Regulation of Respiration and Apoptosis by Cytochrome c Phosphorylation.

描述(申请人提供)：我们最近发现，细胞色素c(Cytc)通过在哺乳动物肝脏和心脏的两个不同位置上的酪氨酸磷酸化而受到细胞信号的直接调节，这一发现是由我们开发的保持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的磷酸化调节了Cytc在细胞凋亡过程中的两个主要功能，即线粒体呼吸和Cytc的释放。我们的长期目标是了解Cytc磷酸化对呼吸和细胞凋亡的影响，并确定针对Cytc的细胞信号通路。在初步数据的支持下，将研究三个特定的目标：1)测试Cytc在体内以不同的组织特异性方式被磷酸化的假说，以及这种磷酸化导致Cytc结构变化的假说；2)测试Cytc的磷酸化导致呼吸受控并阻止其参与细胞凋亡的假说；以及3)系统地鉴定使Cytc磷酸化的线粒体酪氨酸激酶。将从奶牛的肝脏和心脏组织中分离出磷酸化的Cytc，并使用质谱学和蛋白质结晶学对其结构进行表征(目标1)。磷酸化的Cytc将接受功能分析，包括体外呼吸和细胞凋亡测量，并伴随着Cytc基因敲除细胞系的Cytc结构的体内突变研究(AIM 2)。识别作用于细胞色素C的激酶的实验将包括蛋白质组学方法、激酶定位和酵母双杂交研究(目标3)。这项研究有望揭示Cytc，长期以来被认为是呼吸和细胞凋亡的中心分子，受到细胞信号的调节，为理解和控制这两个关键的生物学过程开辟了新的机会。 与公共健康相关：我们的实验室发现，细胞色素c(Cytc)在细胞程序性死亡(细胞凋亡)和细胞呼吸中发挥关键作用，它受到细胞信号网络的调节，细胞信号网络可逆地使Cytc磷酸化。在本研究中，我们将分析磷酸化带来的结构变化，确定磷酸化是否影响Cytc在细胞凋亡和呼吸中的功能，以及在多大程度上影响Cytc的功能，并确定使Cytc磷酸化的激酶。由于细胞凋亡和细胞呼吸参与了许多疾病，如癌症和神经退行性变，这项研究有望有助于更好地了解和控制这些疾病。

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.