Molecular Regulation Mechanism Of MyD88-mediated Signaling Pathway In Miichthys Miiuy

The immune system is a class of defense mechanisms that protect against or eliminate pathogens and sustain the organism in homeostasis.The immune system is generally defined as innate and adaptive immunity,with innate immunity being the first line of defense for most biological organisms against pathogenic invasion.As lower vertebrates,fish are functionally conservative,and although they have both innate and adaptive immune systems just like higher vertebrates,the innate immune system plays a significantly more important role in defending against pathogenic invasion.In this study,Miichthys miiuy were selected as model organisms for the study of immune mechanisms.To establish a conceptual basis for the prophylactic mechanism of innate immune response in Miichthys miiuy as well as fish,so as to reduce the damage caused by diseases to aquatic economic animals.Microbial infection of cells activates an inflammatory response and the innate immune system is the primary defense mechanism for acute inflammation.Pattern recognition receptors（PRRs）,as an important component of an organism’s innate immune response,trigger downstream signals by recognizing pathogen-associated molecular patterns（PAMPs）,which in turn activate a series of antiviral or antimicrobial signals to maintain homeostasis in the organism.These PRRs activate intracellular signaling cascade reactions that evoke the inflammatory mediator transcriptional expression,thereby harmonizing the host clearing away pathogenic agents and inflamed cells.As one of the oldest and most comprehensively studied receptors,TLRs play an important role in immune homeostasis and disease resistance.TLRs generally undergo signaling through adaptor proteins,the most common of which is myeloid differentiation factor 88（MyD88）,which is a central player in innate immune signaling.Except for TLR3,almost every TLR activates the NF-κB signaling pathway through a MyD88-dependent pathway,thereby initiating the immune response.However,dysregulation of MyD88 can also lead to an imbalance in host homeostasis and trigger inflammation-associated diseases.In the innate immune response,MyD88 plays an integral role in the study of the molecular mechanisms of the NF-κB signaling pathway.In this study,we discovered that some proteins regulate the NF-κB signaling pathway by targeting MyD88,which provides new insights into the mechanism of innate immunity in fish.1.Zw10 targets MyD88 autophagic degradation to inhibit NF-κB signaling pathwayFirstly,this study confirmed that Zw10 is a negative regulator of MyD88,then the RT-q PCR experiments were performed,and the results indicated that the Zw10 m RNA level increased under V.harveyi infection.The analysis of the cell proliferation assay results revealed that Zw10 significantly inhibited cell proliferation in the presence of V.harveyi infection.Furthermore,both results of dual luciferase reporter experiments and real-time quantitative PCR assays demonstrated that Zw10 significantly suppressed the expression of inflammatory factors IL-1βand IL-8 downstream of the NF-κB signaling pathway after LPS stimulation.Immunoprecipitation（Co-IP）assays and subcellular localization assays revealed that Zw10 and MyD88 interacted with each other.Meanwhile,Western blot assay analysis showed that Zw10 inhibited the synthesis of MyD88 at the protein level.Next,Zw10 mutant experiments showed that the Zw10-Δ2structural domain（aa 9-160）plays a key role in the degradation of MyD88.Furthermore,the autophagy inhibitors 3-MA and NH₄Cl prevent Zw10 from degrading MyD88.In conclusion,this study identifies that Zw10 negatively regulates the NF-κB signaling pathway by degrading MyD88 through the autophagic pathway.2.Vclb negatively regulates NF-κB signaling pathway by promoting MyD88proteasomal degradationIn the study,genetic analysis revealed that Miichthys miiuy Vclb is highly conserved,suggesting that Vclb has played an important role in the evolution of different species.Dual luciferase reporter assays showed that Vclb downregulated the activity of NF-κB reporter genes and downstream inflammatory factors（IL-1βand IL-8）,and their activity was significantly reduced by LPS stimulation.Meanwhile,Vclb significantly inhibited MyD88-mediated IL-1βand IL-8 activities in a dose-dependent manner.Analysis by Co-IP experiments showed that Vclb and MyD88 interacted at the protein level.Western blot results showed that Vclb effectively inhibited MyD88 expression.Mechanistically,Vclb was found to degrade MyD88 through the proteasome pathway.In summary,this study confirms that Vclb is a negative regulator and mediates the innate immune response;Vclb promotes MyD88 degradation and inhibits the NF-κB signaling pathway in a proteasomal pathway.