GENES CONTROLLING LDL RECEPTOR STRUCTURE AND FUNCTION

控制 LDL 受体结构和功能的基因

• 批准号: 6402656
• 负责人: MONTY KRIEGER
• 金额: $ 26.43万
• 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/6402656
• 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.

该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，这有助于对脂蛋白代谢和动脉粥样硬化提供基本的了解。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的新见解，高尔基体在真核细胞中控制许多整体膜和管腔可溶性蛋白的流动，并参与这些蛋白的翻译后修饰（糖基化，硫酸化）以及糖脂合成中起核心作用。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中克隆到两种细胞质蛋白ldlBp和ldlCp，它们在控制哺乳动物腔内高尔基体加工反应（如糖缀合物合成和重塑）中起关键作用。ldlB和ldlC零突变体在多个腔体高尔基反应中表现出多效性缺陷，导致LDLR和许多其他糖缀合物的合成异常。ldlBp和ldlCp都结合到高尔基体的细胞质表面，是一个非常大的大分子复合物（约950 kD，“ldlCp复合物”）的组成部分。初步研究表明，ldlCp对线虫的发育至关重要。本研究的目的是阐明ldlBp/ldlCp系统从高尔基体细胞质表面调控高尔基体管腔酶活性的机制。我们将1)识别系统的其他成分（额外的复杂成分，高尔基体受体，腔内因子和条件），2)描述ldlBp/ ldlcp -复合物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)表征ldlBp/ ldlcp -高尔基体系统成分的物理和功能相互作用，使用各种技术[免疫化学，蛋白质纯化，配体印迹，生物物理学（EM，离心），克隆，碳水化合物分析，体外细胞器测定，体细胞遗传学等]。该系统的分子表征可能会为LDLR和其他膜和分泌蛋白的合成和加工以及高尔基体在高等真核生物中的结构和功能提供新的见解。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和病理生理（例如，家族性高胆固醇血症，动脉粥样硬化）过程的理解。