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）转运人体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的发展风险直接相关。载脂蛋白（apo）B100基本上是LDL的唯一载脂蛋白组分，并且在人肝脏中是装配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）的必需结构组分。ApoB 100也作为受体介导的多种细胞摄取血浆LDL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以单个分子/颗粒的形式存在，因此，其在血浆中的浓度接近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）风险的强标志物，近年来，apoB已成为预防心血管疾病的关键治疗靶点。然而，目前关于apoB多肽内apoB 100分泌所需的结构元件、指定不同脂质募集的结构域和介导TAG添加到颗粒核心中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apoB 100的上述结构-功能关系。1）载脂蛋白B的667-746位残基与卵黄脂磷蛋白的其他序列或Genbank数据库中的其他序列都不同源，在肝脏中含载脂蛋白B 100的脂蛋白的组装、结构完整性和分泌中起重要作用; 2）新生的含apoB的脂蛋白从富含PL的颗粒到富含TAG的颗粒的细胞内转变需要超过残基1700的氨基酸的翻译; 3）人apoB中高度保守的14-残基1-螺旋基序（残基124-137）可能是MTP介导的TAG加入含apoB的脂蛋白内部导致颗粒核心扩张的关键元件。将分别使用大鼠肝癌McA-RH细胞/原代肝细胞和转基因小鼠模型，通过体外和体内方法表达野生型和突变型全长人apoB 100以及精心选择的apoB截短型文库，按照以下3个特定目的，对这些假设进行检验。 具体目标1。研究apoB中跨越残基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组装和分泌含apoB 100的肝颗粒中的作用。具体目标 2.使用野生型、MTP和PLTP缺陷型McA-RH细胞和培养的原代肝细胞，鉴定apoB中募集各种脂质的结构域，特别是颗粒从富含PL颗粒转变为富含TAG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。通过检测人apoB中保守的14-残基1-螺旋（残基124-137）在MTP介导的过程中的作用，评估TAG添加至新生含apoB颗粒的机制。这将通过在该基序中的关键残基的点突变和评估在体外和体内对含apoB的颗粒的分泌和组成的后续影响来实现。 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apoB 100的结构-功能关系。我们的总体目标是利用这些研究结果，制定策略，以减轻肝脏载脂蛋白B100的过度生产，高脂血症的主要原因，冠状动脉疾病和动脉粥样硬化的发展和进展。 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）水平升高是冠状动脉疾病和动脉粥样硬化发展的主要因素。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，其血浆水平预测动脉粥样硬化的风险。本申请的目的是了解人apoB的结构功能关系，并将该信息应用于旨在减弱含apoB的肝脂蛋白的过度产生和降低动脉粥样硬化风险的策略的开发。