Structure Function Relationship of Human Apolipoprotein B100

人载脂蛋白B100的结构功能关系

• 批准号: 8766562
• 负责人: NASSRIN DASHTI
• 金额: $ 38.95万
• 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 2016-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8766562
• 关键词: Amino Acids; Apolipoproteins; Apolipoproteins B; Atherosclerosis; Attenuated; Blood Circulation; Cell Line; Cells; Cholesterol; Coronary Arteriosclerosis; Databases; Development; 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

ABSTRACT 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 the 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 ¿-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 ¿¿1 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 ¿-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.

摘要 低密度脂蛋白（LDL）转运人体血浆胆固醇的主要部分， 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LDL的主要组分。 专性结构组件的装配和分泌三酰甘油（TAG）丰富的非常低的密度 脂蛋白（VLDL），血浆LDL的前体。ApoB 100也作为受体的主要配体- 通过多种细胞介导血浆LDL的摄取。因此，载脂蛋白B在转运和 血浆胆固醇和TAG的代谢。ApoB作为每个颗粒的单个分子存在，因此，其 血浆中的浓度接近潜在的致动脉粥样硬化脂蛋白颗粒的数量。因为 它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粥样硬化风险的强标志物， 近年来，apoB已成为预防心血管疾病的关键治疗靶点， 疾病然而，目前关于apoB多肽内的结构元件的知识， 是apoB 100分泌所必需的，指定募集不同脂质的结构域，以及关键基序 介导TAG添加到颗粒核心的过程仍然不完全。此应用程序的重点是 在体外和体内两个水平上描述人apoB 100的上述结构-功能关系。 建议的研究由以下假设驱动：1）apoB中的残基667-746，其不是 与卵黄脂磷蛋白中的任何其他序列或Genbank数据库中的任何其他序列同源，发挥 在载脂蛋白B100的组装、结构完整性和分泌中起重要作用。 2）新生的含载脂蛋白B的脂蛋白从富含PL的颗粒到富含TAG的颗粒的细胞内转变 需要翻译超过残基1700的氨基酸; 3）高度保守的14-残基<$-螺旋基序， 人apoB（残基124-137）可以作为MTP介导的TAG加入到 含有apoB的脂蛋白的内部导致颗粒核心膨胀。这些假设将由以下人员进行检验： 野生型和突变体全长人apoB 100的表达以及精心选择的截短的 用大鼠肝癌McA-RH细胞/原代肝细胞，通过体外和体内方法， 和转基因小鼠模型，分别通过以下3个具体目的。 具体目标1。为了研究apoB中跨越残基667-746的独特结构域的作用， 特别是螺旋-环-螺旋基序（残基700-744），和在1的C-末端的残基997-1000 超结构域作为肝载脂蛋白B 100颗粒组装和分泌的关键结构元件 在体外和体内水平。具体目标2。为了确定apoB中募集各种脂质的结构域， 特别是当颗粒从富含PL的颗粒转变为富含TAG的颗粒时，并建立相对作用 使用野生型、MTP-和PLTP-缺陷的McA-RH细胞， 原代肝细胞具体目标3。评估TAG添加至新生含apoB的细胞的机制， 通过测试保守的14个残基的半螺旋，即人apoB中的残基124-137，在这种MTP中的作用， 中介过程。这将通过在该基序中的关键残基的点突变并评估该基序中的突变来实现。 在体外和体内对含apoB的颗粒的分泌和组成的后续影响。 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蛋白质的结构与功能关系 apoB 100.我们的总体目标是利用这些发现来制定策略， 肝脏含apoB 100的脂蛋白的过度产生，这是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。