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首页> 外文期刊>Genes to cells : >Nrf2 degron-fused reporter system: a new tool for specific evaluation of Nrf2 inducers
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Nrf2 degron-fused reporter system: a new tool for specific evaluation of Nrf2 inducers

机译：Nrf2 degron融合的报告系统：一种专门评估Nrf2诱导剂的新工具

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The transcription factor Nrf2 is degraded through the proteasome pathway, but is stabilized in response to oxidative and electrophilic stresses, and activates cytoprotective enzyme genes through binding to the antioxidant/electrophile response element (ARE/EpRE). Nrf2 inducers thus have considerable potential as therapeutic drugs. Although ARE-driven reporters are commonly employed to validate Nrf2 inducers, these reporters are relatively nonspecific. We have generated a new reporter, Nrf2d-LacZ, which may prove to be a better tool for validation of Nrf2 inducers. We made the Nrf2d-LacZ reporter by fusing the N-terminus of Nrf2 harboring a Neh2 degron to beta-galactosidase (LacZ), and compared its activity in immortalized mouse embryo fibroblasts (MEFs) with conventional ARE-luciferase (ARE-Luc) reporters in 293T cells, and in MEFs. Nrf2d-LacZ was degraded in unstressed conditions, but stabilized upon exposure to stresses. LacZ activity was induced by electrophiles in a dose-dependent manner, and the induction was detected much more rapidly compared with ARE-Luc. Nrf2d-LacZ was activated not only by electrophiles but also by a variety of other Nrf2 inducing stresses. Although ARE-Luc was activated by 12-O-Tetradecanoylphorbol 13-acetate in an Nrf2-independent manner, Nrf2d-LacZ was not activated by TPA, thus emphasizing the specificity of the Nrf2d-LacZ reporter system for validation of Nrf2 inducers.
机译：转录因子Nrf2通过蛋白酶体途径降解，但在氧化和亲电子应激中稳定下来，并通过与抗氧化剂/亲电子反应元件（ARE / EpRE）结合而激活细胞保护酶基因。因此，Nrf2诱导剂作为治疗药物具有相当大的潜力。尽管通常使用ARE驱动的报告子来验证Nrf2诱导剂，但这些报告子相对而言是非特异性的。我们生成了一个新的报道分子Nrf2d-LacZ，它可能被证明是验证Nrf2诱导剂的更好工具。我们通过将带有Neh2 degron的Nrf2的N末端与β-半乳糖苷酶（LacZ）融合来制作Nrf2d-LacZ报道基因，并将其在永生小鼠胚胎成纤维细胞（MEF）中的活性与常规ARE荧光素酶（ARE-Luc）报道基因进行比较在293T细胞和MEF中。 Nrf2d-LacZ在无压力的条件下降解，但在暴露于压力下稳定。亲电试剂以剂量依赖性方式诱导LacZ活性，并且与ARE-Luc相比，该诱导的检测速度要快得多。 Nrf2d-LacZ不仅被亲电子试剂激活，而且还被其他各种Nrf2诱导胁迫激活。尽管ARE-Luc被12-O-十四碳酰佛波醇13-乙酸酯以不依赖Nrf2的方式激活，但Nrf2d-LacZ未被TPA激活，因此强调了Nrf2d-LacZ报告系统对Nrf2诱导剂的验证的特异性。

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