Structure Function Relationship of Human Apolipoprotein B100

描述(申请人提供)：低密度脂蛋白(低密度脂蛋白)运输人类血浆胆固醇的主要部分，它们在循环中的水平与动脉粥样硬化性疾病的发展风险直接相关。载脂蛋白B100是低密度脂蛋白中唯一的载脂蛋白成分，在人体肝脏中是组装和分泌富含三酰甘油的极低密度脂蛋白的必需结构成分，极低密度脂蛋白是血浆低密度脂蛋白的前体。ApoB100也是受体介导的多种细胞摄取血浆低密度脂蛋白的主要配体。因此，载脂蛋白B在血浆胆固醇和TAG的运输和代谢中起着重要的作用。载脂蛋白B以单分子形式存在于每个颗粒中，因此，其在血浆中的浓度接近于潜在的致动脉粥样硬化的脂蛋白颗粒的数量。由于载脂蛋白B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)风险的强烈标志物，近年来，载脂蛋白B已成为预防心血管疾病的关键治疗靶点。然而，目前关于apoB多肽中分泌apoB100所需的结构元件、指定不同脂类招募的结构域以及介导标签添加到颗粒核心的关键基序(S)的了解仍然不完整。本应用的重点是在体外和体内水平上描绘人载脂蛋白B100的上述结构与功能的关系。建议的研究是由以下假设驱动的：1)apoB中的667-746残基与Lipovitellin中的任何其他序列或Genbank数据库中的任何其他序列不同源，在肝脏中含apoB100的脂蛋白的组装、结构完整性和分泌中发挥重要作用；2)新生的含apoB的脂蛋白从富含PL的细胞内转变为富含Tag的颗粒需要1700个以上的氨基酸翻译；3)人载脂蛋白B(ApoB)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加成到含apoB的脂蛋白内部导致颗粒核心膨胀的关键元件。这些假设将通过体外和体内表达野生型和突变型全长人apoB100和精心挑选的apoB截短型文库，分别使用大鼠肝癌MCA-RH细胞/原代肝细胞和转基因小鼠模型来验证，目的如下：具体目的1.研究apoB中独特的结构域667-746残基，特别是211超结构域C末端的螺旋-环-螺旋基序(700-744残基)和997-1000残基在体内和体外水平上对肝脏apoB100颗粒组装和分泌的关键结构元件的作用。特定目标 2.利用野生型、MTP和PLTP缺乏的MCA-RH细胞和培养的原代肝细胞，鉴定apoB中招募各种脂质的结构域，特别是颗粒从富含PL向富含Tag的颗粒转变的区域，并建立PLTP和MTP在这一过程中的相对作用。具体目的3.通过检测人类载脂蛋白B中保守的14个残基1-螺旋124-137残基在MTP介导的这一过程中的作用，来评估Tag加到新生的载脂蛋白B颗粒中的机制。这将通过对该基序中关键残基的点突变并在体外和体内评估随后对含apoB颗粒的分泌和组成的影响来实现。我们将利用结构、分子、细胞和体内方法的组合来实现我们的目标。建议的研究旨在更深入地了解apoB100的结构与功能之间的关系。我们的总体目标是利用这些发现来开发策略，以减少含有apoB100的肝脏脂蛋白的过度生产，这是高脂血症以及冠状动脉疾病和动脉粥样硬化的发展和进展的主要原因。 与公众健康相关：血浆中脂质和低密度脂蛋白(LDL)-胆固醇的水平升高，也被称为“坏”胆固醇，是导致冠状动脉疾病和动脉粥样硬化的主要因素。载脂蛋白B是所有致动脉粥样硬化脂蛋白的主要蛋白质组分，其在血浆中的水平预测动脉粥样硬化的风险。这项应用的目的是了解人载脂蛋白B的结构和功能关系，并将这些信息应用于开发旨在减少肝脏载脂蛋白过量生产和降低动脉粥样硬化风险的策略。

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.