Mechanisms of the eukaryotic chaperonin TRiC/CCT and its cochaperone Prefoldin

描述(申请人提供)：许多蛋白质依赖于伴侣才能正确折叠。折叠不正确与许多疾病有关，包括癌症和与衰老相关的神经退行性疾病。虽然大多数伴侣在功能上是多余的，但TIC及其相应的伴侣蛋白在所有王国中都是必不可少的。所有真核生物的伴侣蛋白都由背靠背堆叠的两个环组成，每个环由7-9个亚基组成，形成一个包裹底物的中央空腔。TRIC介导的折叠是由ATP结合和水解驱动的，它们调节其构象周期。虽然在理解更简单的同源低聚古生菌伴侣蛋白的机制方面已经取得了进展，但真核细胞伴侣蛋白trE的折叠机制仍然难以捉摸，该蛋白由8个相邻的亚基组成。重要的是，亚基的分歧提供了独特的折叠特性，允许TIC折叠同源伴侣蛋白无法折叠的重要真核底物。TIC还被异型六聚体辅分子前折叠蛋白辅助，它被认为可以将非自然折叠的底物转移到TIC。这个建议的目的是研究依赖于ATP的TIRE构象循环的机制、功能和结构方面以及TIRE底物识别的机制；我还旨在确定这些TIRE功能是否以及如何被前折叠蛋白调节。弗莱德曼实验室已经在酵母中开发了一种系统，可以用包含致命突变的相同亚基替换任何tric亚基，并拥有丰富的经验和工具来解决体内和体外的这些问题。

DESCRIPTION (provided by applicant): Many proteins are dependent upon chaperones to properly fold. Failure to fold correctly is associated with many diseases including cancer and aging associated neurodegenerative disorders. While most chaperones are redundant in function, TRiC and its homologous chaperonins are essential in all kingdoms. All eukaryotic chaperonins are composed of two rings stacked back to back, each formed by 7-9 subunits that produce a central cavity that encapsulates substrates. TRiC-mediated folding is driven by ATP binding and hydrolysis, which regulate its conformational cycle. While progress has been made in understanding the mechanism of simpler homo-oligomeric archaeal chaperonins, the mechanism of folding by the eukaryotic chaperonin, TRiC, which is composed of eight paralogous subunits, has remained elusive. Importantly, the divergence of the subunits has provided unique folding properties allowing TRiC to fold essential eukaryotic substrates that the homologous chaperonins are incapable of folding. TRiC is also assisted by the hetero-hexameric cochaperone Prefoldin, which is thought to transfer non-natively folded substrates to TRiC. The goal of this proposal is to study mechanistic, functional and structural aspects of the ATP-dependent conformational cycle of TRiC and the mechanisms of TRiC substrate-recognition; I also aim to define whether and how these TRiC functions are modulated by Prefoldin. The Frydman lab has developed a system in yeast that allows replacement of any of the TRiC subunits with the same subunit containing lethal mutations, and has extensive experience and tools to address these questions in vivo and in vitro.