斑马鱼TBK1剪接异构体在病原微生物感染中的作用及其调控机制

TANK-binding kinase 1 (TBK1) plays an important role in the regulation of type I interferon and NF-κB signaling pathways. We have found that zebrafish TBK1 has five forms of splicing isoforms, and TBK1_tv1 and TBK1_tv2 negatively regulate the RLRs-mediated antiviral immune response. However, whether the other three isoforms of TBK1 also participate in immune escape during the pathogens infection is unknown. And TBK1 and its splicing isoforms are involved in the regulation of NF-κB signaling pathway in fish has not been reported at present. This project will study the regulatory roles of TBK1 and its splicing isoforms in the RLRs- and NF-κB signaling pathway, and compare the similarities and differences of TBK1 splicing isoforms in the regulation of immune signal networks, and systematically elucidating the functions of TBK1 and its splicing isoforms in host innate immune response. And then we will not only study the interactions between TBK1 splicing isoforms and the proteins of RLRs- and NF-κB signaling pathways, but also study the influence of protein-protein interactions on pathogens infection. We will uncover the molecular mechanisms of TBK1 and its splicing isoforms in regulating the host immune response. The completion of this project will interpret the new mechanism of TBK1 involved in fish immune regulation and provide theoretical and experimental basis for fish disease prevention and healthy culture.

TANK结合激酶1（TBK1）在调节Ⅰ型干扰素及NF-κB信号通路中发挥着重要作用。我们前期研究发现斑马鱼TBK1存在至少5种形式的剪接异构体，其中TBK1_tv1和TBK1_tv2负调控RLRs介导的抗病毒免疫反应，但另外3种异构体是否也参与了病原微生物感染时的免疫逃逸，以及TBK1及其剪接异构体是否调控鱼类NF-κB信号传导通路目前没有任何报道。本项目将研究TBK1及其剪接异构体在RLRs介导信号通路和NF-κB信号通路中的调控作用，比较这些剪接异构体在免疫网络调控中的异同点，将系统阐明TBK1及其剪接异构体在先天免疫应答中的功能；通过研究TBK1及其剪接异构体与信号通路接头蛋白的相互作用以及蛋白互作对病原微生物感染的影响，将全面阐明TBK1剪接异构体发挥免疫调控功能的分子机制。本项目的完成将诠释鱼类TBK1参与免疫调控的新机制，为鱼类病害防治和健康养殖提供实验和理论依据。

TANK结合激酶1（TBK1）在调节Ⅰ型干扰素及NF-κB信号通路中发挥着重要作用。哺乳动物和鱼类的TBK1存在多种剪接异构体。本项目从斑马鱼和草鱼中都获得了TBK1剪接异构体，斑马鱼TBK1剪接异构体TBK1_tv3与草鱼TBK1剪接异构体gcTBK1_tv3只存在一个氨基酸差异。斑马鱼TBK1_tv3在调控鱼类免疫稳态中起着重要作用，并负调控斑马鱼的抗病毒免疫反应。斑马鱼TBK1_tv3通过与TBK1和IRF3的互作，诱导TBK1和IRF3分别通过泛素-蛋白酶体途径和自噬-溶酶体途径降解。并且TBK1的K251、K256、K271三个氨基酸是被TBK1_tv3泛素化的关键位点。而草鱼TBK1剪接异构体gcTBK1_tv3则抑制草鱼呼肠孤病毒（GCRV）的感染和复制。gcTBK1_tv3与GCRV的NS80和NS38相互作用，在蛋白水平抑制GCRV NS80和NS38的表达。并且gcTBK1_tv3存在自身泛素化，并对NS80的K503和NS38的K328氨基酸进行K48位泛素化，通过泛素-蛋白酶体途径降解NS80和NS38蛋白，然后抑制VIBs的形成，最终抑制GCRV的感染和复制。本项目的研究揭示了鱼类TBK1剪接异构体在调控宿主免疫和病原感染中作用和分子机制。