APOLIPOPROTEIN B EXPRESSION STUDIES

载脂蛋白 B 表达研究

• 批准号: 2210793
• 负责人: MACRAE F LINTON
• 金额: $ 8.24万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-09-01 至 1998-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2210793
• 关键词: apolipoproteins; atherosclerosis; blood lipid; chimeric proteins; disease /disorder model; disulfide bond; frameshift mutation; gene expression; genetically modified animals; laboratory mouse; monoclonal antibody; nucleic acid sequence; site directed mutagenesis; tissue /cell culture; western blottings; apolipoproteins; atherosclerosis; blood lipid; chimeric proteins; disease /disorder model; disulfide bond; frameshift mutation; gene expression; genetically modified animals; laboratory mouse; monoclonal antibody; nucleic acid sequence; site directed mutagenesis; tissue /cell culture; western blottings

As an internal medicine resident, I developed an interest in lipoprotein metabolism and the pathogenesis of atherosclerotic disease. After a year of clinical of clinical training in Endocrinology and Metabolism at UCSF, I began basic research training in Dr. Stephen Young's laboratory at the Gladstone Institute. During the past three years, my research has focused on understanding several different aspects of apolipoprotein (Apo) B genetics and metabolism: 1) the effect of liver transplantation on apo B and apo E phenotype; 2) the expression of truncated apo B species in hepatoma cells; 3) the investigation of a unique allele causing hypobetalipoproteinemia, the apo B86 allele. The apo B86 allele, which contains a one nucleotide deletion (frameshift) in exon 26 of the apo B gene, results in the production of a truncated apo B species, apo B86. Remarkably, genetic and biochemical evidence from both affected family members and cell culture expression studies indicate that the apo B86 allele yields a full-length apo B100 in addition to apo B86. I have determined that the full-length protein is produced from the apo B 86 allele as a result of reading frame restoration, by a mechanism that appears to be unique in human genetics. The first specific aim of this project is to further define this mechanism for reading frame restoration. Site directed mutagenesis will be used to delineate the exact DNA sequence requirements for reading frame restoration. Mutant apo B constructs containing various DNA sequence changes in the region of the apo B86 mutation will be expressed in hepatoma cells. The apo B proteins (truncated vs. full-length) produced by the transformed cell lines will be examined by immunoblot. The mRNA from the transformed cell lines will be examined for evidence of an error in transcription that would restore the proper reading frame. Studies of the apo B86 protein suggest that apo B86 is incapable of associating with apo (a) to form lipoprotein (a) [Lp(a)]. It is widely assumed that apo B100 is linked to apo(a) by a disulfide linkage. Based on our results with apo B86, we hypothesize that one (or more of the carboxyl-terminal cysteines in apo B100 may be involved in forming a disulfide bridge with apo(a). The second specific aim is to use site directed mutagenesis and cell culture expression studies to evaluate the importance of the four carboxyl-terminal cysteines of apo B100 in its association with apo(a) to form Lp(a). Cell culture expression studies of apo B100 have obvious limitations with regard to investigating the role of apo B100 in the pathogenesis of atherosclerosis. Therefore, I plan to develop an animal model in which to test the hypothesis that overexpression of human apo B100 is atherogenic. The third specific aim will be to overexpress human apo B100 in transgenic mice. We plan to investigate the effects of overexpression of human apo B100 in transgenic mice on lipid levels and atherogenesis. In addition, we will develop an apo B86 transgenic mouse as a model in which to study reading frame restoration in the apo B86 allele, and we propose to use transgenic mice to develop allotype specific monoclonal antibodies to human apo B100.

作为居民，我对脂蛋白产生了兴趣 代谢和动脉粥样硬化疾病的发病机理。 一年后 UCSF内分泌和代谢临床培训的临床培训的临床培训， 我开始在Stephen Young博士实验室的基础研究培训 格拉德斯通研究所。 在过去的三年中，我的研究有 专注于理解载脂蛋白的几个不同方面 （APO）B遗传学和代谢：1）肝移植的影响 在apo b和apo e表型上； 2）截短的apo b的表达 肝癌细胞中的物种； 3）对独特等位基因的调查 引起低钙蛋白血症，Apo B86等位基因。 APO B86等位基因，其中包含一个核苷酸缺失（移料） 在Apo B基因的外显子26中，导致截短的产生 Apo B物种，Apo B86。 值得注意的是，遗传和生化证据 来自受影响的家庭成员和细胞培养表达研究 表示Apo B86等位基因在 除Apo B86。 我已经确定全长蛋白是 由于阅读框架，由Apo B 86等位基因产生 通过在人类遗传学中似乎是独一无二的机制恢复。 该项目的第一个具体目的是进一步定义 阅读框架修复的机制。 站点定向诱变将 用于描述阅读确切的DNA序列要求 框架修复。 包含各种DNA的突变Apo B构建体 APO B86突变区域的序列变化将表示 在肝癌细胞中。 Apo B蛋白（截短与全长） 由转化的细胞系产生的将由免疫印迹检查。 将检查来自转化细胞系的mRNA以获取证据 转录错误将恢复正确的阅读框。 对Apo B86蛋白的研究表明Apo B86无能力 与Apo（A）相关联，形成脂蛋白（a）[LP（a）]。 它是广泛的 假设APO B100通过二硫键链接到APO（A）。 基于 关于APO B86的结果，我们假设一个（或更多 Apo B100中的羧基末端半胱氨酸可能参与形成A 带有Apo（A）的二硫键。 第二个特定目的是使用网站 定向诱变和细胞培养表达研究以评估 Apo B100的四个羧基末端半胱氨酸的重要性 与Apo（a）相关联，形成LP（a）。 Apo B100的细胞培养表达研究具有明显的局限性 关于研究Apo B100在发病机理中的作用 动脉粥样硬化。 因此，我计划开发一种动物模型 测试人apo b100的过表达的假设为 动脉粥样硬化。 第三个具体目的是过表达人类apo B100转基因小鼠。 我们计划调查 在脂质水平上的转基因小鼠中人apo B100的过表达， 动脉粥样硬化。 此外，我们将开发Apo B86转基因小鼠 作为研究Apo B86中阅读框架恢复的模型 等位基因，我们建议使用转基因小鼠开发异型 对人Apo B100的特异性单克隆抗体。