Department of Cellular & Integrative Physiology, University of Nebraska Medical Center, Omaha, NE, USA.
Mass Spectrometry & Proteomics Core, University of Nebraska Medical Center, Omaha, NE, USA.
J Physiol. 2020 Dec;598(23):5427-5451. doi: 10.1113/JP280176. Epub 2020 Sep 23.
Nrf2 is a master regulator of endogenous cellular defences, governing the expression of more than 200 cytoprotective proteins, including a panel of antioxidant enzymes. Nrf2 plays an important role in redox haemostasis of skeletal muscle in response to the increased generation of reactive oxygen species during contraction. Employing skeletal muscle-specific transgenic mouse models with unbiased-omic approaches, we uncovered new target proteins, downstream pathways and molecular networks of Nrf2 in skeletal muscle following Nrf2 or Keap1 deletion. Based on the findings, we proposed a two-way model to understand Nrf2 function: a tonic effect through a Keap1-independent mechanism under basal conditions and an induced effect through a Keap1-dependent mechanism in response to oxidative and other stresses.
Although Nrf2 has been recognized as a master regulator of cytoprotection, its functional significance remains to be completely defined. We hypothesized that proteomic/bioinformatic analyses from Nrf2-deficient or overexpressed skeletal muscle tissues will provide a broader spectrum of Nrf2 targets and downstream pathways than are currently known. To this end, we created two transgenic mouse models; the iMS-Nrf2 and iMS-Keap1 , employing which we demonstrated that selective deletion of skeletal muscle Nrf2 or Keap1 separately impaired or improved skeletal muscle function. Mass spectrometry revealed that Nrf2-KO changed expression of 114 proteins while Keap1-KO changed expression of 117 proteins with 10 proteins in common between the groups. Gene ontology analysis suggested that Nrf2 KO-changed proteins are involved in metabolism of oxidoreduction coenzymes, purine ribonucleoside triphosphate, ATP and propanoate, which are considered as the basal function of Nrf2, while Keap1 KO-changed proteins are involved in cellular detoxification, NADP metabolism, glutathione metabolism and the electron transport chain, which belong to the induced effect of Nrf2. Canonical pathway analysis suggested that Keap1-KO activated four pathways, whereas Nrf2-KO did not. Ingenuity pathway analysis further revealed that Nrf2-KO and Keap1-KO impacted different signal proteins and functions. Finally, we validated the proteomic and bioinformatics data by analysing glutathione metabolism and mitochondrial function. In conclusion, we found that Nrf2-targeted proteins are assigned to two groups: one mediates the tonic effects evoked by a low level of Nrf2 at basal condition; the other is responsible for the inducible effects evoked by a surge of Nrf2 that is dependent on a Keap1 mechanism.
Nrf2 是内源性细胞防御的主要调节因子，调控着 200 多种细胞保护蛋白的表达，包括一组抗氧化酶。Nrf2 在骨骼肌的氧化还原稳态中发挥重要作用，以应对收缩过程中活性氧的产生增加。我们利用骨骼肌特异性转基因小鼠模型和无偏倚组学方法，在 Nrf2 或 Keap1 缺失后，揭示了骨骼肌中 Nrf2 的新靶蛋白、下游途径和分子网络。基于这些发现，我们提出了一个双向模型来理解 Nrf2 的功能：在基础条件下通过 Keap1 非依赖性机制产生的基础效应，以及在应对氧化和其他应激时通过 Keap1 依赖性机制产生的诱导效应。
尽管 Nrf2 已被认为是细胞保护的主要调节因子，但它的功能意义仍有待完全确定。我们假设，来自 Nrf2 缺失或过表达骨骼肌组织的蛋白质组/生物信息学分析将提供比目前已知的更广泛的 Nrf2 靶标和下游途径。为此，我们创建了两个转基因小鼠模型；iMS-Nrf2 和 iMS-Keap1，利用这些模型，我们证明了骨骼肌 Nrf2 或 Keap1 的选择性缺失分别损害或改善了骨骼肌功能。质谱分析显示，Nrf2-KO 改变了 114 种蛋白质的表达，而 Keap1-KO 改变了 117 种蛋白质的表达，两组之间有 10 种蛋白质相同。基因本体分析表明，Nrf2 KO 改变的蛋白质参与氧化还原辅酶、嘌呤核糖核苷酸三磷酸、ATP 和丙酸盐的代谢，这些被认为是 Nrf2 的基础功能，而 Keap1 KO 改变的蛋白质参与细胞解毒、NADP 代谢、谷胱甘肽代谢和电子传递链，属于 Nrf2 的诱导效应。经典途径分析表明，Keap1-KO 激活了四条途径，而 Nrf2-KO 没有。通路分析进一步表明，Nrf2-KO 和 Keap1-KO 影响了不同的信号蛋白和功能。最后，我们通过分析谷胱甘肽代谢和线粒体功能来验证蛋白质组学和生物信息学数据。总之，我们发现 Nrf2 靶向蛋白分为两组：一组介导 Nrf2 在基础条件下低水平产生的基础效应；另一组负责由 Keap1 机制依赖的 Nrf2 激增产生的诱导效应。
