Regulation of Nrf2 Signaling

描述（由申请人提供）：抗氧化反应元件介导的基因诱导代表了癌症化学预防策略的重要组成部分，并部分受转录因子Nrf 2调节，Nrf 2本身受Keap 1负调控。初步研究已经确定了一个新的功能性Cys中心，表现出不同的反应性。具体目标将测试Keap 1活性受多个Cys残基调节的假设，这些Cys残基表现出不同的化学反应性，允许化学信号的整合。目的1将鉴定和测量重组Keap 1 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光谱。目的2将确定关键的Keap 1半胱氨酸残基是否调节Nrf 2和Keap 1的Neh 2结构域与巯基反应性亲电试剂反应后的关联。重组Keap 1将用于体外试验，该试验将测量Neh 2 GFP融合蛋白的结合亲和力。将使用定点诱变来验证Keap 1 Cys残基控制Keap 1和Neh 2结构域之间的结合。目的3将确定Nrf 2从Keap 1的释放是否受完整细胞中关键Cys残基的调节。将用表达野生型Keap 1或含有Cys至Ala突变的Keap 1的载体瞬时转染细胞，以验证体内结果。FLAG标记的Keap 1和HA标记的Nrf 2的免疫沉淀免疫印迹将用于测量Nrf 2从Keap 1的结合和释放。使用2D差异凝胶电泳在体内评估突变和野生型FLAG标记的Keap 1的巯基反应性。目的4将确定Keap 1中的关键反应性Cys残基是否调节与Nrf 2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.