Regulation of MD-2 function and expression

描述（由申请人提供）：宿主对内毒素(E)的高度敏感反应是抵御多种入侵革兰氏阴性菌（GNB）的最佳防御所必需的，但当内毒素暴露没有得到充分控制时，也可能导致显著的宿主病理。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（LPS）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的重要作用，将LBP和CD14启动的E识别与TLR4的激活联系起来，并至少在某些情况下（例如气道上皮）决定细胞对E的反应。

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.