Regulation of Nrf2 Signaling

Nrf2 信号传导的调节

• 批准号: 7615551
• 负责人: MICHAEL L. FREEMAN
• 金额: $ 29.79万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-06-11 至 2011-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7615551
• 关键词: Affect; Affinity; Algorithms; Alkylation; Antioxidants; Bacteria; Binding; Biochemical; Cells; Chemical Modifier; Chemicals; Chemistry; Chimeric Proteins; Circular Dichroism; Complex; Data; Differential Scanning Calorimetry; Drug Metabolic Detoxication; Equilibrium; Exhibits; Gene Expression; Genetic; Guanidinium Chloride; Human; Immunoblotting; Immunofluorescence Immunologic; Immunoprecipitation; Individual; Kinetics; Knowledge; Lead; Length; Link; Liquid Chromatography; Measures; Mediating; Modeling; Modification; Molecular; Molecular Conformation; Mutagenesis; Mutate; Mutation; Nuclear; Oxidation-Reduction; Pattern Recognition; Peptides; Phase; Polymers; Precipitation; Process; Proteins; Reaction; Reagent; Recombinants; Regulation; Reporter Genes; Research Personnel; Response Elements; Risk; Signal Transduction; Site; Site-Directed Mutagenesis; Specificity; Structure; Sulfhydryl Compounds; Support System; System; Testing; Two-Dimensional Polyacrylamide Gel Electrophoresis; Ubiquitin; Ubiquitination; Yeasts; base; cancer chemoprevention; carcinogenesis; design; disulfide bond; gel electrophoresis; gene induction; in vitro Assay; in vivo; member; multicatalytic endopeptidase complex; oxidation; sensor; tandem mass spectrometry; transcription factor; ubiquitin-protein ligase; vector

DESCRIPTION (provided by applicant): Antioxidant response element-mediated gene induction represents an important component of cancer chemoprevention strategies and is regulated in part by the transcription factor Nrf2, which itself is negatively regulated by Keap1. Preliminary studies have identified a new functional Cys center that exhibits differential reactivity. The specific aims will test the hypothesis that Keap1 activity is regulated by multiple Cys residues that exhibit differential chemical reactivity, allowing integration of chemical signals. Aim 1 will identify and measure kinetic reactivity of recombinant Keap1 Cys residues towards chemically distinct classes of Phase II inducing agents. The approach will use liquid chromatography- tandem mass spectrometry (LC-MS-MS). LC-MS-MS spectra will be analyzed using a pattern recognition algorithm SALSA. Aim 2 will determine if key Keap1 Cys residues regulate association between the Neh2 domain of Nrf2 and Keap1 following reaction with thiol-reactive electrophiles. Recombinant Keap1 will be used in an in vitro assay that will measure binding affinity of a Neh2 GFP fusion protein. Site directed mutagenesis will be used to verify that Keap1 Cys residues control the association between Keap1 and the Neh2 domain. Aim 3 will determine if release of Nrf2 from Keap1 is regulated by key Cys residues in intact cells. Cells will be transiently transfected with vectors expressing wild type Keap1 or Keap1 containing Cys to Ala mutations in order to verify results in vivo. Immunoprecipitation immunoblotting of FLAG tagged Keap1 and HA tagged Nrf2 will be used to measure association and release of Nrf2 from Keap1. Thiol reactivity of mutated and wild type FLAG tagged Keap1 will be assessed in vivo using 2D Difference Gel Electrophoresis. Aim 4 will determine if key reactive Cys residues in Keap1 regulate conformational changes that are associated with release of Nrf2. Far- and near UV CD will be used to assess secondary and tertiary structure, while conformational stability will be examined using differential scanning calorimetry.

描述（由申请人提供）：抗氧化反应元件介导的基因诱导代表癌症化学预防策略的重要组成部分，并部分受转录因子 Nrf2 调节，而 Nrf2 本身受 Keap1 负调节。初步研究已经确定了一个新的功能性半胱氨酸中心，其表现出不同的反应性。具体目标将测试 Keap1 活性受多个 Cys 残基调节的假设，这些残基表现出不同的化学反应性，从而允许化学信号的整合。目标 1 将鉴定并测量重组 Keap1 Cys 残基对化学上不同类别的 II 期诱导剂的动力学反应性。该方法将使用液相色谱-串联质谱法 (LC-MS-MS)。将使用模式识别算法 SALSA 分析 LC-MS-MS 谱图。目标 2 将确定关键 Keap1 Cys 残基在与硫醇反应性亲电子试剂反应后是否调节 Nrf2 的 Neh2 结构域和 Keap1 之间的关联。重组 Keap1 将用于体外测定，测量 Neh2 GFP 融合蛋白的结合亲和力。定点诱变将用于验证 Keap1 Cys 残基控制 Keap1 和 Neh2 结构域之间的关联。目标 3 将确定 Nrf2 从 Keap1 的释放是否受到完整细胞中关键 Cys 残基的调节。将用表达野生型 Keap1 或含有 Cys 至 Ala 突变的 Keap1 的载体瞬时转染细胞，以验证体内结果。 FLAG 标记的 Keap1 和 HA 标记的 Nrf2 的免疫沉淀免疫印迹将用于测量 Nrf2 从 Keap1 的结合和释放。将使用二维差异凝胶电泳在体内评估突变型和野生型 FLAG 标记的 Keap1 的硫醇反应性。目标 4 将确定 Keap1 中的关键反应性 Cys 残基是否调节与 Nrf2 释放相关的构象变化。远紫外和近紫外 CD 将用于评估二级和三级结构，而构象稳定性将使用差示扫描量热法进行检查。

项目成果

NRF2 deficiency reduces life span of mice administered thoracic irradiation.

• DOI: 10.1016/j.freeradbiomed.2011.05.038
• 发表时间: 2011-09-15
• 期刊: FREE RADICAL BIOLOGY AND MEDICINE
• 影响因子: 7.4
• 作者: Travis, Elizabeth L.;Rachakonda, Girish;Zhou, Xinhui;Korhonen, Katrina;Sekhar, Konjeti R.;Biswas, Swati;Freeman, Michael L.
• 通讯作者: Freeman, Michael L.

Cysteine-based regulation of the CUL3 adaptor protein Keap1.

• DOI: 10.1016/j.taap.2009.06.016
• 发表时间: 2010-04-01
• 期刊: Toxicology and applied pharmacology
• 影响因子: 3.8
• 作者: Sekhar KR;Rachakonda G;Freeman ML
• 通讯作者: Freeman ML