Regulation of the Heat Shock Response in Yeast

热休克反应的特征在于诱导编码蛋白伴侣的基因的转录。 许多压力诱导这种反应，其中最重要的是氧压力。 大多数激活热休克反应的应激导致蛋白质的解折叠和聚集（以及产生氧自由基，超氧阴离子）。 因此，目前的范例表明，在真核生物中的反应的调节发生通过竞争分子伴侣之间的未折叠的蛋白质和热休克转录因子，HSF。 然而，最近的数据表明，HSF响应于超氧阴离子在体外，并建议超氧化物可能是关键的酵母，果蝇和人类的整个细胞的热休克反应。 因此，热休克反应可能更恰当地表征为对超氧化物的反应，蛋白质解折叠是应激的结果，但不是HSF激活的直接触发因素。 本项目的目的是区分未折叠蛋白质或超氧化物是否是功能性的，在体内，触发反应。 实现这一目标的实验包括比较超氧化物和未折叠蛋白质的生产动力学，检查分子伴侣和未折叠蛋白质以及分子伴侣和HSF之间的复合物，检查对蛋白质的氧化损伤，并确定厌氧生长的酵母细胞中的应力的影响。 该项目还将开始追求细胞成分，通过依赖于还原型谷胱甘肽的生化活性抵消超氧化物的影响。 为了补充这些生化方法，将进行遗传分析，以寻找当过表达时与热休克系统相互作用的基因。 除了提供生物化学的遗传确认外，这些实验还将为未来的分析提供一个切入点。

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