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

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.

醌氧化还原酶（NQO1 和 NQ02）是催化解毒的胞质蛋白 醌并防止氧化应激和肿瘤形成。抗氧化反应元件（ARE）和 核因子 Nrf2 调节解毒基因（包括 NQO1 和 NQO2）的协调诱导 对抗氧化剂的反应。 Nrf2 被抑制剂 INrf2 保留在细胞质中。 PKC 磷酸化 Nrf2S40 导致 Nrf2 从 INrf2 中释放。 Nrf2 易位至细胞核，与 ARE 结合，并且 激活基因表达。一旦完成，Nrf2 就会被导出，与 INrf2 结合并降解。 PKC 磷酸化 Nrf2S40 的异构体和 Nrf2 输出的机制仍不清楚。抗氧化剂- Bachl 诱导的核积累导致未知的 Nrf2 诱导基因的抑制 机制。 Nrf2 表达在组织之间存在显着差异，引发了有关组织特异性的问题 Nrf2 具有抗癌作用。 NQO1 基因表达在人体组织中存在未知差异 机制。我们已经培育出在 NQO1-/- 小鼠中表达人类 NQO1 基因的人源化小鼠。 该提案的主要目标是阐明Nrf2调节解毒基因的机制 响应抗氧化剂，确定 Nrf2 在致癌作用中的体内作用并研究 NQO1基因组织特异性表达的机制。计划了四个目标。目标 1 将确定 从抗氧化剂到Nrf2/ARE的信号转导机制。化学抑制剂和 Si RNA 将 用于转染、报告分析、免疫组织化学和免疫沉淀来鉴定 磷酸化 Nrf2S40 以释放 Nrf2 的 PKC 同工型。类似的策略将用于 研究酪氨酸激酶介导的 Nrf2Y568 磷酸化调节核的假设 Nrf2 的导出。 S40/Y568以外位点的Nrf2磷酸化及其在ARE介导的基因中的作用 表达也将被研究。目标2将使用稳定表达四环素的肾癌细胞- 诱导型 Flag-Nrf2 鉴定 Nrf2 在 ARE 介导基因上调中的异二聚体伴侣 表达。此外，实验还将研究抗氧化剂诱导的假设 Bachl 的修饰导致 Bachl 的核积累延迟和 ARE- 介导的基因表达。 Aim 3 将使用 Cre-Lox 系统来生成能够删除 肝脏和皮肤中 Nrf2 基因的外显子 2-4。表达 Nrf2 的条件性敲除小鼠的敏感性 在致癌物和紫外线之后，将无效基因型与表达 Nrf2 的小鼠的基因型进行比较 暴露，发展肝脏和皮肤肿瘤。目标 4 将使用表达人类 NQO1 的人源化小鼠 NQO1-/- 小鼠在 DNasel 超敏反应中以及体内和体外足迹分析中鉴定顺式- 调节组织间 NQO1 基因表达变化的元件和反式作用因子。