Regulation of MD-2 function and expression

描述（由申请方提供）：需要对内毒素（E）高度敏感的宿主反应，以最佳防御许多种入侵的革兰氏阴性菌（GNB），但当暴露于内毒素未得到充分控制时，也可能导致显著的宿主病理学。E信号传导的最大灵敏度需要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（LPS）结合蛋白（LBP），CD 14，MD-2和Toll样受体（TLR）4。最近的观察，包括我们自己的，表明MD-2的重要作用，连接E识别启动LBP和CD 14激活TLR 4和确定，至少在某些设置（如气道上皮），细胞对E。 该项目的长期目标是更好地理解E的先天免疫识别的分子基础以及E识别与促炎反应诱导的耦合。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复合物的分子特性，这些特性是有效的激动剂或拮抗剂功能所必需的; 3）表征MD-2在人气道上皮细胞中表达的调节机制。 我们将使用代谢标记和色谱纯化的E和重组E结合蛋白（LBP，sCD 14和MD-2），以分析蛋白质-E相互作用在低，病理生理相关的E浓度和纯化蛋白质：E复合物进一步的结构和功能分析。将通过人支气管上皮的新鲜组织和原代培养物的mRNA和蛋白质分析来监测MD-2表达，以鉴定MD-2表达的调节介质和机制。 这些研究应产生新的见解有关的结构决定因素主机E相互作用的先天免疫识别和入侵GNB的反应很重要。更好地了解MD-2的结构，功能和表达，应提高对宿主对E的反应性调节的理解，并可能为开发通过调节细胞对E的反应，可能具有预防和/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.