Structure Function Relationship of Human Apolipoprotein B100

人载脂蛋白B100的结构功能关系

• 批准号: 8259964
• 负责人: NASSRIN DASHTI
• 金额: $ 40.84万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-01-01 至 2015-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8259964
• 关键词: Amino Acids; Apolipoproteins; Apolipoproteins B; Atherosclerosis; Attenuated; Blood Circulation; Cell Line; Cells; Cholesterol; Coronary Arteriosclerosis; Databases; Development; Disease; Elements; Genbank; Goals; Helix-Loop-Helix Motifs; Hepatic; Hepatocyte; Human; Hyperlipidemia; In Vitro; Indium; Knowledge; LDL Cholesterol Lipoproteins; Laboratories; Length; Libraries; Ligands; Lipids; Lipoprotein (a); Lipoproteins; Liver; Low-Density Lipoproteins; Mediating; Mediator of activation protein; Metabolism; Modeling; Molecular; N-terminal; Nature; Nucleosome Core Particle; Pathway interactions; Phospholipid Transfer Proteins; Phospholipids; Plasma; Play; Point Mutation; Primary Cell Cultures; Primary carcinoma of the liver cells; Process; Property; Proteins; Publishing; Rattus; Recruitment Activity; Relative (related person); Risk; Risk Marker; Role; Sequence Analysis; Solutions; Specific qualifier value; Structure; Structure-Activity Relationship; Testing; Transgenic Mice; Translations; Very low density lipoprotein; aqueous; base; cardiovascular disorder prevention; high risk; in vivo; lipovitellin; microsomal triglyceride transfer protein; molecular dynamics; molecular mass; mouse model; mutant; particle; polypeptide; receptor; single molecule; therapeutic target; three dimensional structure; uptake

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.

描述（由申请人提供）：低密度脂蛋白（LDL）在人体中运输血浆胆固醇的主要部分，其在循环中的水平与动脉粥样硬化疾病发生的风险直接相关。载脂蛋白 (apo) B100 本质上是 LDL 的唯一载脂蛋白成分，在人类肝脏中，是组装和分泌富含三酰甘油 (TAG) 的极低密度脂蛋白 (VLDL)（血浆 LDL 的前体）的必需结构成分。 ApoB100 还充当多种细胞受体介导的血浆 LDL 摄取的主要配体。因此，apoB 在血浆胆固醇和 TAG 的转运和代谢中起着重要作用。 ApoB 每个颗粒以单个分子形式存在，因此，其在血浆中的浓度近似于潜在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) 风险的强有力标志物，apoB 最近已成为预防心血管疾病的关键治疗靶点。然而，目前关于 apoB100 分泌所需的 apoB 多肽内的结构元件、指定不同脂质募集的结构域以及介导 TAG 添加到颗粒核心的关键基序的知识仍然不完整。本申请的重点是在体外和体内水平上描述人apoB100的上述结构-功能关系。拟议的研究由以下假设驱动：1) apoB 中的残基 667-746 与 lipovitellin 中的任何其他序列或 Genbank 数据库中的任何其他序列均不同源，在肝脏中含有 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脂蛋白在细胞内从富含PL的颗粒转变为富含TAG的颗粒需要翻译残基1700以上的氨基酸； 3) 人apoB中高度保守的14个残基1-螺旋基序（残基124-137）可能作为MTP介导的TAG添加到含apoB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类 apoB100 的表达以及精心挑选的 apoB 截短形式文库进行测试，通过体外和体内方法，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，通过以下 3 个具体目标。 具体目标 1. 研究 apoB 中跨残基 667-746 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 末端的残基 997-1000 作为关键结构元件在体外和体内水平上含有 apoB100 的肝颗粒组装和分泌中的作用。具体目标 2. 使用野生型、MTP 和 PLTP 缺陷的 McA-RH 细胞以及培养的原代肝细胞，鉴定 apoB 中招募各种脂质的结构域，特别是颗粒从富含 PL 的颗粒转变为富含 TAG 的颗粒的区域，并确定 PLTP 和 MTP 在此过程中的相对作用。具体目标 3. 通过测试人 apoB 中保守的 14 个残基 1-螺旋（残基 124-137）在这一 MTP 介导的过程中的作用，评估 TAG 添加到含有新生 apoB 的颗粒的机制。这将通过该基序中关键残基的点突变并评估随后在体外和体内对含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。拟议的研究旨在更深入地了解 apoB100 的结构与功能关系。我们的总体目标是利用这些发现来制定策略，以减轻肝脏含 apoB100 脂蛋白的过量产生，这是高脂血症以及冠状动脉疾病和动脉粥样硬化发生和进展的主要原因。 公共卫生相关性：血浆中脂质和低密度脂蛋白 (LDL) 胆固醇（也称为“坏”胆固醇）水平升高是导致冠状动脉疾病和动脉粥样硬化的主要原因。载脂蛋白 B 是所有致动脉粥样硬化脂蛋白的主要蛋白质成分，其血浆水平可预测动脉粥样硬化的风险。本应用的目的是了解人类 apoB 的结构功能关系，并将这些信息应用于开发旨在减轻肝脏含 apoB 脂蛋白的过量产生并降低动脉粥样硬化风险的策略。