Modifying specific cysteines of the electrophile-sensing human Keap1 disrupt binding to the protein is insufficient to Nrf2 domain Neh2
Modifying specific cysteines of the electrophile-sensing human Keap1 disrupt binding to the protein is insufficient to Nrf2 domain Neh2
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DOI：
10.1073/pnas.0502402102
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发表时间：
2005-07-19
影响因子：
11.1
通讯作者：
Mesecar, AD
中科院分区：
文献类型：
--
作者：
Eggler, AL;Liu, GW;Mesecar, AD
通过上调解毒酶可以降低由活性氧和亲电子试剂引起的癌症和其他退行性疾病的风险。诱导这些保护酶的一个主要机制是通过氧化应激传感器蛋白 Kelch 样 ECH 相关蛋白 1 (Keap1) 和转录因子 NF-E2 相关因子 2 (Nrf2) 激活抗氧化反应元件 (ARE)。在基础条件下，Keap1 通过与其 Neh2 结构域结合将 Nrf2 隔离在细胞质中。已知化学诱导剂（例如萝卜硫素）会与 Keap1 半胱氨酸残基发生反应，从而促进 Nrf2 核积累，从而促进 ARE 激活。 Nrf2 核积累的一个广泛接受的模型是 Keap1 半胱氨酸的修饰直接导致 Keap1-Ntf2 复合物的解离。该模型基于对小鼠蛋白质的研究，并已作为许多研究的实验基础和假设。通过化学、质谱和等温滴定量热法相结合，我们使用一系列 ARE 诱导剂测试了直接解离模型：萝卜硫素、异甘草素、15-脱氧-Delta 12,14-前列腺素-J2、甲萘醌、1-Cl-2,4-二硝基苯和生物素化碘乙酰胺。令人惊讶的是，这些数据表明 Keap1-Nrf2 的直接破坏模型是不正确的。测定了人 Keap1 半胱氨酸的相对反应性。除了为小鼠 Keap1 鉴定出的 5 个相同的半胱氨酸之外，还鉴定出了两个高度反应性且之前未观察到的半胱氨酸。基于这些结果，提出了一个模型，该模型应该有助于理解 Keap1-Nrf2 信号机制。
The risks of cancer and other degenerative diseases caused by reactive oxygen species and electrophiles can be reduced by the up-regulation of detoxifying enzymes. A major mechanism whereby these protective enzymes are induced occurs through activation of the antioxidant response element (ARE) by the oxidative-stress sensor protein Kelch-like ECH-associated protein 1 (Keap1) and the transcription factor NF-E2-related factor 2 (Nrf2). Under basal conditions, Keap1 sequesters Nrf2 in the cytoplasm by binding to its Neh2 domain. Chemical inducers such as sulforaphane are known to react with Keap1 cysteine residues, thereby promoting Nrf2 nuclear accumulation and hence ARE activation. A widely accepted model for Nrf2 nuclear accumulation is that modification of Keap1 cysteines leads directly to dissociation of the Keap1-Ntf2 complex. This model is based on studies with mouse proteins and has served as the experimental basis and hypothesis for numerous investigations. Through a combination of chemical, mass spectrometry, and isothermal titration calorimetry methods, we have tested the direct-dissociation model using a series of ARE inducers: sulforaphane, isoliquiritigenin, 15-deoxy-Delta 12,14-prostaglandin-J2, menadione, 1-Cl-2,4-dinitrobenzene, and biotinylated iodoacetamide. Surprisingly, these data suggest that the direct disruption model for Keap1-Nrf2 is incorrect. The relative reactivity of human Keap1 cysteines was determined. In addition to the same five cysteines identified for mouse Keap1, two highly reactive and previously unobserved cysteines were identified. Based on these results, a model is proposed that should aid in the understanding of Keap1-Nrf2 signaling mechanisms.