Regulation of the Heat Shock Response in Yeast

热休克反应的特征是诱导编码蛋白伴侣的基因转录。许多应激都会诱发这种反应，其中最主要的是氧应激。大多数激活热休克反应的应激会引起蛋白质的展开和聚集（以及产生氧自由基、超氧阴离子）。因此，目前的范式表明，真核生物的反应调节是通过未折叠蛋白和热休克转录因子（HSF）之间对伴侣的竞争发生的。然而，最近的数据表明，HSF在体外对超氧阴离子有反应，并表明超氧阴离子可能对酵母、果蝇和人类全细胞的热休克反应至关重要。因此，热休克反应可以更恰当地描述为对超氧化物的反应，蛋白质展开是应激的结果，而不是HSF激活的直接触发因素。该项目的目的是区分未折叠蛋白或超氧化物是体内反应的功能性触发因素。实现这一目标的实验包括比较超氧化物和未折叠蛋白的产生动力学，检查伴侣蛋白和未折叠蛋白之间的复合物，以及伴侣蛋白和HSF之间的复合物，检查蛋白质的氧化损伤，并确定在厌氧生长的酵母细胞中的应激效应。该项目还将开始寻求通过依赖于还原型谷胱甘肽的生化活性来抵消超氧化物影响的细胞成分。为了补充这些生化方法，将进行遗传分析，以寻找过度表达时与热休克系统相互作用的基因。除了为生物化学提供遗传学上的证实外，这些实验将为将来的分析提供一个突破口

The heat shock response is characterized by the induction of transcription of genes encoding protein chaperones. Many stresses induce this response, foremost among them oxygen stresses. Most stresses that activate the heat shock response cause the unfolding and aggregation of proteins (as well as producing the oxygen radical, superoxide anion). Hence, the current paradigm suggests that regulation of the response in eukaryotes occurs through competition for chaperones between unfolded protein and the Heat Shock Transcription Factor, HSF. However, recent data indicate that HSF responds to superoxide anion in vitro, and suggest that superoxide may be critical for the heat shock response in whole cells of yeast, Drosophila, and humans. Thus, the heat shock response may be more properly characterized as a response to superoxide, with protein unfolding being a consequence of the stress but not the direct trigger of HSF activation. The purpose of this project is to distinguish whether unfolded protein or superoxide is the functional, in vivo, trigger of the response. The experiments to achieve this goal include comparing kinetics of production of superoxide and unfolded protein, examining complexes between chaperones and unfolded protein as well as chaperones and HSF, examining oxidative damage to protein, and determining the effects of stresses in yeast cells that are grown anaerobically. The project will also begin the pursuit of cellular components that counteract the effect of superoxide via biochemical activity that is dependent on reduced glutathione. To complement these biochemical approaches, genetic analyses will be performed to seek genes that, when overexpressed, interact with the heat shock system. In addition to providing genetic confirmation of the biochemistry, these experiments will provide an inroad into future analyses.a