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.

项目概要 胞质伴侣蛋白 CCT 是一种大型蛋白质复合物，在维持 细胞蛋白质组通过协助折叠具有复杂三级结构的众多蛋白质和 不利的折叠轨迹。正确的 CCT 功能对人类视觉至关重要，这一点可以从以下事实证明： CCT 失活突变会导致莱伯先天性黑蒙 (LCA)。 CCT有助于视觉 通过使用 b 螺旋桨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褶皱。其中包括 G 蛋白 b1 (Gb1) 亚基 视觉 G 蛋白转导蛋白，G 蛋白信号传导调节因子 9 (RGS9) 的 G 蛋白 b5 (Gb5) 亚基 二聚体，以及 Bardet-Biedl 综合征 (BBSome) 纤毛运输的 BBS2、BBS7 和 BBS9 亚基 复杂的。在本提案中，我们提供了 CCT 在人类 RPE65 折叠中的新功能的证据， 另一种 b 螺旋桨蛋白，是视觉循环中的关键酶，视觉循环是将全反式转化的过程 光反应的视黄醇产物返回11-顺式视网膜，以再生视紫红质并维持视力。尽管 虽然 CCT 在维持蛋白质组方面的重要性，但我们在分子水平上对 CCT 如何发挥作用知之甚少 协助这些 b 螺旋桨蛋白的折叠，以及这些蛋白的突变如何破坏折叠并导致 疾病。为了解决这一知识差距，我们建议使用高分辨率冷冻电镜来确定 人 RPE65 和 Gb5 折叠中关键中间体的结构。这些结构将告诉我们 分子水平上 CCT 如何与 RPE65 和 Gb5 相互作用以实现其折叠。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以未折叠状态捕获在 CCT 内。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 为基于结构的药物设计奠定基础，为 LCA 和其他视网膜病变引起的疾病创造新疗法 这些突变。