Phagocyte Receptors for Lipid A

描述(申请人提供)：脂类A是脂多糖(内毒素)的活性部分，是一种在革兰氏阴性脓毒症发病机制中重要的细菌产物。脂质A通过特定的受体系统激活吞噬细胞。在LBP和CD14增强内毒素反应的同时，有两种蛋白质是信号转导所必需的：Toll样受体(TLR)4和MD-2。这种多聚体受体利用了所有四个TLR适配器分子：MyD88、MAL、TRAM和TRIF，使TLR4/MD-2具有其他TLR无法比拟的复杂性。过去，我们专注于TLR4激活的三个方面：第一，通过与其他TLR进行比较，我们可以了解到TLR4的哪些方面。第二，MD-2如何与内毒素结合。第三，MAL和电车如何与TLR4互动。在下一个资助阶段，我们的目标是确定TLR4如何达到激活状态(即，传递信号的状态)，并更好地描述含有TIR结构域的接头如何调控基因表达。我们将专注于我们已经成为专家的技术：创建新的细胞系，生产重组TLR，共聚焦和电子显微镜，微阵列分析和转基因小鼠的开发。在目标1中，我们建议确定什么构成活性TLR4/MD-2受体复合体。我们将集中在配体引起的构象变化，以及TLR4/MD-2的二聚化状态。在目标2中，我们将建立适配器分子结合的规则。我们将通过对TLR接头基因敲除小鼠的巨噬细胞进行RNA图谱分析，对接头分子特定介导的下游信号事件进行早就应该进行的全球分析。这些研究将通过实时荧光定量聚合酶链式反应得到验证，并测试规范模型(我们假设该模型有缺陷)，即MyD88/MAL激活促炎基因，而剩余的基因表达通过TRAM/TRIF辅助。我们将分析已知的与MAL和MyD88相关的多态在来自KO小鼠的巨噬细胞系中的功能，并确定这些多态如何影响接头分子的募集。在目标3中，我们将重点研究MD-2，TLR4/MD-2的结合部分，我们已经将其纯化为单体(活性)状态的均一。我们将使用生物物理方法(例如，圆二色谱，FOLIM)来评估配体诱导的MD-2和TLR4的构象变化。我们将结合诱变和正向遗传筛选来鉴定具有结构性活性的MD-2突变体，并通过建立在最近解析的MD-2晶体的基础上，从经验和计算机上确定它们的结构/功能。最后，对MD-2在体内炎症状态中的调节和作用几乎一无所知。我们将通过产生抗鼠MD-2单抗来扩展可用于MD-2的有限试剂的数量。我们还建议设计一种转基因小鼠，其中GFP由MD-2启动子控制，天然MD-2将被标记有FLAG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，我们相信，对TLR4/MD-2生物学的更好理解将导致脓毒症的发病率和死亡率的改善。Toll样受体(TLRs)是白细胞上识别微生物并导致免疫防御和炎症的分子。有许多疾病是由TLR激活引起的，但没有一种疾病比内毒素(脂蛋白A)引起的脓毒症更致命，后者是由TLR4/MD-2受体复合体激活引起的。我们建议了解TLR4/MD-2受体是如何被脂类A激活的，以期改善脓毒症的高死亡率和发病率。

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.