Systematic Analysis of the Regulation of Hsp90 by its cofactors

细胞内的蛋白质动态平衡由分子伴侣和蛋白水解酶组成的一系列分子系统调节。这些系统确保蛋白质折叠到它们的自然状态，并在它们在细胞中的整个生命周期中保持它们的正确构象。伴侣监测系统还确保错误折叠的蛋白质和短暂的调节蛋白质成为降解的目标。HSP90是细胞中主要的、高度保守的伴侣蛋白之一，其功能是帮助蛋白质获得其活性构象。热休克蛋白90也被发现与蛋白质降解有关。它是一种依赖于ATP的伴侣，形成一个结构性二聚体，表现出受ATP结合和水解调节的多种构象。Hsp90的体内和体外活性是由大量的辅因子控制的，这些辅因子针对特定底物的伴侣。酵母中广泛存在的Hsp90辅助因子包括：Sti1(Hop)、Sba1(P23)、CDc37(P50)、Cpr6、Cpr7、Cns1、Aha1、Hch1和Ppt1。HSP90与这些辅因子和靶底物形成特定的复合体。对这些不同的Hsp90-辅因子复合体的组成和相对功能的了解仍然是零散的，主要来自使用部分纯化的蛋白质的重建实验。因此，由于伴侣-辅因子-底物复合体的动态性和异质性，Hsp90的作用机制仍然不清楚。在这项资助申请中，我们建议使用蛋白质组学和生化方法进行系统的分析，以了解Hsp90-辅因子系统是如何建立底物专一性的，从而了解不同的辅因子如何调节Hsp90的活性。为此，将采取几种方法。(1)系统分析正常生长和应力生长条件下热休克蛋白90及其共性因子的相互作用。(2)相互作用研究将通过对数据的广泛计算询问来补充，以揭示决定底物与特定Hsp90辅因子复合体相互作用的共同特征。(3)将进行后续生化研究，以验证生物信息学分析的结果。

Protein homeostasis in the cell is regulated by a wide array of molecular systems consisting of molecular chaperones and proteases. These systems ensure that proteins fold to their native state and maintain their proper conformation throughout their lifetime in the cell. The chaperone surveillance systems also ensure that misfolded proteins and short-lived regulatory proteins are targeted for degradation. Hsp90 is one of the major and most highly conserved chaperones of the cell that functions to assist proteins in attaining their active conformation. Hsp90 has also been found to be involved in protein degradation. It is an ATP-dependent chaperone that forms a constitutive dimer, which exhibits multiple conformations regulated by ATP binding and hydrolysis. The in vivo and in vitro activity of Hsp90 is controlled by a large number of cofactors that target the chaperone to specific substrates. Well-established Hsp90 cofactors in yeast include: Sti1 (Hop), Sba1 (p23), Cdc37 (p50), Cpr6, Cpr7, Cns1, Aha1, Hch1, and Ppt1. Hsp90 forms specific complexes with these cofactors and target substrates. Knowledge of the composition and relative function of each of these different Hsp90-cofactor complexes is still fragmentary and has come largely from reconstitution experiments using partially purified proteins. As a result, the mechanism of Hsp90 function remains elusive due to the dynamic and heterogeneous nature of the chaperone-cofactor-substrate complexes. In this grant application, we propose to carry out a systematic analysis using proteomic and biochemical approaches in order to understand how substrate specificity is established by the Hsp90-cofactor system and, consequently, how the different cofactors regulate Hsp90 activity. To this end, several approaches will be undertaken. (1) Systematic analysis of interactors common to Hsp90 and its cofactors under normal and 'stress' growth conditions will be carried out. (2) The interaction studies will be complimented by extensive computational interrogation of the data to reveal common features that dictate the interaction of a substrate with a specific Hsp90-cofactor complex. (3) Follow up biochemical studies will be carried out to verify the results from the bioinformatic analysis.