【作者】 魏晖；

【导师】 王新泉；

【作者基本信息】 清华大学 ， 生物学， 2015， 博士

【摘要】 白细胞介素18(interleukin 18,IL-18)是白细胞介素1(interleukin 1,IL-1)家族蛋白中的一员,起激动剂的作用。IL-18结合其配体结合受体IL-18Rα和共受体IL-18R?,在细胞膜上形成配体-受体三元信号转导复合物,进而激活下游NF-κB信号通路和MAPK信号通路。IL-18在先天性免疫反应和获得性免疫反应中都有重要作用。人体中过量IL-18的产生会导致多种自身免疫病和慢性炎症性疾病。本论文工作主要研究IL-18识别其受体并组装成信号转导复合物的结构基础。通过大肠杆菌和昆虫细胞分别表达IL-18以及IL-18Rα和IL-18R?的胞外域,通过亲和层析及分子筛纯化蛋白样品,然后将IL-18、IL-18Rα、IL-18R?在体外混合,得到IL-18-IL-18Rα二元复合物的样品以及IL-18-IL-18Rα-IL-18R?三元复合物的样品,然后进行蛋白质晶体生长条件筛选。成功获得IL-18-IL-18Rα二元复合物的晶体后,通过X射线衍射技术,解析出IL-18-IL-18Rα二元复合物分辨率为2.8?的结构。结构分析揭示了IL-18与IL-18Rα相互作用的氨基酸位点,在此基础上,通过定点突变及基于萤光素酶的NF-κB信号检测技术,进一步确定了IL-18上与IL-18Rα相互作用的9个关键氨基酸残基。IL-18和IL-18Rα相互作用界面的分析表明表面电荷互补决定了IL-18和IL-18Rα的特异性识别。此外,通过结构比对,揭示了IL-18天然抑制剂IL-18结合蛋白以及IL-18单克隆中和抗体抑制IL-18活性的结构基础。本论文工作首次报道了IL-18与其受体复合物的晶体结构,结构功能关系研究详细阐明了IL-18与IL-18Rα特异性相互作用的结构基础。之后日本一个研究小组报道了IL-18-IL-18Rα-IL-18R?三元复合物的结构。基于这些研究结果和之前报道的IL-1家族细胞因子IL-1?及IL-33与受体复合物的结构,本论文总结提出一个在IL-1家族中普遍适用的配体与受体组装和激活的结构模型。更多还原

【Abstract】 Interleukin 18(IL-18) is one agonistic member in the IL-1 family that plays important roles in both innate and adaptive immune responses. IL-18 binds its ligand-binding receptor IL-18Rα and co-receptor IL-18R? to form a signaling ternary complex, which initiates downstream NF-κB and MAPK pathways. Overproduction of IL-18 in human is related to several autoimmune diseases and chronic inflammatory disorders.To understand the mechanism how IL-18 recognizes its receptors and assembles the signaling complex, human IL-18 and the ectodomains of IL-18Rαand IL-18R? were expressed in E.coli and insect cells respectively and were purified via affinity chromatography and gel filtration chromatography. The binary complex of IL-18 with IL-18Rαand the ternary complex of IL-18 with IL-18Rα and IL-18R? were reconstituted in vitro and the crystallization conditions were screened. Crystals of IL-18-IL-18Rαbinary complex were successfully obtained and the structure was solved at a resolution of 2.8 ? by X-ray diffraction method. Analysis of the structure determined the interacting residues between IL-18 and IL-18Rα. Via site-specific mutagenesis andNF-κB luciferase reporter system, nine critical residues on IL-18 for the interaction with IL-18Rαwere revealed. It also suggested that surface charge complementarity determines the specific recognition of IL-18 by IL-18Rα. Structural comparison revealed the molecular basis for IL-18 binding protein and monoclonal neutralizing antibody to inhibit the IL-18 activity.The first complex structure of IL-18 with its receptor was reported in this work which clarified the structural basis for the specific recognition of IL-18 by IL-18Rα. Afterwards a Japanese group reported the complex structure of IL-18-IL-18Rα-IL-18R? ternary complex. Based on these results as well as the complex structures of IL-1? and IL-33 with their receptors, a general ligand-receptor assembly andactivation model in the IL-1 family was proposed.更多还原