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: 8102638
• 负责人: BARRY M WILLARDSON
• 金额: $ 37.5万
• 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/8102638
• 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; 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. PUBLIC HEALTH RELEVANCE: Many neurodegenerative diseases are associated with defects in protein folding. This is certainly the case in the photoreceptor cells of the retina, in which misfolding mutations in proteins cause photoreceptor cell death, resulting in retinal degeneration and blindness. This proposal investigates the mechanism of folding and assembly of physiologically important protein complexes that result in diseases when their genes are mutated. One such disease is Bardet-Biedl syndrome, a condition that causes blindness usually by adolescence. An understanding of the way these protein complexes are brought together is necessary to develop treatments that would allow the complexes to assemble and function despite the mutations.

描述（适用提供）：正确蛋白质折叠是维持健康的功能性神经元的关键问题。神经退行性疾病，例如阿尔茨海默氏症，帕金森氏症，亨廷顿和Lou Gehrig氏病（肌萎缩性侧索硬化症或ALS），都是由于神经元中的蛋白质折叠而导致的。蛋白质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。在光感受器细胞中维持蛋白质组稳态的分子链酮系统的一个关键成分是胞质链烯蛋白络合物（CCT，含无讽刺多肽的伴侣蛋白1），可折叠肌动蛋白，微管蛋白和数十个其他胞质蛋白。在这些CCT底物中，是G蛋白�亚基（g¿），它构成了g¿1？1和 G gs9（G蛋白信号传导的调节剂）二聚体是光转导级联的重要组成部分。所有的G oodunits都需要CCT和共伴酮磷素样蛋白1（PHLP1）才能折叠G？并组装成功能二聚体。最近，已经证明了CCT在Bardet-Biedl综合征（BBS）中的额外作用。 BBS是一种睫状功能障碍的遗传疾病，表现出多种病理状况，包括残留变性。 14个BBS蛋白的突变与该疾病有关。其中七个（BBS 1-2、4-5和7-9）形成了一个复合物，称为BBSOME，这对于囊泡运输纤毛至关重要。另外三个（BBS 6、10和12）与CCT亚基同源，并与CCT形成复合物，这些复合物是BBS2和7和BBSOME组装所需的CCT。这些G？和BBS的发现共同指出了CCT在蛋白质复合物组装中的重要作用，该蛋白质复合物在光感受器细胞中执行关键的生理功能。这是拟议研究的基本目标是在分子水平上了解CCT蛋白质复合物组件的机制，以鉴定治疗靶标，以改善折叠过程并治疗像BBS这样的疾病，以提供有效治疗的BBS。具体目的1和2检查了g¿1-CCT和RGS9-g¿5-CCT复合物的结构，以了解CCT如何折叠G？1和G？5和募集RGS9以形成g¿5-rgs9二聚体。具体目标3研究了链蛋白样BBS蛋白和CCT之间的复合物的结构以及BBSOME组装的机理。为了实现这些目的，提出了许多方法，包括位置定向的诱变和基于细胞的结合测定，脉冲练习蛋白复合物的测量，大蛋白质复合物的纯化和冷冻电子显微镜。一支强大的合作者团队已经组装，以提供必要的专业知识，以成功执行拟议的实验。 公共卫生相关性：许多神经退行性疾病与蛋白质折叠的缺陷有关。在视网膜的感光细胞中肯定是这种情况，在蛋白质中折叠突变会导致感光细胞死亡，从而导致残留变性和失明。该提案研究了在物理上重要的蛋白质复合物的折叠和组装机制，这些蛋白质复合物在突变时会导致疾病。一种这样的疾病是Bardet-Biedl综合征，这种病通常会引起青少年失明。对这些蛋白质复合物的结合方式的理解对于开发允许复合物可以组装并起作用目的地突变的治疗方法是必要的。