E3泛素连接酶TRIP负向调控TLR介导的炎症反应及分子机制研究

Innate immunity is the first line to defense against pathogenic microorganisms, lipopolysaccharide on surface of bacteria is recognized by TLR4, then produce large amounts of inflammatory cytokines, such as TNF-α and IL-6. But the study on the mechanism of this pathway is not perfect. Our previous study found that the expression of E3 ligase TRIP increased significantly when macrophages was stimulated by LPS; the specific interference to the TRIP can significantly increase the expression of TNF- α, IL-6 induced by LPS. further research shows, TRIP could combine with phosphokinase TAK1, then promote its degradation. The endotoxin-shock model, RT-PCR, report gene, Co-immunoprecipitation, in vitro ubiquitination and other molecular techniques will be used to systematically investigate the influence of TRIP on TLR4 mediated TNF- α, IL-6 production and TAK1 ubiquitination. The purpose of this study was to elucidate the TRIP function in inflammatory reaction mediated by TLR4, and provide new insight into the TLR4 signal transduction mechanism; To investigate the expression of TRIP can be used as feedback signal regulation of TNF- α, IL-6 production, it will also provide a new way for preventing bacterial infections, endotoxin shock and other ills .

固有免疫细胞表面的TLR会识别细菌的脂多糖（LPS）等，活化NF-kB并产生大量炎症因子，而对该信号通路调控机制的研究并不完善。我们前期研究发现，LPS刺激巨噬细胞后，E3连接酶TRIP的表达量明显增加，对TRIP特异性的干扰可显著增强LPS诱导的TNF-α、IL-6的表达，进一步研究显示，TRIP可与磷酸激酶TAK1发生结合，并促进其降解。我们提出假设，TRIP可能会作为E3泛素连接酶将TAK1进行K48位泛素化并使其降解，进而负向调控TLR4介导的炎症反应。本研究利用内毒素休克模型和RT-PCR、报告基因、免疫共沉淀、体外泛素化等分子技术手段证明这一假说。本研究旨在阐明TRIP调节TLR4介导的炎症反应的分子机制，为TLR4信号转导机制提供新的认识；探讨TRIP可否作为反馈信号调节TNF-α、IL-6的产生，为寻找基因转染防治细菌感染及内毒素休克等疾病提供理论基础。

固有免疫是机体抵抗病原微生物感染的第一道防线，天然免疫细胞感知外界病原微生物后，活化NF-kB并产生大量炎症因子，如TNF-α、IL-6等，而对该信号通路调控机制的研究并不完善。在国家自然基金资助下，我们利用内毒素休克模型和RT-PCR、报告基因、免疫共沉淀、体外泛素化等分子技术手段，系统的研究TRIP调控天然免疫的作用机制。1) 发现LPS刺激巨噬细胞后，E3连接酶TRIP的表达量明显增加，出核入核情况未发生变化； 2）干扰掉TRIP后，LPS诱导的TNF-α、IL-6的表达明显上调，NF-KB磷酸化水平也显著升高; 3) LPS刺激巨噬细胞后，TRIP会与磷酸激酶TAK1发生结合，且具有明显时间梯度；4）体内及体外实验证明，TRIP作为E3泛素连接酶将TAK1进行K48位泛素化及使其降解，进而负向调控TLR4介导的炎症反应。本研究阐明了TRIP调节TLR4介导的炎症反应的分子机制，为TLR4信号转导机制提供新的认识，目前仍有部分结果正在整理投稿中。课题执行期间，作为通讯作者或者主要完成人身份共发表SCI论文9篇（标注课题号的SCI论文3篇），其中影响影响因子大于20的1篇、大于10的2篇。课题负责人获得国家自然基金面上项目1项；项目负责人作为第一作者发表在Nat Immunol上的论文入选F1000 Prime.