Phosphoproteome Analysis Reveals Regulatory Sites in Major Pathways of Cardiac Mitochondria
Phosphoproteome Analysis Reveals Regulatory Sites in Major Pathways of Cardiac Mitochondria
复制标题
DOI：
10.1074/mcp.m110.000117
复制
发表时间：
2011-02-01
影响因子：
7
通讯作者：
Ping, Peipei
中科院分区：
文献类型：
--
作者：
Deng, Ning;Zhang, Jun;Ping, Peipei
线粒体功能在心脏中受到动态调节。特别是，蛋白磷酸化已被证明是一个关键的机制，调节线粒体功能在不同的心血管表型。然而，该器官的位点特异性磷酸化信息仍然很少。因此，我们在线粒体功能通路的背景下对小鼠心脏线粒体磷酸化蛋白质组进行了全面的表征。使用碰撞诱导解离（CID）和电子转移解离（ETD）的互补片段化技术的平台证明了成功鉴定小鼠心脏中总共236个磷酸化位点;其中210个位点是新的。这236个位点被定位到181个磷蛋白和203个磷酸肽。在那些确定的，45磷酸化位点被捕获，只有CID，而185磷酸化位点，包括一个新的修改泛喹啉-细胞色素c还原酶蛋白1（Ser-212），被确定只有ETD，强调了CID和ETD相结合的方法的优势。评价了心肌线粒体磷酸化蛋白质组的生物学意义。我们的研究说明了小鼠心脏线粒体途径中的关键调控位点作为磷酸化调控的靶点，包括电子传递链（ETC）复合物和参与代谢途径（例如三羧酸循环）的酶的组分。此外，钙超载损伤心脏线粒体ETC功能，而增强磷酸化ETC通过应用磷酸酶抑制剂恢复钙衰减ETC复合物I和复合物III的活动，表现出积极的调节ETC功能的磷酸化。此外，对鉴定的磷酸肽基序的计算机分析阐明了参与激酶的分子性质，其包括几种已知的线粒体激酶（例如丙酮酸脱氢酶激酶）以及先前未认识到其线粒体位置的激酶（例如Src）。总之，本文定义的磷酸化事件推进了我们对心脏线粒体生物学的理解，促进了关于线粒体信号网络、代谢途径和心脏功能调节的内在机制的仍然零碎的知识的整合。Molecular & Cellular Proteomics 10：10.1074/mcp. M110.000117，114，2011.
Mitochondrial functions are dynamically regulated in the heart. In particular, protein phosphorylation has been shown to be a key mechanism modulating mitochondrial function in diverse cardiovascular phenotypes. However, site-specific phosphorylation information remains scarce for this organ. Accordingly, we performed a comprehensive characterization of murine cardiac mitochondrial phosphoproteome in the context of mitochondrial functional pathways. A platform using the complementary fragmentation technologies of collision-induced dissociation (CID) and electron transfer dissociation (ETD) demonstrated successful identification of a total of 236 phosphorylation sites in the murine heart; 210 of these sites were novel. These 236 sites were mapped to 181 phosphoproteins and 203 phosphopeptides. Among those identified, 45 phosphorylation sites were captured only by CID, whereas 185 phosphorylation sites, including a novel modification on ubiquinol-cytochrome c reductase protein 1 (Ser-212), were identified only by ETD, underscoring the advantage of a combined CID and ETD approach. The biological significance of the cardiac mitochondrial phosphoproteome was evaluated. Our investigations illustrated key regulatory sites in murine cardiac mitochondrial pathways as targets of phosphorylation regulation, including components of the electron transport chain (ETC) complexes and enzymes involved in metabolic pathways (e.g. tricarboxylic acid cycle). Furthermore, calcium overload injured cardiac mitochondrial ETC function, whereas enhanced phosphorylation of ETC via application of phosphatase inhibitors restored calcium-attenuated ETC complex I and complex III activities, demonstrating positive regulation of ETC function by phosphorylation. Moreover, in silico analyses of the identified phosphopeptide motifs illuminated the molecular nature of participating kinases, which included several known mitochondrial kinases (e.g. pyruvate dehydrogenase kinase) as well as kinases whose mitochondrial location was not previously appreciated (e.g. Src). In conclusion, the phosphorylation events defined herein advance our understanding of cardiac mitochondrial biology, facilitating the integration of the still fragmentary knowledge about mitochondrial signaling networks, metabolic pathways, and intrinsic mechanisms of functional regulation in the heart. Molecular & Cellular Proteomics 10:10.1074/mcp.M110.000117, 114, 2011.