Hong Fei, Freeman Michael L, Liebler Daniel C
Department of Pharmacology and Toxicology, College of Pharmacy, The University of Arizona, Tucson, Arizona 85724, USA.
Chem Res Toxicol. 2005 Dec;18(12):1917-26. doi: 10.1021/tx0502138.
The chemopreventive agent sulforaphane is an isothiocyanate derived from cruciferous vegetables. Sulforaphane exerts cancer chemopreventive effects by inducing antioxidant/electrophile response element (ARE)-regulated phase 2 enzyme and antioxidant genes through activation of the transcription factor nuclear factor-E2-related factor 2 (Nrf2), which is regulated by the thiol-rich sensor protein Kelch-like ECH-associated protein 1 (Keap1). Sulforaphane is an electrophile that can react with protein thiols to form thionoacyl adducts. We hypothesized that, like other electrophilic Nrf2 activators, sulforaphane activates this system through specific modifications of the Keap1 protein. However, thionoacyl adducts are labile to hydrolysis and transacylation reactions, which complicate the identification of the sulforaphane adduct sites on Keap1. In this study, we characterized the stability of sulforaphane thionoacyl adducts and developed a liquid chromatography-tandem mass spectrometry method to map labile sulforaphane adduct sites formed on Keap1 in vitro. Sulforaphane displays a distinctly different pattern of Keap1 modification than previously studied ARE inducers that modify Keap1 by alkylation. Sulforaphane modified Keap1 most readily in the Kelch domain, rather than in the central linker domain, which is targeted by previously characterized ARE inducers. Also, in contrast to previously studied ARE inducers and as reported recently [Zhang, et al. (2005) J. Biol. Chem. 280, 30091-30099], sulforaphane treatment in vivo does not lead to the accumulation of ubiquitinated Keap1. Our observations suggest a novel mechanism for Nrf2 stabilization by sulforaphane-Keap1 thionoacyl adduct formation.
化学预防剂萝卜硫素是一种源自十字花科蔬菜的异硫氰酸酯。萝卜硫素通过激活转录因子核因子E2相关因子2（Nrf2）来诱导抗氧化剂/亲电反应元件（ARE）调控的Ⅱ相酶和抗氧化基因，从而发挥癌症化学预防作用，而Nrf2受富含硫醇的传感蛋白 Kelch样ECH相关蛋白1（Keap1）的调控。萝卜硫素是一种亲电试剂，可与蛋白质硫醇反应形成硫代酰基加合物。我们推测，与其他亲电Nrf2激活剂一样，萝卜硫素通过对Keap1蛋白的特异性修饰来激活该系统。然而，硫代酰基加合物对水解和转酰基反应不稳定，这使得鉴定Keap1上萝卜硫素加合物位点变得复杂。在本研究中，我们表征了萝卜硫素硫代酰基加合物的稳定性，并开发了一种液相色谱-串联质谱法来绘制体外Keap1上形成的不稳定萝卜硫素加合物位点。与之前研究的通过烷基化修饰Keap1的ARE诱导剂相比，萝卜硫素对Keap1的修饰模式明显不同。萝卜硫素最容易在Kelch结构域而非中央连接结构域修饰Keap1，而中央连接结构域是之前表征的ARE诱导剂的作用靶点。此外，与之前研究的ARE诱导剂不同，且如最近报道的[Zhang等人（2005年）《生物化学杂志》280, 30091 - 30099]，体内萝卜硫素处理不会导致泛素化Keap1的积累。我们的观察结果表明，萝卜硫素 - Keap1硫代酰基加合物的形成是Nrf2稳定化的一种新机制。
