Phagocyte Receptors for Lipid A

性状（由申请方提供）：脂质A是脂多糖（LPS，内毒素）的活性部分，脂多糖是革兰氏阴性脓毒症发病机制中重要的细菌产物。脂质A通过确定的受体系统激活吞噬细胞。虽然LBP和CD 14增强LPS应答，但两种蛋白质是信号转导的必需品：Toll样受体（TLR）4和MD-2。这种多聚体受体利用了所有四种TLR接头分子：MyD 88，Mal，TRAM和TRIF，使TLR 4/MD-2具有其他TLR无法比拟的复杂性。在过去，我们关注TLR 4激活的三个方面：首先，我们可以通过将其与其他TLRs进行比较来了解TLR 4。第二，MD-2如何结合LPS。第三，Mal和TRAM如何与TLR 4相互作用。在下一个资助期，我们的目标是确定TLR 4如何达到活性状态（即，转导信号的分子），并更好地描述含有接头的TIR结构域如何调节基因表达。我们将专注于我们已经成为专家的技术：新细胞系的创建，重组TLR的生产，共聚焦和电子显微镜，微阵列分析和转基因小鼠的开发。在目的1中，我们提出确定什么构成了活性TLR 4/MD-2受体复合物。我们将专注于配体诱导的构象变化，以及TLR 4/MD-2的二聚化状态。在目标2中，我们将建立衔接子分子接合的规则。我们将进行一个逾期已久的全球分析下游信号转导事件的衔接分子特异性介导的RNA分析LPS刺激的巨噬细胞从TLR衔接子敲除小鼠。这些研究将通过真实的时间PCR进行验证，并测试MyD 88/Mal激活促炎基因同时通过TRAM/TRIF维持基因表达的经典模型（我们假设该模型是有缺陷的）。我们将分析与Mal和MyD 88相关的已知多态性在来自KO小鼠的巨噬细胞系中的功能，并确定这些多态性如何影响衔接分子的募集。在目标3中，我们将关注MD-2，即TLR 4/MD-2的结合部分，我们已经将其纯化至单体（活性）状态的均一性。我们将使用生物物理方法（例如，圆二色性，FLIM）来评估MD-2和TLR 4中配体诱导的构象变化。我们将结合联合收割机和正向遗传筛选，以确定突变的MD-2是组成型活性，并确定其结构/功能的经验和在电脑上建立在最近解决的MD-2晶体。最后，几乎没有人知道MD-2在体内炎症状态期间的调节和作用。我们将通过产生抗小鼠MD-2 mAb来扩展MD-2可用的有限数量的试剂。我们还提出了一种转基因小鼠，其中GFP是在MD-2启动子的控制下，天然MD-2将与FLAG表位标记。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，TLR 4/MD-2生物学的进一步了解将导致脓毒症的发病率和死亡率的改善。Toll样受体（TLR）是白色血细胞上的分子，可以识别微生物并导致免疫防御和炎症。有许多疾病是由TLR激活引起的，但没有一种比LPS（脂质A）诱导的脓毒症更致命，其是由TLR 4/MD-2受体复合物的激活引起的。我们拟研究LPS（lipid A）是如何激活TLR 4/MD-2受体的，以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.