Control Of Cellular Energy Metabolism

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。在这些研究中，我们已经确定副球菌氧化磷酸化系统具有与哺乳动物线粒体非常相似的光学性质，并且可以通过简单地改变沐浴钾浓度来诱导ATP产生的改变。在线粒体中建立的蛋白质组学筛选工具也被成功地用于评估这些细菌的蛋白质编程。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。我们最初的研究表明，我们可以通过简单地将肌红蛋白添加到我们的拟合阵列中来定量灌注心脏的所有氧化还原敏感发色团。然而，将该技术应用于离体灌注兔心脏发现，与体内血液灌注心脏不同，盐水灌注心脏的肌红蛋白氧合高度不稳定。这些数据表明，在这些条件下，心脏部分缺氧，可能不能反映体内动态。我们不能使用血液灌注系统，因为血红蛋白的光学干扰主导了光学性质。因此，我们设计了一种方法，利用含有全氟碳载氧聚合物的脂质囊泡构建一种无色的人工血液替代品，以改善向灌注心脏的氧气输送。我们已经证明，这种人工供氧系统在灌注心脏中起作用，使供氧量增加了5倍以上。希望这个系统将允许我们将我们分离的线粒体观察推断到工作的完整组织。4)一种被提出的信号氧化磷酸化机制是通过cAMP调节的蛋白激酶A系统通过细胞信号传导。我们使用脂质渗透性cAMP类似物和该信号网络的许多抑制剂，在猪心脏分离的线粒体和粗细胞质浆中评估了这一过程。渗透性cAMP类似物或网络抑制剂未发现显著影响。这些数据表明，cAMP调控网络不是心脏氧化磷酸化的重要急性调节剂。5)常规和非常规位点的蛋白质磷酸化被认为是氧化磷酸化的重要调节因子。氟化物是蛋白质磷酸化的广泛抑制剂之一。我们发现氟化钾（KF）是一种有效的氧化磷酸化抑制剂。我们已经证明KF抑制了完整线粒体中几种氧化磷酸化复合物，这与磷酸化假说一致。此外，我们已经证明，对于复合体V，通过分离纯化的复合体V进行深入的蛋白质组学分析，效果是持久的。我们目前正试图表征导致复合物v上持续KF效应的翻译后修饰。我们不假设KF效应是由于蛋白质磷酸化，因为我们正在筛选所有的翻译后修饰，包括蛋白质-蛋白质相互作用，我们可以使用质谱技术进行检测。KF的可逆作用以及这些作用通过仔细分离复合体V而持续存在的能力表明，这些研究可能揭示了一个翻译后网络，可以修改这一关键氧化磷酸化复合体的功能。

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. In these studies we have established that paracoccus oxidative phosphorylation system has very similar optical properties as the mammalian mitochondria and alterations in ATP production can be induced by simply modifying the bathing potassium concentration. Proteomic screening tools, established in mitochondria, have also been successfully modified to evaluate the protein programming of these bacteria. 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. Our initial studies revealed that we could quantitate all of the redox sensitive chromophores of the perfused heart by simply adding myoglobin to our fitting array. However, applying this technology to the isolated perfused rabbit heart revealed that myoglobin oxygenation was highly labile in the saline perfused heart unlike the blood perfused heart in vivo. These data suggested that the heart was partially hypoxic under these conditions and may not reflect in vivo dynamics. We could not use a blood perfused system since the optical interference from hemoglobin dominated the optical properties. Thus, we devised methods to construct a colorless artificial blood substitute using lipid vesicles containing perfluorocarbon oxygen carrying polymers to improve oxygen delivery to the perfused heart. We have demonstrated that this artificial oxygen delivery system works in the perfused heart increasing oxygen delivery by more than 5 fold. It is hoped that that this system will permit us to extrapolate our isolated mitochondria observations to a working intact tissue. 4) One of the proposed mechanisms of signaling oxidative phosphorylation is via cell signaling through the protein kinase A system modulated by cAMP. We have evaluated this process in porcine heart isolated mitochondria and crude cytosol homogenates using lipid permeable cAMP analogs and numerous inhibitors of this signaling network. No significant effects of the permeable cAMP analogs or network inhibitors could be found. This data suggest that the cAMP regulatory network is not a significant acute modulator of cardiac oxidative phosphorylation. 5) Protein phosphorylation at conventional and non-conventional sites has been proposed as an important modulator of oxidative phosphorylation. One of the broad inhibitors of protein phosphorylation is fluoride. We have found that potassium fluoride (KF) is an effective inhibitor of oxidative phosphorylation. We have shown that KF inhibits several of the oxidative phosphorylation Complexes in intact mitochondria consistent with the phosphorylation hypothesis. In addition, we have shown that with regards to Complex V, the effect is persistent through the isolation of the purified Complex V for in depth proteomic analysis. We are current now attempting to characterize the post-translational modifications responsible for the persistent KF effect on Complex V. We are not assuming the KF effect is due to protein phosphorylation as we are screening for all the post-translational modification, including protein-protein interactions, we can detect using mass spectroscopy techniques. The reversible effects of KF together with the ability of these effects to persist through a careful isolation of Complex V suggest that these studies might reveal a post-translational network modifying the function of this critical Complex of oxidative phosphorylation.