NRF2 Cysteine Residues Are Critical for Oxidant/Electrophile-Sensing, Kelch-Like ECH-Associated Protein-1-Dependent Ubiquitination-Proteasomal Degradation, and Transcription Activation
NRF2 Cysteine Residues Are Critical for Oxidant/Electrophile-Sensing, Kelch-Like ECH-Associated Protein-1-Dependent Ubiquitination-Proteasomal Degradation, and Transcription Activation
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DOI：
10.1124/mol.109.058453
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发表时间：
2009-12-01
影响因子：
3.6
通讯作者：
Ma, Qiang
中科院分区：
文献类型：
--
作者：
He, Xiaoqing;Ma, Qiang
细胞通过激活受体/转录因子核因子-红细胞2相关因子2 （Nrf2）来协调诱导对氧化和其他应激至关重要的细胞保护基因，从而对氧化剂和亲电试剂做出反应。激活包括阻断Nrf2的泛素化-蛋白酶体降解。Kelch-like ECH-associated protein-1 （Keap1）连接区的诱导剂修饰半胱氨酸巯基，将Nrf2聚集到Keap1/Cul3 E3复合体中进行泛素化，这是重要的，但不足以激活Nrf2。在这里，我们发现Nrf2进化上保守的半胱氨酸残基对Nrf2调控至关重要。FlAsH（一种砷基荧光团）和苯larsin oxide （PAO）在体内和体外均能诱导Nrf2靶基因并与Nrf2结合。典型诱导剂砷和叔丁基对苯二酚抑制结合。PAO亲和下拉和个体半胱氨酸对丙氨酸的突变表明，Cys235、Cys311、Cys316、Cys414和Cys506对结合至关重要，并且结合受分子内相互作用调节。为了证实半胱氨酸残基的功能，Nrf2野生型或突变体在Nrf2敲除细胞中表达，以重建Nrf2调控。Nrf2突变体降低了t(1/2)，这与增加与Keap1的结合和突变蛋白的多泛素化呈负相关。值得注意的是，突变体对砷的Nrf2激活和基因诱导没有反应。此外，Cys119、Cys235和Cys506位点的突变阻碍了Nrf2与内源性抗氧化反应元件和辅激活因子cAMP反应元件结合蛋白/p300的结合。研究结果表明，Nrf2半胱氨酸残基对氧化/亲电传感具有重要的调节作用，抑制keap1依赖性泛素化-蛋白酶体降解，促进共激活因子的募集，从而在受体分子上整合化学传感、受体激活和转录激活。
Cells respond to oxidants and electrophiles by activating receptor/transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) to coordinate the induction of cytoprotective genes critical for defense against oxidative and other stresses. Activation involves blocking the ubiquitination-proteasomal degradation of Nrf2. Modification of cysteine thiol groups by inducers in the linker region of Kelch-like ECH-associated protein-1 (Keap1), which congregates Nrf2 into the Keap1/Cul3 E3 complex for ubiquitination, is important but not sufficient for activation of Nrf2. Here we show that evolutionarily conserved cysteine residues of Nrf2 are critical for Nrf2 regulation. FlAsH (an arsenic-based fluorophore) and phenylarsine oxide (PAO) potently induce Nrf2 target genes and bind to Nrf2 in vitro and in vivo. Binding is inhibited by prototypical inducers arsenic and tert-butylhydroquinone. PAO affinity pull-down and mutation of individual cysteine to alanine reveal that Cys235, Cys311, Cys316, Cys414, and Cys506 are critical for binding, and binding is modulated by intramolecular interactions. To corroborate the functions of cysteine residues, Nrf2 wild-type or mutants are expressed in Nrf2 knockout cells to reconstitute Nrf2 regulation. Nrf2 mutants have reduced t(1/2) that inversely correlates with increased binding to Keap1 and polyubiquitination of mutant proteins. It is remarkable that the mutants fail to respond to arsenic for Nrf2 activation and gene induction. Furthermore, mutations at Cys119, Cys235, and Cys506 impede binding of Nrf2 to endogenous antioxidant response element and to coactivator cAMP response element-binding protein-binding protein/p300. The findings demonstrate that Nrf2 cysteine residues critically regulate oxidant/electrophile sensing, repress Keap1-dependent ubiquitination-proteasomal degradation, and promote recruitment of coactivators, such that chemical sensing, receptor activation, and transcription activation are integrated at the receptor molecule.