Protein Folding in the Eukaryotic Cytosol

描述（由申请人提供）：拟议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主要研究者以前的工作，这表明真核细胞质溶胶中的折叠是由与翻译偶联的高度组织化的分子伴侣机制介导的。Frydman博士的工作假设是，新翻译的多肽通过顺序和高度偶联的分子伴侣途径被引导到它们的最终构象。Frydman博士实验室的结果还表明，不同的细胞蛋白亚群表现出不同的伴侣蛋白需求。对于作为所提出的研究的焦点的模型蛋白，折叠途径似乎涉及两类分子伴侣，即小分子伴侣，例如Hsc 70蛋白和GIM/前卵泡素复合物，其通过稳定延伸的多肽起作用，以及分子伴侣TRiC，其凭借其环状结构产生有利于多肽折叠的环境。 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。一般的策略是联合收割机在体外和体内的方法，以获得机制和功能的见解分子伴侣在细胞折叠中的作用。具体而言，主要研究者将：1）定义新生多肽与分子伴侣蛋白相互作用的链长依赖性; 2）评估新合成多肽折叠中分子伴侣的需求; 3）确定介导分子伴侣组分向核糖体结合的新生链募集的机制; 4）定义完整细胞中胞质分子伴侣的底物谱。

DESCRIPTION (provided by applicant): The long-term goal of proposed 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 the principal investigator's previous work, which indicates that folding in the eukaryotic cytosol is mediated by a highly organized chaperone machinery that is coupled to translation. Dr. Frydman's working hypothesis is that the newly translated polypeptides are guided to their final conformation through a sequential and highly coupled chaperone pathway. Results from Dr. Frydman's laboratory also indicate that different subsets of cellular proteins exhibit different chaperone requirements. For the model proteins that are the focus of the proposed research, the folding pathway appears to involve two classes of chaperones, namely small chaperones, such as the Hsc70 proteins and the GIM/prefolclin complex, which act by stabilizing extended polypeptides, and the chaperonin TRiC, which by virtue of its ring-like structure creates an environment that is favorable for polypeptide folding. The objective of this proposal is to elucidate the mechanism by which chaperones mediate the folding of newly synthesized proteins in eukaryotic cells. The general strategy is to combine in vitro and in vivo approaches to obtain mechanistic and functional insights into the role of chaperones in cellular folding. Specifically the principal investigator will: 1) Define the chain-length dependence of the interactions of nascent polypeptides with chaperone proteins; 2) Assess the requirement of molecular chaperones in the folding of newly synthesized polypeptides; 3) Determine the mechanisms that mediate the recruitment of chaperone components to the ribosome-bound nascent chain; 4) Define the substrate spectrum of the cytosolic chaperones in intact cells.