Molecular Mechanism Of Miiuy Croaker Smyd3 Regulating TAK1-Mediated Immune Response

Miichthys Miiuy is an important economic fish in aquaculture,which has not only high nutritional value,but also medicinal value,so M.Miiuy can bring a lot of economic benefits,as more and more people start to breed M.Miiuy,the disease of M.Miiuy is becoming more and more serious,which not only hinders the normal growth of M.Miiuy,but also causes huge economic losses to the breeders.Therefore,in order to reduce the loss of fish caused by diseases,it is necessary to study the immune system of fish deeply,to improve the immunity of fish to pathogenic bacteria or viruses,so as to improve the benefit of fish farmers,the harm caused by disease breeding industry should be solved from the root.In this study,we looked at the innate immune system of M.miiuy,a innate immune system whose cellular recognition do not specifically target pathogens.Unlike adaptive immune system,innate immune system do not provide long-lasting protective immunity,but they have a rapid anti-infective effect in all plants and animals.The innate immune system is also the host’s first line of defense against pathogens.The innate immune system includes three pattern recognition receptor receptors（PRRs）:TLRs,NLRs and RLRs.Toll-like receptor signal transduction includes five adaptor proteins:Myd88,MAL,TRIF,TRAM and SARM.With the exception of TLR3,all signal transductions can be transmitted through Myd88.In the My D88-dependent signaling pathway,it can recruit interleukin-1 receptor-associated kinase（IRAKs）complexes,which comprise four subunits:two active kinases（IRAK1 and IRAK4）and two non-catalytic subunits（IRAK2 and IRAKM）.Phosphorylation of IRAK1 can activate downstream transduction pathways via TRAF6.IRAK-TRAF6 complex induces ubiquitination or deubiquitination of TAK1,thereby activating the NF-κB signaling pathway downstream.Therefore,TAK1 is an indispensable member of NF-κB signaling pathway in innate immune response.It is of great significance to study the mode of action of TAK1 and its related regulatory protein Smyd3.In this paper,we studied that Smyd3 can attenuate the expression of NF-κB downstream by inhibiting the expression of TAK1.Smyd3 negatively regulates the signal transduction of NF-κB pathway by targeting the degradation of TAK1.The conclusions are as follows:1.Overexpression of Smyd3 inhibited the transcription of inflammatory cytokine,in the downstream of NF-κB.Moreover,the reporter gene was activated obviously after the addition of TAK1 and LPS,and Smyd3 still inhibited the reporter gene.2.q RT-PCR showed that Smyd3 could inhibit the expression of downstream inflammatory factors at m RNA level,and RNA interference showed that the expression of downstream inflammatory factors increased after Smyd3 was inhibited.3.Since activation of NF-κB requires the mediation of TAK1,this article also validated whether Smyd3 targets TAK1 to regulate the NF-κB signaling pathway.Dual-luciferin experiments showed that Smyd3 was able to attenuate TAK1-mediated NF-κB signaling pathway,and the inhibitory effect on TAK1-mediated NF-κB signaling pathway also increased with increasing Smyd3 concentration.4.The relationship between Smyd3 protein and TAK1 protein was further studied after it was determined that Smyd3 could inhibit TAK1-mediated NF-κB signaling pathway.Western Blot showed that overexpression of Smyd3 decreased the expression of TAK1,and the expression of TAK1 decreased with the increase of Smyd3 concentration and transfection time.5.Smyd3 was found to interact directly with TAK1 by co-Immunoprecipitation thus affecting TAK1 protein expression.6.To further investigate the domain of Smyd3 interacting with TAK1,the domain of TAK1 was predicted by smart software,and primers were designed to mutate its domain,which revealed that Smyd3 interacts with the STK domain of TAK1,it is the main domain that promotes the degradation of TAK1.7.We used proteasome inhibitor（MG132）,autophagy inhibitor（3-MA）,and lysosome inhibitor（NH₄Cl）to treat cells transfected with TAK1 and Smyd3 or Smyd3-transfected Miichthys Miiuy cells,it was found that MG132 could inhibit the degradation of TAK1 whether exogenous or endogenous.The above experiments indicated that Smyd3 induced the degradation of TAK1 through proteasome pathway.8.To further study the degradation of TAK1.TAK1,Smyd3 and ubiquitin（Ub）plasmids were transfected into HEK293 cells,and Western Blot showed that Smyd3could promote the ubiquitination of TAK1.To specifically investigate the sites of ubiquitination of TAK1,experiments transfected lysine 48 site mutation（K48-Ub）as well as lysine 63 site mutation（K63-Ub）into HEK293;The results showed that Smyd3 could promote the K48-linked ubiquitination of TAK1.9.The methyltransferase inactivating mutant Smyd3-F188A was constructed,the results showed that Smyd3,independent of its methyltransferase activity,promoted TAK1 K48-linked ubiquitination levels and thereby inhibited TAK1-mediated transduction of the inhibitory NF-κB signaling pathway.Taken together,Smyd3,as a negative regulator of the NF-κB signaling pathway,negatively regulates the transduction of the NF-κB signaling pathway by targeting TAK1 and binding to it to enable the K48-linked ubiquitination of TAK1;The expression of IL-8 and IL-1βwas detected by q RT-PCR.This paper provides new data for the study of the innate immune system of teleost,which not only fills the gap of Smyd3 in fish innate immune system,but also enriches the research contents of NF-κB signaling pathway in teleost,these data can also provide a reference for the future study of innate immunity in mammals.