Structure-function studies of endotoxin receptor CD14

描述（由申请人提供）：内毒素如LPS和PGN，来自革兰氏阴性和革兰氏阳性细菌的外细胞壁成分是人类单核细胞和巨噬细胞的有效免疫刺激物，并且与败血症和感染性休克的发病机制有关，这一临床进展每年仅在美国就导致250,000人死亡。作为先天免疫的一部分，炎症反应是通过这些内毒素与关键宿主细胞受体cd14的高亲和力结合而启动的，通过涉及toll样受体的信号转导途径触发多种促炎介质（包括细胞因子）的释放。然而，这种细胞反应的过度激活可能导致促炎介质的过度释放，诱发感染性休克和死亡。已知cd14在先天系统中发挥核心作用，通过调节炎症反应和增强内毒素诱导激活的敏感性。然而，它实现这一目标的分子机制尚不清楚。这些研究的重点是通过CD14的结构/功能研究来了解CD14 -内毒素相互作用的分子细节，这些相互作用负责细胞活化。我们的中心假设是CD14作为一种模式识别受体，通过识别各种内毒素上的特征分子模式并在结构相似但不同的位点结合它们。我们将以以下具体目标来检验我们的假设：1)通过溶液核磁共振光谱测定CD14可溶性形式的高分辨率结构，并绘制内毒素识别所需的CD14功能表位；2)CD14配体复合物的结构表征，揭示CD14结合所需的配体结构特征；3)诱变和生物物理研究，确定与不同内毒素相互作用的关键CD14残基及其在CD14模式识别中的作用。这些研究的完成将为LPS、PGN的有害作用和合理设计潜在的新型LPS、PGN拮抗剂治疗败血症提供结构基础。

DESCRIPTION (provided by applicant): Endotoxins such as LPS and PGN, components of outer cell walls from Gram-negative and Gram-positive bacteria are potent immunostimulators of monocytes and macrophages in humans and have been implicated in the pathogenesis of sepsis and septic shock, a clinical progression that results in 250,000 deaths annually in the United States alone. Inflammatory responses as part of innate immunity are initiated via high-affinity binding of these endotoxins to a key host cell receptor CD 14, triggering release of a variety of pro inflammatory mediators, including cytokines, through a signal transduction pathway involving Toll-like receptors. An overactivation of this cellular response may, however, result in excessive release of the proinflammatory mediators, inducing septic shock and death. CD 14 is known to play a central role in the innate system, by modulating the inflammatory response and enhancing the sensitivity of endotoxininduced activation. However, the molecular mechanisms by which it accomplishes this are not clear. The proposed studies are focused on understanding the molecular details of CD 14-endotoxin interactions responsible for cellular activation by structure/function studies of CD14. Our central hypothesis is that CD14 functions as a pattern recognition receptor by recognition of characteristic molecular patterns on various endotoxins and binding them at structurally similar but distinct sites. We will test our hypothesis with the following specific aims: 1) High-resolution structure determination of a soluble form of CD 14 by solution NMR spectroscopy and mapping functional epitopes on CD 14 necessary for endotoxin recognition, 2) Structural characterization of CD14-ligand complexes to unravel shared ligand structural features required for CD14 binding, 3) Mutagenesis and biophysical studies to identify key CD14 residues involved in interaction with different endotoxins and their role in pattern recognition by CD14. Completion of these studies will provide the structural basis for the deleterious effects of LPS, PGN and rational design of potentially novel LPS, PGN antagonists for treatment of sepsis.