Physiological significance of reactive cysteine residues of keap1 in determining Nrf2 activity
Physiological significance of reactive cysteine residues of keap1 in determining Nrf2 activity
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DOI：
10.1128/mcb.01704-07
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发表时间：
2008-04-01
影响因子：
5.3
通讯作者：
Yamamoto, Masayuki
中科院分区：
文献类型：
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作者：
Yamamoto, Tae;Suzuki, Takafumi;Yamamoto, Masayuki
Keap 1和Cu 13构成了一种独特的泛素E3连接酶，可降解细胞保护基因的关键激活因子Nrf 2。在暴露于氧化剂/亲电体时，该连接酶复合物的酶活性被抑制，并且该复合物不能降解Nrf 2，导致Nrf 2靶基因的转录激活。Keapl具有几个反应性半胱氨酸残基，其在体外与亲电体共价键合。为了阐明每个Keapl半胱氨酸残基在生理条件下的功能意义，我们建立了转基因互补拯救模型。突变体Keapl（C273 A）和/或Keapl（C288 A）蛋白在Keapl敲除小鼠中的转基因表达未能逆转组成型Nrf 2活化，表明位置273和288处的半胱氨酸残基对于Keapl在体内抑制Nrf 2活性是必需的。相反，Keapl（C151 S）保留了阻遏物活性，并且表达该分子的小鼠是存活的。来自Keapl（C151 S）转基因小鼠的小鼠胚胎成纤维细胞在亲电攻击之前和之后均显示Nrf 2靶基因的表达降低，表明Cys 151在促进Nrf 2活化中是重要的。这些结果证明了半胱氨酸残基在体内维持Keap 1功能的关键作用，使得Nrf 2在静止条件下被抑制，并且响应于氧化剂/亲电试剂而活跃。
Keap1 and Cu13 constitute a unique ubiquitin E3 ligase that degrades Nrf2, a key activator of cytoprotective genes. Upon exposure to oxidants/electrophilles, the enzymatic activity of this ligase complex is inhibited and the complex fails to degrade Nrf2, resulting in the transcriptional activation of Nrf2 target genes. Keapl possesses several reactive cysteine residues that covalently bond with electrophiles in vitro. To clarify the functional significance of each Keapl cysteine residue under physiological conditions, we established a transgenic complementation rescue model. The transgenic expression of mutant Keapl(C273A) and/or Keapl(C288A) protein in Keap1] null mice failed to reverse constitutive Nrf2 activation, indicating that cysteine residues at positions 273 and 288 are essential for Keap1 to repress Nrf2 activity in vivo. In contrast, Keapl(C151S) retained repressor activity and mice expressing this molecule were viable. Mouse embryonic fibroblasts from Keapl(C151S) transgenic mice displayed decreased expression of Nrf2 target genes both before and after an electrophilic challenge, suggesting that Cys151 is important in facilitating Nrf2 activation. These results demonstrate critical roles of the cysteine residues in vivo in maintaining Keap1 function, such that Nrf2 is repressed under quiescent conditions and active in response to oxidants/electrophiles.