We previously reported that serine-47 (S47) phosphorylation of cytochrome c (Cytc) in the brain results in lower cytochrome c oxidase (COX) activity and caspase-3 activity in vitro. We here analyze the effect of S47 modification in fibroblast cell lines stably expressing S47E phosphomimetic Cytc, unphosphorylated WT, or S47A Cytc. Our results show that S47E Cytc results in partial inhibition of mitochondrial respiration corresponding with lower mitochondrial membrane potentials (ΔΨ_m) and reduced reactive oxygen species (ROS) production. When exposed to an oxygen-glucose deprivation/reoxygenation (OGD/R) model simulating ischemia/reperfusion injury, the Cytc S47E phosphomimetic cell line showed minimal ROS generation compared to the unphosphorylated WT Cytc cell line that generated high levels of ROS upon reoxygenation. Consequently, the S47E Cytc cell line also resulted in significantly lower cell death upon exposure to OGD/R, confirming the cytoprotective role of S47 phosphorylation of Cytc. S47E Cytc also resulted in lower cell death upon H₂O₂ treatment. Finally, we propose that pro-survival kinase Akt (protein kinase B) is a likely mediator of the S47 phosphorylation of Cytc in the brain. Akt inhibitor wortmannin abolished S47 phosphorylation of Cytc, while the Akt activator SC79 maintained S47 phosphorylation of Cytc. Overall, our results suggest that loss of S47 phosphorylation of Cytc during brain ischemia drives reperfusion injury through maximal electron transport chain flux, ΔΨ_m hyperpolarization, and ROS-triggered cell death.

中文翻译：

---

细胞色素 c 的脑特异性丝氨酸 47 修饰调节细胞色素 c 氧化酶活性，减弱 ROS 产生和细胞死亡：对缺血/再灌注损伤和 Akt 信号传导的影响。

我们之前报道过，大脑中细胞色素c (Cyt c )的丝氨酸 47 (S47) 磷酸化会导致体外细胞色素c氧化酶 (COX) 活性和 caspase-3 活性降低。我们在此分析了 S47 修饰对稳定表达 S47E 磷酸模拟 Cyt c、未磷酸化 WT 或 S47A Cyt c的成纤维细胞系的影响。我们的结果表明，S47E Cyt c会部分抑制线粒体呼吸，从而导致线粒体膜电位 (ΔΨ _m ) 降低和活性氧 (ROS) 产生减少。当暴露于模拟缺血/再灌注损伤的氧糖剥夺/复氧 (OGD/R) 模型时，与在复氧时产生高水平 ROS 的未磷酸化 WT Cyt c细胞系相比，Cyt c S47E 拟磷酸细胞系显示出最少的 ROS 生成。因此，S47E Cyt c细胞系在暴露于 OGD/R 后也导致细胞死亡显着降低，证实了 Cyt c的 S47 磷酸化的细胞保护作用。S47E Cyt c在 H ₂ O ₂处理后也导致较低的细胞死亡。最后，我们提出促生存激酶 Akt（蛋白激酶 B）可能是大脑中Cyt c的 S47 磷酸化的介质。Akt 抑制剂渥曼青霉素消除了 Cyt c的 S47 磷酸化，而 Akt 激活剂 SC79 则维持了 Cyt c的 S47 磷酸化。总体而言，我们的结果表明，脑缺血期间 Cyt c的 S47 磷酸化缺失通过最大电子传递链通量、ΔΨ _m超极化和 ROS 触发的细胞死亡来驱动再灌注损伤。