【作者】 林智杰；

【导师】 沈月全；

【作者基本信息】 南开大学 ， 生物化学与分子生物学， 2013， 博士

【摘要】 Toll样受体(Toll-like receptors,简称TLRs)是一类在脊椎动物和无脊椎动物中广泛分布的模式识别受体,其在宿主抵御病原微生物入侵的自然免疫中发挥了重要作用。TLR通过其胞外结构域识别病原体相关的分子模式,引起自身二聚化激活,从而导致其胞内TIR(Toll/interleukin-1receptor)结构域重新排列组合以募集适配分子来起始细胞质中的信号传导,进而促进炎症因子和抑菌蛋白的表达,并进一步诱发获得性免疫反应。TLR起始信号复合物组装是通过相同类型和不同类型的TIR-TIR结构域之间特异性的相互作用实现的。TIR结构域是一类α/p型球状蛋白,其二级结构元件之间通过变化各异的loop区连接。不同TLR信号通路的特异性依赖于受体募集不同的包含TIR结构域的适配分子。目前已经发现的包含TIR结构域的适配分子共有五个,分别为：MyD88, Mal,TRIF, TRAM和SARM。其中,Mal又被称为TIRAP,它是在TLRs识别病原体入侵而激活之后,协助TLR2和TLR4募集适配分子MyD88进行胞内信号传导的过程中起关键作用的桥联适配分子。细胞内TLR2／4信号传导起始的关键分子机制是TLR2/4,Mal和MyD88三者的TIR结构域的特异性的组装。然而,这些特异性的TIR-TIR相互作用的分子机制目前仍然还是个悬而未决的问题。本论文解析了人源Mal的TIR结构域(简称Mal-TIR)2.40A分辨率的晶体结构。通过结构分析发现Mal-TIR的结构明显地区别于其他的TIR结构域,其含有结构上独特的AB loop,而缺少了仅B螺旋和BB loop这两个结构元件。通过进一步的基于结构的功能研究发现在Mal-TIR的结构表面有两个关键的功能区域：其中一个关键区域是Mal-TIR结构上特征性的AB loop区域,依据定点突变和体外直接相互作用实验证实了Mal-TIR通过AB loop与TLR4和MyD88二者的TIR结构域发生直接相互作用;另一关键区域是Mal的二聚体界面,本文通过生化和细胞的实验证实Mal-TIR以背靠背的方式协助Mal分子在执行功能时组装成轴对称性的二聚体。由此,本论文揭示了Mal在TLR4信号通路中行使桥联适配分子的生物学功能的分子机制——Mal分子自身形成背靠背的二聚体,并通过其两个分子的AB loop协助TLR4募集MyD88。此外,有多项研究表明Mal的自然变异跟一些免疫性疾病有关。其中最受关注的是Mal的单核苷酸突变体S180L,因为S180L突变杂合子的携带者对菌血症,侵袭性肺炎球菌感染,疟疾和过敏性皮炎等传染性免疫疾病具有更强的抵抗力。另一个突变体D96N,有研究表明由于D96N突变影响了对适配分子MyD88的募集和自身的翻译后修饰而导致了Mal的功能缺陷。由此本论文解析了这两个突变体的晶体结构,分别为2.75A和3.10A,为Mal相关的传染性免疫疾病的分子机制研究提供了结构基础。本论文结合x射线晶体学、生物化学和细胞生物学的研究方法,揭示了Mal在TLR4信号通路中执行适配分子功能的结构特征和分子机制,为进一步研究TIR结构域的特异性组装以及免疫性疾病相关的分子机制提供了结构基础。更多还原

【Abstract】 The Toll-like receptors (TLRs) are an important group of pattern recognition receptors (PRRs) that play a critical role in host defense against pathogens throughout the animal kingdom. In response to various extracellular ligands, the ectodomain of a TLR dimerizes, leading to the rearrangement of its cytoplasmic Toll/interleukin-1receptor (TIR) domains to create a signaling platform for the recruitment of adaptor proteins. This rearrangement is followed by the transcription of inflammatory and antimicrobial genes in the initiation of the adaptive immune response. The homo-and heterotypic TIR-TIR domain interactions play a pivotal role in the assembly of the TLR signalosomes and in initiation of the signaling pathway.A TIR domain has a globular fold with α/β secondary structure elements linked together by variable loops. The specificity of TLR signaling depends on the action of different TIR domain-containing adaptor proteins. Five TIR domain-containing adaptor proteins are currently known:myeloid differentiation factor88(MyD88), MyD88adaptor-like protein (Mal), TIR domain-containing adaptor-inducing interferon-b (TRIF), the TRIF-related adaptor molecule (TRAM) and the sterile and HEAT/armadillo (ARM) motif protein (SARM). Mal, which is also identified as the TIR domain-containing adaptor protein (TIRAP) is a crucial adaptor that acts as a bridge to recruit the MyD88molecule to activated TLR4receptors in response to invading pathogens. The specific assembly of theTIR domains of TLR4, Mal and MyD88is responsible for proper signal transduction in the TLR4signaling pathway. However, the molecular mechanism for the specificity of these TIR-TIR domian interactions remains unelusive.In this research, we examined the structure of wild-type Mal-TIR at a resolution of2.4A. Unexpectedly, Mal-TIR exhibits an extraordinarily long AB loop, but no aB helix or BB loop, distinguishing it from other TIR domains. More importantly, the Mal-TIR AB loop is capable of mediating direct binding to the TIR domains of TLR4and MyD88simultaneously. We also found that Mal-TIR can form a back-to-back dimer that may resemble the dimeric assembly of the entire Mal molecule. Our results show the specificity of the TIR domains and the bridging role of the Mal molecule. Recently, several studies have associated natural variants of Mal with various infectious diseases. The most interesting single nucleotide polymorphism (SNP) is S180L in Mal. Heterozygous carriers of this variant are protected against some infectious diseases, including bacteremia, invasive pneumococcal disease, malaria and atopic dermatitis. The D96N mutation has also been shown to impair recruitment of MyD88to the plasma membrane and to influence the post-translational modification of Mal. To further understand the molecular mechanism underlying these associations in the Mal-TIR mutants D96N and S180L, we determined the crystal structures of these mutants at a resolution of2.75A and3.10A respectively, to provide structural information for studying the molecular mechanism of infectious diseases association.In present study, we use the methods of X-ray crystalgraphy, biochemistry and cell biology to reveal the molecular basis of the bridging role of Mal in connecting TLR4and MyD88, and to provide structural basic for the further study of the specificity of TIR domain assembly and the molecular mechanism of infectious diseases association.更多还原