Role of CCT assisted functions in cellular activities

胞质伴侣蛋白CCT在真核细胞包括哺乳动物细胞的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。然而，伴侣机制的细节仍然知之甚少。在此，我们通过RNAi介导的哺乳动物细胞中CCT的敲除，以及CCT对底物蛋白的体外特异性识别，研究了CCT依赖的细胞功能;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-折叠的蛋白并阻止其聚集。通过详细的分析，我们证明了CCT特异性结合疏水β链的方式不同于E.此外，我们发现CCT通过RNAi介导的敲低和基于载体的过表达实验防止聚谷氨酰胺扩展蛋白的聚集和毒性，所述聚谷氨酰胺扩展蛋白已知聚集在富含p-折叠的结构中。聚集过程的分析表明，CCT在聚集的早期可溶性阶段阻止聚集。因此，CCT是一种分子伴侣，其通过防止易聚集蛋白（包括疏水性β-折叠）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易聚集蛋白的毒性。

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.