SPOP Promotes Degradation Of MyD88 To Suppress The Innate Immune Response

Toll-like receptors are the most widely used pattern-recognition receptors that are involved in the recognition of bacterial,fungal and virus.Upon recognition of the pathogens,MyD88 converts signals from most TLRs to activate downstream pathways and immune response.The homeostasis of MyD88 determines whether the host can trigger immune response during pathogen infection.Ubiquitinationmediated degradation is an important way of regulating protein levels.SPOP is the adaptor of Cul3-SPOP-Rbx1 ligase complex and determines the substrate specificity.SPOP plays an important role in regulating muscle development,cell cycle regulation,cancer development.At present,it is not clear about the regulatory relationship between SPOP and immune response.The purpose of our research is to reveal the mechanism through which SPOP regulates MyD88 protein homeostasis and the host antiinfection ability.The main conclusions are as follows:1.SPOP interacted with MyD88.The interaction between them was validated by immunoprecipitation and western blot in chicken embryonic fibroblasts(DF1).The interaction was mediated by SPOP MATH domain and MyD88 INT domain and immunofluorescence showed that the two proteins co-localized in the cytoplasm.Besides,the interaction was also observed in human embryonic kidney cells(293T)and Chinese hamster ovary cells(CHO),indicating the conservation amoung different species.2.SPOP promoted the degradation of MyD88 in ubiquitin-proteasome-dependent way.Exogenously expressed SPOP efficiently decreased the protein level of MyD88 in a dose-dependent manner.Knockdown of endogenous SPOP led to an increase in MyD88 abundance in DF1.Meanwhile,MyD88 protein level decreased after SPOP transfection in A549 and CHO.However,the mRNA level of MyD88 remained unchanged when the expression of SPOP was altered,indicating that SPOP regulated MyD88 degradation in post-translation level rather than transcription level.After inhibitors of ubiquitin-proteasome way treatment,the observed decease in MyD88 by SPOP was rescued,showing that SPOP promoted the degradation of MyD88 in a ubiquitin-proteasome-dependent way.3.SPOP promoted K48-linked polyubiquitination of MyD88.Overexpression of SPOP increased the ubiquitination level of MyD88 in chickens.After co-transfection of SPOP and ubiquitin mutant(K48 or K63),SPOP enhanced the K48-linked rather than the K63-linked ubiquitination of MyD88.There were 17 lysine sites in MyD88 amino acid sequence.Regulatory effect of SPOP on MyD88 abolished when three lysine sites(118,124 and 143)were mutant,showing that SPOP promoted the degradation of MyD88 through the ubiquitination of these three lysine sites.4.SPOP negatively regulated NF-κB signaling pathway by targeting MyD88.First,the SPOP expression was dynamic changes after pathogen infection,demonstrating that SPOP participated in the immune response.Second,SPOP overexpression efficiently decreased the production of NF-κB activity and IL-1β,IL-8 expression.The results were converse when SPOP was knockdown.To clarify the specificity of the SPOP regulation on immune response through MyD88,SPOP and MyD88 or MyD88 downstream molecule TRAF6 were co-transfected.The results showed SPOP overexpression inhibited MyD88 mediated NF-κB activation,while NF-κB activation mediated by overexpression of TRAF6 was not inhibited,demonstrating that the manipulation of NF-κB signaling pathway of SPOP was dependent on MyD88.5.SPOP enhanced host defenses against Salmonella infection.Spop expression in the spleen was negatively correlated with Il-1β,Il-8,IgA production and bacterial loads when 7-day-old chicken were infected with Salmonella.We constructed Spop conditional knockout mouse model.Spop knockdown mice were more susceptible to Salmonella compared with control: the survival time of Spop knockout mice was shorter after intraperitoneal injection with Salmonella;the bacterial loads in liver and mortality rate were significantly higher;the concentration of inflammatory factors in serum was significantly higher.After LPS stimulation,the bone marrow macrophage from Spop knockout mice produced more inflammatory factors and the phosphorylation of IRAK4 was higher,indicating that immune response was more intense in Spop knockout mice.Ratio of immune cells in peripheral blood varied considerably between two groups,and the neutrophilia was significantly higher in knockout mice.In summary,our research revealed that SPOP interacted with TLR adaptor MyD88,systematically explored the molecular mechanism of SPOP-mediated negative regulation of MyD88 protein level and NF-κB pathway,and proved the crucial role of SPOP in maintaining innate immune homeostasis and disease resistance by knockout animal model.