Regulation of NAD(P)H:Quinone Oxidoreductases

NAD(P)H:醌氧化还原酶的调节

• 批准号: 7495935
• 负责人: Anil Kumar Jaiswal
• 金额: $ 25.5万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-07-01 至 2010-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7495935
• 关键词: Antioxidants; Applications Grants; Benzo(a)pyrene; Binding; Biological Assay; Carcinogens; Cell Line; Cell Nucleus; Cells; Chemicals; Cysteine; Cytoplasm; Development; Dominant-Negative Mutation; Drug Metabolic Detoxication; Elements; Enzyme Gene; Enzymes; Epidermis; Exons; Exposure to; Flavoproteins; Gene Expression; Genes; Genetic Transcription; Genotype; Goals; Hour; Human; Hypersensitivity; Immunohistochemistry; Immunoprecipitation; In Vitro; Knockout Mice; Liver; Malignant Epithelial Cell; Maps; Mediating; Modification; Mus; NAD(P)H dehydrogenase (quinone) 1, human; NF-E2-related factor 2; NQO1 gene; NRH - quinone oxidoreductase2; Neoplasms; Nuclear; Nuclear Export; Nuclear Import; Nuclear Localization Signal; Nuclear Protein; Nuclear Proteins; Nuclear Translocation; Oxidation-Reduction; Oxidative Stress; Pattern; Phosphorylation; Phosphotransferases; Plasmids; Protein Binding; Protein Dephosphorylation; Protein Isoforms; Protein Tyrosine Kinase; Proteins; Quinones; RNA; Regulation; Renal carcinoma; Reporter; Repression; Response Elements; Role; Signal Transduction; Site; Site-Directed Mutagenesis; Skin; Skin Neoplasms; Sodium Dodecyl Sulfate-PAGE; Spectrophotometry; System; Testing; Tetracycline; Tetracyclines; Tissue-Specific Splicing; Tissues; Trans-Activators; Transfection; Transgenic Mice; UV carcinogenesis; Up-Regulation; Variant; benzoquinone; carcinogenesis; gene induction; human tissue; in vivo; inhibitor/antagonist; interest; leptomycin B; research study; response; src-Family Kinases; ultraviolet; Antioxidants; Applications Grants; Benzo(a)pyrene; Binding; Biological Assay; Carcinogens; Cell Line; Cell Nucleus; Cells; Chemicals; Cysteine; Cytoplasm; Development; Dominant-Negative Mutation; Drug Metabolic Detoxication; Elements; Enzyme Gene; Enzymes; Epidermis; Exons; Exposure to; Flavoproteins; Gene Expression; Genes; Genetic Transcription; Genotype; Goals; Hour; Human; Hypersensitivity; Immunohistochemistry; Immunoprecipitation; In Vitro; Knockout Mice; Liver; Malignant Epithelial Cell; Maps; Mediating; Modification; Mus; NAD(P)H dehydrogenase (quinone) 1, human; NF-E2-related factor 2; NQO1 gene; NRH - quinone oxidoreductase2; Neoplasms; Nuclear; Nuclear Export; Nuclear Import; Nuclear Localization Signal; Nuclear Protein; Nuclear Proteins; Nuclear Translocation; Oxidation-Reduction; Oxidative Stress; Pattern; Phosphorylation; Phosphotransferases; Plasmids; Protein Binding; Protein Dephosphorylation; Protein Isoforms; Protein Tyrosine Kinase; Proteins; Quinones; RNA; Regulation; Renal carcinoma; Reporter; Repression; Response Elements; Role; Signal Transduction; Site; Site-Directed Mutagenesis; Skin; Skin Neoplasms; Sodium Dodecyl Sulfate-PAGE; Spectrophotometry; System; Testing; Tetracycline; Tetracyclines; Tissue-Specific Splicing; Tissues; Trans-Activators; Transfection; Transgenic Mice; UV carcinogenesis; Up-Regulation; Variant; benzoquinone; carcinogenesis; gene induction; human tissue; in vivo; inhibitor/antagonist; interest; leptomycin B; research study; response; src-Family Kinases; ultraviolet

Quinone Oxidoreductases (NQO1 and NQ02) are cytosolic proteins that catalyze detoxification of quinones and protect against oxidative stress and neoplasia. Antioxidant response element (ARE) and nuclear factor Nrf2 regulate coordinated induction of detoxifying genes including NQO1 and NQO2 in response to antioxidants. Nrf2 is retained in the cytoplasm by inhibitor INrf2. PKC phosphorylation of Nrf2S40 results in the release of Nrf2 from INrf2. Nrf2 translocates to the nucleus, binds to ARE, and activate gene expression. Once this is done, Nrf2 is exported out, binds to INrf2 and degrade. The PKC isoform(s) that phosphorylates Nrf2S40 and the mechanism of Nrf2 export remains unknown. Antioxidant- induced nuclear accumulation of Bachl leads to repression of Nrf2-induced genes by unknown mechanism. Nrf2 expression varies significantly among tissues, raising questions regarding tissue specific Nrf2 protection against carcinogenesis. NQO1 gene expression varies among human tissues by unknown mechanism. We have generated humanized mice that express human NQO1 gene in NQO1-/- mice. The major goals of this proposal are to elucidate the mechanism of Nrf2 regulation of detoxifying genes in response to antioxidants, determine in vivo role of Nrf2 in carcinogenesis and investigate the mechanism of tissue specific expression of NQO1 gene. Four aims are planned. Aim 1 will determine the mechanism of signal transduction from antioxidants to Nrf2/ARE. The chemical inhibitors and Si RNA will be used in transfection, reporter assays, immunohistochemistry and immunoprecipitation to identify the PKC isoform(s) that phosphorylate Nrf2S40 for release of Nrf2. Similar strategies will be used to investigate the hypothesis that tyrosine kinase-mediated phosphorylation of Nrf2Y568 regulates nuclear export of Nrf2. The Nrf2 phosphorylation at sites other than S40/Y568 and its role in ARE-mediated gene expression will also be investigated. Aim 2 will use kidney carcinoma cells stably expressing tetracycline- inducible Flag-Nrf2 to identify the heterodimeric partner of Nrf2 in upregulation of ARE-mediated gene expression. In addition, the experiments will investigate the hypothesis that antioxidant induced modification(s) of Bachl leads to delayed nuclear accumulation of Bachl and negative regulation of ARE- mediated gene expression. Aim 3 will use Cre-Lox system to generate mice that demonstrate deletion of exon 2-4 of Nrf2 gene in liver and skin. The sensitivity of conditional Nrf2 knockout mice expressing the null genotype will be compared with that of mice expressing Nrf2, following carcinogen and ultraviolet exposure, to develop liver and skin tumors. Aim 4 will use humanized mice that express human NQO1 in NQO1-/- mice in DNasel hypersensitivity and in vivo.and in vitro footprinting assays to identify the cis- element(s) and trans-acting factor(s) that regulate variations in NQO1 gene expression among the tissues.

喹酮氧化还原酶（NQO1和NQ02）是胞质蛋白，催化解毒的解毒 奎因酮并预防氧化应激和肿瘤。抗氧化剂响应元件（AS）和 核因子NRF2调节包括NQO1和NQO2在内的排毒基因的协调诱导 对抗氧化剂的反应。 NRF2由抑制剂INRF2保留在细胞质中。 PKC的磷酸化 NRF2S40导致NRF2从INRF2释放。 NRF2易位到核，结合为IS，并且 激活基因表达。完成此操作后，将NRF2导出，结合到INRF2并降解。 PKC 磷酸化NRF2S40和NRF2导出机理的同工型仍然未知。抗氧化剂 诱导的BACHL核积累导致未知抑制NRF2诱导的基因 机制。 NRF2表达在组织之间有很大变化，提出了有关组织特异性的问题 NRF2防止致癌作用。 NQO1基因表达在人体组织之间通过未知 机制。我们已经产生了在NQO1 - / - 小鼠中表达人NQO1基因的人性化小鼠。 该提案的主要目标是阐明排毒基因的NRF2调节机制 响应抗氧化剂，确定NRF2在癌变中的体内作用，并研究了 NQO1基因组织特异性表达的机理。计划四个目标。 AIM 1将确定 从抗氧化剂到NRF2/AR的信号转导机制。化学抑制剂和Si RNA将 用于转染，报告基因测定，免疫组织化学和免疫沉淀以鉴定 PKC同工型磷酸化NRF2S40释放NRF2。类似的策略将用于 研究酪氨酸激酶介导的NRF2Y568磷酸化的假设调节核 NRF2的导出。 S40/Y568以外的其他位点的NRF2磷酸化及其在read介导的基因中的作用 表达也将被研究。 AIM 2将使用稳定表达四环素的肾脏癌细胞 - 可诱导的FLAG-NRF2识别NRF2的异二聚体伴侣在介导的基因上调中 表达。此外，实验将研究抗氧化剂诱导的假设 BACHL的修饰导致Bachl的核积累延迟，而对 介导的基因表达。 AIM 3将使用Cre-Lox系统生成显示删除的小鼠 肝脏和皮肤中NRF2基因的外显子2-4。表达有条件的NRF2敲除小鼠的灵敏度 无效的基因型将与表达NRF2的小鼠进行比较，癌变和紫外线 暴露，发展肝脏和皮肤肿瘤。 AIM 4将使用在 NQO1 - / - 小鼠在DNASEL超敏反应和体内和体外足迹测定法中，以鉴定顺式 - 元素和反式作用因子（S）调节组织中NQO1基因表达的变化。