REGULATION OF GLUTATHIONE SYNTHESIS IN OXIDATIVE STRESS

谷胱甘肽(GSH)是抗氧化防御的重要组成部分。 醌类化合物产生的氧化应激促进合成和酶 谷氨酰半胱氨酸合成酶(GCS)活性。这在一定程度上是由于 导致GCS催化(重)亚单位转录增加。 GCS的调节(LIGH)亚单位的mRNA也增加。这个 GCS酶活性的增加增强了细胞的能力 增加GSH的合成，这可能有助于产生更强的抵抗力 对苯二酚和环境毒素造成的氧化应激，如 二氧化氮(NO2)。 这项调查的目的是确定监管机制 GCS合成增加在氧化应激中的作用。为了这些 研究中，我们将使用氧化应激与上皮细胞的体外模型 来自大鼠和人肺的线条。具体目标是：(1) 确定导致GCS增加的功能监管要素 通过鉴定其启动子/增强子区域来响应对苯二酚的反应 GCS的催化(重)亚单位与功能基序的剖析(S) 调节GSCs-HS的表达，检测可能的氧化应激反应 利用报告基因研究GCS-HS区启动子/增强子元件 构造，并确定是否转录和/或翻译 调节(光)亚基与催化亚基协调 亚单位。(2)确定参与氧化信号转导的机制 随着GCS的增强和持续上升，通过表征 氧化应激反应转录因子参与GCS-HS和 检测细胞内H_2O_2谷胱甘肽氧化还原状态的关系 (GSH/GSSG)、谷胱甘肽偶联物和谷胱甘肽-蛋白质混合物 二硫化物对合成GCS重亚单位和轻亚单位mRNAs、蛋白质和 酶活性。(3)确定GSH升高与 合成和增强对氧化应激的抵抗能力 二氧化氮和活性醛产生的氧化应激 GSH和GCS的增加类似于对苯二酚的观察，以及NO2， 反应性醛和苯二酚可诱导自身耐受性或杂交 宽容。长远目标是药理学的发展 提高谷胱甘肽的方法，以提供抗氧化保护 压力。这可能延伸到其他病理，如炎症，在这些情况下 氧化应激是其中的一个组成部分。

Glutathione (GSH) is an essential component of antioxidant defense. Oxidative stress generated by quinones elevates synthesis and enzymatic activity of gamma-glutamylcysteine synthetase (GCS). This is due, in part to an increase in transcription of the catalytic (heavy) subunit of GCS. The mRNA for the regulatory (light) subunit of GCS is also increased. The increase in GCS enzymatic activity enhances the ability of cells to increase GSH synthesis, which may aid in producing greater resistance to oxidative stress from quinones as well as environmental toxins, such as nitrogen dioxide (NO2). The goal of this investigation is to determine the mechanisms of regulation and roles of the increased synthesis of GCS in oxidative stress. For these studies, we will use in vitro models of oxidant stress with epithelial cell lines derived from rat and human lungs. The specific aims are: (1) To identify the functional regulatory elements responsible for increased GCS in response to quinones by characterizing the promoter/enhancer region of the catalytic (heavy) subunit of GCS and dissecting the functional motif(s) modulating GSCS-HS expression, examining putative oxidative stress response elements of the promoter/enhancer of GCS-HS region using reporter constructs, and determining whether transcription and/or translation of the regulatory (light) subunit is coordinated with that of the catalytic subunit. (2) To determine the mechanism of oxidant signaling involved with the enhancement and sustained elevation of GCS by characterizing the oxidative stress responsive transcription factors involved with GCS-HS and examining the relationship of cellular H2O2 glutathione redox status (GSH/GSSG), glutathione-conjugates, and glutathione-protein mixed disulfides to synthesis of GCS heavy and light subunit mRNAs, proteins and enzymatic activity. (3) Determine the relationship between increased GSH synthesis and enhanced resistance to oxidative stress by examining whether oxidative stress from nitrogen dioxide and reactive aldehydes produce similar increases in GSH and GCS as observed with quinones and whether NO2, reactive aldehydes, and quinones can induce self tolerance or cross tolerance. The long range goal is the development of pharmacological approaches to elevating GSH that will provide protection against oxidative stress. This may extend to other pathologies, such as inflammation, where oxidative stress is a component.