ZBP1诱导一条新的细胞程序性坏死信号通路. 在病毒入侵时，ZBP1通过RIPK3和MLKL激活细胞程序性坏死，剔除感染的细胞，是防御病毒的重要机制。但ZBP1细胞坏死通路上游调控机制和信号蛋白却不清楚。本申请项目利用IP—LC-MS/MS技术，发现了三个新的调控ZBP1细胞坏死通路的信号蛋白PKR、TRAF4和RNF34. 免疫沉淀实验发现它们在细胞内与ZBP1结合，形成复合物；PKR和TRAF4利用不同机制促进ZBP1通过RHIM结构域聚合，它们与ZBP1共转染均增强293T细胞死亡。而RNF34泛素化降解ZBP1，调控ZBP1蛋白稳定性。PKR和TRAF家族都是先天免疫的重要调控蛋白。PKR识别结合病毒的dsRNA，可能给ZBP1传递信号启动细胞坏死通路；TRAF4则可能通过泛素化调控ZBP1的功能。后续工作将深入研究新发现的信号蛋白调控ZBP1细胞程序性坏死通路的分子机制。

ZBP1 induces a new necrotic cell death pathway. Upon viral infection, ZBP1 binds to RIPK3 and activates MLKL-mediated necrotic cell death to inhibit virus replication, which is a critical antiviral defense mechanism. However, the upstream signaling and mechanism that control the activation of the ZBP1 cell death pathway are not understood. In this proposal, using IP—LC-MS/MS analysis we identified three novel signaling proteins that regulate ZBP1 cell death pathway: PKR, TRAF4 and RNF34. Immunoprecipitation experiments showed that PKR and TRAF4 interact with ZBP1 in 293T cells. PKR and TRAF4 induced the polymerization of ZBP1 via its RHIM domain and enhanced ZBP1-induced cell death. These results suggest that PKR and TRAF4 activate the ZBP1 cell death pathway. In contrast, expression of RNF34 degraded ZBP1 and downregulated the protein level of ZBP1. RNF34 might negatively regulate the ZBP1 cell death pathway. PKR is an important PRR protein (pathogen-recognition receptors). PKR detects viral infection with its dsRNA binding domain. TRAF family proteins have critical roles in the TNF and IL-1/TLR signaling pathway. Based on the notion that both PKR and TRAF family proteins have important functions in the innate immunity to mediate defense mechanisms against viral infection, our finding that links the function of PKR and TRAF with ZBP1 is interesting, which suggests that PKR and TRAF might play important roles in the ZBP1 antiviral cell death pathway. In future study, we will explore the CRISPR/Cas9 technique and other methods to fully investigate the mechanisms by which PKR, TRAF4 and RNF34 regulate the ZBP1 cell death pathway.