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.

胞质伴侣蛋白CCT在包括哺乳动物细胞在内的真核细胞中蛋白质折叠中起重要作用。 CCT具有由八个不同亚基发挥的高度进化的伴侣功能。但是，伴侣机制的细节仍然鲜为人知。在这里，我们通过使用RNAi介导的CCT在哺乳动物细胞中的CCT敲除CCT依赖性细胞功能，除了CCT特异性识别底物蛋白的体外识别纯系统（一种无细胞的体外翻译系统），我们建立了CCT，该系统建立了CCT，该系统既可以特异性地识别富含β-Sheet的蛋白质并预测蛋白质的蛋白质并预测其聚集。 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基于向量的过度膨胀实验。对聚合过程的分析表明，CCT可防止聚集在聚集的早期稳定阶段。这是CCT是一种分子链酮，通过防止包括疏水性β-折叠的聚集蛋白的聚集来促进蛋白质的生产性折叠，并通过调节聚集过程来保护细胞免受聚集蛋白的毒性的保护。