Genome-wide Function and Substrate Recognition of the Chaperone Prefoldin

描述(申请人提供)：伴侣有助于许多蛋白质的正确折叠并防止蛋白质聚集，蛋白质聚集越来越被认为是人类疾病(如阿尔茨海默氏症)的致病机制。Prefoldin是一种蛋白质伴侣，已知在新翻译的肌动蛋白和微管蛋白的折叠中发挥作用。我们希望了解前折叠蛋白的整体细胞功能，并确定它如何识别其底物。对肌动蛋白和微管蛋白的前折叠蛋白功能的研究表明，它结合新生多肽并将其隔离，同时将它们运送到TIC/CCT伴侣蛋白。前折叠蛋白有六个同源亚基，每个亚基都有一个长的螺旋状突起。某些亚基的末端与肌动蛋白和微管蛋白结合，这些底物中的每一种都需要确定的残基来进行这种相互作用。因此，前折叠蛋白结合是特异的，可能涉及不同亚基对不同靶点的组合结合。我们的具体目标是：1)确定前折叠蛋白的细胞功能。我们将使用与伴侣突变的基因相互作用的全基因组测量来确定哪些过程受到前折叠蛋白的影响。2)确定新的前折叠蛋白靶点并检测TIC/CCT依赖性。我们将通过利用伴侣蛋白突变和其靶点之间的上位性特征模式，找到依赖于前折叠蛋白的额外蛋白质。然后，我们将测试候选底物与伴侣的直接物理相互作用，并确定伴侣功能的丧失是否会影响它们在体内的折叠。3)了解前折叠蛋白如何与其各种无关的底物结合。我们将使用基于已解决的前折叠蛋白结构的缺失和点突变来确定特定靶蛋白所需的前折叠蛋白的哪些区域。我们将使用前折叠蛋白功能的遗传测试以及前折叠蛋白结合的生化测量。蛋白质聚集在许多人类疾病中起着重要作用，包括阿尔茨海默氏症。蛋白质伴侣与新产生的蛋白质结合，帮助它们折叠，同时防止它们聚集。我们想知道伴侣蛋白前折叠蛋白是如何与特定的靶蛋白结合的，以及这在正常细胞功能中有什么作用。

DESCRIPTION (provided by applicant): Chaperones assist in the proper folding of many proteins and protect against protein aggregation, which is increasingly recognized as a pathogenic mechanism in human diseases such as Alzheimer's. Prefoldin is a protein chaperone that is known to play a role in the folding of newly-translated actin and tubulin. We wish to understand the overall cellular function of prefoldin and determine how it recognizes its substrates. Studies of prefoldin function on actin and tubulin suggest that it binds nascent peptides and sequesters them while delivering them to the TRiC/CCT chaperonin. Prefoldin has six homologous subunits, each of which has a long, coiled-coil projection. The tips of certain subunits bind to actin and tubulin, and defined residues in each of these substrates are necessary for this interaction. Thus, prefoldin binding is specific ' and may involve a combinatorial binding of different subunits for different targets. Our specific aims are: 1) Determine the cellular functions of prefoldin. We will use whole-genome measurements of genetic interactions with chaperone mutations to determine which processes are affected by prefoldin. 2) Identify new prefoldin targets and test for TRiC/CCT dependence. We will find additional proteins that depend on prefoldin by using the characteristic pattern of epistasis between mutations in a chaperone and its target. We will then test candidate substrates for direct physical interaction with the chaperone and determine whether loss of chaperone function affects their folding in vivo. 3) Understand how prefoldin binds its various, unrelated substrates. We will use deletions and point mutants based on the solved structure of prefoldin to determine which regions of prefoldin are required by specific target proteins. We will use genetic tests for prefoldin function as well as biochemical measurements of prefoldin binding. Protein aggregation plays a major role in many human diseases, including Alzheimer's. Protein chaperones bind to newly-produced proteins and help them to fold while preventing them from aggregating. We want to know how the chaperone prefoldin binds specific target proteins and what role this has in normal cell function.