Center for Molecular Medicine and Genetics, Wayne State University, Detroit, Michigan, USA.
Department of Biochemistry, Microbiology, and Immunology, Wayne State University, Detroit, Michigan, USA.
FASEB J. 2019 Dec;33(12):13503-13514. doi: 10.1096/fj.201901120R. Epub 2019 Sep 28.
Cytochrome (Cyt) is a multifunctional protein that operates as an electron carrier in the mitochondrial electron transport chain and plays a key role in apoptosis. We have previously shown that tissue-specific phosphorylations of Cyt in the heart, liver, and kidney play an important role in the regulation of cellular respiration and cell death. Here, we report that Cyt purified from mammalian brain is phosphorylated on S47 and that this phosphorylation is lost during ischemia. We have characterized the functional effects using phosphorylated Cyt purified from pig brain tissue and a recombinant phosphomimetic mutant (S47E). We crystallized S47E phosphomimetic Cyt at 1.55 Å and suggest that it spatially matches S47-phosphorylated Cyt, making it a good model system. Both S47-phosphorylated and phosphomimetic Cyt showed a lower oxygen consumption rate in reaction with isolated Cyt oxidase, which we propose maintains intermediate mitochondrial membrane potentials under physiologic conditions, thus minimizing production of reactive oxygen species. S47-phosphorylated and phosphomimetic Cyt showed lower caspase-3 activity. Furthermore, phosphomimetic Cyt had decreased cardiolipin peroxidase activity and is more stable in the presence of HO. Our data suggest that S47 phosphorylation of Cyt is tissue protective and promotes cell survival in the brain.-Kalpage, H. A., Vaishnav, A., Liu, J., Varughese, A., Wan, J., Turner, A. A., Ji, Q., Zurek, M. P., Kapralov, A. A., Kagan, V. E., Brunzelle, J. S., Recanati, M.-A., Grossman, L. I., Sanderson, T. H., Lee, I., Salomon, A. R., Edwards, B. F. P, Hüttemann, M. Serine-47 phosphorylation of cytochrome in the mammalian brain regulates cytochrome oxidase and caspase-3 activity.
细胞色素 (Cyt) 是一种多功能蛋白，作为线粒体电子传递链中的电子载体发挥作用，并在细胞凋亡中发挥关键作用。我们之前已经表明，心脏、肝脏和肾脏中 Cyt 的组织特异性磷酸化在调节细胞呼吸和细胞死亡方面起着重要作用。在这里，我们报告从哺乳动物大脑中纯化的 Cyt 在 S47 上发生磷酸化，并且这种磷酸化在缺血期间丢失。我们使用从猪脑组织中纯化的磷酸化 Cyt 和重组磷酸模拟突变体 (S47E) 来表征其功能影响。我们在 1.55 Å处结晶了 S47E 磷酸模拟 Cyt，并提出它在空间上与 S47 磷酸化 Cyt 匹配，使其成为一个很好的模型系统。S47 磷酸化 Cyt 和磷酸模拟 Cyt 与分离的 Cyt 氧化酶反应时的耗氧率均较低，我们提出在生理条件下，这会维持中间的线粒体膜电位，从而最小化活性氧的产生。S47 磷酸化 Cyt 和磷酸模拟 Cyt 的 caspase-3 活性均较低。此外，磷酸模拟 Cyt 的心磷脂过氧化物酶活性降低，并且在 HO 存在下更稳定。我们的数据表明，Cyt 的 S47 磷酸化具有组织保护作用，并促进大脑中的细胞存活。
