Control Of Cellular Energy Metabolism

这些研究的目的是利用现代系统生物学方法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、代谢组学、蛋白质结构、翻译后修饰、微创代谢率信息和光谱学等方面使用筛选方法。这个项目的一个主要假设是，执行氧化磷酸化的多蛋白复合物的活性以某种方式协调，以平衡细胞中ATP的产生率和利用率。这导致了观察到的代谢稳态，即使在工作量发生重大变化时，细胞中做功的势能也保持在接近恒定的水平。在过去的一年中，我们取得了以下主要发现：1)我们扩大了线粒体在完整跳动心脏中功能的透射光谱学研究，包括对灌注的小鼠心脏的研究。老鼠心脏的小尺寸需要一种新的光学收集系统，以最小的光学伪影检测通过心脏传输的光。我们开发了一种方法，将完整的心脏放置在积分球的中心，允许对所有离开心脏的光进行采样，很少或没有平移运动伪影。与墨菲博士的团队一起，我们已经证明了线粒体在缺血再灌注条件下的代谢状态，并在该临床相关方案中揭示了线粒体膜电位和细胞色素氧化还原状态的新信息。2)在离体灌注心脏中使用外源性血管扩张剂，我们已经证明了一种矛盾的小动脉收缩的存在，这种收缩使离体心脏处于部分缺氧状态，尽管它有血流储备，但它没有使用。这些数据表明，正常的血流与代谢需求匹配的调节机制在这个主要的心功能模型系统中被破坏了。另一种血管扩张剂是血管一氧化氮，它也是一种被认为是氧化磷酸化的抑制剂。我们测试了微摩尔浓度的一氧化氮对离体灌注兔和小鼠心脏的影响，发现对细胞色素氧化还原状态没有抑制作用。我们证明，通过肌红蛋白氧化的一氧化氮代谢，加上一个高活性的肌红蛋白还原酶，是一个强大的一氧化氮代谢途径，可能限制血管一氧化氮对线粒体功能的影响。为了扩大我们对线粒体代谢调节的分析，我们已经扩大了我们的研究，研究线粒体的祖先，简单的细菌。我们已经开始研究被认为最接近线粒体起源的分离细菌，反硝化副球菌（PD）。这些研究的目标是揭示这种细菌的急性能量转换调节，然后在哺乳动物线粒体中寻找类似的机制。随着人们对微生物组的兴趣日益浓厚，这些研究也应该为尚未广泛研究的细菌能量代谢的急性调节提供新的见解。在此期间，我们已经证明了先前描述的哺乳动物心脏中的代谢稳态，即ATP和线粒体质子动机在增加工作量时的恒定自由能，存在于PD中。这是通过测量细菌膜电位、跨膜pH梯度、基因编码的细胞内pH探针以及细胞色素的氧化还原状态来完成的。我们已经证明，细胞能量转换的这种特性在广泛的生物系统中是保守的，并且希望，细菌系统将提供关于线粒体如何被驯化以在人类心脏和其他组织中执行这项任务的见解。

The purpose of these studies is to establish a better understanding of the energy metabolism of biological tissues using modern system biology approaches. Towards this goal, the laboratory concentrates on the use of screening approaches in proteomics, metabolomics, protein structure, post-translational modifications, minimally invasive metabolic rate information and optical spectroscopy. One of the major hypothesizes in this program is that the activity of the multi-protein Complexes that perform Oxidative Phosphorylation are coordinated in some fashion to balance the rate of ATP production with utilization in the cell. This results in the observed metabolic homeostasis where the potential energy for doing work is maintained near constant in the cell even during major alterations in workload. The following major findings were made over the last year: 1)We have expanded our transmission optical spectroscopy investigation of the functioning of mitochondria in the intact beating heart to include studies on the perfused mouse heart. The small size of the mouse heart required a new optical collection system to detect the light transmitted through the heart with minimal optical artifacts. We developed an approach where the intact heart is placed in the center of an integrating sphere permitting the sampling of all of the light leaving the heart with little or no translational motion artifacts. With Dr. Murphys group we have demonstrated the metabolic state of the mitochondria during ischemia reperfusion conditions and revealed new information on the mitochondrial membrane potential and cytochrome redox state during this clinically relevant protocol. 2) Using exogenous vasodilators in the isolated perfused heart, we have demonstrated the existence of a paradoxical arteriole contraction that puts the isolated heart in partial hypoxia despite the fact that it has flow reserve it is not using. These data implied that the normal regulatory mechanisms of matching blood flow with metabolic demand is disrupted in this major model system of cardiac function. 3) Another vasodilator is vascular nitric oxide, that is also a putative inhibitor of oxidative phosphorylation. We tested the impact of micromolar concentrations of nitric oxide on the isolated perfused rabbit and mouse heart and found no inhibitory action on the cytochrome redox state. We demonstrated that the metabolism of nitric oxide through the oxidation of myoglobin, coupled to a highly active myoglobin reductase, is a powerful nitric oxide metabolizing pathway likely limiting the impact of vascular nitric oxide on mitochondrial function . 3) To broaden our analysis of metabolic regulation in the mitochondria we have expanded our studies to study the ancestors of mitochondria, simple bacteria. We have initiated studies on isolated bacteria believed to be closest to the mitochondrial origins, paracoccus denitrificans(PD). The goal of these studies is to unravel acute energy conversion regulation in this bacterium and then look for similar mechanisms in mammalian mitochondria. With the growing interest in the microbiome, these studies should also provide new insight into the acute regulation of bacterial energy metabolism that has not been extensively studied. We have demonstrated in this period that the previously described metabolic homeostasis described in the mammalian heart, that is constant free energy available in ATP as well as the mitochondria proton motive during increases is workload, exists in PD. This was done with measures of the bacteria membrane potential, transmembrane pH gradient, with a genetically encoded intracellular pH probe, along with the redox state of the cytochromes. We have demonstrated that this property of energy conversion by the cell is conserved over a wide range of biological systems, and hopefully, the bacterial system will provide insights into how the mitochondria were domesticated to perform this task in the human heart and other tissues.