Mechanisms of assembly of photoreceptor G protein complexes

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。阿尔茨海默氏症、帕金森氏症、亨廷顿氏症和卢格里克病(肌萎缩侧索硬化症或ALS)等神经退行性疾病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会导致视网膜感光细胞的神经元退化。许多严重的视网膜病变与视紫红质和其他光感受器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平衡的分子伴侣系统的一个关键组成部分是胞浆伴侣蛋白复合体(CCT)，它折叠肌动蛋白、微管蛋白和其他几十种胞质蛋白。在这些CCT底物中有G蛋白亚基(G？)，它形成G？1？1和 G？5-RGS9(G蛋白信号调节因子)二聚体是光转导级联的重要组成部分。所有的G？亚基都需要CCT和辅助分子伴侣--硫代蛋白样蛋白1(PhLP1)来折叠G？，并组装成功能性二聚体。最近，CCT在Bardet-Biedl综合征(BBS)中的另一个作用被证明。BBS是一种睫状体功能障碍的遗传性疾病，表现为包括视网膜变性在内的多种病理状态。14种BBS蛋白的突变与这种疾病有关。其中7个(BBS 1-2，4-5和7-9)形成一个复合体，称为BBSome，它是囊泡向纤毛运输所必需的。另外三个(BBS 6、10和12)与CCT亚基同源，并与CCT形成BBS2和7折叠和组装BBSome所需的复合体。总而言之，G？和BBS的这些发现指出了CCT在组装蛋白质复合体中的重要作用，这些蛋白质复合体在光感受器细胞中发挥关键的生理功能。因此，拟议研究的基本目标是在分子水平上了解CCT组装蛋白质复合体的机制，以确定改善折叠过程的治疗靶点，并治疗诸如BBS等尚无有效治疗方法的疾病。特定目标1和2检查G？1-CCT和RGS9-G？5-CCT复合体的结构，以了解CCT如何折叠G？1和G？5，并招募RGS9形成G？5-RGS9二聚体。具体目的3研究伴侣素样BBS蛋白与CCT的结合结构及BBSome的组装机制。为了实现这些目标，人们提出了许多方法，包括定点突变和基于细胞的结合分析、蛋白质复合体的脉冲追逐组装测量、大分子蛋白质复合体的纯化和冷冻电子显微镜。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。 公共卫生相关性：许多神经退行性疾病与蛋白质折叠缺陷有关。视网膜的感光细胞当然就是这种情况，蛋白质的错误折叠突变会导致感光细胞死亡，导致视网膜退化和失明。这项建议研究了重要的生理蛋白质复合体的折叠和组装机制，当它们的基因突变时，这些复合体会导致疾病。其中一种疾病是Bardet-Biedl综合征，这种疾病通常在青春期就会导致失明。了解这些蛋白质复合体是如何聚集在一起的，对于开发治疗方法是必要的，这种治疗方法将允许这些复合体在发生突变的情况下组装并发挥作用。

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.