REGULATION OF GLUTATHIONE SYNTHESIS IN OXIDATIVE STRESS

描述(改编自调查人员摘要)。这项提案的重点是 调控新生血管生成的信号转导机制 在适应氧化剂和其他物质的过程中合成谷胱甘肽(GSH) 亲电者。谷氨酰半胱氨酸合成酶(GCS)，限速 谷胱甘肽从头合成中的酶由两个亚基组成。两个GCS基因 转录上调是对来自氧化剂和 亲电的外源生物。尽管如此， 两个GCS亚基蛋白可能导致动力学改变和/或减少 酶的稳定性。γ-谷氨酰转肽酶是一种胞外酶 通过分解细胞外GSH，为GSH的合成提供底物。 GGT的酶活性和mRNA转录也增加 氧化剂。虽然在过去的几年里已经学到了很多关于 谷胱甘肽增加适应的机制，即控制 GCS和GGT对转录、转录后和 翻译后水平在很大程度上仍未得到解决。调查人员建议 为了在三个目标中检验以下假设：1-为 GCS基因的转录激活与H_2O_2产生 化合物通过重叠的途径发生；2-GCS调节 亚基(GCS-LS)也可以稳定催化亚基，防止蛋白质降解 AS调节动力学；3-GGT通过活性氧进行转录 是通过激活特定的信号通路和顺式作用介导的 元素(S)被过氧化氢激活。这些假说将用这些试剂进行检验。 (2，3-二甲氧基-1，4-萘二酚(DMNQ)，4-羟基壬烯醛(4HNE)，和 β-萘黄酮(BNF)引起的浓度和孵化时间 这两个GCS亚基的转录增加。要使用的方法包括， 显性负性和结构性活性信号成分，激酶 检测，体内足迹，报告构建，四环素调控 反义基因转导及活性氧、谷胱甘肽测定 氧化和α-不饱和醛。 这些目标将通过使用已建立的经过修改的细胞模型来实现 需要确定结论是独一无二的还是普遍适用的。 他们研究的长期目标是了解 谷胱甘肽的合成及其药理调控的潜力 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.