The Regulation Of ZBP1-induced Cell Death By PKR

ZBP1 is one of the intracellular nucleic acid receptors that recognize IAV nucleic acid species when cells are invaded by influenza A virus.ZBP1 then interacts with RIP1 and RIP3 kinases through their shared and conserved RHIM domains.When RIP3 is present,ZBP1 can bind to RIP1 and Caspase-8 to induce apoptosis and can also trigger MLKL-mediated programmed cell necrosis through RIP3.In the absence of RIP3,ZBP1 only binds to RIP1 to induce apoptosis.Therefore,ZBP1,RIP1 and RIP3 play important roles in these cell death pathways,but the detailed molecular mechanisms that regulate these cell death pathways are still unclear.To further investigate the molecular mechanisms,we found that PKR binds to ZBP1 by CO-IP combined with mass spectrometry arid bioinformatics analysis in 293T cells.And the interaction between ZBP1 and PKR was confirmed by immunoprecipitation experiment.Furthermore,we found that PKR can promote ZBP1-mediated cell death,and specifically we found that PKR promotes ZBP1-RIP 1-mediated apoptosis and ZBP1-RIP3-mediated cell necrosis.In addition,we found that expression of PKR makes ZBP1,RIP1 and RIP3 proteins more insoluble,probably through inducing the oligomerizaton of these proteins.Immunofluorescence experiments show that PKR changes the localization of ZBP1 from a diffuse distribution into a high-density spot-like particle structure.When the amino acids of the 64th nucleic acid binding site and the kinase activity site at position 296 of PKR are mutated,its effect on ZBP1 is lost.The change in the localization of ZBP1 induced by PKR is the reason why the ZBP1 protein is transferred from soluble to less soluble in the supernatant of cell extracts.We speculate that in this structure PKR promotes the binding of ZBP1 to RIP1 and the binding of ZBP1 to RIP3,which increases cell death.We established a model of PKR in the process of ZBP1-mediated death,which contributes to the understanding of the regulatory mechanism of ZBP1-mediated cell death and provides a theoretical basis for the treatment of infectious diseases such as IAV.