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

基本信息

批准号：
9236889
负责人：
MAIK HUETTEMANN
金额：
$ 36.74万
依托单位：
WAYNE STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-09-08 至 2021-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9236889
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 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

项目摘要

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.

概括细胞色素 c (Cytc) 在线粒体呼吸和 2 型呼吸中发挥核心作用细胞凋亡。我们开发了新的方案来纯化线粒体蛋白，同时维持其生理调节特性和翻译后修饰，我们发现 Cytc 在心脏和心脏中的不同酪氨酸残基上被磷酸化肝组织。最近，我们在纯化的 Cytc 上绘制了另外两个磷酸化位点来自牛和大鼠肾脏的 Thr28 和 Ser47，这是本次资助的主要重点应用。有强有力的初步数据支持，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传导对 Cytc 的调节正常和病理条件下的途径。作为实现我们目标的第一步，我们将检验四个具体假设：1）检验肾脏 Cytc 可以被 Thr28 和 Ser47 被磷酸化，这些修饰会影响基本的分子的性质； 2) 检验 Cytc Thr28 和 Ser47 的假设磷酸化影响线粒体呼吸和细胞凋亡； 3) 鉴定激酶和控制 Cytc 磷酸化的磷酸酶； 4) 展示对小鼠的生理作用。将从牛和大鼠中分离磷酸化的 Cytc 肾脏和拟磷突变体 Cytc 将从中过度表达和纯化细菌，然后使用质谱、光谱进行结构表征方法和蛋白质晶体学（目标 1）。磷酸化和拟磷化突变体 Cytc 将接受一系列全面的功能分析，包括体外呼吸和细胞凋亡测量，以及通过体内研究拟磷化 Cytc 在 Cytc 敲除细胞中稳定表达（目标 2）。接下来我们将铺设探索信号通路和识别激酶的基础工作使用亲和纯化/质谱技术作用于 Cytc 的磷酸酶（目标 3）。最后，我们将重新引入拟磷酸化和不可磷酸化的突变体将 Cytc 转入 Cytc 敲除小鼠中，研究 Cytc 修饰和调节的效果动物水平（目标 4）。我们预计这项研究将揭示 Cytc，一种蛋白质，做出生死决定，受到细胞信号传导的调节，开启新的了解线粒体呼吸和细胞凋亡的机会，并在呼吸和细胞凋亡失调的病理条件下控制它。