The Identification Of Proteins Interacting With DAI By Co-immunoprecipitation And Mass Spectrometry

Cells can utilize the intracellular DNA/RNA sensors to recognize the DNA or RNA released into cytoplasm by infected microbial pathogens,most of which are viruses,and then activate the downstream interferon-related signaling pathway as well as NF-κB signaling pathway to promote the production of type I interferon and other inflammatory factors,resulting in immunoreaction to inhibit the replication and spread of virus.In some cases,cells take the alternative strategy of triggering cell death to defense against pathogen infection.DAI(ZBP1),a interferon inducible protein,is one of the intracellular DNA sensors.Recent research demonstrated that in addition to recognition of DNA,DAI harbors the capacity of binding RNA from some pathogens.Under the condition of influenza A virus(IAV)infection,DAI can bind viral RNA and subsequently mediate apoptosis or necrosis depending on RIP3(RIPK3).DAI bound viral RNA can also induce apoptosis independent of RIP3.However,the precise molecular mechanism remains unclear.In order to illucidate the underlying mechanism of this biological process,we performed immunoprecipitaion and mass spectrometry in HEK293T cells where RIP3 is not expressed.By means of bioinformatics analysis,we identified PKR(eif2ak2)as a DAI interacting protein,and verified the interaction by CO-IP.Furthermore,our studies suggested that overexpression of full length DAI as well as its truncations 1-194 or 195-429 can induce apoptosis in HEK293T cells.The overexpression of PKR accelerates the apoptosis triggered by DAI.In addition,we investigated the influence of PKR and DAI on NF-κB transcriptional activity,and found that overexpression of PKR or DAI alone promotes the NF-kB activity to different degrees.Surprisingly,when overexpressed together,PKR and DAI restrained the NF-κB activity.Further investigation is needed to reveal the underlying mechanism.Overall,this study enhances our understanding of the cell apoptosis signaling pathway mediated by DAI against virus invasion,and provided a new therapeutic target for the clinical treatment of antiviral therapy.