The Construction And Screening Of An ER Protein CDNA Library For Innate Immune Function

As the first line of host defense,innate immunity is critical for the defense against exogenous microbes.CytosolicRNA/DNA sensing recognizes patho-gen-associated molecular patterns(PAMPs)via pattern recognition receptors(PRRs)and elicits downstream immune pathways to respond against invaded and infected pathogens.For example,cytosolicds RNA is recognized by RIG-I-Like receptors(RLRs),and DNA in the cytosol is perceived by the DNA sensor cGAS.Facilitated by mitochondrial-associated MAVS endoplasmicreticulum-located STING,nucleicacids recognition results in the activation of TBK1 and/or IKKε.TBK1/IKKε are then autophosphorylated,and modify STING,and transcription factor IRF3.IRF3 dimerizes and enters the nucleus and triggers the production of type I interferons and proinflammatory cytokines.The cGAS-STING signaling axis is used to detect a variety of cytosolicDNAs,which often function as the carrier of information for pathogen invasion and tissue damage.Therefore,innate DNA sensing functions as a pivotal mechanism in the sterile inflammatory response,cellular senescence,autoimmunity,neurodegenerative,and antitumor immunity.STING is an evolutionary conserved endoplasmicreticulum(ER)transmembrane protein.Upon cGAMP binding,STING exits the ER and translocates into ER-Golgi intermediate compartments(ERGIC)and Golgi apparatus,where TBK1 is recruited and activated.However,the molecular mechanism underlying the translocation of STING from the endoplasmicreticulum to the Golgi apparatus has not been elucidated.In an attempt to understand this elusive event during the activation of cGAS-STING signaling,we established a cDNA library expressing ER proteins,setting it as a basis to decipher the function of individual ER protein in STING biology.A list of human ER proteins containing 235 members was compiled according to the information of Protein Atlas.org and cloned from two cDNA libraries(Invitrogen ORF LITE Clones and the Human ORFeome 5.1 cDNA library),by employing the p DEST-N-Flag-HA vector and the Gateway system.Expression of 170 ER proteins was validated in mammalian cells and was screened in the IRF3-responsive reporter assays(5×ISRE-lucand IFNβ-lucreporters)under activation of STING signaling.Three ER proteins including ERQ63,ERQ78,and ERQ124 were found to robustly regulate the cGAS-STING pathway,and their effects were further validated by siRNA interference.Among them,ERQ63 interacted strongly with STING,and depleting the endogenous expression of ERQ63 led to a delay in the formation of STING aggregation.Collectively,this study offers an overall perspective for the involvement of ER proteins in cGAS-STING biology and sets a solid basis to investigate the translocation and activation of STING in the future.