权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Hsp90 Chaperone Machine Structure and Function

Hsp90伴侣机结构及功能

基本信息

批准号：
7175334
负责人：
AVROM J. CAPLAN
金额：
$ 26.71万
依托单位：
ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
依托单位国家：
美国
项目类别：
财政年份：
2005
资助国家：
美国
起止时间：
2005-02-01 至 2008-01-31
项目状态：
已结题

项目摘要

DESCRIPTION (provided by applicant): Hsp90 is a molecular chaperone whose function is largely restricted to folding of signal transducing proteins, such as protein kinases and transcription factors. Hsp90 does not function by itself, but in association with many different co-chaperones or helpers. The goal of our studies is to understand the role of Hsp90 as a chaperone machine involved in protein kinase folding. Studies over the past decade have demonstrated general principles of Hsp90 function and organization, but little is known about their function in protein kinase folding, which also requires the chaperone called Cdc37. In addition, there are many novel co-chaperones that were not assigned roles in the original description of Hsp90 action. Our studies address these areas where knowledge is lacking in three specific aims. In aim 1 we will determine the chaperone requirements for protein kinase folding. We will first determine which yeast kinases interact with Hsp90 and Cdc37 using physical binding and functional assays. Binding will be determined by Western blot analysis of Hsp90 and Cdc37 that associate with individual kinases. Kinases that require Cdc37 for activity will be screened in a cdc37 mutant yeast strain. This analysis will reveal the extent to which chaperones interact with the yeast kinome. We will then determine whether co-chaperones that function with Hsp90 act in the same way for all kinases or whether they exhibit specificity for different kinases. The effect of stress on chaperone interactions will also be determined. In aim 2 we will determine the composition of the Hsp90 chaperone machine. Hsp90 exists in a series of sub-complexes containing different co-chaperones. In the first part of this aim we will characterize the composition of these sub-complexes and in the second part of the aim we will determine which Hsp90 co-chaperones exist in complexes with a protein kinase. We will then test our hypothesis that Stil and Cdc37 participate in assembly of different Hsp90:kinase complexes. In aim 3 we analyze the function of chaperones and co-chaperones in protein kinase folding and activation. In the first sub-aim we will assay protein kinase conformation and stability in different co-chaperone mutant strains by limited proteolysis and pulse chase analysis. In the second sub-aim we will assay for chaperone activity of novel co-chaperones that are important for protein kinase folding. Finally, we will distinguish between the role of Cdc37 in Cdk folding and cyclin binding.

描述（由申请人提供）：Hsp90是一种分子伴侣，其功能主要限于折叠信号转导蛋白，如蛋白激酶和转录因子。Hsp90本身并不起作用，而是与许多不同的辅助分子或辅助分子一起起作用。我们的研究目的是了解热休克蛋白90作为一个伴侣机器参与蛋白激酶折叠的作用。过去十年的研究已经证明了Hsp90功能和组织的一般原理，但对其在蛋白激酶折叠中的功能知之甚少，这也需要称为Cdc37的伴侣蛋白。此外，还有许多新的辅助分子伴侣在最初的Hsp90作用描述中没有被分配角色。我们的研究针对这些领域的知识是缺乏三个具体目标。在目标1中，我们将确定蛋白激酶折叠的分子伴侣要求。我们将首先确定哪些酵母激酶与热休克蛋白90和Cdc37使用物理结合和功能测定相互作用。结合将通过与单个激酶相关的Hsp90和Cdc37的蛋白质印迹分析来确定。将在cdc37突变酵母菌株中筛选需要Cdc37活性的激酶。该分析将揭示分子伴侣与酵母激酶组相互作用的程度。然后，我们将确定是否与热休克蛋白90功能的辅分子伴侣以相同的方式为所有激酶或他们是否表现出不同的激酶的特异性。还将确定压力对分子伴侣相互作用的影响。在目标2中，我们将确定Hsp90分子伴侣机器的组成。热休克蛋白90存在于一系列含有不同辅分子伴侣的亚复合物中。在这个目标的第一部分中，我们将表征这些子复合物的组成，并且在该目标的第二部分中，我们将确定哪些Hsp90共分子伴侣存在于与蛋白激酶的复合物中。然后，我们将测试我们的假设，斯蒂尔和Cdc37参与组装不同的热休克蛋白90：激酶复合物。目的3分析分子伴侣和辅分子伴侣在蛋白激酶折叠和激活中的作用。在第一个子目标中，我们将通过有限蛋白水解和脉冲追踪分析来分析不同共伴侣突变株中蛋白激酶的构象和稳定性。在第二个子目标中，我们将测定对蛋白激酶折叠重要的新型共分子伴侣的分子伴侣活性。最后，我们将区分Cdc37在Cdk折叠和细胞周期蛋白结合中的作用。