Graduate Program in Pharmaceutical Science, Center for Cancer Prevention Research, Ernest-Mario School of Pharmacy, Rutgers, The State University of New Jersey, Piscataway, NJ 08854, USA.
Antioxid Redox Signal. 2013 Jul 10;19(2):89-101. doi: 10.1089/ars.2012.4586. Epub 2012 Sep 17.
Nuclear factor-erythroid-related factor 2 (Nrf2) is a critical transcriptional factor that is used in regulating cellular defense against oxidative stress. This study is aimed at investigating new interacting protein partners of Nrf2 using One-strep tag pull-down coupled with LTQ Orbitrap LC/MS/MS, and at examining the impact on Nr2 signaling by the newly identified IQ motif containing GTPase activating protein 1 (IQGAP1).
Using the One-strep tag pull-down and LTQ Orbitrap LC/MS/MS, we identified IQGAP1 as a new Nrf2 interacting partner. Direct interactions between IQGAP1 and Nrf2 proteins were verified using in vitro glutathione S-transferase (GST) pull-down, transcription/translation assays, and in vivo utilizing Nrf2 overexpressing cells. Coexpression of Dsredmono-IQGAP1 and eGFP-Nrf2 increased the stability of eGFP-Nrf2 and enhanced the expression of Nrf2-target gene heme oxygenase-1 (HO-1). To confirm the functional role of IQGAP1 on Nrf2, knock-downed IQGAP1 using siIQGAP1 attenuated the expression of endogenous Nrf2, HO-1 proteins, and Nrf2-target genes GSTpi, GCLC, and
NAD(P)H: quinone oxidoreductase 1 (NQO-1). Furthermore, the stability of Nrf2 was dramatically decreased in IQGAP1-deficient mouse embryonic fibroblast (MEF) cells. Since IQGAP1 signaling could be mediated by calcium, treating the cells with calcium showed the translocation of IQGAP1/Nrf2 complex into the nucleus, suggesting that IQGAP1 may play a critical role in Nrf2 stability. Interestingly, consistent with calcium signaling for IQGAP1, treating the cells with calcium functionally enhanced Nrf2-mediated antioxidant responsive element-transcription activity and enhanced the expression of the endogenous Nrf2-target gene HO-1.
In the aggregate, our current study identifies and functionally characterizes a new Nrf2 partner protein IQGAP1, which may contribute to Nrf2's regulation of antioxidant enzymes such as HO-1.
IQGAP1 may play a critical role in the stability and transactivation of Nrf2.
核因子-红细胞相关因子 2(Nrf2)是一种关键的转录因子,用于调节细胞对氧化应激的防御。本研究旨在使用 One-strep 标签下拉结合 LTQ Orbitrap LC/MS/MS 来鉴定 Nrf2 的新相互作用蛋白伴侣,并研究新鉴定的含 IQ 基序的 GTP 酶激活蛋白 1(IQGAP1)对 Nr2 信号的影响。
使用 One-strep 标签下拉和 LTQ Orbitrap LC/MS/MS,我们鉴定出 IQGAP1 是 Nrf2 的一个新的相互作用伙伴。使用体外谷胱甘肽 S-转移酶(GST)下拉、转录/翻译测定以及利用 Nrf2 过表达细胞进行体内实验,验证了 IQGAP1 与 Nrf2 蛋白之间的直接相互作用。Dsredmono-IQGAP1 和 eGFP-Nrf2 的共表达增加了 eGFP-Nrf2 的稳定性,并增强了 Nrf2 靶基因血红素加氧酶-1(HO-1)的表达。为了确认 IQGAP1 在 Nrf2 上的功能作用,使用 siIQGAP1 敲低 IQGAP1 减弱了内源性 Nrf2、HO-1 蛋白和 Nrf2 靶基因 GSTpi、GCLC 和 NAD(P)H:醌氧化还原酶 1(NQO-1)的表达。此外,IQGAP1 缺陷型小鼠胚胎成纤维细胞(MEF)中 Nrf2 的稳定性显著降低。由于 IQGAP1 信号可以通过钙介导,用钙处理细胞显示 IQGAP1/Nrf2 复合物转位到核内,表明 IQGAP1 可能在 Nrf2 稳定性中起关键作用。有趣的是,与 IQGAP1 的钙信号一致,用钙处理细胞在功能上增强了 Nrf2 介导的抗氧化反应元件转录活性,并增强了内源性 Nrf2 靶基因 HO-1 的表达。
总的来说,我们当前的研究鉴定并功能表征了 Nrf2 的一个新的伴侣蛋白 IQGAP1,它可能有助于 Nrf2 对抗氧化酶如 HO-1 的调节。
IQGAP1 可能在 Nrf2 的稳定性和反式激活中起关键作用。