Regulation of Respiration and Apoptosis by Cytochrome c Phosphorylation.

描述（由申请人提供）：我们最近发现细胞色素c（Cytc）通过哺乳动物肝脏和心脏中两个不同位点上的酪氨酸磷酸化直接受细胞信号传导调节，这一发现是通过我们开发的保留蛋白质生理磷酸化状态的分离技术实现的。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过程中的Cytc释放。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）检验Cytc在体内以独特的组织特异性方式磷酸化并且这种磷酸化导致Cytc结构变化的假设; 2）检验Cytc磷酸化导致呼吸控制并阻止其参与凋亡的假设;和3）系统地鉴定使Cytc磷酸化的线粒体酪氨酸激酶。将从牛肝脏和心脏组织中分离磷酸化Cytc，并使用质谱和蛋白质晶体学进行结构表征（Aim 1）。将对磷酸化的Cytc进行功能测定，包括体外呼吸和细胞凋亡测量，同时在Cytc敲除细胞系中使用Cytc构建体进行体内诱变研究（Aim 2）。实验，以确定激酶的细胞色素将包括蛋白质组学的方法，激酶定位，酵母双杂交研究（目标3）。这项研究有望揭示，长期以来被认为是呼吸和凋亡中的中心分子的Cytc受到细胞信号传导的调节，为理解和控制这两个关键的生物过程开辟了新的机会。 公共卫生相关性：我们的实验室发现，细胞色素c（Cytc），这在程序性细胞死亡（凋亡）和细胞呼吸中起着关键作用，是由细胞信号网络，可逆磷酸化Cytc调节。在这项研究中，我们将分析磷酸化所带来的结构变化，确定磷酸化是否以及在多大程度上影响细胞凋亡和呼吸的功能，并确定磷酸化细胞色素c的激酶。由于细胞凋亡和细胞呼吸参与许多疾病，如癌症和神经退行性疾病，这项研究有望导致更好地理解和控制这些疾病。

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.