Ctenopharyngodon Idella TRAF6 Up-Regulates IFN1 Expression By Activating IRF5

TRAF6(Tumor necrosis factor receptor related factor 6,TRAF6)is a new hotspot in the study of immunology in the new era.In known signaling pathways,it is both a linker factor and mediated by certain receptors;it is also a RING structural E3 ubiquitinase,which participates in the ubiquitin-protein modification system and TLRs Receptor-mediated signaling process,so TRAF6 must have a special mission in the body,but the molecular mechanism research of TRAF6 in grass carp is not yet perfect.Interferon regulatory factor 5(Interferon regulatory factor 5,IRF5)and TRAF6 can also participate in TLR-mediated cell signaling pathways,and IRF5 is the intermediate hub and key factor of TLR4 signaling pathway,which can effectively activate and regulate the transcriptional regulation of IFN Cellular immune system activity to suppress tumor progression.IRF5 is in a low activity state in the cell,and it needs to be activated before it is transferred to the nucleus and regulates the expression of interferon.However,whether TRAF6 can activate IRF5 and the molecular mechanism of activation in grass carp is unclear.Interferon(IFN)is a group of stress proteins released by host cells in response to pathogen damage;its expression is regulated by IRFs.For example,IFN1,which plays a vital role in the innate immunity of vertebrates,can be regulated by IRF5.However,the research on the transmission mechanism of LPS-IRF5-IFN signaling pathway in grass carp needs to be further studied.In other words,the molecular mechanism by which TRAF6 regulates IFN expression in fish is currently unclear.Similarly,the molecular mechanism of the relationship and effect between TRAF6 and IRF5 is uncertain.In this study,we explored how grass carp(Ctenopharyngodon idella)TRAF6 triggers innate immunity by activating IRF5.We found that CiTRAF6,CiIRF5 and CiIFN1 were significantly up-regulated in LPS-stimulated CIK cells,and the expression of CiTRAF6 increased earlier than the expression of CiIRF5 and CiIFN1.These findings indicate that CiTRAF6,CiIRF5,and CiIFNI have important functions in grass carp,and CiTRAF6 may induce the expression of CiIFN1 in fish.In experiments overexpressing CiTRAF6 or CiIRF5 alone and overexpressing CiTRAF6 and CiIRF5 simultaneously,we found that CiIFN1 expression,CiIFN1 promoter activity and CO cell viability were significantly up-regulated,and reached the highest when co-overexpressed,but in gene silencing experiments They are all significantly reduced.This indicates that CiTRAF6 and/or CiIRF5 regulate the expression of CiIFN1,and this regulation does not depend on the stimulation of external pathogens.Localization analysis found that under normal circumstances,CiTRAF6 and CiIRF5 are located in the cytoplasm.After 24 hours of LPS stimulation,we observed the translocation of CiIRF5 into the nucleus,which is consistent with our results of protein nuclear translocation sequence analysis.It shows that CiTRAF6 only exists in the cytoplasm and plays a role in the cytoplasm;CiIRF5 must inevitably undergo a state transition before entering the nucleus,such as activation.GST pull down and Co-IP experiments show that CiTRAF6 interacts with CiIRF5.Colocalization analysis also showed that CiTRAF6 binds to CiIRF5 in the cytoplasm,which implies that there is some interaction between CiTRAF6 and CiIRF5.Moreover,overexpression of CiTRAF6 increased endogenous CiIRF5 expression,promoting its ubiquitination and nuclear translocation.In summary,in the cytoplasm,CiTRAF6 binds to CiIRF5,and then activates CiIRF5,thereby causing CiIRF5 to be transferred into the nucleus,and promoting the up-regulation of CiIFN1 expression.