The TLR4/MD-2 signaling pathway in gonoccocal disease

淋病奈瑟菌可引起多种疾病综合征，包括盆腔炎性疾病 [PID]。产后疼痛会导致慢性盆腔疼痛、宫外孕和不孕症。由N。 淋病的共同特点是由炎性细胞介导的强烈炎症。这种炎症是 主要是内毒素(LOS)与内毒素受体复合体TLR4和MD- 2.内毒素受体的成分在十年前就已确定，但至今仍知之甚少，1) 脂质A与MD-2的结合如何导致活性受体复合体的形成，以及，2)如何 信号随后被传递，导致产生促炎介质，如TNFa 和IL-IP。在这项提案中，我们描述了确定MD-2一旦与脂质A结合， 获得激活TLR4的能力。这种方法建立在我们成功提纯MD-2的基础上，MD-2是一种小的 含有7个半胱氨酸残基的分子，有形成无效多聚体的恶名。我们计划 在没有和存在激活配体的情况下，以及在TLR4存在的情况下，拆分MD-2的结构 为了确定TLR4/MD-2的哪些构象变化可以诱导信号传导。然后我们将集中我们的 参与细胞信号转导的TLR4相关接头分子的能量。我们之前已经分析了5个 称为MAI的接头蛋白中的单核苷酸多态(SNPs)(由TLR2和 TLR4)。两个SNP：S180L和D96N是人们非常感兴趣的。作为NIH资助的另一个项目的一部分，我们有 开始产生携带这些病变的小鼠，并正在筛查患者样本中是否存在D96N。 MAI敲除小鼠和敲入小鼠携带相当于D96N或S180L的小鼠将在 Ingalls博士建立的小鼠GC感染模型(PI，项目2)。我们将进行类似的分子遗传学研究 在MyD88中已知的6个SNP中，与MAI相互作用的下游适配器和一个重要的适配器 至少8个TLR。如果任何一个SNPs表现出表型，我们将产生敲入小鼠和 筛选患者样本以确定相关性。最后，我们将尝试定义MAI和 MyD88通过生化手段，最终试图使MAL/MyD88二聚体共结晶。

Neisseria gonorrhoeae causes a variety of disease syndromes including pelvic iniflammatory disease [PID]. PID can lead to chronic pelvic pain, ectopic pregnancy and infertility. Those syndromes caused by N. gonorrhoeae have, in common, intense inflammation mediated by inflammatory cells. This inflammation is primarily the result of the interaction of neisserial LPS (LOS) v\/ith the LPS receptor complex: TLR4 and MD- 2. The components of the LPS receptor were identified a decade ago, yet it is still poorly understood, 1) how the binding of lipid A to MD-2 results in the formation of an active receptor complex, and, 2) how a signal is subsequently transmitted resulting in the production of proinflammatory mediators such as TNFa and IL-ip. In this proposal, we describe plans to determine how MD-2, once bound to lipid A, acquires the ability to activate TLR4. The approach builds upon our success in purifying MD-2, a small molecule with 7 cysteine residues that has a notorious tendency to form inactive multimers. We plan to resolve the structure of MD-2 in the absence and presence of activating ligand, and in the presence of TLR4 to determine what conformational changes in TLR4/MD-2 induce signaling. We shall then focus our energies on TLR4-related adapter molecules involved in cell signaling. We have previously analyzed 5 single nucleotide polymorphisms (SNPs) in the adapter protein known as Mai (used by both TLR2 and TLR4). Two SNPs are of great interest: S180L and D96N. As part of another NIH funded project, we have begun to generate mice carrying these lesions and are screening patient samples for the presence of D96N. Mai knock out mice and knock-in mice carrying the mouse equivalent of D96N or S180L will be tested in the mouse model of GC infection by Dr. Ingalls (PI, project 2). We will perform similar molecular genetic studies of the 6 known SNPs in MyD88, the downstream adapter that interacts with Mai and an important adapter for at least 8 of the TLRs. Should any of the SNPs display a phenotype, we will generate knock-in mice and screen patient samples to determine relevancy. Finally, we will attempt to define the interaction of Mai and MyD88 by biochemical means, culminating in an attempt to co crystallize the Mal/MyD88 dimer.