Regulation of MD-2 function and expression

MD-2 功能和表达的调节

• 批准号: 6760722
• 负责人: JERROLD P WEISS
• 金额: $ 36.88万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-06-01 至 2009-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6760722
• 关键词: Adenoviridae; CD14 molecule; clinical research; endotoxins; enzyme linked immunosorbent assay; gene expression; genetic regulation; human tissue; immune response; lipopolysaccharides; monocyte; protein protein interaction; protein structure function; recombinant proteins; respiratory epithelium; surface antigens; tissue /cell culture; toll like receptor; transfection /expression vector

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.

描述（由申请人提供）：需要对内毒素（E）的高度敏感宿主反应，以最佳防御对许多入侵革兰氏阴性细菌（GNB），但当当暴露于内毒素受到充分控制时，还可以导致明显的宿主病理。 E信号传导的最大敏感性需要E与几个细胞外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。最近的观察值，包括我们本身的c. and c. signition 4。 TLR4并至少在某些环境（例如气道上皮）中确定细胞对E的反应性。 该项目的长期目标是更好地理解对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。我们的直接重点是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）鉴定有效激动剂或拮抗剂功能所需的E：MD-2复合物的分子特性； 3）调节人类气道上皮细胞中MD-2表达的机制的表征。 我们将使用代谢标记的和色谱纯化的E和重组E绑定蛋白（LBP，SCD14和MD-2）来分析低，病原体相关的E浓度的蛋白质-E相互作用，并以纯净的纯净纯度纯化蛋白质：E蛋白质：E复合物以进行进一步的结构和功能分析。 MD-2表达将通过mRNA和新鲜组织和人支气管上皮的原发性培养的蛋白质分析来监测，以鉴定MD-2表达调节的介体和机制。 这些研究应产生有关主机相互作用的结构决定因素对先天免疫识别和对入侵GNB的反应重要的新见解。对MD-2的结构，功能和表达的更好理解应改善对E宿主对E的调控的理解，并可能为开发化合物的开发铺平道路，通过调节细胞对E的细胞反应可能具有预防性和/或治疗性应用。