Screening And Identification Of Proteins Interacting With Porcine Cyclic GMP-AMP Synthase

CyclicGMP-AMP synthase(cGAS)is a newly identified cytosolic DNA sensor that recognizes DNA activation and catalyses the generation of cGAMP,which in turn activates the downstream adapter protein STING,induces production of type I interferons(IFNs)and initiates host defense.In addition,cGAS can also recognize DNA from the host cells and is activated abnormally,which may lead to immune damage and autoimmune diseases.Therefore,activation of cGAS is strictly regulated by the organism.Although various post-translational regulatory mechanisms of cGAS in human and mouse have been studied,how the porcine cGAS(pcGAS)activation is regulated remains largely unclear.In order to understand the regulatory mechanisms of pcGAS activation,we first constructed a prokaryotic expression vector of pcGAS and expressed recombinant pcGAS in Escherichia coli(BL21/DE3).Soluble recombinant pcGAS proteins were obtained by optimizing the expression temperature,IPTG concentration,and induction time.The recombinant protein pcGAS were then purified by affinity chromatography using a nickel column to reach high purity.The biological activity of the purified pcGAS were further verified by using an in vitro enzymatic reaction to detect the ability of recombinant pcGAS to synthesis of 2’3’-cGAMP and induction of type I IFNs.To screen and identify molecules that may interact with pcGAS,the purified recombinant pcGAS were coated on a CM5 Chip through amino coupling as bait protein to screen potential interaction proteins in porcine alveolar macrophage lysates by using Bia CORE system for interactions of biological macromolecules.As a result,61 potential proteins that may interact with pcGAS were identified by liquid chromatograph mass spectrometer(LC/MS).GO enrichment analysis showed that the identified proteins distributed in exosomes,nuclei,extracellular regions,and cytoplasm,and were functionally involved in regulation of gene transcription,protein translation,RNA metabolism,autophagy and apoptosis.The interaction of pcGAS and two high possible proteins 14-3-3 protein beta/alpha(YWHAB)and fatty acid-binding protein 5(FABP5)were confirmed by immunofluorescence and co-immunoprecipitation(Co-IP).The results showed that pcGAS co-localized interacted with YWHAB and FABP5 in cytoplasm and interacted with YWHAB and FABP5 in cells,suggesting that the interacting molecules identified in this study are reliable.In summary,we have successfully obtained soluble recombinant pcGAS with high purity and biological activity.Moreover,61 pcGAS interacting proteins that were previously unappreciated were identified by Bia CORE and LC/MS method and two of them were further confirmed immunofluorescence and Co-IP.Hence,the results in this study provided a theoretical basis for deep understanding of regulatory mechanisms of pcGAS activation and potential targets for antiviral drugs.