Molecular mechanism of apolipoprotein binding to lipopolysaccharides

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闻名。它们对健康和疾病，特别是心血管疾病的重要性已得到充分证实。载脂蛋白是先天免疫系统的重要组成部分，这一点越来越清楚。在这种作用下，载脂蛋白具有结合和中和脂多糖（LPS）的能力，这些脂多糖大量存在于革兰氏阴性菌的外膜中。当在血液循环中释放时，LPS会引起感染性休克，这是重症监护病房的主要死亡原因。在目前的建议中，我们的目标是为载脂蛋白- lps相互作用提供分子基础。为了实现这一目标，我们将使用无脊椎动物载脂蛋白III （apoLp-III）作为模型，因为该蛋白具有丰富的结构信息。人类apoA-I将被用来补充我们的研究。据推测，apoLp-III是一种模式识别蛋白，结合并中和多种微生物入侵者的细胞壁成分，最明显的是LPS。该蛋白的柔性a-螺旋结构可适应大的构象变化，是促进LPS结合相互作用的关键特征。利用重组载脂蛋白和各种LPS变体，结合分子生物学、生物化学和生物物理分析，在溶液中研究其结合相互作用。研究计划包括以下具体目标。(i)对LPS/apoLp-III复合物进行全面的生物物理表征，以深入了解载脂蛋白-LPS结合相互作用。（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需要一个大的蛋白质构象改变将研究使用分子光谱与单色氨酸和双半胱氨酸突变蛋白。（iii）由于电荷在LPS与人类apoA-I的结合相互作用中起着重要作用，因此将确定apoA-I中LPS结合所必需的关键赖氨酸残基。使用定点诱变方法，作为载脂蛋白- lps结合相互作用一部分的赖氨酸残基将被识别。综上所述，通过使用一个完善的模型蛋白来研究可交换载脂蛋白与人类载脂蛋白ai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。这一知识可用于改进治疗和制定治疗革兰氏阴性败血症的新策略。

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.