Hsp90 Chaperone Machine Structure and Function

Hsp90伴侣机结构及功能

• 批准号: 7007307
• 负责人: AVROM J. CAPLAN
• 金额: $ 27.51万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-02-01 至 2009-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7007307
• 关键词: cell cycle proteins; conformation; cyclins; heat shock proteins; molecular chaperones; protein binding; protein folding; protein kinase; protein protein interaction; protein structure function; proteomics; western blottings; yeast two hybrid system

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（申请人提供）：Hsp90是一种分子伴侣，其功能主要限于信号转导蛋白的折叠，例如蛋白激酶和转录因子。 Hsp90 本身并不发挥作用，而是与许多不同的共伴侣或帮助者联合发挥作用。我们研究的目的是了解 Hsp90 作为参与蛋白激酶折叠的伴侣机器的作用。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，蛋白激酶折叠也需要称为 Cdc37 的伴侣。此外，还有许多新颖的辅助伴侣在 Hsp90 作用的原始描述中并未指定角色。我们的研究针对三个具体目标缺乏知识的领域。在目标 1 中，我们将确定蛋白激酶折叠的伴侣要求。我们将首先使用物理结合和功能测定来确定哪些酵母激酶与 Hsp90 和 Cdc37 相互作用。结合将通过与各个激酶相关的 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 才能发挥活性的激酶。该分析将揭示伴侣与酵母激酶组相互作用的程度。然后我们将确定与 Hsp90 一起发挥作用的共伴侣是否对所有激酶都以相同的方式起作用，或者它们是否对不同的激酶表现出特异性。压力对伴侣相互作用的影响也将被确定。在目标 2 中，我们将确定 Hsp90 伴侣机的组成。 Hsp90 存在于一系列包含不同共伴侣的子复合物中。在该目标的第一部分中，我们将表征这些子复合物的组成，在该目标的第二部分中，我们将确定哪些 Hsp90 共伴侣存在于与蛋白激酶的复合物中。然后我们将检验我们的假设，即 Stil 和 Cdc37 参与不同 Hsp90:激酶复合物的组装。在目标 3 中，我们分析了蛋白激酶折叠和激活中伴侣和辅助伴侣的功能。在第一个子目标中，我们将通过有限的蛋白水解和脉冲追踪分析来测定不同共伴侣突变株中的蛋白激酶构象和稳定性。在第二个子目标中，我们将测定对蛋白激酶折叠很重要的新型辅助伴侣的伴侣活性。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。