Genetic and biochemical analysis of the Hsp90 system in Saccharomyces cerevisiae

热休克蛋白Hsp90是一种全长90kodalton的热休克蛋白，在所有真核生物中起着维持蛋白稳定的重要作用。我的研究重点是了解Hsp90的作用机制，以及辅助伴侣蛋白和翻译后修饰(PTM)对其调控的基础。HSP90伴随着数百种“客户”蛋白质的构象变化，这些变化是配体结合、催化活性或组装成大分子复合体所必需的。HSP90是一种同源二聚体ATPase，由ATP驱动的客户端激活周期受到一组辅助伴侣蛋白和翻译后修饰的严格调控。与Hsp90 ATPase循环相关的全球构象变化如何最终驱动客户蛋白激活仍然是该领域的一个悬而未决的问题。我的研究计划专注于阐明Hsp90客户激活周期的机制，特别强调刺激ATP水解的两个辅助伴侣，Aha1p和Hch1p，以及调节它们作用的PTM。我的计划由三个主题组成，使用不同的模式和技术提供不同的培训机会。1)利用酵母遗传学研究Hsp90的Aha1p/Hch1p调控。Hsp90系统在所有真核生物中都高度保守，酿酒酵母是Hsp90研究中应用最广泛的模型。我们收集了一组表达不同Hsp90和Hsp90协同伴侣突变体的酵母菌株。我们将集中在一组这些菌株上，以表征Aha1p和Hch1p的体内作用。我们将在生长分析以及客户特定的功能分析中测试这些菌株。改变Hsp90机器系统的不同结构元素或调节组件(辅助伴侣和翻译后修饰)的影响可以在酵母模型系统中进行剖析。2)研究Aha1p和Hch1p对Hsp90 ATPase活性的调节作用。我们利用纯化的Hsp90、Hsp90共伴侣蛋白和模拟客户蛋白，在体外分析了ATP水解和客户激活的动力学。含有突变的HSP90或HSP90辅伴蛋白在酵母中被提纯，并在各种体外试验中进行研究。我们测量了Hsp90对ATP的水解以及辅伴蛋白介导的客户激活的动力学参数。这些体外策略使我们能够确定我们在酵母中观察到的表型的分子基础。3)Hsp90复合体的物理特征我们与其他专门从事核磁共振、结晶学、小角光散射和荧光技术的研究小组合作，询问Hsp90酶活性和生物活性的结构基础。我的项目的这一方面不仅产生了对Hsp90生物学的重要科学见解，而且还为我的学员提供了宝贵的培训机会，因为它涉及到广泛的合作。

The 90 kiloDalton heat shock protein, Hsp90, plays a fundamental and essential role in maintaining proteostasis in all eukaryotes. My research program is focused on understanding the mechanism of action Hsp90, and the basis for its regulation by co-chaperone proteins and post-translational modifications (PTMs). Hsp90 chaperones conformational changes in hundreds of 'client' proteins that are required for things like ligand binding, catalytic activity, or assembly into macromolecular complexes. Hsp90 is a homodimeric ATPase and the ATP-driven client activation cycle is tightly regulated by a cohort of co-chaperone proteins and post-translational modifications. How the global conformational changes associated with the Hsp90 ATPase cycle ultimately drive client protein activation remains an open question in the field. My research program is focused on elucidating the mechanics of the Hsp90 client activation cycle with particular emphasis on two co-chaperones that stimulate ATP hydrolysis, Aha1p and Hch1p, and PTMs that modulate their action. My program is comprised of three themes that provide diverse training opportunities using different models and techniques. 1) Characterize Aha1p/Hch1p regulation of Hsp90 using yeast genetics The Hsp90 system is highly conserved in all eukaryotes and Saccharomyces cerevisiae is the most widely used model for Hsp90 studies. We have a collection of yeast strains expressing different Hsp90 and Hsp90 co-chaperone mutants. We will focus on a set of these strains to characterize the in vivo action of Aha1p and Hch1p. We will test these strains in growth assays as well as in client-specific functional assays. The impact of alteration different structural elements of the Hsp90 machine system or regulatory components (co-chaperones and post-translational modifications) can be dissected in the yeast model system. 2) Characterize regulation of the Hsp90 ATPase activity by Aha1p and Hch1p We use purified Hsp90, Hsp90 co-chaperones, and model client proteins to analyze the kinetics of ATP hydrolysis and client activation in vitro. Hsp90 or Hsp90 co-chaperones harbouring mutations that confer growth defects or client-specific activation defects in yeast are purified and studied in a variety of in vitro assays. We measure kinetic parameters of ATP hydrolysis by Hsp90 as well as co-chaperone-mediated activation of client activation. These in vitro strategies allow us to determine the molecular underpinnings of the phenotypes we observe in yeast. 3) Physically characterize Hsp90 complexes We collaborate with other research groups specializing in NMR, crystallography, small-angle light scattering, and fluorescence techniques to interrogate the structural basis for the enzymatic and biological activity of Hsp90. This aspect of my program not only yields important scientific insights into Hsp90 biology but also provides valuable training opportunities for my trainees because of the extensive collaborations it involves.