REGULATION OF GLUTATHIONE SYNTHESIS IN OXIDATIVE STRESS

描述（改编自研究者摘要）。该提案的重点是 调节从头增加的信号转导机制 谷胱甘肽（GSH）的合成过程中发生的适应氧化剂和其他 亲电体γ-谷氨酰半胱氨酸合成酶（GCS）， 还原型谷胱甘肽从头合成中的酶由两个亚基组成。两个GCS基因 在对氧化剂应激的反应中被转录上调， 亲电性异生物质尽管如此， 两种GCS亚基蛋白可导致动力学改变和/或降低 酶的稳定性。γ-谷氨酰转肽酶（GGT）是一种胞外酶， 通过分解细胞外GSH为GSH合成提供底物。 GGT酶活性和mRNA转录也响应于 氧化剂虽然在过去几年里， GSH的机制增加适应，信号机制， GCS和GGT对转录、转录后和 翻译后水平仍然在很大程度上没有得到解决。研究人员建议， 为了在三个目标中测试以下假设：1- 通过亲电体和H2 O2产生的GCS基因的转录激活 化合物通过重叠途径发生; 2-GCS调节 亚基（GCS-LS）稳定催化亚基，防止蛋白水解，以及 作为调节动力学; 3-GGT转录活性氧物种 是通过激活特异性信号通路和顺式作用介导的 被H2 O2激活的元素。这些假设将使用代理进行测试 （2，3-二甲氧基-1，4-萘醌（DMNQ）、4-羟基壬烯醛（4 HNE）和 β-萘酚酮（BNF））的浓度和孵育时间， 两种GCS亚基的转录增加。使用的方法包括， 显性负性和组成性活性信号传导成分，激酶 检测，体内足迹法，报告基因构建，四环素调控 反义转染和活性氧物质、谷胱甘肽的测量 氧化和α-不饱和醛。 这些目标将通过使用已建立的细胞模型进行修改， 需要确定结论是独特的还是普遍适用的。 他们研究的长期目标是了解 谷胱甘肽合成及其药理学操作的潜力，以提高 合成这种重要的细胞成分。

DESCRIPTION (Adapted from the Investigators abstract). This proposal focuses on the signal transduction mechanisms regulating the increase in de novo glutathione (GSH) synthesis that occurs during adaptation to oxidants and other electrophiles. Gamma-Glutamylcysteine synthetase (GCS), the rate limiting enzyme in de novo GSH synthesis, is composed of two subunits. Both GCS genes are transcriptionally up-regulated in response to stress from oxidants and electrophilic xenobiotics. Nonetheless, a mismatch between expression of the two GCS subunit proteins may result in altered kinetics and/or decreased stability of the enzyme. gamma-Glutamyl transpeptidase (GGT) is an exoenzyme that provides substrates for GSH synthesis by breaking down extracellular GSH. GGT enzymatic activity and mRNA transcription also increases in response to oxidants. While much has been learned over the past few years regarding mechanisms of GSH increases in adaptation, the signaling mechanisms that govern regulation of GCS and GGT on the transcriptional, post-transcriptional and post-translational levels remain largely unresolved. The investigators propose to test the following hypotheses in three aims: 1- that signaling for the transcriptional activation of GCS genes by electrophiles and H2O2-generating compounds occurs through overlapping pathways; 2- that the GCS regulatory subunit (GCS-LS) stabilizes the catalytic subunit against proteolysis as well as regulates the kinetics; 3- that GGT transcription by reactive oxygen species is mediated through activation specific signaling pathways and cis acting element(s) activated by H2O2. These hypotheses will be tested using the agents (2,3-dimethoxy-1,4-naphthoquinone (DMNQ), 4-hydroxynonenal (4HNE), and beta-naphthoflavone (BNF)) at concentrations and incubation times that cause increased transcription of both GCS subunits. Methods to be used include, dominant negative and constitutively active signaling components, kinase assays, in vivo footprinting, reporter construct, tetracycline regulated antisense transfection, and measurement of reactive oxygen species, glutathione oxidation and alpha-unsaturated aldehydes. These aims will be carried out by using established cell models modified as needed to determine whether the conclusions are unique or generally applicable. The long-range goal of their research is an understanding of the regulation of GSH synthesis and the potential for its pharmacological manipulation to enhance synthesis of this essential cellular constituent.