Mitochondrial matrix phosphoproteome: Effect of extra mitochondrial calcium
Mitochondrial matrix phosphoproteome: Effect of extra mitochondrial calcium
复制标题
DOI：
10.1021/bi052475e
复制
发表时间：
2006-02-28
期刊：
影响因子：
2.9
通讯作者：
Balaban, RS
中科院分区：
文献类型：
--
作者：
Hopper, RK;Carroll, S;Balaban, RS
通过磷酸化或去磷酸化对线粒体蛋白的翻译后修饰在参与调节能量代谢和线粒体诱导的细胞凋亡的许多细胞信号传导途径中起重要作用。在这里，我们提出了一个磷酸化的线粒体基质蛋白质组学筛选和开始建立蛋白磷酸化急性相关的钙离子（Ca 2+）信号在猪心脏线粒体。通过凝胶电泳-质谱法检测到45个磷酸化蛋白质的Pro-Q Diamond染色，而更多的Pro-Q Diamond染色的蛋白质逃避质谱检测。时间依赖性的P-32掺入完整的线粒体证实了广泛的基质蛋白磷酸化，并揭示了这一过程的动态性质。检测到的蛋白质类别包括所有线粒体呼吸链复合物，以及参与中间代谢的酶，如丙酮酸脱氢酶（PDH）、柠檬酸合酶和酰基辅酶A脱氢酶。这些数据表明，线粒体基质的磷酸化蛋白质组是广泛的和动态的。Ca 2+以前已被证明可以激活各种细胞色素酶，促进活性氧（ROS）的产生，并通过细胞色素c的释放启动细胞凋亡。为了评估Ca 2+信号网络，评估了足以释放细胞色素c的Ca 2+挑战对线粒体磷酸蛋白质组的影响。锰超氧化物歧化酶（MnSOD）以及先前表征的PDH中观察到新的Ca 2+诱导的去磷酸化。MnSOD的Ca 2+剂量依赖性去磷酸化与活性的2倍最大增加相似;在没有Ca 2+的情况下，ROS产生既不诱导去磷酸化也不诱导活性变化。这些数据表明，使用磷酸化蛋白质组筛选确定线粒体信号转导途径，并揭示了新的途径，在MnSOD的水平上的线粒体功能的Ca 2+修饰。
Post-translational modification of mitochondrial proteins by phosphorylation or dephosphorylation plays an essential role in numerous cell signaling pathways involved in regulating energy metabolism and in mitochondrion-induced apoptosis. Here we present a phosphoproteomic screen of the mitochondrial matrix proteins and begin to establish the protein phosphorylations acutely associated with calcium ions (Ca2+) signaling in porcine heart mitochondria. Forty-five phosphorylated proteins were detected by gel electrophoresis-mass spectrometry of Pro-Q Diamond staining, while many more Pro-Q Diamond-stained proteins evaded mass spectrometry detection. Time-dependent P-32 incorporation in intact mitochondria confirmed the extensive matrix protein phosphoryation and revealed the dynamic nature of this process. Classes of proteins that were detected included all of the mitochondrial respiratory chain complexes, as well as enzymes involved in intermediary metabolism, such as pyruvate dehydrogenase (PDH), citrate synthase, and acyl-CoA dehydrogenases. These data demonstrate that the phosphoproteome of the mitochondrial matrix is extensive and dynamic. Ca2+ has previously been shown to activate various dehydrogenases, promote the generation of reactive oxygen species (ROS), and initiate apoptosis via cytochrome c release. To evaluate the Ca2+ signaling network, the effects of a Ca2+ challenge sufficient to release cytochrome c were evaluated on the mitochondrial phosphoproteome. Novel Ca2+-induced dephosphorylation was observed in manganese superoxide dismutase (MnSOD) as well as the previously characterized PDH. A Ca2+ dose-dependent dephosphorylation of MnSOD was associated with an similar to 2-fold maximum increase in activity; neither the dephosphorylation nor activity changes were induced by ROS production in the absence of Ca2+. These data demonstrate the use of a phosphoproteome screen in determining mitochondrial signaling pathways and reveal new pathways for Ca2+ modification of mitochondrial function at the level of MnSOD.