Kaspar James W, Jaiswal Anil K
Department of Pharmacology and Experimental Therapeutics, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA.
J Biol Chem. 2010 Jul 9;285(28):21349-58. doi: 10.1074/jbc.M110.121863. Epub 2010 May 7.
The INrf2 (Keap1)/Cul3-Rbx1 complex constantly degrades Nrf2 under normal conditions. When a cell encounters oxidative or electrophilic stress, Nrf2 dissociates from the INrf2/Cul3-Rbx1 complex and translocates into the nucleus. In the nucleus, Nrf2 activates a myriad of antioxidant and defensive genes that protect cells. Nrf2 is then exported out of the nucleus and degraded. INrf2 serves as a substrate adaptor to link Nrf2 to Cul3 and Rbx1. Cul3 and Rbx1 make up the ubiquitin ligase complex that is responsible for the ubiquitination and degradation of Nrf2. Previously we have shown a feedback autoregulatory loop between Nrf2 and INrf2 indicating that Nrf2 regulates INrf2 by controlling its transcription. Here we are extending this research by demonstrating the presence of another feedback autoregulatory loop between Cul3-Rbx1 and Nrf2. Experiments using Hepa-1 and HepG2 cells indicate that Nrf2 controls its own degradation by regulating expression and induction of Cul3-Rbx1 genes. Treatment with the antioxidant tert-Butylhydroquinone (t-BHQ) leads to induction of Cul3-Rbx1 genes. Mutagenesis and transfection experiments identified an antioxidant response element in the forward and reverse strands of the proximal Cul3 and Rbx1 promoters, respectively, that Nrf2 binds and regulates expression and antioxidant induction of the Cul3-Rbx1 genes. In addition, short interfering RNA inhibition and overexpression of Nrf2 led to a respective decrease and increase in Cul3-Rbx1 gene expression. The increase in Cul3-Rbx1 leads to ubiquitination and degradation of Nrf2. These data suggest that Nrf2 regulates Cul3-Rbx1 by controlling regulation of expression and induction of Cul3-Rbx1. The induction of Cul3-Rbx1 control Nrf2 by increasing degradation.
在正常条件下，INrf2（Keap1）/Cul3-Rbx1复合物持续降解Nrf2。当细胞遭遇氧化或亲电应激时，Nrf2从INrf2/Cul3-Rbx1复合物上解离并转位进入细胞核。在细胞核中，Nrf2激活众多保护细胞的抗氧化和防御基因。随后Nrf2被输出细胞核并降解。INrf2作为底物衔接蛋白，将Nrf2与Cul3和Rbx1连接起来。Cul3和Rbx1组成泛素连接酶复合物，负责Nrf2的泛素化和降解。此前我们已证明Nrf2与INrf2之间存在反馈自调节环，表明Nrf2通过控制其转录来调节INrf2。在此，我们通过证明Cul3-Rbx1与Nrf2之间存在另一个反馈自调节环来扩展这项研究。使用Hepa-1和HepG2细胞进行的实验表明，Nrf2通过调节Cul3-Rbx1基因的表达和诱导来控制自身的降解。用抗氧化剂叔丁基对苯二酚（t-BHQ）处理会导致Cul3-Rbx1基因的诱导。诱变和转染实验分别在近端Cul3和Rbx1启动子的正向和反向链中鉴定出一个抗氧化反应元件，Nrf2与之结合并调节Cul3-Rbx1基因的表达和抗氧化诱导。此外，Nrf2的短发夹RNA抑制和过表达分别导致Cul3-Rbx1基因表达的降低和增加。Cul3-Rbx1的增加导致Nrf2的泛素化和降解。这些数据表明，Nrf2通过控制Cul3-Rbx1的表达调节和诱导来调节Cul；3-Rbx1。Cul3-Rbx1的诱导通过增加降解来控制Nrf2。
