萝卜硫素对 Keap1 半胱氨酸残基的修饰,Chemical Research in Toxicology

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萝卜硫素对 Keap1 半胱氨酸残基的修饰
Chemical Research in Toxicology ( IF 3.7 ) Pub Date : 2011-03-10 00:00:00 , DOI: 10.1021/tx100389r
Chenqi Hu ₁ , Aimee L Eggler , Andrew D Mesecar , Richard B van Breemen

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通过修饰 Kelch 样 ECH 相关蛋白 1 (Keap1) 半胱氨酸激活转录因子 NF-E2 相关因子 2 (Nrf2)，导致抗氧化反应元件 (ARE) 上调，是一种重要机制对活性氧物质和异生亲电子试剂的细胞防御。萝卜硫素存在于十字花科蔬菜（如西兰花）中，是一种有效的天然 ARE 激活剂，可通过修饰 Keap1 半胱氨酸残基发挥作用，但关于这些半胱氨酸残基中哪些会发生反应，体外和体内数据存在矛盾。尽管大多数生物学数据表明 C151 的修饰对于萝卜硫素作用至关重要，但最近一些使用质谱法的研究未能将 C151 确定为 Keap1 萝卜硫素反应的位点。我们使用质谱法和修订后的样品制备方案协调了这些相互矛盾的数据，并确认 C151 确实是最容易被萝卜硫素修饰的 Keap1 半胱氨酸之一。之前基于质谱的研究在样品制备过程中使用碘乙酰胺衍生游离半胱氨酸巯基，导致 Keap1 包括 C151 上的高反应性和可逆半胱氨酸残基损失萝卜硫素。通过从协议中省略碘乙酰胺并减少样品制备时间，我们基于质谱的研究现在证实了以前基于细胞的研究，这些研究表明萝卜硫素与 Keap1 的至少四个半胱氨酸残基反应，包括 C151。

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Modification of Keap1 Cysteine Residues by Sulforaphane

Activation of the transcription factor NF-E2-related factor-2 (Nrf2) through modification of Kelch-like ECH-associated protein 1 (Keap1) cysteines, leading to up-regulation of the antioxidant response element (ARE), is an important mechanism of cellular defense against reactive oxygen species and xenobiotic electrophiles. Sulforaphane, occurring in cruciferous vegetables such as broccoli, is a potent natural ARE activator that functions by modifying Keap1 cysteine residues, but there are conflicting in vitro and in vivo data regarding which of these cysteine residues react. Although most biological data indicate that modification of C151 is essential for sulforaphane action, some recent studies using mass spectrometry have failed to identify C151 as a site of Keap1 sulforaphane reaction. We have reconciled these conflicting data using mass spectrometry with a revised sample preparation protocol and confirmed that C151 is indeed among the most readily modified cysteines of Keap1 by sulforaphane. Previous mass spectrometry-based studies used iodoacetamide during sample preparation to derivatize free cysteine sulfhydryl groups causing the loss of sulforaphane from highly reactive and reversible cysteine residues on Keap1 including C151. By omitting iodoacetamide from the protocol and reducing sample preparation time, our mass spectrometry-based studies now confirm previous cell-based studies which showed that sulforaphane reacts with at least four cysteine residues of Keap1 including C151.

更新日期：2011-03-10

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