Regulation of MD-2 function and expression

描述(由申请人提供)：对内毒素(E)的高度敏感的宿主反应是对抗多种入侵的革兰氏阴性细菌(GNB)的最佳防御所必需的，但当暴露于内毒素没有得到充分控制时，也可能导致严重的宿主病理。E信号的最大敏感性需要E与几种细胞外和细胞表面宿主蛋白的有序相互作用，包括：脂多糖(LBP)结合蛋白(LBP)、CD14、MD-2和Toll样受体(TLR)4。最近的观察，包括我们自己的观察，表明MD-2具有重要作用，将LBP和CD14启动的E识别与TLR4的激活联系起来，并至少在某些环境下(如呼吸道上皮)决定细胞对E的反应性。 该项目的长期目标是更好地了解E的先天免疫识别的分子基础，以及E识别与诱导促炎反应的耦合。我们目前的重点将是MD-2的结构、功能和表达，包括：1)进一步表征E-MD-2相互作用的结构决定因素；2)鉴定E：MD-2复合体的分子性质，这是发挥强大的激动剂或拮抗剂功能所必需的；以及3)表征(S)调节人呼吸道上皮细胞MD-2表达的机制。 我们将使用代谢性标记和层析纯化的E和重组E结合蛋白(LBP、sCD14和MD-2)来分析低浓度、病理生理相关的E蛋白与E的相互作用，并为进一步的结构和功能分析准备纯化蛋白质：E复合体。将通过对新鲜组织和原代培养的人支气管上皮进行mRNA和蛋白质分析来监测MD-2的表达，以确定MD-2表达的调节介质和机制。 这些研究应该对宿主-E相互作用的结构决定因素产生新的见解，这些结构决定因素在先天性免疫识别和对入侵的GNB的反应中非常重要。更好地了解MD-2的结构、功能和表达将有助于理解宿主对E的反应性调节，并可能为开发通过调节细胞对E的反应而具有预防和/或治疗应用的化合物铺平道路。

DESCRIPTION (provided by applicant): Highly sensitive host responses to endotoxin (E) are needed for optimal defense against many species of invading Gram-negative bacteria (GNB) but can also lead to significant host pathology when exposure to endotoxin is not adequately controlled. Maximum sensitivity of E signaling requires the ordered interaction of E with several extracellular and cell surface host proteins, including: lipopolysaccharide (LPS)-binding protein (LBP), CD14, MD-2 and Toll-Like Receptor (TLR) 4. Recent observations, including our own, indicate an essential role for MD-2, linking E recognition initiated by LBP and CD14 to activation of TLR4 and determining, at least in certain settings (e.g. airway epithelium), cellular responsiveness to E. The long-term goals of this project are to better understand the molecular basis of innate immune recognition of E and of the coupling of E recognition to induction of pro-inflammatory responses. Our immediate focus will be the structure, function and expression of MD-2, including: 1) further characterization of the structural determinants of E-MD-2 interactions; 2) identification of molecular properties of E:MD-2 complexes required either for potent agonist or antagonist function; and 3) characterization of mechanism(s) regulating MD-2 expression in human airway epithelial cells. We will use metabolically labeled and chromatographically purified E and recombinant E-binding proteins (LBP, sCD14 and MD-2) to analyze protein-E interactions at low, patho-physiologically relevant E concentrations and to preparatively purify protein:E complexes for further structural and functional analyses. MD-2 expression will be monitored by mRNA and protein analyses of fresh tissue and primary cultures of human bronchial epithelium to identify mediators and mechanisms of regulation of MD-2 expression. These studies should yield novel insights concerning the structural determinants of host-E interactions important in innate immune recognition and response to invading GNB. A better understanding of the structure, function and expression of MD-2 should improve understanding of the regulation of host responsiveness to E and could pave the way for development of compounds that, by modulating cellular responses to E, may have prophylactic and/or therapeutic applications.