Critical Cysteine Residues of Kelch-Like ECH-Associated Protein 1 in Arsenic Sensing and Suppression of Nuclear Factor Erythroid 2-Related Factor 2
Critical Cysteine Residues of Kelch-Like ECH-Associated Protein 1 in Arsenic Sensing and Suppression of Nuclear Factor Erythroid 2-Related Factor 2
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DOI：
10.1124/jpet.109.160465
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发表时间：
2010-01-01
影响因子：
3.5
通讯作者：
Ma, Qiang
中科院分区：
文献类型：
--
作者：
He, Xiaoqing;Ma, Qiang
砷激活核因子红细胞2相关因子2（Nrf 2）诱导II相和抗氧化基因。在这里，我们分析了砷Kelch样ECH相关蛋白1（Keap 1）半胱氨酸巯基相互作用在Nrf 2激活。基于砷的Nrf 2激活剂，荧光双砷标记试剂（FlAsH）和氧化苯胂（PAO），被用来探测砷与Keap 1的结合。在FlAsH与纯化的Keap 1结合时观察到强荧光。用砷、叔丁基对苯二酚（tBHQ）或2，3-二巯基丙醇预处理显著降低了荧光信号。PAO亲和珠在体外和从hepa 1c 1c 7细胞中有效地拉下Keap 1。砷、tBHQ、游离PAO或镉阻断Keap 1下拉。此外，砷和游离PAO显着降低纯化或内源性Keap 1的游离巯基含量。因此，砷、FlAsH和PAO以及tBHQ和镉以类似的方式与Keap 1半胱氨酸硫醇结合。Keap 1的所有结构域都能与PAO结合，其中连接子区域的结合活性最高。砷Keap 1相互作用的功能进行了评估，在重建系统，模仿内源性Nrf 2的调节。接头区Cys 273或Cys 288的突变导致Nrf 2蛋白的高水平基础表达。Cys 151突变可阻断砷对Nrf 2的激活作用。C273 A、C288 A或C151 A的过表达改变了Nrf 2靶基因的基础表达和砷诱导的表达。该研究表明Cys 273和Cys 288在Keap 1抑制Nrf 2中起重要作用，Cys 151在砷反应中起关键作用。我们的研究结果支持一个模型，其中砷结合到不同的Keap 1半胱氨酸残基，以调节不同的功能，在Nrf 2信号转导。
Arsenic activates nuclear factor erythroid 2-related factor 2 (Nrf2) to induce phase II and antioxidative genes. Here we analyzed arsenic-Kelch-like ECH-associated protein 1 (Keap1) cysteine thiol interaction in Nrf2 activation. Arsenic-based Nrf2 activators, fluorescent biarsenical labeling reagent (FlAsH) and phenylarsine oxide (PAO), were used to probe binding of arsenic to Keap1. Strong fluorescence was observed on binding of FlAsH to purified Keap1. Pretreatment with arsenic, tert-butylhydroquinone (tBHQ), or 2,3-dimercaptopropanol significantly reduced the fluorescent signal. PAO affinity beads effectively pulled down Keap1 in vitro and from hepa1c1c7 cells. Arsenic, tBHQ, free PAO, or cadmium blocked Keap1 pull-down. Furthermore, arsenic and free PAO significantly reduced the free thiol contents of purified or endogenous Keap1. Thus, arsenic, FlAsH, and PAO, as well as tBHQ and cadmium, bind to Keap1 cysteine thiols in a similar fashion. All the domains of Keap1 bound PAO, and the linker region exhibited the highest binding activity. The function of arsenic-Keap1 interaction was evaluated in a reconstituted system that mimics endogenous Nrf2 regulation. Mutation of Cys273 or Cys288 in the linker region resulted in high level basal expression of Nrf2 protein. Mutation of Cys151 abolished Nrf2 activation by arsenic. Overexpression of C273A, C288A, or C151A altered the basal and arsenic-induced expression of Nrf2 target genes. The study shows an important role of Cys273 and Cys288 in the suppression of Nrf2 by Keap1 and a critical function of Cys151 in arsenic responsiveness. Our findings support a model in which arsenic binds to different sets of Keap1 cysteine residues to regulate divergent functions in Nrf2 signal transduction.