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神经元的关键问题。神经退行性疾病如阿尔茨海默氏病、帕金森氏病、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。蛋白质组稳态的类似破坏引起视网膜感光细胞的神经元变性。许多严重的视网膜病变与视紫红质和其他感光蛋白的错误折叠突变有关。维持感光细胞中蛋白质组稳态的分子伴侣系统的关键组分是胞质伴侣蛋白复合物（CCT，包含无尾多肽1的伴侣蛋白），其折叠肌动蛋白、微管蛋白和数十种其他胞质蛋白。在这些CCT底物中，G蛋白亚基（G）形成G 1？1和 G？5-RGS 9（G蛋白信号调节器）二聚体，是光转导级联反应的重要组成部分。所有的G亚基都需要CCT和辅助分子伴侣蛋白1（PhLP 1）来折叠G亚基并组装成功能性二聚体。最近，CCT在Bardet-Biedl综合征（BBS）中的额外作用已得到证实。BBS是一种遗传性睫状体功能障碍疾病，表现出包括视网膜变性在内的多种病理状态。14种BBS蛋白的突变与该疾病有关。其中7个（BBS 1-2，4-5和7-9）形成一个复合物，称为BBSome，这是必不可少的囊泡运输到纤毛。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BBS 2和7折叠以及BBSome组装所需的。在一起，这些G和BBS的发现指出CCT在光感受器细胞中执行关键生理功能的蛋白质复合物的组装中起重要作用。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，以确定治疗靶点，改善折叠过程，治疗无法获得有效治疗的疾病，如BBS。具体目的1和2检查G <$1-CCT和RGS 9-G <$5-CCT复合物的结构，以了解CCT如何折叠G <$1和G <$5并募集RGS 9形成G <$5-RGS 9二聚体。具体目标3研究了伴侣蛋白样BBS蛋白与CCT之间的复合物的结构以及BBSome组装的机制。为了实现这些目标，提出了许多方法，包括定点诱变和基于细胞的结合试验，脉冲追逐组装测量的蛋白质复合物，纯化的大蛋白质复合物和冷冻电子显微镜。已经组建了一个强大的合作者团队，为成功执行拟议的实验提供必要的专业知识。