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型中起着核心作用凋亡。我们已经开发了新方案来纯化线粒体蛋白，而保持其生理调节特性和翻译后修饰，我们发现CYTC在心脏中不同的酪氨酸残基上磷酸化肝组织。最近，我们在纯化的CytC上绘制了两个磷酸化位点来自牛和老鼠肾脏，Thr28和Ser47，这是这笔赠款的主要重点应用。在强大的初步数据的支持下，我们的总体假设是CYTC 磷酸化调节CYTC的两个主要功能，线粒体呼吸和凋亡。我们的长期目标是通过细胞信号了解CYTC的调节在正常和病理条件下的途径。作为朝着我们目标的第一步将检验四个特定假设：1）检验肾脏CYTC的假设在Thr28和Ser47上磷酸化，并且这些修饰会影响基本分子的特性； 2）检验CYTC THR28和SER47的假设磷酸化会影响线粒体呼吸和凋亡。 3）识别激酶和控制CYTC磷酸化的磷酸酶； 4）证明小鼠的生理作用。磷酸化的CYTC将从牛和大鼠中分离出来肾脏和磷酸化突变体CYTC将过表达并从中纯化细菌，然后使用质谱，光谱法进行结构表征方法和蛋白质晶体学（AIM 1）。磷酸化和磷酸化突变体CYTC将进行一系列全面的功能分析体外呼吸和凋亡测量以及通过体内研究在CYTC基因敲除细胞中稳定表达的磷酸化CYTC（AIM 2）。我们接下来要躺探索信号通路并识别激酶的地面工作使用亲和力纯化/质谱技术对CYTC作用的磷酸酶（目标3）。最后，我们将重新引入磷酸化和非磷酸化突变体 CYTC进入CYTC敲除小鼠，以研究CYTC修饰和调节的影响动物级别（目标4）。我们希望这项研究将揭示CYTC，一种蛋白质做出生命和死亡决策，受细胞信号的调节，开放新的理解线粒体呼吸和凋亡的机会，以及在病理条件下控制呼吸和凋亡失调的病理条件。