Mechanisms of assembly of photoreceptor G protein complexes

光感受器G蛋白复合物的组装机制

• 批准号: 8448264
• 负责人: BARRY M WILLARDSON
• 金额: $ 35.63万
• 依托单位: BRIGHAM YOUNG UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-05-03 至 2015-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8448264
• 关键词: Actins; Adolescence; Alzheimer' s Disease; Amyotrophic Lateral Sclerosis; Area; Attention; Bardet-Biedl Syndrome; Binding; Binding Sites; Biochemical; Biological Assay; Blindness; Cell Culture Techniques; Cell Death; Cells; Cilia; Complex; Cryoelectron Microscopy; Data; Defect; Disease; Endoplasmic Reticulum; Functional disorder; GTP-Binding Protein Regulators; GTP-Binding Proteins; Genes; Goals; Hereditary Disease; Homeostasis; Huntington Disease; Link; Maps; Measurement; Membrane Proteins; Methods; Modeling; Molecular; Molecular Chaperones; Mutate; Mutation; Nerve Degeneration; Neurodegenerative Disorders; Neurons; Parkinson Disease; Pathology; Photoreceptors; Phototransduction; Physiologic pulse; Physiological; Process; Proteins; Proteome; Publishing; RGS Proteins; Recruitment Activity; Retina; Retinal Degeneration; Retinal Diseases; Rhodopsin; Role; Site-Directed Mutagenesis; Structural Protein; Structure; System; Testing; Time; Tubulin; Variant; Vesicle; Work; base; chaperonin; cytosolic chaperonin; design; dimer; effective therapy; human disease; improved; in vivo; mutant; phosducin-like protein; polypeptide; protein complex; protein folding; protein misfolding; public health relevance; research study; therapeutic target; tool; trafficking

DESCRIPTION (provided by applicant): Proper protein folding is a key issue in maintaining healthy, functional neurons. Neurodegenerative diseases such as Alzheimer's, Parkinson's, Huntington's and Lou Gehrig's disease (amyotrophic lateral sclerosis or ALS) all result from protein misfolding in neurons. Similar disruptions of proteome homeostasis cause neuronal degeneration of the photoreceptor cells of the retina. Many severe retinopathies have been linked to misfolding mutations in rhodopsin and other photoreceptor proteins. A key component of molecular chaperone system that maintains proteome homeostasis in photoreceptor cells is the cytosolic chaperonin complex (CCT, Chaperonin Containing Tailless polypeptide 1) which folds actin, tubulin and dozens of other cytosolic proteins. Among these CCT substrates are the G protein ¿ subunits (G¿) which form the G ¿1?1 and G¿5-RGS9 (Regulator of G protein Signaling) dimers that are essential components of the phototransduction cascade. All G¿ subunits require CCT and the co-chaperone phosducin-like protein 1 (PhLP1) to fold G¿ and assemble into functional dimers. Very recently, an additional role for CCT in Bardet-Biedl syndrome (BBS) has been demonstrated. BBS is a genetic disease of ciliary dysfunction displaying multiple pathological conditions including retinal degeneration. Mutations in 14 BBS proteins have been associated with the disease. Seven of them (BBS 1-2, 4-5 and 7-9) form a complex, termed the BBSome, which is essential for vesicle trafficking to cilia. Three others (BBS 6, 10 and 12) are homologous to CCT subunits and form complexes with CCT that are required for the folding of BBS2 and 7 and assembly of the BBSome. Together, these G¿ and BBS findings point to an important role for CCT in the assembly of protein complexes that perform key physiological functions in photoreceptor cells. Thus, the underlying goal of the proposed studies is to understand at the molecular level the mechanisms of protein complex assembly by CCT in order to identify therapeutic targets to improve the folding process and treat diseases like BBS for which effective treatments are not available. Specific Aims 1 and 2 examine the structures of the G¿1-CCT and RGS9-G¿5-CCT complexes in order to understand how CCT folds both G¿1 and G¿5 and recruits RGS9 to form the G¿5-RGS9 dimer. Specific Aim 3 investigates the structure of the complex between the chaperonin-like BBS proteins and CCT and the mechanism of BBSome assembly. To accomplish these aims, a number of methods are proposed including site-directed mutagenesis and cell-based binding assays, pulse-chase assembly measurements of protein complexes, purification of large protein complexes and cryo-electron microscopy. A strong team of collaborators has been assembled to provide the necessary expertise to successfully execute the proposed experiments.

描述（由申请人提供）：正确的蛋白质折叠是维持健康、功能性神经元的关键问题，例如阿尔茨海默氏症、帕金森氏症、亨廷顿氏病和卢伽雷氏病（肌萎缩侧索硬化症或 ALS）都是由神经元中的蛋白质错误折叠引起的。蛋白质组稳态的破坏导致视网膜感光细胞的神经元变性，许多严重的视网膜病变与此有关。视紫红质和其他光感受器蛋白的错误折叠突变是维持光感受器细胞蛋白质组稳态的分子伴侣系统的关键组成部分，它是胞质伴侣蛋白复合物（CCT，含有无尾多肽的伴侣蛋白1），它折叠肌动蛋白、微管蛋白和许多其他胞质蛋白。这些 CCT 底物是 G 蛋白 ¿形成 G¿ 的亚基 (G¿) 1?1 和 G?? 5-RGS9（G 蛋白信号传导调节剂）二聚体是光转导级联的重要组成部分 All G¿亚基需要 CCT 和共伴侣磷酸蛋白样蛋白 1 (PhLP1) 来折叠 G¿最近，CCT 在 Bardet-Biedl 综合征 (BBS) 中的另一个作用已被证实，这是一种与 14 种 BBS 蛋白突变相关的睫状功能障碍遗传性疾病。其中 7 个（BBS 1-2、4-5 和 7-9）形成一个复合体，称为 BBSome，它对于囊泡运输至关重要。其他三个（BBS 6、10 和 12）与 CCT 亚基同源，并与 CCT 形成复合物，这是 BBS2 和 7 的折叠以及 BBSome 组装在一起所需的。和 BBS 的研究结果指出，CCT 在执行感光细胞关键生理功能的蛋白质复合物组装中发挥着重要作用，因此，拟议研究的根本目标是在分子水平上了解 CCT 组装蛋白质复合物的机制。为了确定治疗靶点以改善折叠过程并治疗诸如 BBS 之类无法获得有效治疗的疾病，具体目标 1 和 2 检查 G¿ 的结构。 1-CCT 和 RGS9-G¿ 5-CCT 复合物，以了解 CCT 如何折叠两个 G¿ 1 和 G¿ 5并招募RGS9组成G¿ 5-RGS9 二聚体。特定目标 3 研究伴侣蛋白样 BBS 蛋白和 CCT 之间的复合物结构以及 BBSome 组装机制。为了实现这些目标，提出了多种方法，包括定点诱变和基于细胞的方法。结合测定、蛋白质复合物的脉冲追踪组装测量、大型蛋白质复合物的纯化和冷冻电子显微镜已经组建了一个强大的合作团队，为成功执行提供必要的专业知识。所提议的实验。