Phagocyte Receptors for Lipid A

脂质 A 的吞噬细胞受体

基本信息

批准号：
7465775
负责人：
Douglas T Golenbock
金额：
$ 47.08万
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：
1997
资助国家：
美国
起止时间：
1997-01-01 至 2013-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7465775
关键词：
Aftercare Agonist Agreement Antibodies Avidity Bacillus (bacterium)Back Bacteria Baculoviruses Binding Biochemical Biological Assay Biology Blood Breeding CD14 gene Cell Line Cell surface Cells Characteristics Chimeric Proteins Circular Dichroism Co-Immunoprecipitations Collaborations Complex Computer Simulation Condition Confocal Microscopy Custom Cytochalasins Cytometry Cytoplasmic Tail Depth Dimerization Disease E 5531 Electron Microscopy Endotoxins Energy Transfer Engineering Epitopes Erythrocytes Event Exposure to Extracellular Domain Fluorescence Resonance Energy Transfer Funding Gene Expression Gene Expression Regulation Generations Genes Genetic Polymorphism Genetic Screening Goals Golgi Apparatus Gram-Negative Bacteria Green Fluorescent Proteins Health Image Immune In Vitro Individual Infection Inflammation Inflammatory Injection of therapeutic agent Insecta Interferon Type I Interferons Interleukin-6 Internal Ribosome Entry Site Kinetics Knock-in Mouse Knockout Mice Label Lead Learning Leukocytes Libraries Ligands Ligation Lipid A Lipids Lipopolysaccharides Liver Malaria Mammalian Cell Measures Mediating Membrane Microarray Analysis Microbe Microscopy Modeling Molecular Conformation Morbidity - disease rate Mus Mutagenesis NF-kappa B Netherlands Numbers Pathogenesis Pathway interactions Phagocytes Phagocytosis Phagolysosome Polymerase Chain Reaction Production Proteins Puncture procedure RNA Reagent Receptor Activation Recombinants Recruitment Activity Recycling Regulation Reporter Research Design Resistance Resources Respiratory syncytial virus Respiratory syncytial virus RSV proteins Role Scanning Transmission Electron Microscopy Procedures Sepsis Signal Pathway Signal Transduction Single Nucleotide Polymorphism Spleen Streptococcus pneumoniae plY protein Structure Surface System TLR4 gene TNF gene Techniques Testing Thinking Time Toll-like receptors Transgenic Mice Transgenic Organisms Uncertainty Universities Virus Wild Type Mouse Work Zeocin chemokine crosslink cytokine human wyatt protein improved in vivo inhibitor/antagonist interest macrophage mortality mouse wyatt protein mutant mutein 2 novel prevent promoter receptor respiratory response toll-like receptor 4 tool

项目摘要

DESCRIPTION (provided by applicant): Lipid A is the active moiety of lipopolysaccharide (LPS, endotoxin), a bacterial product important in the pathogenesis of Gram-negative sepsis. Lipid A activates phagocytes through a defined receptor system. While LBP and CD14 enhance LPS responses, two proteins are obligatory for signal transduction: Toll-like receptor (TLR) 4 and MD-2. This multimeric receptor utilizes all four TLR-adapter molecules: MyD88, Mal, TRAM and TRIF, giving TLR4/MD-2 a complexity that is unrivaled by other TLRs. In the past, we focused on three aspects of TLR4 activation: first, what we could learn about TLR4 by comparing it to other TLRs. Second, how MD-2 binds LPS. Third, how Mal and TRAM interact with TLR4. In the next funding period, our goal is to determine how TLR4 achieves an active state (i.e., one that transduces signals) and to better delineate how TIR domain containing adapters modulate gene expression. We will focus on techniques with which we have become expert: the creation of novel cell lines, the production of recombinant TLRs, confocal and electron microscopy, microarray analysis and the exploitation of transgenic mice. In the Aim 1, we propose to identify what constitutes an active TLR4/MD-2 receptor complex. We will focus on conformational changes induced by ligands, as well as the dimerization status of TLR4/MD-2. In Aim 2, we will establish the rules for adapter molecule engagement. We will perform a much-overdue global analysis of downstream signaling events specifically mediated by adapter molecules by RNA profiling LPS-stimulated macrophages from TLR adapter knockout mice. These studies will be validated by real time PCR, and test the canonical model (which we hypothesize to be flawed) that MyD88/Mal activates proinflammatory genes while the remained of gene expression is subserved via TRAM/TRIF. We will analyze the functions of known polymorphisms associated with Mal and MyD88 in macrophage cell lines derived from KO mice, and determine how these polymorphisms influence adapter molecule recruitment. In Aim 3, we will focus on MD-2, the binding portion of TLR4/MD-2, which we have purified to homogeneity in its monomeric (active) state. We will use biophysical approaches (e.g., circular dichroism, FLIM) to assess ligand-induced conformational changes in MD-2 and TLR4. We will combine mutagenesis and forward genetic screening to identify mutants of MD-2 that are constitutively active, and determine their structure/function both empirically and in silico by building on the recently resolved crystal for MD-2. Finally, virtually nothing is known about the regulation and role of MD-2 during inflammatory states in vivo. We will expand upon the limited number of reagents available for MD-2 by generating anti-mouse MD-2 mAbs. We also propose to engineer a transgenic mouse in which GFP is under the control of the MD-2 promoter and natural MD-2 will be epitope tagged with FLAG. We will determine which cells produce MD-2 and how much is produced during inflammation. Ultimately, we believe that an improved understanding of TLR4/MD-2 biology will lead to an amelioration of the morbidity and mortality of sepsis. Toll-like receptors (TLRs) are molecules on white blood cells that recognize microbes and lead to immune defense and inflammation. There are many diseases caused by TLR activation, but none is more deadly than LPS (lipid A) induced sepsis, which is caused by activation of the TLR4/MD-2 receptor complex. We propose to learn how the TLR4/MD-2 receptor is activated by LPS (lipid A), in order that the high mortality and morbidity of sepsis can be ameliorated.

描述（由申请人提供）：脂质A是脂多糖（LPS，内毒素）的活性部分，这是一种对革兰氏阴性败血症发病机理重要的细菌产物。脂质A通过定义的受体系统激活吞噬细胞。尽管LBP和CD14增强了LPS响应，但两个蛋白具有信号转导的强制性：Toll样受体（TLR）4和MD-2。该多聚体受体利用了所有四个TLR-apapter分子：MyD88，MAL，TRAM和TRIF，使TLR4/MD-2具有由其他TLR无与伦比的复杂性。过去，我们专注于TLR4激活的三个方面：首先，通过将其与其他TLR进行比较，我们可以了解到TLR4。其次，MD-2如何结合LPS。第三，MAL和TRAM如何与TLR4相互作用。在下一个资金期间，我们的目标是确定TLR4如何实现活性状态（即传递信号的状态），并更好地描述包含适配器的TIR域如何调节基因表达。我们将重点关注我们已成为专家的技术：新型细胞系，重组TLR的产生，共聚焦和电子显微镜，微阵列分析以及转基因小鼠的开发。在目标1中，我们建议确定什么构成活性TLR4/MD-2受体复合物。我们将重点关注配体引起的构象变化以及TLR4/MD-2的二聚化状态。在AIM 2中，我们将建立适配器分子参与的规则。我们将通过RNA分析LPS刺激的巨噬细胞从TLR适配器基因敲除小鼠进行的，对衔接子分子专门介导的下游信号事件进行了彻底的全球分析。这些研究将通过实时PCR来验证，并测试MyD88/MAL激活促炎基因的规范模型（我们假设是有缺陷的），而剩余的基因表达则通过TRAM/TRIF进行了供应。我们将分析与MAL和MYD88相关的已知多态性的功能，在源自KO小鼠的巨噬细胞系中，并确定这些多态性如何影响衔接子分子的募集。在AIM 3中，我们将重点放在MD-2（TLR4/MD-2的结合部分）上，我们已将其纯化具有其单体（主动）状态的同质性。我们将使用生物物理方法（例如，圆形二色性，FLIM）来评估配体诱导的MD-2和TLR4构象变化。我们将结合诱变和正向遗传筛选，以鉴定成组成型活性的MD-2的突变体，并通过在最近解决的MD-2上建立在最近分辨的晶体上，从经验上和计算机确定它们的结构/功能。最后，实际上，关于MD-2在体内炎症状态中的调节和作用几乎一无所知。我们将通过产生抗小鼠MD-2 mABS来扩展MD-2可用的试剂数量有限。我们还建议设计一种转基因小鼠，其中GFP在MD-2启动子的控制之下，天然MD-2将被标记为标记的表位。我们将确定哪些细胞产生MD-2，以及在炎症过程中产生多少。最终，我们认为，对TLR4/MD-2生物学的理解得到了改善，将导致对败血症的发病率和死亡率的改善。 Toll样受体（TLR）是识别微生物并导致免疫防御和炎症的白细胞上的分子。 TLR激活引起许多疾病，但没有比LPS（脂质A）诱发的败血症更致命，这是由TLR4/MD-2受体复合物的激活引起的。我们建议学习如何通过LPS（Lipid A）激活TLR4/MD-2受体，以便可以改善败血症的高死亡率和发病率。