Structure Function Relationship of Human Apolipoprotein B100

摘要 低密度脂蛋白(LDL)转运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性疾病的发展风险直接相关。 载脂蛋白B100基本上是低密度脂蛋白中唯一的载脂蛋白成分，在人类肝脏中是一种 富含三酰甘油(TAG)极低密度的组装和分泌所必需的结构成分 脂蛋白(VLDL)，血浆低密度脂蛋白的前体。ApoB100也是受体的主要配体- 多种细胞对血浆低密度脂蛋白的介导摄取。因此，载脂蛋白B在运输和运输中起着基础性作用。 血浆胆固醇和TAG的代谢。载脂蛋白B是以每个粒子的单分子形式存在的，因此，它的 血浆中的浓度接近于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性脂蛋白代谢中起着关键作用，是冠状动脉风险的有力标志 近年来，载脂蛋白B已成为预防心血管疾病的重要治疗靶点 疾病。然而，目前关于载脂蛋白B多肽结构元件的知识 是分泌载脂蛋白B100所必需的，这是指定不同脂类招募的结构域，以及关键基序(S) 粒子核心的中介标记添加仍然是不完整的。此应用程序的重点是 从体外和体内两个水平描述了人apoB100的上述结构与功能的关系。 拟议的研究是由以下假设驱动的：1)apoB中的667-746残基，它们不是 与卵黄蛋白脂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，发挥作用 在含apoB100的脂蛋白的组装、结构完整性和分泌中起重要作用 肝脏；2)含载脂蛋白的新生脂蛋白从富含PL的颗粒向富含Tag的颗粒的细胞内转变 需要翻译超过1700个残基的氨基酸；3)高度保守的14-残基-螺旋基序 人apoB(残基124-137)可能在MTP介导的Tag加成中起关键作用。 含有载脂蛋白的脂蛋白的内部，导致颗粒核心膨胀。这些假设将通过以下方式进行验证 人apoB100野生型和突变型全长表达及精选截短文库的构建 用大鼠肝癌MCA-RH细胞/原代肝细胞体外和体内研究载脂蛋白B的形态 和转基因小鼠模型，分别由以下3个特定目的。 具体目的1.研究apoB中独特的结构域跨越残基667-746的作用， 特别是螺旋-环-螺旋基序(残基700-744)和C-末端的残基997-1000 超结构域在肝脏载脂蛋白B100颗粒组装和分泌中的关键结构元件 在体外和体内水平上。具体目的2.鉴定载脂蛋白B中招募各种脂质的结构域， 特别是在粒子从富PL粒子过渡到富标签粒子的情况下，并建立相对角色 在此过程中，使用野生型、MTP和PLTP缺陷的MCA-RH细胞并培养 原代肝细胞。具体目标3.评估含新生载脂蛋白B的标签添加的机制 通过测试人类apoB中保守的14-残基-螺旋，124-137残基，在这个MTP- 调解过程。这将通过对该基序中关键残基的点突变和评估 在体外和体内对含载脂蛋白B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。 建议的研究旨在更深入地了解结构与功能之间的关系。 载脂蛋白B100。我们的总体目标是利用这些发现来制定策略，以减少 肝脏中含有apoB100的脂蛋白产生过多，这是导致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。

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.