Synthesis and Application of Novel Apolipoprotein Mimetics

描述（由申请人提供）：载脂蛋白A-I （apoA-I）是高密度脂蛋白（HDL）的主要蛋白质成分，通过多种生物机制逆转动物动脉粥样硬化。血浆盘状膜水平升高（前？）高密度脂蛋白颗粒富含apoa - 1，可预防人类动脉粥样硬化和冠心病。人类apoa - 1由10个两亲性（或两亲性）组成？-螺旋（8个22-米和2个11-米），它们共同作用，结合脂质，产生一系列高密度脂蛋白颗粒。有趣的是，18元两亲性，A类？-螺旋肽Ac-DWFKAFYDKVAEKFKEAF-NH2 （4F）可以模拟apoA-I的许多功能特性。apoa - 1和4F分子之间的巨大差异提出了关于结构与功能的重要问题，因为我们可能想知道：(1)一个简单的、孤立的螺旋是如何发挥如此良好的功能的；(2)将这种类型的多个螺旋合并到一个单一的分子实体中会产生什么效果。这个建议的一个主要目的是设计具有多重两亲性的分子。-附着在分子支架上的螺旋，系统地覆盖2-8个螺旋亚基。我们建议设计、合成、表征和探索能够模拟apoA-I的新型化学物质。这些纳米材料，由不同的支架组成，承载多个，两亲性，a级？-螺旋肽，在天然高密度脂蛋白（6-12纳米）的大小域中具有适当的尺寸，将在体外进行物理表征和生物学评估，单独并以高密度脂蛋白样纳米颗粒（即纳米脂质）的形式脂化。感兴趣的结构将构成二聚体、三聚体、四聚体、六聚体和具有线性和支链拓扑结构的八聚体。我们计划研究这些用磷脂包装的设计材料，如(R)-(+)-1-棕榈酰-2-油基-sn-甘油-3-磷脂（POPC）或大豆卵磷脂，以重组HDL （rHDL）颗粒的形式。研究将在低脂（盘状）和富脂（球形）状态下对自组装纳米脂进行研究，并将其与4F和天然apoA-I进行比较，以了解其结构和功能。我们计划通过体外生物测定来评估衍生的高密度脂蛋白样纳米盘的潜在抗动脉粥样硬化特性，例如培养细胞中的高密度脂蛋白颗粒重塑和胆固醇外排。我们计划使用蛋白质组学技术（例如MALDI-TOF MS）分析我们的设计纳米脂暴露于血浆后获得的蛋白质成分。脂质学研究应提供以下方面有价值的信息：(1)纳米脂颗粒稳定和形态的相关因素；(2)纳米脂颗粒的结构-功能特性；(3)与HDL相关的蛋白-脂质关联。我们的研究结果可能为通过动脉粥样硬化斑块在体内的消退来治疗动脉粥样硬化的潜在药物建立一个平台。

DESCRIPTION (provided by applicant): Apolipoprotein A-I (apoA-I), the major protein component of high-density lipoprotein (HDL), regresses atherosclerosis in animals by several biological mechanisms. Elevated plasma levels of discoidal (pre-?) HDL particles, which are rich in apoA-I, protect against atherosclerosis and coronary heart disease in humans. Human apoA-I is comprised of 10 amphiphilic (or amphipathic) ?-helices (eight 22-mers and two 11-mers) that act together to bind lipids and engender a range of HDL particles. Interestingly, the 18-mer amphiphilic, class A, ?-helical peptide Ac-DWFKAFYDKVAEKFKEAF-NH2 (4F) can mimic many of apoA-I's functional properties. The dramatic difference between the apoA-I and 4F molecules raises important questions about structure vs. function in that we may wonder: (1) how a simple, isolated helix can function so well and (2) what would be the effect of incorporating multiple helices of this type into a single molecular entity. A principal aim of this proposal is to devise molecules with a multiplicity of amphiphilic ?-helices that are attached to a molecular scaffold, covering a range of 2-8 helical subunits systematically. We propose to design, synthesize, characterize, and explore novel chemical species that can mimic apoA-I. These nanomaterials, composed of a distinct scaffold bearing multiple, amphiphilic, class A ?-helical peptides, of appropriate dimensions in the size domain of native HDL (6-12 nm), will be physically characterized and biologically evaluated in vitro, alone and lipidated in the form of HDL-like nanoparticles (i.e. nanolipids). The structures of interest will constitute dimers, trimers, tetramers, hexamers, and octamers with linear and branched chain topologies. We plan to investigate these designer materials packaged with phospholipid, such as (R)-(+)-1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) or soy lecithin, in the form of recombinant HDL (rHDL) particles. Studies will be conducted on the self-assembled nanolipids in lipid-poor (discoidal) and lipid-rich (spherical) states, with and without cholesterol present, to gain insight into structure and function, in comparison with 4F and native apoA-I. We plan to assess the potential antiatherogenic properties of derived HDL-like nanodiscs via in vitro bioassays, such as HDL particle remodeling and cholesterol efflux from cells in culture. We plan to analyze our designer nanolipids for acquired protein components after their exposure to plasma by using proteomics techniques (e.g. MALDI-TOF MS). This lipidology research should provide valuable information on: (1) factors involved in nanolipid particle stabilization and morphology, (2) structure-function properties of nanolipid particles, and (3) protein-lipid association that is relevant to HDL. Our results may establish a platform for potential therapeutic agents to treat atherosclerosis by the regression of atherosclerotic plaque in vivo. PUBLIC HEALTH RELEVANCE: In human studies, elevated plasma levels of apoA-I, the major protein in HDL, correlated inversely with the development of coronary heart disease. We propose to design, synthesize, and explore specific molecular entities that can mimic apoA-I. This project could provide a blueprint for new drugs to combat heart disease.