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

项目总结 胞浆伴侣蛋白CCT是一种大的蛋白质复合体，在维持细胞周期中起着不可或缺的作用。 细胞蛋白质组通过协助许多具有复杂三级结构的蛋白质和 不利的折叠轨迹。正确的CCT功能对人类视觉至关重要，这一事实证明了 CCT基因失活突变可导致Leber先天性巨结肠(LCA)。CCT有助于视觉 通过b-螺旋桨折叠细胞骨架蛋白肌动蛋白和微管蛋白以及一些蛋白质的过程 在视觉过程中具有基本功能的折叠。其中包括G蛋白b1(GB1)亚单位 视觉G蛋白转导蛋白，G蛋白b5(GB5)亚单位，G蛋白信号转导9调节因子(RGS9) 二聚体以及Bardet-Biedl综合征(BBSome)纤毛运输的BBS2、BBS7和BBS9亚单位 很复杂。在这个建议中，我们提出了CCT在人类RPE65折叠中的新功能的证据， 另一种b-螺旋桨蛋白是视觉周期中的关键酶，这一过程将全反式 视黄醇产物的光反应回到11顺式视网膜，再生视紫红质，维持视力。尽管 CCT在维持蛋白质组中的重要性，我们在分子水平上对CCT如何 协助这些b-螺旋桨蛋白的折叠，以及这些蛋白质中的突变如何破坏折叠并导致 疾病。为了解决这一知识差距，我们建议使用高分辨率冷冻EM来确定 人RPE65和GB5折叠过程中关键中间体的结构。这些结构将告诉我们 分子水平CCT如何与RPE65和GB5相互作用以使它们能够折叠。此外，我们建议 确定与CCT结合的RPE65和GB5折叠突变体的结构，以了解这些突变是如何破坏 折叠轨迹并将突变蛋白以未折叠状态捕获到CCT内。这些研究将提供新的 洞察导致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和其他由以下原因引起的视网膜病变创造新的疗法 这些突变。

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.