Structure Function Relationship of Human Apolipoprotein B100

人载脂蛋白B100的结构功能关系

• 批准号: 8585878
• 负责人: NASSRIN DASHTI
• 金额: $ 38.76万
• 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/8585878
• 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

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.

抽象的 低密度脂蛋白（LDL）运输人类血浆胆固醇的主要部分及其水平 在循环中，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，在人肝脏中是 三甘油（TAG）组装和分泌的强制性结构成分 - 富密度非常低 脂蛋白（VLDL），血浆LDL的前体。 APOB100也是接收器的主要配体 - 通过多种细胞介导的血浆LDL摄取。那样，Apob在运输中起着基本作用， 血浆胆固醇和标签的代谢。 APOB以每个粒子为单个分子存在，因此，它 血浆中的浓度近似于潜在的动脉粥样硬化脂蛋白颗粒的数量。由于 它在动脉粥样硬化脂蛋白的代谢中的关键作用，并成为冠状动脉风险的强烈标志 最近，APOB是预防心血管的关键治疗靶点（CAD） 疾病。但是，关于APOB多肽内的结构元素的当前知识 APOB100的分泌所需 在粒子核心中添加的介导标签仍然不完整。该应用的重点是 描述了在体外和体内水平上人类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，这不是 与Lipovitellin或GenBank数据库中的任何其他序列中的任何其他序列同源 在组装，结构完整性和含APOB100脂蛋白的分泌中的重要作用 肝; 2）含有新生APOB的脂蛋白从富含PL的颗粒到富含标记的颗粒的细胞内过渡 需要翻译1700年居住地以外的氨基酸； 3）在 人类APOB（残基124-137）可能是MTP介导的TAG添加到该的关键因素 含APOB的脂蛋白的内部导致粒子核膨胀。这些假设将通过 野生型和突变体全长人APOB100的表达以及精心选择的截短库 使用大鼠MCA-RH细胞/原发性肝细胞，通过体外和体内方法的APOB形式 分别通过以下3个特定目的分别通过转基因小鼠模型。 特定目标1。研究跨越残差667-746在APOB中的唯一域的作用， 特别是螺旋环螺旋序（残基700-744），并保留997-1000在»1的C末端 超祖因作为含有肝APOB100颗粒的组装和分泌的关键结构元素 在体外和体内水平上。特定目的2。确定APOB中募集各种脂质的域， 特别是粒子从富含PL的粒子转变为富含标记的粒子，并建立相对角色 在此过程中，使用野生型，MTP和PLTP缺乏MCA-RH细胞的PLTP和MTP和MTP的培养 原发性肝细胞。特定目的3。评估添加到新生APOB的TAG添加机理 通过测试构成14分helix的作用粒子，在人类Apob中残留124-137，在此MTP-中 中介过程。这将通过此基序中的关键保留点突变来实现，并评估 随后对体外和体内含APOB的颗粒的分泌和组成的影响。 我们将利用结构，分子，细胞和体内方法的结合来实现我们的目标。 拟议的研究旨在获得对对结构功能关系的更深入的了解 APOB100。我们的总体目标是利用这些发现来发展策略以减轻 含有肝APOB100的脂蛋白的过量生产，这是高脂血症的主要原因和 冠状动脉疾病和动脉粥样硬化的发展和进展。