Structure Function Relationship of Human Apolipoprotein B100

描述（由申请人提供）：低密度脂蛋白（LDL）运输人类血浆胆固醇的主要部分，其在血液循环中的水平与动脉粥样硬化性疾病的发生风险直接相关。载脂蛋白（apo） B100本质上是LDL的唯一载脂蛋白成分，在人体肝脏中是富含三甘油酯（TAG）的极低密度脂蛋白（VLDL）的组装和分泌的必需结构成分，VLDL是血浆LDL的前体。ApoB100也是多种细胞受体介导的血浆LDL摄取的主要配体。因此，载脂蛋白ob在血浆胆固醇和TAG的转运和代谢中起着基础作用。载脂蛋白ob以单个分子/颗粒的形式存在，因此，其在血浆中的浓度近似于潜在的致动脉粥样硬化脂蛋白颗粒的数量。由于其在致动脉粥样硬化脂蛋白代谢中的关键作用以及作为冠状动脉疾病（CAD）风险的强大标记物，近年来，载脂蛋白ob已成为预防心血管疾病的关键治疗靶点。然而，目前关于载脂蛋白ob多肽中分泌载脂蛋白100所需的结构元件，指定募集不同脂质的结构域，以及介导TAG添加到颗粒核心的关键基序的知识仍然不完整。本应用程序的重点是在体外和体内水平上描述人类apoB100的上述结构-功能关系。提出的研究基于以下假设：1)载脂蛋白ob中的残基667-746与脂蛋白蛋白或Genbank数据库中的任何其他序列都不同源，在肝脏中含载脂蛋白100的脂蛋白的组装、结构完整性和分泌中发挥重要作用；2)含载脂蛋白的新生脂蛋白在细胞内从富含pl的颗粒转化为富含tag的颗粒需要翻译残基1700以外的氨基酸；3)人类载脂蛋白ob中高度保守的14个残基1-螺旋基序（残基124-137）可能是mtp介导的TAG添加到含载脂蛋白内部导致颗粒核心扩张的关键元件。这些假设将通过野生型和突变型全长人apoB100的表达以及精心挑选的apoB100截断形式文库，分别通过体外和体内方法，使用大鼠肝癌McA-RH细胞/原代肝细胞和转基因小鼠模型，通过以下3个特定目的进行验证。具体目标研究apoB中横跨667-746残基的独特结构域，特别是螺旋-环-螺旋基序（残基700-744）和211超结构域c端997-1000残基在体外和体内水平上作为肝脏含apob100颗粒组装和分泌的关键结构元件的作用。特定的目标

DESCRIPTION (provided by applicant): Low density lipoproteins (LDL) transport the major portion of plasma cholesterol in humans, and their levels in circulation are directly correlated with the risk for the development of atherosclerotic diseases. Apolipoprotein (apo) B100 is essentially the only apolipoprotein component of LDL and in human liver is an obligatory structural component for the assembly and secretion of triacyglycerol (TAG)-rich very low density lipoproteins (VLDL), the precursors of plasma LDL. ApoB100 also serves as a major ligand for the receptor- mediated uptake of plasma LDL by a variety of cells. Thus, apoB plays a fundamental role in the transport and metabolism of plasma cholesterol and TAG. ApoB is present as a single molecule per particle and therefore, its concentration in the plasma approximates the number of potential atherogenic lipoprotein particles. Because of its pivotal role in the metabolism of atherogenic lipoproteins and as a strong marker for the risk of coronary artery disease (CAD), recently, apoB has been a key therapeutic target for the prevention of cardiovascular diseases. However, current knowledge with respect to the structural elements within apoB polypeptide which are required for the secretion of apoB100, domains that specify recruitment of different lipids, and key motif(s) that mediate TAG addition into the core of the particle is still incomplete. The focus of this application is to delineate th above structure-function relationship of human apoB100 at both the in vitro and in vivo levels. Proposed studies are driven by the following hypotheses: 1) Residues 667-746 in apoB, which are not homologous to any other sequence in lipovitellin or any other sequence in the Genbank database, play an important role in the assembly, structural integrity, and secretion of apoB100-containing lipoproteins in the liver; 2) The intracellular transition of nascent apoB-containing lipoprotein from a PL-rich to a TAG-rich particle requires translation of amino acids beyond residue 1700; 3) The highly conserved 14-residue 1-helix motif in human apoB (residues 124-137) may serve as a key element in the MTP-mediated addition of TAG into the interior of apoB-containing lipoproteins resulting in particle core expansion. These hypotheses will be tested by expression of wild-type and mutant full-length human apoB100 and a carefully selected library of truncated forms of apoB by both in vitro and in vivo approaches, using rat hepatoma McA-RH cells/primary hepatocytes and transgenic mouse models, respectively, by the following 3 Specific Aims. Specific Aim 1. To investigate the roles of the unique domain spanning residues 667-746 in apoB, specifically the helix-loop-helix motif (residues 700-744), and residues 997-1000 at the C-terminus of 211 superdomain as key structural elements in the assembly and secretion of hepatic apoB100-containing particles at both in vitro and in vivo levels. Specific Aim 2. To identify the domains in apoB that recruit various lipids, specifically where the particle transitions from a PL-rich to a TAG-rich particle, and establish the relative roles of PLTP and MTP in this process, using wild-type, MTP- and PLTP-deficient McA-RH cells, and cultured primary hepatocytes. Specific Aim 3. To assess the mechanism of TAG addition to nascent apoB-containing particle by testing the role of the conserved 14-residue 1-helix, residues 124-137 in human apoB, in this MTP- mediated process. This will be achieved by point mutations of key residues in this motif and assessing the subsequent effects on the secretion and composition of the apoB-containing particles both in vitro and in vivo. We will utilize a combination of structural, molecular, cellular, and in vivo approaches to achieve our goals. Proposed studies are aimed at gaining a more in-depth understanding of the structure-function relationship of apoB100. Our overall goal is to exploit these findings toward the development of strategies to attenuate the overproduction of hepatic apoB100-containing lipoproteins, a major cause of hyperlipidemia and the development and progression of coronary artery disease and atherosclerosis. PUBLIC HEALTH RELEVANCE: Elevated levels of lipids and low density lipoprotein (LDL)-cholesterol, also known as the "bad" cholesterol, in plasma are major contributors to the development of coronary artery disease and atherosclerosis. Apolipoprotein B is the major protein component of all atherogenic lipoproteins and its levels in plasma predicate the risk for atherosclerosis. The goal of this application is to understand the structure function relationship of human apoB and to apply this information to the development of strategies aimed at attenuating the overproduction of hepatic apoB- containing lipoproteins and decreasing the risk of atherosclerosis.