The TLR4/MD-2 signaling pathway in gonoccocal disease

淋球菌疾病中的 TLR4/MD-2 信号通路

基本信息

批准号：
7764287
负责人：
Douglas T Golenbock
金额：
$ 29.27万
依托单位：
UNIV OF MASSACHUSETTS MED SCH WORCESTER
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-09-25 至 2014-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7764287
关键词：
Acute Affect Affinity Chromatography Appearance Back Backcrossings Bacterial Infections Baculoviruses Binding Biochemical Biology Bone Marrow Breeding Budgets C-terminal CD14 Antigen Cations Cell Line Cell surface Cells Circular Dichroism Cleaved cell Co-Immunoprecipitations Collaborations Complex Confocal Microscopy Crystallography Cysteine Cytoplasmic Tail DNA Data Defect Disease Distal Ectopic Pregnancy Endotoxins Engineering Escherichia coli Event Extracellular Protein Funding Future Gel Chromatography Genetic Genetic Polymorphism Genitourinary system Goals Gonorrhea Gram-Negative Bacteria Growth Habits Human I-kappa B Proteins IL6 gene IRAK4 gene Immunity Immunoglobulin G Immunomodulators In Vitro Inbred BALB C Mice Incubated Individual Infection Infertility Inflammation Inflammatory Insecta Interferons Interleukin-1 Receptors Ion-Exchange Chromatography Procedure Knock-in Mouse Knock-out Knockout Mice Lead Lesion Ligand Binding Ligands Lipid A Lipids Lipopolysaccharides Measures Mediating Mediator of activation protein Membrane Molecular Molecular Conformation Molecular Genetics Molecular Sieve Chromatography Molecular Structure Mus Nature Neisseria gonorrhoeae Outcome Pathway interactions Patients Pelvic Inflammatory Disease Pelvic Pain Pelvis Phase Phenotype Phosphorylation Physiological Play Post-Translational Protein Processing Production Proteins Recruitment Activity Reporter Robin bird Role Sampling Screening procedure Signal Pathway Signal Transduction Signaling Molecule Single Nucleotide Polymorphism Speed Structure Syndrome System TLR1 gene TLR2 gene TLR4 gene Technology Testing Thrombin Toll-like receptors Transfection Tubal Pregnancy United States National Institutes of Health Upper arm Vaccine Therapy Vagina Variant Woman Work adapter protein base chronic pelvic pain congenic breeding cytokine dimer high risk human TYRP1 protein in vivo interest lipooligosaccharide macrophage mouse model novel prevent receptor receptor function response sexually active small molecule success toll-like receptor 4

项目摘要

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.

淋病奈瑟氏菌引起多种疾病综合征，包括骨盆疾病 [pid]。 PID会导致慢性骨盆疼痛，异位妊娠和不育。那些由n引起的综合征。淋病通常具有由炎症细胞介导的强烈炎症。这种炎症是主要是奈瑟氏LPS（LOS）V \/ITH的相互作用的结果：LPS受体复合物：TLR4和MD- 2。十年前确定了LPS受体的成分，但仍然很少了解，1）脂质A与MD-2的结合如何导致活性受体复合物的形成，以及2）A 随后传播信号，导致产生促炎性介体（例如TNFA）和il-ip。在此提案中，我们描述了确定MD-2曾经绑定到脂质A的计划，获得激活TLR4的能力。这种方法基于我们在净化MD-2的成功基础上，这是一个小的具有7种半胱氨酸残基的分子，具有形成非活性多聚体的臭名昭著的趋势。我们计划在不存在和存在激活配体的情况下解决MD-2的结构，并存在TLR4 确定TLR4/MD-2中哪些构象变化诱导信号传导。然后我们将集中精力与细胞信号传导有关的TLR4相关衔接子分子上的能量。我们以前已经分析了5 衔接蛋白中的单核苷酸多态性（SNP）称为MAI（由TLR2和TLR2使用 TLR4）。两个SNP引起了极大的兴趣：S180L和D96N。作为另一个由NIH资助的项目的一部分，我们有开始产生携带这些病变的小鼠，并正在筛查患者样品以在存在D96N的情况下。 MAI将携带小鼠等效的D96N或S180L的小鼠敲出小鼠和敲门小鼠将在 Ingalls博士的GC感染的小鼠模型（PI，项目2）。我们将进行类似的分子遗传研究在MyD88中的6个已知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SNP都显示表型，我们将生成敲门型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，最终试图结晶MAL/MYD88二聚体。