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，该实验室专注于蛋白质组学和翻译后修饰中筛选方法的使用。在过去的一年里，我们取得了以下主要发现：1)我们之前已经证明，线粒体基质中的蛋白质磷酸化是广泛的。利用最小干扰的蓝色天然凝胶电泳法，我们已经证明了大部分磷酸化线粒体氧化磷酸化复合体的激酶活性都存在于复合体中。也就是说，当不同的复合体通过蓝色天然电泳法纯化时，复合体内的蛋白激酶活性保持不变。这意味着线粒体激酶在组成氧化磷酸化复合体的蛋白质组合中被分割。抗体筛选表明，这些复合体中不存在典型的胞浆蛋白激酶(PKA、PKC等)，这表明这些复合体本身具有独特的蛋白激酶活性。进一步的研究表明，这些蛋白的磷酸化是酸敏感的，这表明它们可能是早期细菌蛋白磷酸化的信号通路，涉及天冬氨酸和组氨酸的自动磷酸化。2)有一种假说认为，代谢活性波动较大的组织(如心脏和肌肉)通过翻译后修饰保留线粒体的电子传递活性，而更恒定的组织(如肝脏)的大部分酶活性是活跃的和可用的。我们还提出，这种关系适用于不同大小的动物，这些动物的心脏由于基础活动的相对差异而具有非常高的动态范围(人、大动物)或低的动态范围(小鼠、大鼠、小动物)。我们已经证明线粒体复合体V和IV的活性确实遵循这一概念，在猪的心脏和骨骼肌中受到抑制，而在猪肝脏中完全活跃。相比之下，持续活跃的小鼠心脏和肝脏具有基本相同的酶活性。一种中间动物，兔子，显示出介于猪和老鼠之间的中间值。这些研究表明，在静息条件下，氧化磷酸化酶存在内在的翻译后节流，可能防止势能和相关的活性氧物种的积累。

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 previously shown that protein phosphorylation in the mitochondria matrix is extensive. Using minimally disruptive blue native gel electrophoresis we have demonstrated that much of the kinase activity phosphorylating mitochondria oxidative phosphorylation complexes was identified to exist within the Complex itself. That is, when the different complexes were purified by blue native electrophoresis, the protein kinase activity within the complex was maintained. This implies that mitochondrial kinases are compartmentalized in the protein assemblies making up the oxidative phosphorylation complexes. Antibody screening suggested that typical cytosolic protein kinases (PKA, PKC etc) are not present in these complexes, suggesting that the complexes themselves have unique protein kinase activity. Further studies have revealed that these protein phosphorylations are acid sensitive suggesting they may represent early bacterial protein phosphorylation signaling pathways involving the autophosphorylation of aspartate and histidine. 2) A hypothesis has been developed that tissues with large swings in metabolic activity (i.e. heart and muscle) hold mitochondrial electron transport activity in reserve via post translational modifications while more constant activity tissues (i.e. liver) have most of their enzymatic activity active and available. We also propose that this relationship holds in animals of different sizes where the heart has very high (man, large animals) or low (mouse, rat, small animals) dynamic range due to their relative differences in basal activity. We have demonstrated that the activity of mitochondria Complex V and IV indeed follow this notion, being suppressed in heart and skeletal muscle of pigs while fully active in the porcine liver. In contrast, the constantly active mouse heart and liver have essentially identical enzyme activities. An intermediate animal, the rabbit, showed intermediate values between the pig and mouse. These studies suggest that an inherent post-translational throttle on oxidative phosphorylation enzymes is present likely preventing the buildup of potential energy and associated reactive oxygen species under resting conditions.