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）：抗氧化剂元件介导的基因诱导是癌症化学预防策略的重要组成部分，部分受转录因子NRF2的调节，该因子NRF2本身受KEAP1负调控。初步研究已经确定了一个新的功能性CYS中心，该中心表现出差异反应性。具体目的将检验以下假设：KEAP1活性受多种Cys残基的调节，这些残基表现出差异化学反应性，从而允许整合化学信号。 AIM 1将识别并测量重组Keap1 Cys残基对化学不同类别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）。 LC-MS-MS光谱将使用模式识别算法莎莎分析。 AIM 2将确定与硫醇反应性电力反应后，Keap1 Cys残基是否调节NRF2和KEAP1的NEH2结构域之间的关联。重组KEAP1将用于体外测定法，该测定方法将测量NEH2 GFP融合蛋白的结合亲和力。定位的诱变将用于验证KEAP1 CYS残基控制Keap1与NEH2域之间的关联。 AIM 3将确定从KEAP1释放NRF2是否受完整细胞中的关键CYS残基的调节。细胞将用表达野生型Keap1或含有Cys的cys cys Ala突变的矢量瞬时转染，以便在体内验证结果。标记为KEAP1和HA标记的NRF2的FLAG标记的免疫沉淀免疫印迹将用于测量KEAP1的NRF2的关联和释放。突变和野生型标记为Keap1的硫醇反应性将在体内使用2D差异凝胶电泳评估。 AIM 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。遥远和近紫外CD将用于评估次级和第三纪结构，而构象稳定性将使用差扫描量热法检查。

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