Genetic and biochemical analysis of the Hsp90 system in Saccharomyces cerevisiae

热休克蛋白90（Hsp 90）是一种分子量为90千道尔顿的热休克蛋白，在维持真核生物的蛋白质稳态中起着重要的作用。我的研究项目主要集中在了解Hsp 90的作用机制，以及其通过共伴侣蛋白和翻译后修饰（PTM）进行调节的基础。Hsp 90伴侣蛋白在数百种“客户”蛋白中的构象变化，这些蛋白是配体结合，催化活性或组装成大分子复合物等所需的。热休克蛋白90是一种同源二聚体ATP酶，ATP驱动的客户端激活周期受到一组共伴侣蛋白和翻译后修饰的严格调控。与Hsp 90 ATP酶循环相关的全局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开放问题。我的研究计划的重点是阐明热休克蛋白90客户端激活周期的机制，特别强调刺激ATP水解，Aha 1 p和Hch 1 p，以及调节其作用的PTM。我的课程由三个主题组成，使用不同的模型和技术提供多样化的培训机会。* 1)使用酵母遗传学表征Hsp 90的Aha 1 p/Hch 1 p调节 * Hsp 90系统在所有真核生物中高度保守，酿酒酵母是Hsp 90研究中最广泛使用的模型。我们收集了表达不同Hsp 90和Hsp 90共伴侣突变体的酵母菌株。我们将集中在一组这些菌株的特点在体内的行动Aha 1 p和Hch 1 p。我们将在生长测定以及客户特定的功能测定中测试这些菌株。可以在酵母模型系统中剖析改变Hsp 90机器系统的不同结构元件或调节组分（共分子伴侣和翻译后修饰）的影响。*2）表征Aha 1 p和Hch 1 p对Hsp 90 ATP酶活性的调节 * 我们使用纯化的Hsp 90、Hsp 90共分子伴侣和模型客户蛋白来分析ATP水解和客户激活的体外动力学。纯化了Hsp 90或Hsp 90共分子伴侣，并在多种体外测定中研究了其突变，所述突变赋予酵母中的生长缺陷或客户特异性活化缺陷。我们测量的动力学参数ATP水解热休克蛋白90以及共分子伴侣介导的激活客户端激活。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)Hsp 90复合物的物理特性 * 我们与其他专门从事NMR、晶体学、小角度光散射和荧光技术的研究小组合作，以探究Hsp 90的酶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Hsp 90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. **