Molecular Mechanisms of Mitochondrial Ca2+ Transport

线粒体 Ca2 运输的分子机制

• 批准号: 9000157
• 负责人: Gyorgy Hajnoczky
• 金额: $ 35.35万
• 依托单位: THOMAS JEFFERSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-02-01 至 2017-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9000157
• 关键词: Affect; Affinity; Amino Acids; Animal Model; Binding; Bioenergetics; C-terminal; Calcium; Calcium Oscillations; Calcium Signaling; Cell Death; Cell Respiration; Cell Survival; Cells; Cellular Metabolic Process; Cellular Stress Response; Charge; Complement; Complex; Development; Disease; Dominant-Negative Mutation; Drug Targeting; EF Hand Motifs; EF-Hand Domain; Employee Strikes; Energy Metabolism; Evolution; Fasting; Hepatic; Hepatocyte; Hereditary Disease; Homeostasis; Human; Injection of therapeutic agent; Injury; Inner mitochondrial membrane; Ischemia; Knockout Mice; Link; Liver; Measurement; Mediating; Methods; Mitochondria; Modeling; Molecular; Mouse Cell Line; Mus; Mutation; Myopathy; Nerve Degeneration; Neurons; Paper; Pathway interactions; Phenotype; Physiological; Physiology; Protein Isoforms; Proteins; Publications; Publishing; Regulation; Reperfusion Therapy; Reporting; Role; Signal Transduction; Site; Stress; System; Testing; Time; Tissues; VDAC1 gene; Virus; Work; base; calcium uniporter; cell injury; driving force; feeding; fluorescence imaging; human disease; in vivo; new therapeutic target; novel; paralogous gene; uptake

Mitochondrial Ca2+ handling is central to cellular calcium homeostasis, energy metabolism and cell survival/cell death mechanisms. Impaired mitochondrial Ca2+ transport is also thought to be an important contributor in the development of a variety of diseases. After decades of frustrating ambiguity about the molecules mediating mitochondrial Ca2+ uptake, in 2010-2011, MICU1, an EF-hand domain protein, and MCU were identified as an indispensible regulator and pore forming component of the Ca2+ uniporter, respectively. Now, it is becoming possible to tackle the fundamental questions about the molecular mechanisms that underlie a sophisticated Ca2+ transport system and the physiological significance of MICU1, MCU and other uniporter components. We have started to investigate the mode of action of MICU1, and contrary to the first report, we found that MICU1 is not required to allow Ca2+ uptake through the uniporter but, instead, is critical to keep the uniporter closed at low cytoplasmic [Ca2+] ([Ca2+]c) concentrations and to support cooperative activation of the uniporter as the [Ca2+]c increases. These studies have been recently published in a widely recognized paper in Cell Metabolism. To further the study of the physiological significance of MICU1 we have generated a conditional MICU1 knockout mouse that has a striking phenotype and will provide several models for the proposed study. Our hypotheses are that (1) MICU1 employs its C-terminal cluster of positively charged amino acid residues to interact with the DIME domain of the intermembrane space loop of MCU, highlighting a site that might be a novel drug target, (2) MICU1 and MICU2 differently bind Ca2+ and/or Mg2+ with their EF-hands, providing a possible mechanisms for their distinct effects on the cooperative activation of the MCU, and (3) MICU1 is required in vivo for effective decoding of calcium signals in terms of oxidative metabolism in hepatocytes under fed and fasting conditions. To explore the role of MICU1 in Ca2+ handling we will apply advanced fluorescence imaging approaches, including some novel methods combined with assessment of cell bioenergetics and the activity of cell survival regulating pathways in the same models. The proposal relies on the use of a powerful targeting system of MICU1 in both cell lines and mouse tissues. The proposed work will uncover the mechanism of the sophisticated Ca2+- and time-dependent control of the uniporter and will expose MICU1's role in keeping mitochondria healthy and responsive to Ca2+.

线粒体Ca 2+处理是细胞钙稳态、能量代谢和细胞存活/细胞的核心 死亡机制受损的线粒体Ca 2+转运也被认为是一个重要的贡献者， 各种疾病的发展。经过几十年令人沮丧的关于分子介导的模糊性， 线粒体Ca 2+摄取，在2010-2011年，MICU 1，EF-手结构域蛋白，和MCU被确定为一个 Ca 2+单向转运体的不可缺少的调节剂和成孔组分。现在，它正在成为 有可能解决关于复杂的生物学行为背后的分子机制的基本问题， 钙转运系统及MICU 1、MCU等单向转运体组分的生理意义。我们 我已经开始调查MICU 1的行动方式，与第一次报告相反，我们发现MICU 1 不需要允许Ca 2+通过单向转运体摄取，但相反，对于保持单向转运体关闭至关重要。 低细胞质[Ca 2 +]（[Ca 2 +]c）浓度，并支持协同激活单向转运蛋白， [Ca2[C]上升。这些研究最近发表在《细胞》杂志上一篇广受认可的论文中 新陈代谢.为了进一步研究MICU 1的生理学意义，我们产生了一个条件性的 MICU 1基因敲除小鼠具有显著的表型，将为拟议的研究提供几种模型。 我们的假设是：（1）MICU 1利用其C-末端带正电荷的氨基酸残基簇， 与MCU的膜间空间环的DIME结构域相互作用，突出显示可能是一个 新的药物靶点，（2）MICU 1和MICU 2以其EF-手不同地结合Ca 2+和/或Mg 2+， 它们对MCU的协同激活的不同影响的可能机制，以及（3）MICU 1是 在肝细胞氧化代谢方面，在体内有效解码钙信号所需的 进食和禁食条件。为了探索MICU 1在Ca 2+处理中的作用，我们将应用先进的 荧光成像方法，包括一些新的方法结合评估细胞 生物能量学和细胞存活调节途径的活性。该提案依赖于 在细胞系和小鼠组织中使用MICU 1的强大靶向系统。拟议的工作将 揭示复杂的Ca 2+和时间依赖性控制的uniporter的机制，并将暴露 MICU 1在保持线粒体健康和对Ca 2+反应中的作用。