GENES CONTROLLING LDL RECEPTOR STRUCTURE AND FUNCTION

控制 LDL 受体结构和功能的基因

• 批准号: 6129420
• 负责人: MONTY KRIEGER
• 金额: $ 26.16万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-05-01 至 2004-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6129420
• 关键词: Golgi apparatus; N acetylglucosaminidase; confocal scanning microscopy; electron microscopy; enzyme activity; fluorescence microscopy; galactosyltransferases; gene expression; genetic regulation; human subject; immunocytochemistry; immunoelectron microscopy; immunoprecipitation; laboratory rabbit; low density lipoprotein receptor; molecular cloning; protein purification; protein structure function; receptor mediated endocytosis; western blottings

The long term goal of the proposed study is to define the molecular details of low density lipoprotein (LDL) receptor- mediated endocytosis, which can help provide fundamental understanding of lipoprotein metabolism and atherosclerosis. In addition, this work may contribute new insights into the general mechanisms underlying the structure and function of the Golgi apparatus, which plays a central role in controlling the flow of many integral membrane and lumenal soluble proteins through eukaryotic cells and also participates in the posttranslational modifications of these proteins (glycosylation, sulfation) as well as in glycolipid synthesis. Two cytoplasmic proteins, ldlBp and ldlCp, which were identified by expression cloning from recessive LDL receptor (LDLR)-defective Chinese hamster ovary (CHO) cell mutants, ldlB and ldlC, play critical roles in controlling intralumenal Golgi processing reactions (e.g., glycoconjugate synthesis and remodeling) in mammals. The ldlB and ldlC null mutants exhibit pleiotropic defects in multiple lumenal Golgi reactions, which result in the abnormal synthesis the LDLR and many other glycoconjugates. Both ldlBp and ldlCp bind to the cytoplasmic surface of the Golgi and are components of a very large macromolecular complex (approximately 950 kD, 'ldlCp complex'). Preliminary studies indicate that ldlCp is essential for development in the worm C. elegans. The goal of this proposal is to elucidate the mechanism underlying the control of Golgi lumenal enzymatic activities by the ldlBp/ldlCp system from the cytoplasmic surface of the Golgi. We will 1) identify additional components of the system (additional complex components, Golgi receptor, intralumenal factors and conditions), 2) describe the effects of the ldlBp/ldlCp-complex system on the structure, composition, and function of the Golgi in normal and mutant cells, and 3) characterize the physical and functional interactions of the components of the ldlBp/ldlCp-Golgi system, using a variety of techniques [immunochemistry, protein purification, ligand blotting, biophysics (EM,centrifugation), cloning, carbohydrate analysis, in vitro organelle assays, somatic cell genetics, and others]. It is likely that the molecular characterization of this system will provide fundamental new insights into the synthesis and processing of the LDLR and other membrane and secreted proteins, as well as the structure and function of the Golgi in higher eukaryotes. This should further our understanding of the complex physiologic and pathophysiologic (e.g., familial hypercholesterolemia, atherosclerosis) processes which are based on the function and dysfunction of the LDLR.

这项拟议研究的长期目标是确定低密度脂蛋白(LDL)受体介导的内吞作用的分子细节，这有助于提供对脂蛋白代谢和动脉粥样硬化的基本了解。此外，这项工作可能有助于对高尔基体结构和功能的一般机制提供新的见解，高尔基体在控制许多完整的膜和腔可溶性蛋白质通过真核细胞的流动中发挥核心作用，并参与这些蛋白质的翻译后修饰(糖基化、硫酸化)以及糖脂的合成。从隐性低密度脂蛋白受体(LDLR)缺陷的中国仓鼠卵巢(CHO)细胞突变体ldlB和ldlC的表达克隆中鉴定出两个细胞质蛋白ldlBp和ldlCp，它们在哺乳动物腔内高尔基体加工反应(如糖共轭合成和重塑)中起关键作用。LdlB和ldlC缺失突变体在多腔高尔基反应中表现出多效性缺陷，导致LDLR和许多其他糖结合物的异常合成。LdlBp和ldlCp都与高尔基体细胞质表面结合，是一个非常大的大分子复合体(约950kD)的组成部分，称为ldlCp复合体。初步研究表明，ldlCp对线虫的发育是必需的。本研究的目的是从高尔基体细胞质表面阐明ldlBp/ldlCp系统控制高尔基体腔内酶活性的机制。我们将1)确定系统的其他组件(额外的复杂组件、高尔基受体、腔内因素和条件)，2)描述ldlBp/ldlCp-复合系统对正常和突变细胞中高尔基体的结构、组成和功能的影响，以及3)利用各种技术[免疫化学、蛋白质纯化、配基和杂交、生物物理学(EM、离心法)、克隆、碳水化合物分析、体外细胞器分析、体细胞遗传学等]表征ldlBp/ldlCp-Golgi系统组件的物理和功能相互作用。该系统的分子表征可能为LDLR和其他膜蛋白和分泌性蛋白的合成和加工，以及高等真核生物高尔基体的结构和功能提供新的基础。这将进一步加深我们对基于LDLR功能和功能障碍的复杂生理学和病理生理学(如家族性高胆固醇血症、动脉粥样硬化)过程的理解。