LIPOPROTEIN(A) SYNTHESIS IN BABOON HEPATOCYTES

狒狒肝细胞中脂蛋白(A) 的合成

基本信息

批准号：
2460005
负责人：
Robert E LANFORD
金额：
$ 29.86万
依托单位：
TEXAS BIOMEDICAL RESEARCH INSTITUTE
依托单位国家：
美国
项目类别：
财政年份：
1993
资助国家：
美国
起止时间：
1993-08-15 至 1999-07-31
项目状态：
已结题

项目摘要

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.

几乎一半的死亡是由冠心病 (CHD) 造成的在西方国家。诸多环境因素已被认为会增加患这种疾病的风险，即吸烟、高脂肪饮食，缺乏运动。然而，强大的遗传影响也存在。脂蛋白 Lp(a) 的血浆水平非常高可遗传，并且 Lp(a) 水平升高是一个很强的独立风险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 通过二硫键连接额外的高分子量糖蛋白，apo(a)。载脂蛋白 (a) 是由肝脏合成，并以多种基因决定的形式存在同种型的大小从 400 kDa 到大于 800 kDa 不等。等离子 Lp(a) 水平从小于 1 到大于 100 mg/dl，并且同种型大小和同工型之间存在负相关趋势血浆浓度。尽管其临床意义重大，但实际上对于调节 Lp(a) 生成速率的因素一无所知并从循环中除去。因此，非常需要一个体外系统分析调节合成的因素这种高度致动脉粥样硬化脂蛋白的分泌。狒狒表现相似人类血浆 Lp(a) 和 apo(a) 水平的特征同工型大小。我们开发了一种无血清培养基，可维持长时间培养高度分化的狒狒肝细胞，最近，我们展示了该系统的实用性分析 Lp(a) 的形态发生。本次活动的第一个具体目标建议检查获得的肝细胞中 apo(a) 的合成情况来自表达高水平和低水平的基因选择的狒狒血浆 Lp(a)。将检查等位基因变异的影响 apo(a) mRNA 转录水平、apo(a) 多肽比率 apo(a) 的合成、胞内成熟动力学以及 apo(a) 细胞内降解水平。第二个具体目标是对 apo(a) 的细胞内成熟进行深入分析包括蛋白质折叠、与内质伴侣的关联网状结构，以及 apo(a) 与反式蛋白的关联高尔基。第三个具体目标是审查的性质和 apo(a) 和 apoB 相互作用形成 Lp(a) 的要求。在第四个具体目标中，具有潜在治疗益处的化合物将在体外检查降低 Lp(a) 浓度的作用作用机制。最终的具体目标是发展不朽适合分析 apo(a) 的肝细胞系代谢。