Control Of Cellular Energy Metabolism

细胞能量代谢的控制

• 批准号: 8939787
• 负责人: Robert Balaban
• 金额: $ 132.8万
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8939787
• 关键词: Acute; Bacteria; Behavior; Biological; Biological Models; Calcium; Cardiac; Cells; Complex; Creatine; Cyclic AMP; Cytochromes; DNA; Data; Diffusion; Energy Metabolism; Enzymes; Goals; Growth; Heart; Hypoxia; Investigation; Ischemia; Knock-out; Laboratories; Laboratory Study; Lead; Mammals; Metabolic; Metabolic Activation; Methods; Mitochondria; Mitochondrial Matrix; Modeling; Monitor; Mus; Myoglobin; NADH; Optics; Organelles; Oryctolagus cuniculus; Oxidation-Reduction; Oxidative Phosphorylation; Oxygen; Paracoccus bacteria; Paracoccus denitrificans; Performance; Post-Translational Protein Processing; Process; Production; Protein Biosynthesis; Proteomics; Rattus; Regulation; Reperfusion Therapy; Rest; Saline; Scanning; Signal Transduction; Skeletal Muscle; Spectrum Analysis; Structure; System; Testing; Tissues; Work; base; biological systems; body system; chromophore; cytochrome c oxidase; in vivo; insight; light scattering; novel; operation; parallel processing; respiratory; screening

The purpose of these studies is to establish a better understanding of the energy metabolism of biological tissues. Towards this goal, the laboratory concentrates on the use of screening approaches in proteomics and post-translational modifications. The following major findings were made over the last year: 1) We have continued to develop a non-destructive optical spectroscopy method using a center mounted integrating sphere and a rapid scanning spectroscopy system to monitor the redox sensitive chromophores of mitochondrial oxidative phosphorylation minimizing light scattering effects. Using this approach we have established characterized all of the redox chromophores in the mitochondria and begun to establish the regulation of reducing equivalent distribution within the network. We have described that both the activation, with calcium, and deactivation, ischemia reperfusion in intact heart, that all of the Complexes of oxidative phosphorylation are modified in concert. These data imply that the activity of these Complexes is orchestrated together in the intact mitochondria. The mechanisms responsible for the coordination of the Complex activities are still under investigation. Our working hypothesis is based on the fact that the mitochondria evolved from early symbiotic bacteria retaining many of bacteria protein synthesis processes and even DNA. We speculate that the signaling mechanisms within the mitochondria may also be closer to bacterial signaling systems than the more familiar eukaryotic systems. 2) To test the bacterial signaling hypothesis stated above, we have initiated studies on isolated bacteria believed to be closest to the mitochondrial origins, paracoccus denitrificans. Our initial studies have demonstrated that the respiratory rate, or ATP production rate, can be acutely modulated using the volume regulatory processes in these bacteria. Using this approach we have surprisingly demonstrated that the bacteria up-regulate metabolic capacity acutely with increases in work demand based on the increase in mitochondrial NADH with work transitions. This effect seems to parallel processes we have observed in the mammalian mitochondria. We are continuing to characterize the basic energy metabolism of paracoccus during growth and volume regulatory processes using our proteomic and non-invasive optical approaches. It is hoped that this simplified system will provide insights into the regulation of mitochondrial oxidative phosphorylation, in the coming year. 3) Our previous work on the regulation of oxidative phosphorylation has concentrated on isolated mitochondria that we have extrapolated to in vivo conditions. We are now moving our non-invasive optical studies of the chromophores of oxidative phosphorylation into the study of the isolated perfused heart. We have demonstrated that a homemade artificial oxygen delivery system, based on perflurocarbons, works in the perfused heart increasing oxygen delivery by more than 5 fold. Spectroscopic studies demonstrate that this increase in oxygen delivery to the classical perfused hearts of rat and rabbits increases the oxygenation of both the myoglobin as well as the mitochondria, based on the absorbance of cytochrome oxidase. These data suggest that using the classical saline perfused heart my introduce hypoxia in the perfused heart models. It is hoped that that this system will permit us to extrapolate our isolated mitochondria observations to a working intact tissue over the next year. 4) We completed our studies on cAMP and have reached the conclusion that mitochondrial matrix cAMP doe not impact the capability of the mitochondria to generate ATP nor change the redox poise of the cytochrome chain. We believe this make matrix cAMP a very unlikely candidate in the acute regulation of mitochondrial ATP production. 5) We demonstrated with Dr. Finkel and Murphy that knocking out the putative mitochondrial Ca transporter, MCU, had minimal cardiac performance effects and only impacted the peak performance of the skeletal muscle. This was consistent with our previous work on the allometry of cardiac energetics that demonstrated that the mouse heart was near maximal performance even at rest, not requiring the calcium activation system, while the skeletal muscle still had considerable dynamic range that required full metabolic activation at peak power. These data are consistent with other knockout studies in our lab over the last 2 years where we have removed the two major facilitated diffusion systems, myoglobin and creatine, and also only found effects at peak power in the skeletal muscles of the mice. These results suggest that major enzyme systems are present in mice that only support small increments in peal performance that might have significant evolutionary survival advantages for this small mammal.

这些研究的目的是为了更好地了解生物组织的能量代谢。为了实现这一目标，实验室专注于在蛋白质组学和翻译后修饰中使用筛选方法。在过去的一年中，我们取得了以下主要发现：1)我们继续开发一种非破坏性的光学光谱方法，使用中心安装的积分球和快速扫描光谱系统来监测线粒体氧化磷酸化的氧化还原敏感发色团，最大限度地减少光散射效应。利用这种方法，我们已经确定了线粒体中所有氧化还原发色团的特征，并开始建立网络中减少等效分布的调节。我们已经描述了在完整心脏中，钙的激活和缺血再灌注的失活，所有氧化磷酸化复合物都被一致地修饰。这些数据表明，这些复合物的活性在完整的线粒体中被协调在一起。负责协调复杂活动的机制仍在调查之中。我们的工作假设是基于线粒体从早期共生细菌进化而来的事实，线粒体保留了许多细菌蛋白质合成过程甚至DNA。我们推测，线粒体内的信号机制也可能比更熟悉的真核生物系统更接近细菌信号系统。2)为了验证上述细菌信号传导假说，我们对被认为最接近线粒体起源的分离细菌——反硝化副球菌（paracoccus反硝化副球菌）进行了研究。我们的初步研究表明，呼吸速率或ATP生产速率可以通过这些细菌的体积调节过程进行急性调节。使用这种方法，我们已经令人惊讶地证明，基于线粒体NADH随着工作转换的增加，细菌随着工作需求的增加而急剧上调代谢能力。这种效应似乎与我们在哺乳动物线粒体中观察到的过程相似。我们将继续使用我们的蛋白质组学和非侵入性光学方法来表征副球菌在生长和体积调节过程中的基本能量代谢。希望这个简化的系统将在未来的一年里为线粒体氧化磷酸化的调节提供见解。3)我们之前关于氧化磷酸化调控的工作主要集中在分离的线粒体上，我们已经推断出体内条件。我们现在正将氧化磷酸化发色团的非侵入性光学研究转移到离体灌注心脏的研究中。我们已经证明了一种自制的人工供氧系统，基于全氟碳化合物，在灌注的心脏中工作，将供氧量增加了5倍以上。光谱研究表明，基于细胞色素氧化酶的吸收，向大鼠和家兔经典灌注心脏输送的氧气增加了肌红蛋白和线粒体的氧合。这些数据提示，采用经典生理盐水灌注心脏可以在灌注心脏模型中引入缺氧。希望这个系统将允许我们在明年将我们分离的线粒体观察外推到一个工作的完整组织。4)我们完成了对cAMP的研究，得出了线粒体基质cAMP不会影响线粒体生成ATP的能力，也不会改变细胞色素链的氧化还原平衡的结论。我们认为，这使得基质cAMP在线粒体ATP产生的急性调节中不太可能成为候选物。5)我们与Finkel博士和Murphy一起证明，敲除假定的线粒体Ca转运蛋白MCU对心脏性能的影响最小，仅影响骨骼肌的峰值性能。这与我们之前关于心脏能量异速测量的研究一致，该研究表明，即使在休息时，小鼠心脏也接近最大性能，不需要钙激活系统，而骨骼肌仍然具有相当大的动态范围，需要在峰值功率下进行完全代谢激活。这些数据与我们实验室在过去两年中进行的其他基因敲除研究一致，在这些研究中，我们移除了两种主要的促进扩散系统，肌红蛋白和肌酸，并且也只在小鼠骨骼肌的峰值功率下发现了效果。这些结果表明，小鼠体内存在主要的酶系统，这些酶系统只支持珍珠性能的微小增加，这可能对这种小型哺乳动物具有显著的进化生存优势。