LIPOPROTEIN(A) SYNTHESIS IN BABOON HEPATOCYTES

狒狒肝细胞中脂蛋白(A) 的合成

• 批准号: 3369359
• 负责人: Robert E LANFORD
• 金额: $ 27万
• 依托单位: TEXAS BIOMEDICAL RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-08-15 至 1998-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3369359
• 关键词: Golgi apparatus; alleles; animal population genetics; antimetabolites; apolipoproteins; baboons; blood lipoprotein biosynthesis; cell line; endoplasmic reticulum; genetic transcription; immunoprecipitation; liver cells; low density lipoprotein; molecular chaperones; northern blottings; protein degradation; protein folding; protein isoforms; secretion; tissue /cell culture

Coronary heart disease (CHD) is responsible for almost half of all deaths in western countries. A number of environmental factors have been identified as increasing the risk of this disease, i.e., smoking, high fat diet, and lack of exercise. However, a strong genetic influence exists as well. The plasma levels of lipoproteins(s), Lp(a), are highly heritable, and increased Lp(a) levels are a strong, independent risk factor atherosclerosis, the major cause of CHD. Lp(a) consist of low density lipoprotein (LDL) in which apoB is disulfide-linked to an additional high molecular weight glycoprotein, apo(a). Apo(a) is synthesized by the liver and exists as a number of genetically determined isoforms that vary in size from 400 to greater than 800 kDa. The plasma levels of Lp(a) vary from less than 1 to greater than 100 mg/dl, and there is a tendency for an inverse correlation between isoform size and plasma concentration. Despite its clinical significance, virtually nothing is known about factors which regulate rates of Lp(a) production and removal from the circulation. A great need therefore exists for an in vitro system to analyze the factors regulating the synthesis and secretion of this highly atherogenic lipoprotein. Baboons show similar characteristics to humans in terms of plasma levels of Lp(a) and apo(a) isoform sizes. We have developed a serum-free medium that maintains highly differentiated baboon hepatocytes in culture for extended times, and, recently, we have demonstrated the utility of this system for analyzing the morphogenesis of Lp(a). The first specific aim of this proposal is to examine the synthesis of apo(a) in hepatocytes obtained from genetically selected baboons expressing high and low levels of plasma Lp(a). The influence of allelic variation will be examined for the level of apo(a) mRNA transcription, the rate of apo(a) polypeptide synthesis, the kinetics of intracellular maturation of apo(a), and the level of apo(a) intracellular degradation. The second specific aim is to conduct an in depth analysis of the intracellular maturation of apo(a) including protein folding, association with chaperones in the endoplasmic reticulum, and the association of apo(a) with proteins in the trans- Golgi. The third specific aim is to examine the nature of and requirements for the interaction between apo(a) and apoB to form Lp(a). In the fourth specific aim, compounds of potential therapeutic benefit in lowering Lp(a) concentrations will be examined in vitro for their mechanism of action. The final specific aim is to develop immortalized hepatocyte cell lines that are suitable for analysis of apo(a) metabolism.

冠心病（CHD）几乎造成所有死亡的一半 在西方国家。 许多环境因素已经 被确定为增加这种疾病的风险，即吸烟，高 脂肪饮食和缺乏运动。 但是，强烈的遗传影响 也存在。 脂蛋白的血浆水平（a）高度 可遗传和提高的LP（a）水平是强大，独立的风险 因子动脉粥样硬化，是冠心病的主要原因。 LP（a）由低 密度脂蛋白（LDL），其中APOB二硫化物连接至 其他高分子量糖蛋白Apo（a）。 apo（a）是 由肝脏合成并作为许多遗传确定 从400到800 kDa的同工型不等。 血浆 LP（a）的水平从小于1到大于100 mg/dl，并且 同工型大小和 血浆浓度。 尽管具有临床意义，但实际上 关于调节LP（a）生产率的因素，尚无了解 并从循环中删除。 因此，存在一个巨大的需求 体外系统以分析调节合成的因素 这种高度动脉粥样硬化脂蛋白的分泌。 狒狒显示相似 从LP（A）和APO（a）的血浆水平方面，人类的特征 同工型。 我们已经开发了一种无血清的培养基 长时间的培养文化中高度分化的狒狒肝细胞， 最近，我们已经证明了该系统的实用性 分析LP（a）的形态发生。 第一个具体目的 建议是检查获得的肝细胞中apo（a）的合成 从遗传选择的狒狒表达高水平和低水平 等离子体LP（A）。 将检查等位基因变异的影响 APO（A）mRNA转录的水平，Apo（a）多肽的速率 合成，Apo（A）细胞内成熟的动力学和 Apo（a）细胞内降解的水平。 第二个具体目标是 对Apo的细胞内成熟进行深入分析（a） 包括蛋白质折叠，与内质中的伴侣关联 网状，以及Apo（a）与反式的蛋白质的关联 高尔基。 第三个具体目的是检查和 APO（a）和APOB之间相互作用的要求，以形成LP（a）。 在第四个特定目标中，潜在的治疗益处的化合物 在降低LP（a）的浓度将在体外检查其 作用机理。 最终的具体目的是发展不朽 适合分析Apo（A）的肝细胞细胞系 代谢。