Phagocyte Receptors for Lipid A

描述（由申请人提供）：革兰氏阴性细菌性败血症仍然是美国发病率和死亡率的重要原因。革兰氏阴性脓毒症发生时，细菌膜释放内毒素（脂多糖，LPS）并与吞噬细胞和其他脂多糖敏感细胞表面的信号受体结合。虽然可能还会发现其他受体成分，但LPS反应机制的基本成分的名称现在是已知的。这些包括lps结合蛋白（LBP）、CD14、Toll样受体（TLR） 4、MD-2和一个或多个含有“Toll，白细胞介素1，抗性”（TIR）结构域的适配蛋白。待验证的假设代表了当前的教条：脓毒症始于lbp介导的LPS对CD14的呈现。CD14向TLR4/MD-2复合物呈递LPS。LPS与TLR4结合导致受体二聚化和MyD88/Mal复合物的募集，从而启动NF-KB和IRF信号转导途径。这些转录因子驱动细胞因子的产生，导致败血症。这一教条的许多方面需要重新评估和改进。例如，虽然初步数据证实LPS直接与MD-2结合，并提示TLR4也被结合，但TLR4/MD-2在被LPS结合后形成比二聚体大得多的多聚体。除了MyD88和Mal/TIRAP外，至少还有另外两个适配分子TRAM（我们最近发现的）和TRIF参与TLR4通路。我们提出了4个具体目标：1)表征LPS与TLR4和MD-2的结合。2)确定TLR4/MD-2复合体是否通过产生“信号/ some”簇激活信号转导。目的是精确量化lps刺激细胞中受体的大小和信号体的组成。3)评估5种T/R结构域适配器分子在TLR4信号转导中的作用。这个目标结合了生物化学、分子遗传学和共聚焦显微镜来定义适配器在LPS刺激中的作用。最后，4)通过生成和表征TRAM靶向缺失小鼠，表征新发现的适配分子TRAM在LPS信号转导中的作用。这只老鼠的细胞将被测试对LPS和其他微生物产物的反应。此外，我们将用沙门氏菌挑战这种敲除动物，以确定TRAM表达是否和热是否有助于宿主防御。

DESCRIPTION (provided by applicant): Gram-negative bacterial septicemia remains an important cause of morbidity and mortality in the United States. Gram-negative sepsis begins when bacterial membranes shed endotoxin (lipopolysacchadde, LPS)and engage signaling receptors on the surface of phagocytes and other LPS-sensitive cells. Although additional receptor components may yet be discovered, the names of the basic components of the LPS response machinery are now known. These include LPS-binding protein (LBP), CD14, Toll-like receptor (TLR) 4, MD-2 and one or more "Toll, interleukin 1, resistance" (TIR)-domain containing adapter proteins. The hypothesis to be tested represents the current dogma: sepsis begins with LBP-mediated presentation of LPS to CD14. CD14 presents LPS to the TLR4/MD-2 complex. LPS binding to TLR4 results in receptor dimerization and recruitment of MyD88/Mal complexes resulting in the initiation of the NF-KB and IRF signal transduction pathways. These transcription factors drive cytokine production, resulting in septicemia. Many aspects of this dogma need considerable reassessment and refinement. For example, while preliminary data confirm that LPS directly binds to MD-2, and suggest that TLR4 is also bound, TLR4/MD-2 forms large multimers- much larger than dimmers- after being bound by LPS. In addition to MyD88 and Mal/TIRAP, at least two other adapter molecules, TRAM (which we recently discovered) and TRIF, are involved in the TLR4 pathway. We propose 4 specific aims: 1) To characterize the binding of LPS to TLR4 and MD-2. 2) To determine if the TLR4/MD-2 complex activates signal transduction by creating 'signa/osome' clusters. The aim is meant to precisely quantify receptor size and composition of the signalosome in LPS-stimulated cells. 3) To assess the roles of the five T/R domain containing adapter molecules in TLR4 signal transduction. This aim combines aspects of biochemistry, molecular genetics and confocal microscopy to define the role of adapters in LPS stimulation. And finally, 4) to characterize the role of TRAM, a newly discovered adapter molecule, in LPS signal transduction by generating and characterizing a mouse with a targeted deletion in TRAM. Cells from this mouse will be tested for responses to LPS and other microbial products. In addition, we will challenge this knockout animal with Salmonella to determine if and hot TRAM expression contributes to host defense.