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）的主要蛋白组分，通过几种生物学机制使动物动脉粥样硬化消退。盘状（前？）富含apoA-I的HDL颗粒可以预防人类的动脉粥样硬化和冠心病。人apoA-I由10个两亲性（或两亲性）？螺旋（8个22聚体和2个11聚体），共同作用结合脂质并产生一系列HDL颗粒。有趣的是，18聚体两亲性，A类，？-螺旋肽Ac-DWFKAFYDKVAEKFKEAF-NH 2（4F）可以模拟apoA-I的许多功能特性。apoA-I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、孤立的螺旋如何能够如此良好地发挥作用，以及（2）将这种类型的多个螺旋并入单个分子实体会产生什么影响。这个建议的一个主要目的是设计具有多重两亲性？连接到分子支架上的螺旋，系统地覆盖2-8个螺旋亚基的范围。我们建议设计，合成，表征和探索新的化学物种，可以模拟apoA-I。这些纳米材料，由一个独特的支架轴承多个，两亲性，A类？在天然HDL的尺寸域（6-12 nm）中具有适当尺寸的螺旋肽将在体外单独和以HDL样纳米颗粒（即纳米脂质）形式脂化进行物理表征和生物学评价。感兴趣的结构将构成具有直链和支链拓扑结构的二聚体、三聚体、四聚体、六聚体和八聚体。我们计划以重组HDL（rHDL）颗粒的形式研究这些用磷脂包装的设计材料，例如（R）-（+）-1-棕榈酰-2-油酰-sn-甘油-3-磷酸胆碱（POPC）或大豆卵磷脂。将对贫脂（盘状）和富脂（球形）状态下的自组装纳米脂质进行研究，与4F和天然apoA-I相比，存在和不存在胆固醇，以深入了解结构和功能。我们计划通过体外生物测定评估衍生的HDL样纳米盘的潜在抗动脉粥样硬化特性，例如HDL颗粒重塑和培养细胞中的胆固醇流出。我们计划通过使用蛋白质组学技术（例如MALDI-TOF MS）分析我们设计的纳米脂质在暴露于血浆后获得的蛋白质组分。该脂质学研究应提供以下方面的有价值信息：（1）涉及纳米脂质颗粒稳定和形态的因素，（2）纳米脂质颗粒的结构-功能特性，以及（3）与HDL相关的蛋白质-脂质缔合。我们的研究结果可能为潜在的治疗药物建立一个平台，通过在体内消退动脉粥样硬化斑块来治疗动脉粥样硬化。

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.