Role and Regulation of INrf2

INrf2的作用和调节

• 批准号: 8416899
• 负责人: Anil Kumar Jaiswal
• 金额: $ 32.74万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-04-08 至 2015-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8416899
• 关键词: Antioxidants; Apoptosis; Applications Grants; Benzo(a)pyrene; Binding; Biological Assay; Carcinogen exposure; Carcinogens; Cell Fractionation; Cell Nucleus; Cell Survival; Cells; Cessation of life; Chemicals; Chemoprevention; Chemoprotective Agent; Complex; Cytosol; Deletion Mutation; Development; Elements; Enzymes; Epidermis; Event; Exposure to; Family member; Feedback; Gene Expression; Generations; Genes; Genistein; Genotype; Goals; Heat-Shock Proteins 90; Heat-Shock Response; Homodimerization; Immunoprecipitation; Investigation; Knockout Mice; Link; Liver; Liver neoplasms; Malignant Neoplasms; Maps; Mass Spectrum Analysis; Mediating; Modification; Mus; Mutagenesis; Mutase; Neoplasms; Nuclear; Nuclear Export; Nuclear Import; Nutrient; Oxidative Stress; Phosphoglycerate Mutase; Phosphorylation; Phosphorylation Site; Play; Predisposition; Prevention; Protein Dephosphorylation; Proteins; RBX1 gene; Radiation; Regulation; Reporter; Role; Serine; Signal Transduction; Site-Directed Mutagenesis; Skin; Skin Neoplasms; Stress; Structure; Susceptibility Gene; System; Testing; Threonine; Trans-Activators; Transfection; Tyrosine; Ubiquitination; Xenobiotics; base; cytotoxicity; in vivo; public health relevance; response; sensor; stressor; tumor; ubiquitin ligase

DESCRIPTION (provided by applicant): INrf2:Nrf2 acts as a sensor for oxidative/electrophilic stress. INrf2 functions as an adaptor that interacts with Nrf2 and brings closer to ubiquitin ligase Cul3-Rbx1 for ubiquitination and degrading of Nrf2. Under basal conditions, INrf2/Cul3-Rbx1 complex is constantly degrading Nrf2. Nrf2 dissociates from the INrf2 in response to antioxidant and other stressors and translocates into the nucleus leading to activation of a myriad of genes that protect cells against stress and neoplasia. Immunoprecipitation and mass spectra analysis demonstrated interaction of INrf2 also with Phosphoglyerate mutase family member 5 (PGAM5)/BclXL/Bcl2, heat shock HSP90 and methylosome subunit pICln for unknown functions. Mass spectra analysis of immunoprecipitated INrf2 from antioxidant untreated and treated cells demonstrated phosphorylation of specific serine, threonine and tyrosine residues that might control INrf2 functions but remains unknown. Antioxidant treatment of cells led to genestein sensitive nuclear export of INrf2, Cul3 and Rbx1 presumably to allow nuclear import of Nrf2 without threat of degradation. However, the mechanism of export remains obscure. A feedback auto-regulatory loop between Nrf2 and INrf2 controls their cellular abundance. Interestingly, the Cul3 and Rbx1 gene expression is coordinately induced with INrf2 in response to antioxidant raising questions if Cul3 and Rbx1 are part of the INrf2-Nrf2 autoragulatory loop. The major goals of this proposal are to study role and regulation of INrf2. To this effect, we propose four specific aims. Aim 1 will investigate INrf2 interaction with PGAM5/BclXL/Bcl2, HSP90, and pICln and its significance in cellular protection and cell survival/death. The domain deletion, transfection, immunoprecipitation, Western analysis, reporter and apoptosis assays will be used in these studies. Aim 2 will elucidate the role of phosphorylation in control of homodimerization and stability of INrf2, nuclear localization, interaction with Cul3-Rbx1 and Nrf2 and degradation of Nrf2, antioxidant-induced release of Nrf2 from INrf2 and activation of gene expression, and interaction of INrf2 with PGAM5/BclXL/Bcl2, HSP90 and pICln. Sitedirected mutagenesis along with transfection and reporter assays, immunoprecipitation and Western analysis will be used to achieve the goals of this aim. Aim 3 will continue with generation of INrf2 conditional knockout mice that do not express INrf2 protein in liver and epidermis of skin. The Cre-Lox system will be used to generate these mice. In addition, compare the sensitivity of conditional INrf2 knockout mice expressing the null genotype with that of mice expressing INrf2, following carcinogen exposure, to develop skin and liver tumors. Aim 4 will test the hypothesis that Cul3 and Rbx1 are part of the auto-regulatory loop between INrf2 and Nrf2. Deletion mapping/transfection and band/super shift/ChIP assays will determine that ARE element and Nrf2 that regulate INrf2 also regulates antioxidant induction of Cul3 and Rbx1 genes. Co-transfection and ubiquitination assays will elucidate the role of coordinated induction of Cul3-Rbx1 in ubiquitination and degradation of Nrf2.

描述（由申请人提供）：INrf2:Nrf2充当氧化/亲电子应激的传感器。 INrf2 充当与 Nrf2 相互作用的接头，并更接近泛素连接酶 Cul3-Rbx1，以实现 Nrf2 的泛素化和降解。在基础条件下，INrf2/Cul3-Rbx1 复合物不断降解 Nrf2。 Nrf2 响应抗氧化剂和其他应激源而从 INrf2 解离，并易位到细胞核中，从而激活大量保护细胞免受应激和肿瘤形成的基因。免疫沉淀和质谱分析表明，INrf2 还与磷酸甘油酸变位酶家族成员 5 (PGAM5)/BclXL/Bcl2、热休克 HSP90 和甲基小体亚基 pICln 相互作用，但功能未知。对未经抗氧化剂处理和经过抗氧化剂处理的细胞进行免疫沉淀的 INrf2 的质谱分析表明，特定丝氨酸、苏氨酸和酪氨酸残基的磷酸化可能控制 INrf2 功能，但仍未知。细胞的抗氧化处理导致基因斯坦敏感的 INrf2、Cul3 和 Rbx1 核输出，可能允许 Nrf2 的核输入而没有降解的威胁。然而，出口机制仍不清楚。 Nrf2 和 INrf2 之间的反馈自动调节回路控制着它们的细胞丰度。有趣的是，如果 Cul3 和 Rbx1 是 INrf2-Nrf2 自调节环的一部分，则 Cul3 和 Rbx1 基因表达与 INrf2 协同诱导，以响应抗氧化剂引发的问题。该提案的主要目标是研究 INrf2 的作用和调控。为此，我们提出四个具体目标。目标 1 将研究 INrf2 与 PGAM5/BclXL/Bcl2、HSP90 和 pICln 的相互作用及其在细胞保护和细胞存活/死亡中的意义。这些研究将使用结构域缺失、转染、免疫沉淀、Western 分析、报告基因和细胞凋亡测定。目标 2 将阐明磷酸化在控制 INrf2 同源二聚化和稳定性、核定位、与 Cul3-Rbx1 和 Nrf2 的相互作用以及 Nrf2 的降解、抗氧化剂诱导的 Nrf2 从 INrf2 中释放和激活基因表达以及 INrf2 的相互作用中的作用与 PGAM5/BclXL/Bcl2、HSP90 和 pICln。定点诱变以及转染和报告分析、免疫沉淀和蛋白质印迹分析将用于实现这一目标。目标3将继续产生INrf2条件性敲除小鼠，其肝脏和皮肤表皮不表达INrf2蛋白。 Cre-Lox 系统将用于生成这些小鼠。此外，还比较了表达无效基因型的条件性 INrf2 敲除小鼠与表达 INrf2 的小鼠在暴露于致癌物质后发展皮肤和肝脏肿瘤的敏感性。目标 4 将检验以下假设：Cul3 和 Rbx1 是 INrf2 和 Nrf2 之间自动调节循环的一部分。缺失作图/转染和条带/超位移/ChIP 测定将确定调节 INrf2 的 ARE 元件和 Nrf2 也调节 Cul3 和 Rbx1 基因的抗氧化诱导。共转染和泛素化检测将阐明 Cul3-Rbx1 协同诱导在 Nrf2 泛素化和降解中的作用。