Control of Cell Respiration and Apoptosis by Phosphorylation of Cytochrome c

通过细胞色素 c 磷酸化控制细胞呼吸和凋亡

• 批准号: 9982332
• 负责人: MAIK HUETTEMANN
• 金额: $ 35.37万
• 依托单位: WAYNE STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-08 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9982332
• 关键词: Affect; Affinity Chromatography; Amino Acids; Animals; Apoptosis; Applications Grants; Bacteria; Binding; Candidate Disease Gene; Cattle; Cell Death; Cell Line; Cell Respiration; Cells; Cellular Stress; Cessation of life; Crystallography; Cytoprotection; Data; Diabetes Mellitus; Disease; Electron Transport; Electrons; Functional disorder; Generations; Glutamates; Goals; Heart; In Vitro; Kidney; Knock-out; Knockout Mice; Laboratories; Lead; Left; Life; Liver; Malignant Neoplasms; Maps; Mass Spectrum Analysis; Measurement; Mediating; Membrane Potentials; Metabolic; Methods; Mitochondria; Mitochondrial Proteins; Modeling; Modification; Mus; Nerve Degeneration; Null Lymphocytes; Organ; Oxidative Phosphorylation; Oxygen; Pathologic; Pathway interactions; Pharmacology; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphorylation Site; Phosphotransferases; Physiological; Play; Positioning Attribute; Post-Translational Protein Processing; Process; Production; Property; Protein Dephosphorylation; Proteins; Protocols documentation; Rattus; Reaction; Reactive Oxygen Species; Regulation; Research; Respiration; Role; Signal Pathway; Signal Transduction; Signaling Molecule; Stress; Structure; Techniques; Testing; Tissues; Transgenic Mice; Transgenic Organisms; Tyrosine; Variant; Water; Work; X-Ray Crystallography; adenylate kinase; apoptotic protease-activating factor 1; base; cytochrome c; cytochrome c oxidase; experience; experimental study; genetic approach; in vivo; mitochondrial membrane; mutant; novel; overexpression; prevent; stress state

SUMMARY Cytochrome c (Cytc) plays a central role in mitochondrial respiration and type 2 apoptosis. We have developed new protocols to purify mitochondrial proteins while maintaining their physiological regulatory properties and posttranslational modifications, and we discovered that Cytc is phosphorylated on distinct tyrosine residues in heart and liver tissue. Recently, we have mapped two more phosphorylation sites on Cytc purified from cow and rat kidneys, Thr28 and Ser47, which are the primary focus of this grant application. Supported by strong preliminary data it is our overall hypothesis that Cytc phosphorylation regulates the two main functions of Cytc, mitochondrial respiration and apoptosis. Our long term goal is to understand the regulation of Cytc by cell signaling pathways under normal and pathological conditions. As a first step towards our goal we will test four specific hypotheses: 1) to test the hypothesis that kidney Cytc can be phosphorylated on Thr28 and Ser47, and that these modifications affect the basic properties of the molecule; 2) to test the hypothesis that Cytc Thr28 and Ser47 phosphorylation affects mitochondrial respiration and apoptosis; 3) to identify kinases and phosphatases that control Cytc phosphorylation; and 4) to demonstrate the physiological effect in mice. Phosphorylated Cytc will be isolated from cow and rat kidneys and phosphomimetic mutant Cytc will be overexpressed and purified from bacteria, followed by structural characterization using mass spectrometry, spectroscopic methods, and protein crystallography (Aim 1). Phosphorylated and phosphomimetic mutant Cytc will be subjected to a comprehensive set of functional analyses including in vitro respiration and apoptosis measurements, and by in vivo studies with phosphomimetic Cytc stably expressed in Cytc knockout cells (Aim 2). We will next lay the ground work to explore the signaling pathways and identify kinases and phosphatases that act on Cytc using affinity purification/mass spectrometry techniques (Aim 3). Finally, we will reintroduce phosphomimetic and non-phosphorylatable mutant Cytc into Cytc knockout mice to study the effect of Cytc modification and regulation at the animal level (Aim 4). We expect that this research will reveal that Cytc, a protein that makes life and death decisions, is subject to regulation by cell signaling, opening new opportunities for the understanding of mitochondrial respiration and apoptosis, and to control it in pathological conditions in which respiration and apoptosis are dysregulated.

摘要 细胞色素c(Cytc)在线粒体呼吸和2型呼吸中起核心作用。 细胞凋亡。我们开发了新的方案来纯化线粒体蛋白，同时 维持它们的生理调节特性和翻译后修饰， 我们发现Cytc在心脏和心脏中不同的酪氨酸残基上被磷酸化 肝组织。最近，我们在纯化的Cytc上又定位了两个磷酸化位点 来自牛和大鼠的肾脏，Thr28和Ser47，这是这笔赠款的主要重点 申请。在强劲的初步数据支持下，我们的总体假设是Cytc 磷酸化调节Cytc的两个主要功能，线粒体呼吸和 细胞凋亡。我们的长期目标是了解细胞信号对Cytc的调控。 正常和病理条件下的通路。作为迈向目标的第一步，我们 将测试四个具体的假设：1)测试肾脏细胞可以 Thr28和Ser47上的磷酸化，这些修饰会影响基本的 分子的性质；2)检验Cytc Thr28和Ser47假设 磷酸化影响线粒体呼吸和细胞凋亡；3)识别激酶 和控制Cytc磷酸化的磷酸酶；以及4)展示 对小鼠的生理影响。将从牛和大鼠中分离出磷酸化的Cytc 肾脏和仿磷突变体Cytc将从 细菌，然后使用质谱学进行结构表征，光谱 方法和蛋白质结晶学(目标1)。磷酸化和仿磷化 突变的Cytc将接受一套全面的功能分析，包括 体外呼吸和细胞凋亡的测量，并通过体内研究 拟磷脂酶C在Cytc基因敲除细胞中稳定表达(目标2)。我们接下来要做的是 基础工作是探索信号通路并识别激酶和 利用亲和纯化/质谱学技术研究作用于细胞色素C的磷酸酶 (目标3)。最后，我们将重新介绍拟磷化和非磷酸化突变体 将Cytc导入Cytc基因敲除小鼠，研究Cytc的修饰和调控作用 动物层面(目标4)。我们预计这项研究将揭示细胞色素C，一种可以 做出生死决定，受制于细胞信号的调节，打开新的 了解线粒体呼吸和细胞凋亡的机会，以及 在呼吸和细胞凋亡失调的病理条件下控制它。

项目成果

High Sucrose Diet-Induced Subunit I Tyrosine 304 Phosphorylation of Cytochrome c Oxidase Leads to Liver Mitochondrial Respiratory Dysfunction in the Cohen Diabetic Rat Model.

• DOI: 10.3390/antiox13010019
• 发表时间: 2023-12-21
• 期刊: Antioxidants (Basel, Switzerland)