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Role of CCT assisted functions in cellular activities

CCT 辅助功能在细胞活动中的作用

基本信息

批准号：
18570175
负责人：
KUBOTA Hiroshi
金额：
$ 2.57万
依托单位：
Kyoto University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2006
资助国家：
日本
起止时间：
2006 至 2007
项目状态：
已结题

项目摘要

Cytosolic chaperonin CCT plays an important role in protein folding in eukaryotic cells including mammalian cells. CCT has a highly evolved chaperoning function exerted by eight different subunits. However, details of the chaperone mechanism are still poorly understood. Here, we studied CCT-dependent cellular functions by using RNAi-mediated knockdown of CCT in mammalian cells, in addition to CCT specific recognition of substrate proteins in vitro.Using PURE system, a cell-free in vitro translation system, we found that CCT specifically recognizes proteins rich in β-sheet and prevents their aggregation. By detailed analysis, we demonstrated that CCT specifically binds hydrophobic β-strands in a manner distinct from that of the E. coli homologue GroEL, and facilitates productive folding of substrate proteins.Moreover, we found that CCT prevents aggregation and toxicity of polyglutamine-expansion proteins, which are known to aggregate in p-sheet rich structures, by RNAi-mediated knockdown and vector-based overexperssion experiments. Analysis of aggregation process suggested that CCT prevents aggregation in an early soluble stage of aggregation. Thus, CCT is a molecular chaperone that facilitates productive folding of proteins by preventing aggregation of aggregation-prone proteins including hydrophobic β-sheets and that protects cells from the toxicity of aggregation-prone proteins by modulating aggregation process.

细胞质伴侣蛋白CCT在包括哺乳动物细胞在内的真核细胞的蛋白质折叠中起重要作用。CCT具有高度进化的伴随功能，由8个不同的亚基发挥作用。然而，伴侣机制的细节仍然知之甚少。在这里，我们研究了CCT依赖的细胞功能，通过rnai介导的哺乳动物细胞中CCT的敲除，以及CCT在体外对底物蛋白的特异性识别。使用PURE系统（一种无细胞体外翻译系统），我们发现CCT特异性识别富含β-sheet的蛋白质并阻止它们聚集。通过详细的分析，我们证明了CCT以一种不同于大肠杆菌同源物GroEL的方式特异性地结合疏水β-链，并促进底物蛋白的生产性折叠。此外，通过rnai介导的敲低和基于载体的过表达实验，我们发现CCT可以阻止聚谷氨酰胺扩增蛋白的聚集和毒性，已知聚谷氨酰胺扩增蛋白聚集在p-sheet丰富的结构中。聚集过程分析表明，CCT在聚集的早期可溶阶段阻止聚集。因此，CCT是一种分子伴侣，通过阻止包括疏水β-片在内的易聚集蛋白的聚集，促进蛋白质的生产折叠，并通过调节聚集过程保护细胞免受易聚集蛋白的毒性。