Department of Biochemistry and Molecular Biology, University of Southern Denmark, DK-5230 Odense M, Denmark.
Mol Cell Proteomics. 2011 Jan;10(1):M110.000299. doi: 10.1074/mcp.M110.000299. Epub 2010 Sep 10.
Mitochondria play a central role in energy metabolism and cellular survival, and consequently mitochondrial dysfunction is associated with a number of human pathologies. Reversible protein phosphorylation emerges as a central mechanism in the regulation of several mitochondrial processes. In skeletal muscle, mitochondrial dysfunction is linked to insulin resistance in humans with obesity and type 2 diabetes. We performed a phosphoproteomics study of functional mitochondria isolated from human muscle biopsies with the aim to obtain a comprehensive overview of mitochondrial phosphoproteins. Combining an efficient mitochondrial isolation protocol with several different phosphopeptide enrichment techniques and LC-MS/MS, we identified 155 distinct phosphorylation sites in 77 mitochondrial phosphoproteins, including 116 phosphoserine, 23 phosphothreonine, and 16 phosphotyrosine residues. The relatively high number of phosphotyrosine residues suggests an important role for tyrosine phosphorylation in mitochondrial signaling. Many of the mitochondrial phosphoproteins are involved in oxidative phosphorylation, tricarboxylic acid cycle, and lipid metabolism, i.e. processes proposed to be involved in insulin resistance. We also assigned phosphorylation sites in mitochondrial proteins involved in amino acid degradation, importers and transporters, calcium homeostasis, and apoptosis. Bioinformatics analysis of kinase motifs revealed that many of these mitochondrial phosphoproteins are substrates for protein kinase A, protein kinase C, casein kinase II, and DNA-dependent protein kinase. Our results demonstrate the feasibility of performing phosphoproteome analysis of organelles isolated from human tissue and provide novel targets for functional studies of reversible phosphorylation in mitochondria. Future comparative phosphoproteome analysis of mitochondria from healthy and diseased individuals will provide insights into the role of abnormal phosphorylation in pathologies, such as type 2 diabetes.
线粒体在能量代谢和细胞存活中发挥着核心作用，因此线粒体功能障碍与许多人类疾病有关。可逆蛋白磷酸化是调节多种线粒体过程的核心机制之一。在肥胖和 2 型糖尿病患者的骨骼肌中，线粒体功能障碍与胰岛素抵抗有关。我们对从人类肌肉活检中分离的功能性线粒体进行了磷酸蛋白质组学研究，旨在全面了解线粒体磷酸蛋白。我们结合了一种高效的线粒体分离方案和几种不同的磷酸肽富集技术以及 LC-MS/MS，在 77 种线粒体磷酸蛋白中鉴定出 155 个独特的磷酸化位点，包括 116 个磷酸丝氨酸、23 个磷酸苏氨酸和 16 个磷酸酪氨酸残基。相对较高数量的磷酸酪氨酸残基表明酪氨酸磷酸化在线粒体信号转导中具有重要作用。许多线粒体磷酸蛋白参与氧化磷酸化、三羧酸循环和脂质代谢，即被认为与胰岛素抵抗有关的过程。我们还对参与氨基酸降解、导入和转运、钙稳态和细胞凋亡的线粒体蛋白中的磷酸化位点进行了分配。激酶基序的生物信息学分析表明，这些线粒体磷酸蛋白中的许多是蛋白激酶 A、蛋白激酶 C、酪蛋白激酶 II 和 DNA 依赖性蛋白激酶的底物。我们的研究结果表明，从人体组织中分离的细胞器进行磷酸蛋白质组学分析是可行的，并为线粒体可逆磷酸化的功能研究提供了新的靶标。未来对健康和患病个体的线粒体进行比较磷酸蛋白质组学分析，将深入了解异常磷酸化在 2 型糖尿病等疾病中的作用。
