Protein Folding in the Eukaryotic Cytosol

描述（由申请人提供）：我们研究的长期目标是了解真核细胞中蛋白质折叠的生物化学和细胞生物学。拟议的研究将集中在折叠事件，因为它们发生在核糖体在多肽合成过程中，并将检查分子伴侣在折叠过程中的作用。理解蛋白质从核糖体中出现时的折叠所需的概念框架源于我们以前的工作，这表明真核细胞胞质溶胶中的折叠是由物理和功能上与翻译相关的分子伴侣网络介导的。该建议的目的是阐明分子伴侣介导真核细胞中新合成蛋白质折叠的机制。为了深入了解这一过程，我们确定了关键问题，这将使我们能够理解从头折叠，即：（i）如何以及何时伴侣接触新兴的新生链？(ii)分子伴侣结合与从头折叠的关系是什么？(iii)不同的分子伴侣系统对细胞内的整体折叠有什么贡献？(iv)伴侣是如何与翻译机器互动的？我们回答这些问题的一般策略是联合收割机在体外和体内的方法，以获得分子伴侣在细胞折叠中的作用的机制和功能的见解。前两个具体目标将分析模型蛋白在体内和无细胞翻译裂解物中的折叠，这些裂解物忠实地代表了完整的胞质溶胶。由于我们的体内分析表明，不同的蛋白质表现出不同的伴侣的要求，第三个具体的目标将检查不同的伴侣细胞折叠的贡献，并将确定不同的分子伴侣的底物光谱。最后，我们的第四个具体目标将探讨分子伴侣和翻译机制之间的相互作用。

DESCRIPTION (provided by applicant): The long-term goal of our research is to understand the biochemistry and cell biology of protein folding in eukaryotic cells. The proposed research will focus on folding events as they occur at the ribosome during synthesis of a polypeptide and will examine the role of molecular chaperones in the folding process. The conceptual framework required to understand the folding of proteins as they emerge from the ribosome originates from our previous work, which indicates that folding in the eukaryotic cytosol is mediated by a chaperone network that is physically and functionally linked to translation. The objective of this proposal is to elucidate the mechanism by which chaperones mediate the folding of newly synthesized proteins in eukaryotic cells. To gain insight into this process we identified critical questions that will allow us to understand de novo folding, namely: (i) how and when do chaperones contact the emerging nascent chains?; (ii) what is the relevance of chaperone-binding for de novo folding?; (iii) what is the contribution of different chaperone systems to overall folding in the cell? (iv) how do chaperones interact with the translation machinery? Our general strategy to answer these questions is to combine in vitro and in vivo approaches to obtain mechanistic and functional insights into the role of chaperones in cellular folding. The first two specific aims will analyze the folding of model proteins in vivo and in cell-free translation lysates that faithfully represent the intact cytosol. Since our in vivo analysis indicates that different proteins exhibit distinct chaperone requirements, the third specific aim will examine the contribution of different chaperones to cellular folding and will identify the substrate spectrum of different molecular chaperones. Finally, our fourth specific aim will explore the interaction between chaperones and the translational machinery.