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组分，是人体单核细胞和巨噬细胞的强效免疫刺激剂，并与脓毒症和脓毒性休克的发病机制有关，脓毒症和脓毒性休克是一种临床进展，仅在美国每年就导致250，000例死亡。作为先天免疫的一部分的炎症反应通过这些内毒素与关键宿主细胞受体CD 14的高亲和力结合而引发，从而通过涉及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，诱导败血性休克和死亡。已知CD 14通过调节炎症反应和增强内毒素诱导的活化的敏感性在先天系统中发挥中心作用。然而，它实现这一点的分子机制尚不清楚。拟定研究的重点是通过CD 14的结构/功能研究了解负责细胞活化的CD 14-内毒素相互作用的分子细节。我们的中心假设是，CD 14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，作为模式识别受体发挥作用。我们将通过以下具体目标来检验我们的假设：1）通过溶液NMR光谱法和绘制内毒素识别所需的CD 14上的功能表位来高分辨率确定可溶形式的CD 14的结构，2）CD 14-配体复合物的结构表征以揭示CD 14结合所需的共有配体结构特征，3）突变和生物物理学研究，以鉴定参与与不同内毒素相互作用的关键CD 14残基及其在CD 14模式识别中的作用。这些研究的完成将为LPS、PGN的毒性作用和合理设计治疗脓毒症的潜在新型LPS、PGN拮抗剂提供结构基础。

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.