Regulation of Respiration and Apoptosis by Cytochrome c Phosphorylation.

细胞色素 c 磷酸化对呼吸和细胞凋亡的调节。

• 批准号: 8306157
• 负责人: MAIK HUETTEMANN
• 金额: $ 28.59万
• 依托单位: WAYNE STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-08-01 至 2015-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8306157
• 关键词: Affect; Affinity; Amino Acids; Antibodies; Apoptosis; Apoptotic; Biological Assay; Biological Process; Cattle; Cell Line; Cell Respiration; Cells; Cellular Stress; Computing Methodologies; Crystallography; Data; Disease; Electron Transport; Electrons; Enzyme Kinetics; Epitopes; Equilibrium; Glutamates; Goals; Heart; Human; In Vitro; Knock-out; Laboratories; Lead; Liver; Malignant Neoplasms; Mass Spectrum Analysis; Measurement; Membrane Potentials; Metabolism; Methods; Mitochondria; Modeling; Molecular; Mutagenesis; Nerve Degeneration; Null Lymphocytes; Oxidation-Reduction; Oxygen; Oxygen Consumption; Pathway interactions; Phenylalanine; Phosphorylation; Phosphorylation Site; Phosphotransferases; Physiological; Play; Production; Protein Dephosphorylation; Protein Tyrosine Kinase; Proteins; Proteomics; Protocols documentation; Reaction; Reactive Oxygen Species; Regulation; Research; Respiration; Role; Signal Pathway; Signal Transduction; Site; Stress; Techniques; Testing; Tissues; Transgenic Organisms; Tyrosine; Tyrosine Phosphorylation; Variant; Water; X-Ray Crystallography; Yeasts; base; cytochrome c; cytochrome c oxidase; design; in vivo; mitochondrial membrane; mutant; overexpression; prevent; public health relevance; research study; respiration regulation; respiratory; response; yeast two hybrid system

DESCRIPTION (provided by applicant): We recently discovered that cytochrome c (Cytc) is directly regulated by cell signaling via tyrosine phosphorylation on two distinct sites in mammalian liver and heart, a finding made possible by isolation techniques developed by us that preserve the physiological phosphorylation status of the protein. It is our overall hypothesis that Cytc phosphorylation regulates the two main functions of Cytc, mitochondrial respiration and Cytc release during apoptosis. Our long term goal is to understand the effect of Cytc phosphorylation on respiration and apoptosis and to identify the cell signaling pathways directed to Cytc. Supported by preliminary data, three specific aims will be investigated: 1) to test the hypotheses that Cytc is phosphorylated in vivo in a distinct tissue-specific manner and that this phosphorylation results in structural changes of Cytc; 2) to test the hypotheses that phosphorylation of Cytc leads to controlled respiration and prevents its participation in apoptosis; and 3) to systematically identify mitochondrial tyrosine kinases that phosphorylate Cytc. Phosphorylated Cytc will be isolated from cow liver and heart tissue and structurally characterized using mass spectrometry and protein crystallography (Aim 1). Phosphorylated Cytc will be subjected to functional assays including in vitro respiration and apoptosis measurements, accompanied by mutagenesis studies in vivo with Cytc constructs in a Cytc knockout cell line (Aim 2). Experiments to identify kinases that act on Cytc will include a proteomic approach, kinase localization, and yeast two-hybrid studies (Aim 3). This research is expected to reveal that Cytc, long recognized as a central molecule in respiration and apoptosis, is subject to regulation by cell signaling, opening new opportunities for the understanding and control of those two key biological processes. PUBLIC HEALTH RELEVANCE: Our laboratory discovered that Cytochrome c (Cytc), which plays a key role in programmed cell death (apoptosis) and cellular respiration, is regulated by cell signaling networks that reversibly phosphorylate Cytc. In this study, we will analyze the structural changes brought about by phosphorylation, determine whether and to what extent phosphorylation affects the functions of Cytc in apoptosis and respiration, and identify the kinases that phosphorylate Cytc. Since apoptosis and cellular respiration are involved in many diseases such as cancer and neurodegeneration, this study is expected to lead to better understanding and control of such diseases.

描述（由申请人提供）：我们最近发现，细胞色素c（CYTC）通过酪氨酸磷酸化在哺乳动物肝脏和心脏的两个不同位点上直接受到细胞信号的调节，这一发现是由我们开发的隔离技术使蛋白质的生理磷酸化状态所开发的。我们的总体假设是，CYTC磷酸化调节凋亡过程中CYTC，线粒体呼吸和CYTC释放的两个主要功能。我们的长期目标是了解CYTC磷酸化对呼吸和凋亡的影响，并确定针对CYTC的细胞信号通路。在初步数据的支持下，将研究三个具体目标：1）测试CYTC以不同组织特异性方式磷酸化的假设，并且这种磷酸化会导致CYTC的结构变化； 2）检验的假设，即CyTC的磷酸化导致控制呼吸并防止其参与凋亡； 3）系统地识别磷酸化Cytc的线粒体酪氨酸激酶。磷酸化的CYTC将从牛肝和心脏组织中分离出来，并在结构上使用质谱和蛋白质晶体学（AIM 1）进行结构表征。磷酸化的CYTC将接受功能测定法，包括体外呼吸和凋亡测量结果，并伴有在CYTC敲除细胞系中的CYTC构建体的体内诱变研究（AIM 2）。鉴定在CYTC上起作用的激酶的实验将包括蛋白质组学方法，激酶定位和酵母两杂化研究（AIM 3）。预计这项研究将表明，长期以来被认为是呼吸和凋亡中的中央分子的CYTC受细胞信号的调节，为理解和控制这两个关键生物学过程的新机会提供了调节。 公共卫生相关性：我们的实验室发现，在程序性细胞死亡（凋亡）和细胞呼吸中起关键作用的细胞色素C（CYTC）受细胞信号网络的调节，这些细胞信号网络可逆地磷酸化CYTC。在这项研究中，我们将分析磷酸化带来的结构变化，确定磷酸化是否以及在何种程度上影响CYTC在凋亡和呼吸中的功能，并确定磷酸化CYTC的激酶。由于凋亡和细胞呼吸涉及许多疾病，例如癌症和神经变性，因此这项研究有望使人们更好地理解和控制此类疾病。