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: 8399726
• 负责人: PAUL Michiel WEERS
• 金额: $ 10.28万
• 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 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8399726
• 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; Environment; Escherichia coli; 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; System; Training; Transport Process; Tryptophan; United States National Institutes of Health; Variant; Work; apolipophorin III; base; flexibility; improved; insight; lipid transport; microbial; molecular mass; mortality; mutant; protein structure; protein structure function; stoichiometry; undergraduate student

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.

描述（由申请人提供）：载脂蛋白是丰富的血清蛋白，并以其在脂质传输过程中的作用而闻名。它们在健康和疾病中的重要性，尤其是心血管疾病。越来越清楚的是，载脂蛋白是先天免疫系统的重要组成部分。在这一角色中，载脂蛋白具有结合和中和脂多糖（LPS）的能力，这些能力大量存在于革兰氏阴性细菌的外膜中。在流通中释放时，LP会引起化粪池休克，这是重症监护病房中的主要死亡原因。在当前的建议中，我们旨在为载脂蛋白LPS相互作用提供分子基础。为此，我们将使用无脊椎动物载脂蛋白III（Apolp-III）作为模型，因为该蛋白质可用于大量结构信息。人类APOA-I将被用来补充我们的研究。假设Apolp-III是一种模式识别蛋白，结合和中和微生物入侵者的各种细胞壁成分，最明显的是LP。该蛋白质的柔性A螺旋结构可容纳大构象变化，并且是促进LPS结合相互作用的关键特征。使用重组载脂蛋白和各种LPS变体，将使用分子生物学，生化和生物物理分析的结合在溶液中研究结合相互作用。研究计划包括以下特定目标。 （i）LPS/apolp-III复合物的彻底生物物理表征，以获得对载脂蛋白LPS结合相互作用的见解。 （ii）阐明LPS碳水化合物在结合相互作用中的作用。 LPS碳水化合物的重要性和对大蛋白构象变化的需求将使用单个色氨酸和双半胱氨酸突变蛋白的分子光谱进行研究。 （iii）由于电荷在与人apoA-I的LPS结合相互作用中起着重要作用，因此将确定LPS结合所需的APOA-I中的关键赖氨酸残基。使用位置定向的诱变方法，将鉴定为载脂蛋白LPS结合相互作用的一部分的赖氨酸残基。总之，通过采用良好的模型蛋白来实现可交换载脂蛋白与人类ApoaI结合的结构功能关系，将获得载脂蛋白LPS相互作用的分子机制的重要见解。这些知识可用于改善治疗，并制定新的策略来治疗革兰氏阴性败血症。