Center for Molecular Medicine and Genetics, Wayne State University, Detroit, MI, 48201, USA.
Department of Biochemistry, Microbiology and Immunology, Wayne State University, Detroit, MI, 48201, USA.
Sci Rep. 2019 Nov 1;9(1):15815. doi: 10.1038/s41598-019-52101-z.
Cytochrome c (Cytc) is a multifunctional protein, acting as an electron carrier in the electron transport chain (ETC), where it shuttles electrons from bc complex to cytochrome c oxidase (COX), and as a trigger of type II apoptosis when released from the mitochondria. We previously showed that Cytc is regulated in a highly tissue-specific manner: Cytc isolated from heart, liver, and kidney is phosphorylated on Y97, Y48, and T28, respectively. Here, we have analyzed the effect of a new Cytc phosphorylation site, threonine 58, which we mapped in rat kidney Cytc by mass spectrometry. We generated and overexpressed wild-type, phosphomimetic T58E, and two controls, T58A and T58I Cytc; the latter replacement is found in human and testis-specific Cytc. In vitro, COX activity, caspase-3 activity, and heme degradation in the presence of HO were decreased with phosphomimetic Cytc compared to wild-type. Cytc-knockout cells expressing T58E or T58I Cytc showed a reduction in intact cell respiration, mitochondrial membrane potential (∆Ψ), ROS production, and apoptotic activity compared to wild-type. We propose that, under physiological conditions, Cytc is phosphorylated, which controls mitochondrial respiration and apoptosis. Under conditions of stress Cytc phosphorylations are lost leading to maximal respiration rates, ∆Ψ hyperpolarization, ROS production, and apoptosis.
细胞色素 c（Cytc）是一种多功能蛋白，作为电子传递链（ETC）中的电子载体，在那里它将电子从 bc 复合物转移到细胞色素 c 氧化酶（COX），并在从线粒体释放时作为 II 型细胞凋亡的触发因子。我们之前表明，Cytc 以高度组织特异性的方式受到调节：从心脏、肝脏和肾脏中分离出的 Cytc 在 Y97、Y48 和 T28 上分别发生磷酸化。在这里，我们分析了新的 Cytc 磷酸化位点 threonine 58 的影响，我们通过质谱法在大鼠肾脏 Cytc 中映射了该位点。我们生成并过表达了野生型、磷酸模拟 T58E 以及两个对照，T58A 和 T58I Cytc；后者替换存在于人类和睾丸特异性 Cytc 中。在体外，与野生型相比，COX 活性、存在 HO 时的 caspase-3 活性和血红素降解在磷酸模拟 Cytc 中降低。与野生型相比，表达 T58E 或 T58I Cytc 的 Cytc 敲除细胞的完整细胞呼吸、线粒体膜电位（∆Ψ）、ROS 产生和凋亡活性降低。我们提出，在生理条件下，Cytc 发生磷酸化，从而控制线粒体呼吸和凋亡。在应激条件下，Cytc 磷酸化丢失导致最大呼吸速率、∆Ψ 超极化、ROS 产生和凋亡。
