Protection against electrophile and oxidant stress by induction of the phase 2 response: Fate of cysteines of the Keap1 sensor modified by inducers
Protection against electrophile and oxidant stress by induction of the phase 2 response: Fate of cysteines of the Keap1 sensor modified by inducers
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DOI：
10.1073/pnas.0307301101
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发表时间：
2004-02-17
影响因子：
11.1
通讯作者：
Talalay, P
中科院分区：
文献类型：
--
作者：
Wakabayashi, N;Dinkova-Kostova, AT;Talalay, P
诱导一个2期基因家族编码的蛋白质，保护免受亲电体和活性氧中间体的损害，是潜在的一个主要策略，降低癌症和慢性退行性疾病的风险。许多2期基因受上游抗氧化反应元件（ARE）的调控，这些元件是亮氨酸拉链转录因子Nrf 2的靶点。在基础条件下，Nrf 2主要存在于细胞质中，与其富含半胱氨酸、含有Kelch结构域的伴侣keap 1结合，keap 1本身锚定在肌动蛋白细胞骨架上并抑制Nrf 2活性。诱导剂通过修饰Keap 1的25个半胱氨酸残基中的两个（C273和C288）来破坏Keap 1-Nrf 2复合物。C273和C288的关键作用是通过以下方式确定的：（i）当用甲磺酸地塞米松处理纯化的重组Keap 1并通过质谱分析地塞米松修饰的胰蛋白酶肽时，它们具有高反应性，以及（ii）用表达Keap 1的半胱氨酸至丙氨酸突变体的构建体转染keap 1和nrf 2基因缺陷的小鼠胚胎成纤维细胞，以及测量共转染的Nrf 2抑制ARE-荧光素酶报道基因的能力。Keap 1与诱导剂的反应导致分子间二硫键的形成，可能在一个Keap 1分子的C273和第二个Keap 1分子的C288之间。通过诱导剂处理的细胞提取物的2D PAGE，以及通过证明Keap 1的C273 A和C288 A突变体单独不能抑制ARE-荧光素酶报告基因的Nrf 2激活，这些突变体构建体的等量混合物恢复了阻遏物活性，获得了形成这种二聚体的证据。
Induction of a family of phase 2 genes encoding for proteins that protect against the damage of electrophiles and reactive oxygen intermediates is potentially a major strategy for reducing the risk of cancer and chronic degenerative diseases. Many phase 2 genes are regulated by upstream antioxidant response elements (ARE) that are targets of the leucine zipper transcription factor Nrf2. Under basal conditions, Nrf2 resides mainly in the cytoplasm bound to its cysteine-rich, Kelch domain-containing partner keap1, which is itself anchored to the actin cytoskeleton and represses Nrf2 activity. Inducers disrupt the Keap1-Nrf2 complex by modifying two (C273 and C288) of the 25 cysteine residues of Keap1. The critical role of C273 and C288 was established by (i) their high reactivity when purified recombinant Keap1 was treated with dexamethasone mesylate and the dexamethasone-modified tryptic peptides were analyzed by mass spectrometry, and (ii) transfection of keap1 and nrf2 gene-deficient mouse embryonic fibroblasts with constructs expressing cysteine to alanine mutants of Keap1, and measurement of the ability of cotransfected Nrf2 to repress an ARE-luciferase reporter. Reaction of Keap1 with inducers results in formation of intermolecular disulfide bridges, probably between C273 of one Keap1 molecule and C288 of a second. Evidence for formation of such dimers was obtained by 2D PAGE of extracts of cells treated with inducers, and by the demonstration that whereas C273A and C288A mutants of Keap1 alone could not repress Nrf2 activation of the ARE-luciferase reporter, an equal mixture of these mutant constructs restored repressor activity.