Genetic and biochemical analysis of the Hsp90 system in Saccharomyces cerevisiae

90千道尔顿热休克蛋白Hsp90在维持所有真核生物的蛋白质平衡中起着基础和必要的作用。我的研究重点是了解Hsp90的作用机制，以及其通过co-chaperone蛋白和翻译后修饰（post-translational modification, PTMs）调控的基础。Hsp90伴随着数百种“客户”蛋白的构象变化，这些蛋白质是配体结合、催化活性或组装成大分子复合物所必需的。***Hsp90是一种同源二聚体atp酶，atp驱动的客户端激活周期受到一系列共伴侣蛋白和翻译后修饰的严格调控。与Hsp90 atp酶循环相关的全局构象变化最终如何驱动客户蛋白激活仍然是该领域的一个悬而未决的问题。***我的研究项目集中于阐明Hsp90客户端激活周期的机制，特别强调两种促进ATP水解的共伴侣，Aha1p和Hch1p，以及调节其作用的ptm。我的项目由三个主题组成，提供不同的培训机会，使用不同的模型和技术。***1)利用酵母遗传学表征Hsp90的Aha1p/Hch1p调控*** Hsp90系统在所有真核生物中高度保守，酿酒酵母是研究Hsp90最广泛使用的模型。我们收集了表达不同Hsp90和Hsp90共伴侣突变体的酵母菌株。我们将集中在一组这些菌株来表征Aha1p和Hch1p的体内作用。我们将在生长试验以及客户特定功能试验中测试这些菌株。改变Hsp90机器系统的不同结构元件或调控元件（共伴侣和翻译后修饰）的影响可以在酵母模型系统中进行剖析。***2)表征Aha1p和Hch1p对Hsp90 ATP酶活性的调控***我们使用纯化的Hsp90、Hsp90共伴侣和模型客户蛋白来分析ATP水解和客户活化的体外动力学。在酵母中，Hsp90或Hsp90共伴侣携带突变，赋予生长缺陷或客户特异性激活缺陷，被纯化并在各种体外试验中进行研究。我们测量了Hsp90水解ATP的动力学参数以及协同伴侣介导的客户端活化的激活。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) Hsp90配合物的物理表征***我们与其他研究小组合作，专门从事核磁共振，晶体学，小角度光散射和荧光技术，以探究Hsp90酶和生物活性的结构基础。我的项目的这一方面不仅对Hsp90生物学产生了重要的科学见解，而且由于它涉及广泛的合作，也为我的学员提供了宝贵的培训机会。**

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. **