Regulation of Nrf2 Signaling

描述(申请人提供)：抗氧化反应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调控，而转录因子Nrf2本身受Keap1负调控。初步研究已经确定了一个新的功能Cys中心，它表现出不同的反应性。具体的目标将检验Keap1活性受多个半胱氨酸残基调控的假设，这些残基表现出不同的化学反应能力，从而允许化学信号的整合。目标1将鉴定和测量重组Keap1半胱氨酸残基对化学上不同类别的第二相诱导剂的动力学反应性。该方法将使用LC-MS-MS(LC-MS-MS)。LC-MS-MS光谱将使用模式识别算法SALSA进行分析。目的2将确定关键的Keap1半胱氨酸残基在与硫醇反应的亲电体反应后是否调节Nrf2的Neh2结构域与Keap1之间的结合。重组的Keap1将用于体外实验，以测量Neh2 GFP融合蛋白的结合亲和力。定点突变将用于验证Keap1半胱氨酸残基控制Keap1和Neh2结构域之间的联系。AIM 3将确定Keap1释放Nrf2是否受完整细胞中关键半胱氨酸残基的调节。为了在体内验证结果，将瞬时地将表达野生型Keap1或含有Cys to Ala突变的Keap1的载体导入细胞。FLAG标记的Keap1和HA标记的Nrf2的免疫沉淀免疫印迹将被用来测量Nrf2从Keap1的结合和释放。突变和野生型FLAG标记的Keap1的硫醇反应性将在体内使用2D差异凝胶电泳法进行评估。目标4将确定Keap1中关键的反应性半胱氨酸残基是否调节与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。

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.