Hydroxynonenal induces glutathione synthesis through JNK

羟基壬烯醛通过 JNK 诱导谷胱甘肽合成

• 批准号: 7422483
• 负责人: HENRY Jay FORMAN
• 金额: $ 0.52万
• 依托单位: UNIVERSITY OF CALIFORNIA, MERCED
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-22 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7422483
• 关键词: 4 hydroxynonenal; Address; Affinity Chromatography; Air Pollutants; Anabolism; Antibodies; Antioxidants; Be++ element; Beryllium; Binding; Biological Assay; Catalytic Domain; Cell Extracts; Cells; Chromatin; Complex; Condition; DNA; Dominant-Negative Mutation; Elements; Enzyme Induction; Enzymes; Epithelial Cells; Excision; Exposure to; FarGo; GCLC gene; GCLM gene; Gene Expression; Genes; Genetic Transcription; Glutamate-Cysteine Ligase; Glutathione; Goals; Hour; Human; Hydrogen Peroxide; Immunoprecipitation; In Situ; Indium; Inflammation; Injury; Interphase Cell; Investigation; Ligase Gene; Lipid Peroxidation; Luciferases; Lung; Lung diseases; MAPK14 gene; MAPK8 gene; Mass Spectrum Analysis; Measurement; Measures; Membrane; Metabolism; Nuclear Protein; Nuclear Proteins; Oxidants; Oxidative Stress; Pathologic Processes; Pathway interactions; Peptides; Phosphotransferases; Physiological; Post-Translational Protein Processing; Protein Overexpression; Proteins; Range; Rate; Regulation; Reporter; Research Personnel; Rest; Reverse Transcriptase Polymerase Chain Reaction; Role; Signal Pathway; Signal Transduction; Signaling Protein; Stress; System; Tissues; Transcription Factor AP-1; Transcription factor genes; Western Blotting; Xenobiotics; chromatin immunoprecipitation; design; human GCLC protein; inhibitor/antagonist; lung injury; oxidation; programs; protein folding; response; stress activated protein kinase; transcription factor

DESCRIPTION (provided by applicant): This competing renewal application focuses on induction of glutamate cysteine ligase (GCL), the rate limiting enzyme in glutathione (GSH) synthesis, by the lipid peroxidation product, 4-hydroxy-2-nonenal (HNE) through activation of EpRE and TRE cis elements and the JNK signaling pathway. A common response to sublethal exposure to oxidants and electrophiles is an increase in GSH concentration, which is critical to adaptation to greater stress. This increase is dependent on the increased expression of the catalytic and modulatory subunits of glutamate cysteine ligase (GCL, also called (-glutamylcysteine synthetase), the first enzyme in de novo GSH synthesis. HNE in healthy human plasma is 0.3-0.7 micromolar, but increases more than tenfold in inflammatory diseases and exposure to air pollutants. Although HNE can be toxic, at concentrations found in tissues under oxidative stress, it is a potent inducer of GSH, both GCL subunit genes, and other Phase II enzymes. We recently found that transcription factor switching appears to regulate GCL and that inhibition of JNK by a permeable dominant negative peptide completely inhibited GCL induction by HNE while neither ERK or p38MAPK inhibitors were effective. Our hypothesis is that induction of both human GCL genes by HNE occurs through alteration of the compositions of the EpRE and AP-1 binding complexes from those that repress to those that activate transcription. We also hypothesize that the alteration of composition of the EpRE and AP-1 binding complexes is mediated through HNE activation of the JNK signaling pathway. Using human bronchial epithelial cells (HBE1) for all studies, the specific aims of this proposal are to: 1- determine the composition of the complexes binding to the EpRE that regulate GCLC, GCLM, NQO1, and GST4A4 in response to HNE; 2- determine the mechanism of HNE by which HNE causes changing composition of the EpRE binding complexes; 3- determine how the changes in AP-1 complexes in response to HNE relate to the increase of GCL gene transcription; and 4- determine how HNE activates the JNK pathway. The approaches include DNA affinity chromatography, HPLC tandem mass spectrometry, immunodepletion EMSA, chromatin immunoprecipitation assays, Western blotting, protein translocation, protein phosphorylation measurements. Understanding the molecular basis of HNE induction of GCL to assist development of rational design of a drug with a less potential toxicity is the long range goal.

描述（由申请人提供）：本竞争性更新申请的重点是通过激活 EpRE 和 TRE 顺式元件以及 JNK 信号通路，通过脂质过氧化产物 4-羟基-2-壬烯醛 (HNE) 诱导谷氨酸半胱氨酸连接酶 (GCL)，谷胱甘肽 (GSH) 合成中的限速酶。亚致死接触氧化剂和亲电子试剂的常见反应是谷胱甘肽浓度增加，这对于适应更大的压力至关重要。这种增加取决于谷氨酸半胱氨酸连接酶（GCL，也称为（-谷氨酰半胱氨酸合成酶）的催化和调节亚基表达的增加，它是谷胱甘肽从头合成的第一种酶。健康人血浆中的 HNE 为 0.3-0.7 微摩尔，但在炎症性疾病和接触空气污染物时会增加十倍以上。 可能有毒，在氧化应激下的组织中发现的浓度，它是 GSH、GCL 亚基基因和其他 II 期酶的有效诱导剂。我们最近发现转录因子转换似乎可以调节 GCL，并且渗透性显性负性肽对 JNK 的抑制完全抑制了 HNE 诱导的 GCL，而 ERK 或 p38MAPK 抑制剂均无效。我们的假设是 HNE 对人类 GCL 基因的诱导是通过改变 EpRE 和 AP-1 结合复合物的组成从抑制转录的复合物变为激活转录的复合物来实现的。我们还假设 EpRE 和 AP-1 结合复合物组成的改变是通过 JNK 信号通路的 HNE 激活介导的。所有研究均使用人支气管上皮细胞（HBE1），该提案的具体目标是 1-确定与 EpRE 结合的复合物的组成，该复合物响应 HNE 调节 GCLC、GCLM、NQO1 和 GST4A4； 2-确定HNE的机制，HNE通过该机制引起EpRE结合复合物的组成发生变化； 3-确定AP-1复合物响应HNE的变化如何与GCL基因转录的增加相关； 4-确定 HNE 如何激活 JNK 通路。 这些方法包括 DNA 亲和层析、HPLC 串联质谱、免疫耗竭 EMSA、染色质免疫沉淀测定、蛋白质印迹、蛋白质易位、蛋白质磷酸化测量。了解 HNE 诱导 GCL 的分子基础，以协助开发合理设计潜在毒性较小的药物是长期目标。