Study of Nitric Oxide Chemistry/Biochemistry in S. Cerevisiae

酿酒酵母一氧化氮化学/生物化学的研究

• 批准号: 0096380
• 负责人: Jon Fukuto
• 金额: $ 30万
• 依托单位: University of California-Los Angeles
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-03-01 至 2004-02-29
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0096380&HistoricalAwards=false
• 关键词: Study; Nitric; Oxide; Chemistry; Biochemistry

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.

由于多种生理原因，哺乳动物细胞会产生一氧化氮 (NO)。 例如，它可以充当血管介质、细胞毒性/细胞抑制物质和神经递质。 与 NO 的许多作用相关的生物化学大部分是从纯化学和/或体外系统推断出来的。 迄今为止，由于体外或体内哺乳动物系统的限制，确定NO生物活性的密切化学细节一直很困难。 然而，酿酒酵母提供了一个理想的实验系统，可以在整个细胞环境中描述 NO 化学反应。 由于酿酒酵母在厌氧或有氧条件下生长和存活，因此可以检查双氧对 NO 与特定蛋白质的化学相互作用的影响。 酵母基因操作的简便性使得能够以可预测且重要的方式控制细胞内条件。 因此，可以检查NO在多种细胞条件下的作用。 此外，由于许多蛋白质关系已被充分阐明，因此可以通过检查对酵母细胞生物学的总体影响来监测NO对蛋白质活性的改变。 该项目涉及对 NO 与两种酵母金属代谢蛋白 Ace1（铜响应转录因子）和 Fre1（跨膜金属还原酶）之间的化学和生化相互作用进行系统检查。 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：金属硫醇盐蛋白和血红素蛋白。 NO-蛋白质相互作用的详细细节也将使用化学模型系统和纯化的蛋白质进行检查。 通过这种方式，可以验证 NO 的细胞生物化学。