Study of Nitric Oxide Chemistry/Biochemistry in S. Cerevisiae

由于多种生理原因，哺乳动物细胞会产生一氧化氮（NO）。例如，它可以作为血管介质，细胞毒性/细胞抑制剂和神经递质。与一氧化氮的许多作用相关的生物化学在很大程度上是从纯化学和/或体外系统中推断出来的。到目前为止，由于体外或体内哺乳动物系统的限制，确定一氧化氮生物活性的内部化学细节一直很困难。然而，酵母酿酒酵母提供了一个理想的实验系统，其中可以在全细胞环境中描述NO化学。由于酿酒酵母在厌氧或好氧条件下生长和存活，因此可以研究双氧对NO与特定蛋白质的化学相互作用的影响。酵母菌基因操作的便利性使其能够以可预测和重要的方式控制细胞内条件。因此，一氧化氮在各种细胞条件下的作用可以被检查。此外，由于许多蛋白质的关系已经完全阐明，可以通过检查酵母细胞生物学的整体影响来监测NO对蛋白质活性的改变。该项目包括对NO与两种酵母金属代谢蛋白Ace1（一种铜反应转录因子）和Fre1（一种跨膜金属还原酶）的化学和生化相互作用进行系统研究。选择这些蛋白质进行研究是因为它们代表了已知与NO相互作用的两类重要蛋白质；金属硫化物蛋白和血红素蛋白。no -蛋白质相互作用的细节也将使用化学模型系统和纯化蛋白质进行研究。通过这种方式，可以验证NO的细胞生化。

Nitric oxide (NO) is made in mammalian cells for a variety of physiological reasons. For example, it can serve as a vascular mediator, a cytotoxic/cytostatic species and a neurotransmitter. The biological chemistry associated with many of the actions of NO is, for the most part, extrapolated from purely chemical and/or in vitro systems. Thus far, determining the intimate chemical details responsible for the biological activity of NO has been difficult due to the limitations of in vitro or in vivo mammalian systems. However, the yeast Saccharomyces cerevisiae offers an ideal experimental system in which NO chemistry can be delineated in a whole cell environment. Due to the fact that S. cerevisiae grows and survives under anaerobic or aerobic conditions, the effect of dioxygen on the chemical interaction of NO with specific proteins can be examined. The ease of genetic manipulation of yeast allows the ability to control intracellular conditions in predictable and important ways. Thus, the effects of NO under a variety of cellular conditions can be examined. Moreover, since many of the protein relationships have been fully elucidated, alteration of protein activity by NO can be monitored by examining an overall effect on yeast cell biology. This project involves a systematic examination of the chemical and biochemical interaction of NO with two yeast metal metabolism proteins, Ace1 (a copper-responsive transcription factor) and Fre1 (a membrane spanning metal reductase). These proteins were chosen for study since they represent two important classes of proteins which are known interact with NO; metal-thiolate proteins and heme proteins. The intimate details of the NO-protein interactions will also be examined using chemical model systems and purified proteins. In this way, the cellular biochemistry of NO can be validated.