Sites of alkylation of human Keap1 by natural Chemoprevention agents
Sites of alkylation of human Keap1 by natural Chemoprevention agents
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DOI：
10.1016/j.jasms.2007.09.015
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发表时间：
2007-12-01
影响因子：
3.2
通讯作者：
van Breemen, Richard B.
中科院分区：
文献类型：
--
作者：
Luo, Yan;Eggler, Aimee L.;van Breemen, Richard B.
在基础条件下，胞浆蛋白Keap1（Kelch样ECH相关蛋白1）与转录因子核因子-E-2相关因子2（Nrf2）的相互作用导致细胞保护基因的低水平表达，其启动子区域含有抗氧化反应元件（ARE）。人 Keap1 的 27 个半胱氨酸硫氢基中的一个或多个烷基化被认为会导致 Nrf2 核积累，通过 ARE 上调细胞保护基因表达，并预防退行性疾病，例如癌症。因此，鉴定这些半胱氨酸残基对特定亲电子试剂最具反应性应该有助于阐明这种癌症预防机制，也称为化学预防。为了解决这个问题，使用液相色谱-串联质谱 (LC-MS/MS) 和圆柱形离子阱质谱仪，以及 LC-MS/MS 和混合线性离子阱 FT ICR 质谱仪，对亲电子试剂 1-biotinamido-4-(4'-[马来酰亚胺乙基-环己烷]-甲酰氨基) 丁烷烷基化的 Keap1 的胰蛋白酶消化物进行了初步分析。由于FT ICR仪器提供了更完整的肽测序覆盖范围，并能够识别更多烷基化的半胱氨酸残基，因此在后续的Keap1被三种可上调ARE、黄腐酚、异甘草素和10-姜烯酚的亲电子天然产物烷基化的研究中，仅使用该仪器。在 Keap1 的各个半胱氨酸残基中，C151 对这三种亲电子试剂的反应性最强。这些体外结果与体内实验的证据一致，并表明 C151 是化学预防剂对 Keap1 进行烷基化的最重要位点，化学预防剂通过 Nrf2 激活 ARE 发挥作用。
Under basal conditions, the interaction of the cytosolic protein Keap1 (Kelch-like ECH-associated protein 1) with the transcription factor nuclear factor-E-2-related factor 2 (Nrf2) results in a low level of expression of cytoprotective genes whose promoter region contains the antioxidant response element (ARE). Alkylation of one or more of the 27 cysteine sulfhydryl groups of human Keap1 is proposed to lead to Nrf2 nuclear accumulation, to upregulation of cytoprotective gene expression by the ARE, and to prevention of degenerative diseases, such as cancer. Therefore, identification of the most reactive of these cysteine residues toward specific electrophiles should help clarify this mechanism of cancer prevention, also known as chemoprevention. To address this issue, preliminary analyses of tryptic digests of Keap1 alkylated by the model electrophile 1-biotinamido-4-(4'-[maleimidoethyl-cyclohexane]-carboxamido) butane were carried out using liquid chromatographic-tandem mass spectrometry (LC-MS/MS) with a cylindrical ion trap mass spectrometer and also using LC-MS/MS with a hybrid linear ion trap FT ICR mass spectrometer. Because the FT ICR instrument provided more complete peptide sequencing coverage and enabled the identification of more alkylated cysteine residues, only this instrument was used in subsequent studies of Keap1 alkylation by three electrophilic natural products that can upregulate the ARE, xanthohumol, isoliquiritigenin, and 10-shogaol. Among the various cysteine residues of Keap1, C151 was most reactive toward these three electrophiles. These in vitro results agree with evidence from in vivo experiments, and indicate that C151 is the most important site of alkylation on Keap1 by chemoprevention agents that function by activating the ARE through Nrf2.