virus.Further studies confirmed that Crlf3 inhibits transcription of interferon and interferon-stimulating factors,and the effect is more pronounced in the presence of SCRV.After silencing Crlf3 by specific si RNA,the q RT-PCR results showed that the expression of interferon and interferon-stimulating factors increased.Subsequently,the effects of Crlf3 on the activity of promoters such as IRF3,ISRE,and IFN1 were investigated.It was found that Crlf3 could inhibit promoter activity of IRF3,ISRE,and IFN1.Subsequently,the target molecules that Crlf3 may regulate were screened.The co-IP experiment showed that Crlf3 protein could interact with MAVS,MITA,TBK1,and IRF3 proteins.Subsequent studies showed that Crlf3 could promote the degradation of TBK1 at the protein level,but had no effect on other target molecules.Further studies also found that Crlf3 can inhibit the phosphorylation of MITA and IRF3 by promoting the degradation of TBK1.After analyzing the structure of Crlf3 protein,it was found that when the N-terminal domain of Crlf3 was mutated,Crlf3 could not interact with TBK1 at the protein level and promote its degradation.When the STK domain of TBK1 was mutated,TBK1 could not interact with Crlf3.When studying the degradation mechanism,the experimental results showed that Crlf3 could promote the degradation of TBK1,but this process could be blocked by proteasome inhibitors.Therefore,it was determined that Crlf3 promoted the degradation of TBK1 through ubiquitin-proteasome pathway.In further studies,Crlf3 was found to promote the K48-link polyubiquitination of TBK1.In conclusion,Crlf3 promoted the degradation of TBK1 ubiquitin-proteasome pathway through K48-linked polyubiquitination in RLRs antiviral immune signaling pathway.