Role of Glutaredoxin in Metabolic Oxidative Stress

描述（由申请人提供）：拟议研究项目的长期目标是了解细胞对独特肿瘤微环境（低糖浓度、慢性缺氧和低细胞外pH）反应的分子机制。我们之前观察到，葡萄糖剥夺会增加细胞内过氧化氢的浓度。它还激活SAPK（应激活化蛋白激酶）信号转导途径。在这项拨款申请中，我们假设谷胱甘肽（GRX），一种氧化还原调节蛋白，识别代谢氧化应激并触发ASK1-MEK-MAPK信号转导通路。指导假说认为，葡萄糖剥夺提高了细胞内活性氧（ROSs）水平，并增加了氧化谷胱甘肽水平。GRX在活性中心含有两个具有氧化还原活性的半胱氨酸残基（Cys-Pro-Tyr-Cys），通过与氧化分子（如氧化谷胱甘肽）催化硫-二硫交换反应来识别代谢氧化应激。氧化后的GRX与ASK1（凋亡信号调节激酶1）分离。GRX与ASK1的分离导致ASK1的激活，随后激活ASK1- mek - mapk信号转导通路。本项目的具体目的是研究(1)GRX如何识别代谢氧化应激；(2)GRX在ASK1-MEK-MAPK信号转导通路中的作用；(3)GRX与硫氧还蛋白（TRX）在葡萄糖剥夺过程中协同调节ASKI激活。第一个目的的拟议研究采用定点诱变在氧化还原活性位点以及其他半胱氨酸残基处创建点突变体。这些研究将说明半胱氨酸残基如何参与代谢氧化应激的识别。第二个目标将采用分子遗传学和生化技术来阐明GRX在ASK1-MEK-MAPK信号转导通路中的作用。第三个目标将使用免疫复合物激酶测定来评估细胞内谷胱甘肽剥夺、细胞外氧化谷胱甘肽治疗、抑制GRX或TRX基因表达对ASK1激活的影响。我们相信，研究肿瘤细胞代谢氧化应激反应的机制将有助于深入了解肿瘤细胞如何识别代谢氧化应激并启动信号转导。

DESCRIPTION (provided by applicant): The long-term goal of the proposed research project is to understand the molecular mechanisms of cellular responses to the unique tumor microenvironment (low glucose concentration, chronic hypoxia, and low extracellular pH). We previously observed that glucose deprivation increases the intracellular concentration of hydroperoxide. It also activates the SAPK (stress activated protein kinase) signal transduction pathway. In this grant proposal, we postulate that glutaredoxin (GRX), a redox-regulatory protein, recognizes the metabolic oxidative stress and triggers the ASK1-MEK-MAPK signal transduction pathway. The guiding hypothesis is that glucose deprivation raises the intracellular level of reactive oxygen species (ROSs) and increases the level of oxidized glutathione. GRX, which contains two redox-active half-cystine residues (Cys-Pro-Tyr-Cys) in an active center, recognizes metabolic oxidative stress through catalysis of thiol-disulfide interchange reactions with oxidized molecules such as oxidized glutathione. The oxidized GRX dissociates from ASK1 (apoptosis signal regulating kinase 1). The dissociation of GRX from ASK1 results in the activation of ASK1 and subsequently activates the ASK1-MEK-MAPK signal transduction pathway. The specific aims of this project are to examine (1) how GRX recognizes the metabolic oxidative stress, (2) the role of GRX in the ASK1-MEK-MAPK signal transduction pathway, (3) cooperation between GRX and thioredoxin (TRX) to regulate ASKI activation during glucose deprivation. The proposed studies for the first aim employ site-directed mutagenesis to create a point mutant at the redox-active site as well as at other cysteine residues. These studies will illustrate how half-cystine residues are involved in the recognition of metabolic oxidative stress. The second aim will employ molecular genetics and biochemical techniques to elucidate the involvement of GRX in the ASK1-MEK-MAPK signal transduction pathway. The third aim will use the immune complex kinase assay to assess the effect of intracellular glutathione deprivation, extracellular oxidized glutathione treatment, inhibition of GRX or TRX gene expression on ASK1 activation. We believe that investigating the mechanisms of metabolic oxidative stress responses in tumor cells will provide insight into how tumor cells recognize metabolic oxidative stress and initiate signal transduction.