Molecular mechanism of apolipoprotein binding to lipopolysaccharides

载脂蛋白与脂多糖结合的分子机制

• 批准号: 7761161
• 负责人: PAUL Michiel WEERS
• 金额: $ 10.76万
• 依托单位: CALIFORNIA STATE UNIVERSITY LONG BEACH
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-01-01 至 2013-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7761161
• 关键词: Acetylation; Address; Apolipoprotein A-I; Apolipoprotein E; Apolipoproteins; Apolipoproteins A; Apolipoproteins C; Area; Bacteria; Binding; Binding Proteins; Biochemical; Biological Models; Blood; Blood Circulation; Carbohydrates; Cardiovascular Diseases; Cause of Death; Cell Wall; Cessation of life; Charge; Cholesterol; Collaborations; Complement; Complex; Cysteine; Development; Disease; Electron Transport Complex III; Environment; Fluorescence Spectroscopy; Foundations; Funding; Gram-Negative Bacteria; Grant; Health; Homeostasis; Human; Immune; Immune system; Infection; Intensive Care Units; Invaded; Invertebrates; Investigation; Knowledge; Lipid A; Lipid Binding; Lipids; Lipopolysaccharides; Lipoproteins; Lysine; Membrane; Modeling; Molecular; Molecular Biology; Molecular Conformation; Natural Immunity; Nature; Particle Size; Pattern Recognition; Play; Process; Protein Binding; Proteins; Publications; Recombinants; Research; Role; Sepsis; Septic Shock; Serum; Serum Proteins; Shock; Side; Site-Directed Mutagenesis; Solutions; Spectrum Analysis; Structure; Structure-Activity Relationship; Students; System; Training; Transport Process; Tryptophan; Variant; Work; apolipophorin III; base; flexibility; improved; insight; lipid transport; microbial; molecular mass; mortality; mutant; protein structure; protein structure function; public health relevance; stoichiometry

DESCRIPTION (provided by applicant): Apolipoproteins are abundant serum proteins and well known for their role in lipid transport processes. Their importance in health and disease, in particular cardiovascular disease has been well established. It has become increasingly clear that apolipoproteins are an important component of the innate immune system. In that role, apolipoproteins have the ability to bind and neutralize lipopolysaccharides (LPS), which are abundantly present in the outer membrane of Gram-negative bacteria. When released in the circulation, LPS cause septic shock, a major cause of death in intensive care units. In the current proposal we aim to provide a molecular basis for the apolipoprotein-LPS interaction. To accomplish this, we will use invertebrate apolipophorin III (apoLp-III) as a model, since a wealth of structural information is available for this protein. Human apoA-I will be employed to complement our studies. It is hypothesized that apoLp-III is a pattern recognition protein, binding and neutralizing a variety of cell wall components of microbial invaders, most noticeably LPS. The flexible a-helical structure of the protein accommodates for large conformational changes, and is a key feature that facilitates the LPS binding interaction. Using recombinant apolipoprotein and various LPS variants, the binding interaction will be studied in solution using a combination of molecular biology, biochemical and biophysical analysis. The research plan includes the following specific aims. (i) A thorough biophysical characterization of the LPS/apoLp-III complex to gain insight in the apolipoprotein-LPS binding interaction. (ii) Elucidate the role of LPS-carbohydrate in the binding interaction. The importance of LPS carbohydrate and the need for a large protein conformational change will be investigated using molecular spectroscopy with single tryptophan and double cysteine mutant proteins. (iii) Since charge plays an important role in the LPS binding interaction with human apoA-I, key lysine residues in apoA-I necessary for LPS binding will be identified. Using a site-directed mutagenesis approach, lysine residues which are part of the apolipoprotein-LPS binding interaction will be identified. In conclusion, by employing a well established model protein for the structure-function relationship of exchangeable apolipoproteins in conjunction with human apoAI, important insights in the molecular mechanism of apolipoprotein-LPS interaction will be obtained. This knowledge can be used to improve treatment and develop new strategies to treat Gram-negative sepsis. PUBLIC HEALTH RELEVANCE: Bacterial sepsis is a common threat causing more than 200,000 fatalities each year in the US. Apolipoproteins, well known for their role in lipid and cholesterol transport, are likely to play a vital role in innate immunity, by neutralizing lipopolysaccharides released from invading bacteria which are responsible for sepsis which often results in shock and death. The proposed research aims to understand the molecular basis of the protective role of apolipoproteins, thereby providing a foundation for improving the treatment of bacterial sepsis.

描述(申请人提供)：载脂蛋白是一种丰富的血清蛋白，因其在脂质转运过程中的作用而广为人知。它们在健康和疾病，特别是心血管疾病中的重要性已经得到很好的证实。载脂蛋白是先天免疫系统的重要组成部分，这一点已经变得越来越清楚。在这一作用中，载脂蛋白具有结合和中和内毒素的能力，内毒素大量存在于革兰氏阴性细菌的外膜中。当释放到循环中时，内毒素会引起感染性休克，这是重症监护病房死亡的主要原因。在目前的提议中，我们的目标是为载脂蛋白-内毒素的相互作用提供分子基础。为了实现这一点，我们将使用无脊椎动物载脂蛋白III(apoLp-III)作为模型，因为该蛋白质具有丰富的结构信息。人类载脂蛋白A-I将用于补充我们的研究。假设apoLp-III是一种模式识别蛋白，结合并中和微生物入侵者的各种细胞壁成分，最引人注目的是脂多糖。蛋白质的柔性α-螺旋结构适应较大的构象变化，是促进内毒素结合相互作用的关键特征。利用重组载脂蛋白和各种内毒素突变体，结合分子生物学、生化和生物物理分析，在溶液中研究结合作用。研究计划包括以下具体目标。(I)对脂多糖/apoLp-III复合体进行彻底的生物物理表征，以深入了解载脂蛋白与脂多糖的结合作用。(Ii)阐明内毒素-碳水化合物在结合作用中的作用。将使用单色氨酸和双半胱氨酸突变蛋白的分子光谱学来研究内毒素碳水化合物的重要性和需要大的蛋白质构象变化。(Iii)由于电荷在内毒素与人载脂蛋白A-I的结合作用中起重要作用，因此将确定载脂蛋白A-I中与内毒素结合所需的关键赖氨酸残基。使用定点突变方法，将鉴定作为载脂蛋白-脂多糖结合作用一部分的赖氨酸残基。总之，通过使用一个成熟的模型蛋白来研究可交换载脂蛋白的结构-功能关系，结合人类载脂蛋白AI，将获得关于载脂蛋白-内毒素相互作用的分子机制的重要见解。这些知识可以用来改进治疗和开发治疗革兰氏阴性败血症的新策略。 与公共卫生相关：细菌败血症是一种常见的威胁，在美国每年导致20多万人死亡。载脂蛋白以其在脂质和胆固醇运输中的作用而闻名，它可能通过中和入侵细菌释放的脂多糖而在先天免疫中发挥重要作用，这些脂多糖是导致败血症的原因，败血症通常会导致休克和死亡。这项研究旨在了解载脂蛋白保护作用的分子基础，从而为改进细菌败血症的治疗提供基础。