Phosphoproteome analysis of isoflurane-protected heart mitochondria:: phosphorylation of adenine nucleotide translocator-1 on Tyr194 regulates mitochondrial function
Phosphoproteome analysis of isoflurane-protected heart mitochondria:: phosphorylation of adenine nucleotide translocator-1 on Tyr194 regulates mitochondrial function
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DOI：
10.1093/cvr/cvn161
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发表时间：
2008-10-01
影响因子：
10.8
通讯作者：
Zaugg, Michael
中科院分区：
文献类型：
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作者：
Feng, Jianhua;Zhu, Min;Zaugg, Michael
目的线粒体蛋白的可逆磷酸化在呼吸功能、能量代谢和线粒体介导的细胞死亡的调节中是必不可少的。我们假设，线粒体蛋白磷酸化在心脏保护中起着至关重要的作用，在预处理和后处理，两个最有效的抗缺血therapy.Methods和results使用磷酸化蛋白质组学方法，我们调查了Wistar大鼠心脏线粒体中的磷酸化蛋白质的配置文件保护的药理学预处理和后处理引起的异氟烷。使用磷酸化-Ser/Thr/Tyr特异性抗体通过二维蓝色天然凝胶电泳偶联蛋白质印迹法检测61个斑点，并通过基质辅助激光解吸/电离飞行时间质谱法鉴定其中45个斑点。与氧化磷酸化，能量代谢，伴侣蛋白，载体功能相关的11个蛋白点在其磷酸化状态下表现出显着的变化时，保护线粒体与未保护的。使用磷酸肽富集协议，然后液相色谱-MS/MS，26个潜在的磷酸化位点被确定在19个蛋白质。其中，在腺嘌呤核苷酸转运体-1（ANT 1）的Tyr（194）残基处检测到一个新的磷酸化位点。免疫沉淀证实了保护和未保护线粒体之间ANT磷酸化的变化。ANT在Tyr（194）处磷酸化的生物学意义用酵母中的定点诱变进一步测试。取代酪氨酸（194）与苯丙氨酸，模仿非磷酸化状态，导致在抑制酵母生长的非发酵碳源，这意味着在这个残基的磷酸化在调节ANT功能和细胞呼吸的关键作用。结论我们的分析强调了磷酸化蛋白质组在心脏线粒体的调节功能，揭示了一种新的，潜在的生物能量学和心脏保护之间的联系。
Aims Reversible phosphorylation of mitochondrial proteins is essential in the regulation of respiratory function, energy metabolism, and mitochondrion-mediated cell death. We hypothesized that mitochondrial protein phosphorylation plays a critical role in cardioprotection during pre and postconditioning, two of the most efficient anti-ischaemic therapies.Methods and results Using phosphoproteomic approaches, we investigated the profiles of phosphorylated proteins in Wistar rat heart mitochondria protected by pharmacological pre and postconditioning elicited by isoflurane. Sixty-one spots were detected by two-dimensional blue-native gel electrophoresis-coupled Western blotting using a phospho-Ser/Thr/Tyr-specific antibody, and 45 of these spots were identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Eleven protein spots related to oxidative phosphorylation, energy metabolism, chaperone, and carrier functions exhibited significant changes in their phosphorylation state when protected mitochondria were compared with unprotected. Using a phosphopeptide enrichment protocol followed by liquid chromatography-MS/MS, 26 potential phosphorylation sites were identified in 19 proteins. Among these, a novel phosphorylation site was detected in adenine nucleotide translocator-1 (ANT1) at residue Tyr(194). Changes in ANT phosphorylation between protected and unprotected mitochondria were confirmed by immunoprecipitation. The biological significance of ANT phosphorylation at Tyr(194) was further tested with site-directed mutagenesis in yeast. Substitution of Tyr(194) with Phe, mimicking the non-phosphorylated state, resulted in the inhibition of yeast growth on non-fermentable carbon sources, implying a critical role of phosphorylation at this residue in regulating ANT function and cellular respiration.Conclusions Our analysis emphasizes the regulatory functions of the phosphoproteome in heart mitochondria and reveals a novel, potential link between bioenergetics and cardioprotection.