Structural basis for chaperone-dependent folding of beta-propeller proteins essential for vision

视觉必需的β-螺旋桨蛋白的伴侣依赖性折叠的结构基础

• 批准号: 10441543
• 负责人: BARRY M WILLARDSON
• 金额: $ 38.59万
• 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 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10441543
• 关键词: 11 cis Retinal; ATP phosphohydrolase; Actins; Address; Affinity Chromatography; All-Trans-Retinol; Arrhythmia; Back; Bardet-Biedl Syndrome; Binding; Biochemical; Biochemistry; Biological Assay; Cells; Cognition Disorders; Complex; Coupled; Cryoelectron Microscopy; Cytoskeletal Proteins; Defect; Disease; Drug Design; Enzymes; Foundations; GTP-Binding Protein Regulators; GTP-Binding Proteins; Goals; Human; Knowledge; Lead; Learning; Leber' s amaurosis; Light; Link; Molecular; Molecular Chaperones; Mus; Mutation; Natural regeneration; Patients; Photoreceptors; Phototransduction; Play; Positioning Attribute; Process; Proteins; Proteome; RGS Proteins; RGS9 protein; Resolution; Retinal Diseases; Rhodopsin; Role; Set protein; Signaling Protein; Structure; Structure of retinal pigment epithelium; Techniques; Technology; Tertiary Protein Structure; Transducin; Tubulin; Vision; Visual; Work; base; chaperonin CCT; cytosolic chaperonin; dimer; experience; experimental study; gene augmentation therapy; insight; mutant; novel therapeutics; phosducin-like protein; protein complex; protein folding; response; stem; visual cycle; visual process

PROJECT SUMMARY The cytosolic chaperonin CCT is a large protein complex that plays an indispensable role in maintaining the cellular proteome by assisting in the folding of numerous proteins with complex tertiary structures and unfavorable folding trajectories. Proper CCT function is vital to human vision as evidenced by the fact that inactivating mutations in CCT cause Leber Congenital Amaurosis (LCA). CCT contributes to the visual process by folding the cytoskeletal proteins actin and tubulin as well as a number of proteins with b-propeller folds that have essential functions in the visual process. These include the G protein b1 (Gb1) subunit of the visual G protein transducin, the G protein b5 (Gb5) subunit of the regulator of G protein signaling 9 (RGS9) dimer, and the BBS2, BBS7 and BBS9 subunits of the Bardet-Biedl syndrome (BBSome) ciliary transport complex. In this proposal, we present evidence for a new function of CCT in the folding of human RPE65, another b-propeller protein that is a key enzyme in the visual cycle, the process that converts the all-trans retinol product of the light response back to 11-cis retinal to regenerate rhodopsin and maintain vision. Despite the importance of CCT in maintaining the proteome, we know very little at the molecular level about how CCT assists in the folding of these b-propeller proteins and how mutations in these proteins disrupt folding and cause disease. To address this gap in knowledge, we propose to use high resolution cryo-EM to determine the structures of key intermediates in the folding of human RPE65 and Gb5. These structures will tell us at the molecular level how CCT interacts with RPE65 and Gb5 to enable their folding. Furthermore, we propose to determine the structure of RPE65 and Gb5 folding mutants bound to CCT to learn how the mutations disrupt the folding trajectory and trap the mutant proteins inside CCT in an unfolded state. These studies will provide new insight into the molecular defects that cause misfolding of the RPE65 and Gb5 mutants and will establish a foundation for structure-based drug design to create new therapies for LCA and other retinopathies caused by these mutations.

项目摘要 胞质伴侣蛋白CCT是一种大型蛋白质复合物，在维持该维护方面起着必不可少的作用 细胞蛋白质组通过辅助用复杂的三级结构和 不利的折叠轨迹。适当的CCT功能对人类的视力至关重要，这是事实证明的 CCT中的灭活突变导致Leber先天性症（LCA）。 CCT有助于视觉 通过折叠细胞骨架蛋白肌动蛋白和微管蛋白以及许多蛋白质的过程 在视觉过程中具有重要功能的折叠。其中包括G蛋白B1（GB1）亚基 视觉G蛋白转霉素，G蛋白信号传导调节剂的G蛋白B5（GB5）亚基9（RGS9） 二聚体以及Bardet-Biedl综合征（BBSOME）睫状传输的BBS2，BBS7和BBS9亚基 复杂的。在此提案中，我们提供了CCT在人类RPE65折叠中的新功能的证据， 另一个是视觉周期中关键酶的B螺旋桨蛋白，转化全型的过程 光反应回到11-cis视网膜的视网膜产物，以再生视紫红质并保持视力。尽管 CCT在维持蛋白质组方面的重要性，我们在分子水平上对CCT的了解很少 有助于折叠这些B核蛋白以及这些蛋白质中的突变如何破坏折叠并导致 疾病。为了解决知识的这一差距，我们建议使用高分辨率的冷冻EM来确定 关键中间体在人RPE65和GB5的折叠中的结构。这些结构会告诉我们 分子水平CCT与RPE65和GB5相互作用以使其折叠。此外，我们建议 确定与CCT结合的RPE65和GB5折叠突变体的结构，以了解突变如何破坏 折叠轨迹并将突变蛋白捕获在CCT中以展开状态。这些研究将提供新的 深入了解导致RPE65和GB5突变体错误折叠的分子缺陷，并将建立一个 基于结构的药物设计基础，为LCA和其他视网膜病变创建新疗法 这些突变。