Quantitative Non-canonical Amino Acid Tagging (QuaNCAT) Proteomics Identifies Distinct Patterns of Protein Synthesis Rapidly Induced by Hypertrophic Agents in Cardiomyocytes, Revealing New Aspects of Metabolic Remodeling.
Quantitative Non-canonical Amino Acid Tagging (QuaNCAT) Proteomics Identifies Distinct Patterns of Protein Synthesis Rapidly Induced by Hypertrophic Agents in Cardiomyocytes, Revealing New Aspects of Metabolic Remodeling.
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DOI：
10.1074/mcp.m115.054312
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发表时间：
2016-10
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影响因子：
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通讯作者：
Proud CG
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作者：
Liu R;Kenney JW;Manousopoulou A;Johnston HE;Kamei M;Woelk CH;Xie J;Schwarzer M;Garbis SD;Proud CG
心肌细胞在特定的病理或生理条件下发生生长和重塑。在前者中，心肌生长是心力衰竭的危险因素，更快的蛋白质合成是推动心肌细胞生长的主要因素。我们的目标是量化不同的促肥大刺激对ARVC中特定蛋白质合成的快速影响，并确定这种影响是由mRNA丰度的改变还是特定mRNAs的翻译引起的。心肌细胞的蛋白质合成率非常低，在研究特定蛋白质合成的变化方面提出了一个具有挑战性的问题，这也适用于其他未分裂的原代细胞。为了研究特定蛋白质在这些细胞中的积累速度，我们开发了一种优化的定量非规范氨基酸标记LC/MS蛋白质组学方法来标记和选择性地丰富这些原代细胞中新合成的蛋白质，同时消除了预先存在的和高度丰富的非同位素标记多肽的抑制作用。我们的数据显示，经典的病理(苯肾上腺素；PE)和最近发现的胰岛素刺激(也有助于病理性心肌肥厚(INS)的发展)都增加了与糖酵解、Krebs循环和β-氧化有关的蛋白质的合成，以及肌节成分。然而，与PE相比，胰岛素能更大程度地促进许多代谢酶的合成。使用一种新的验证方法，我们证实了PE和胰岛素确实上调了选定候选基因的合成。所有被研究的蛋白质的合成都被通过哺乳动物靶标雷帕霉素复合体1的信号上调，而它们的mRNA水平没有变化，这表明翻译控制在肥大刺激的快速效应中的关键作用。TAC可上调大鼠心脏中PKM2的表达。这种异构体具有特定的调节特性，因此这一发现表明它可能参与代谢重塑，也可以作为一种新的候选生物标志物。翻译因子eEF1的水平在TAC过程中也增加了，可能有助于更快的细胞质量积累。有趣的是，这两个候选者在妊娠或运动诱导的CH中没有上调，表明PKM2和eEF1是病理CH的特异性标记物。我们预计，这里描述的方法学将对其他研究原代细胞中蛋白质合成的研究人员有价值。
Cardiomyocytes undergo growth and remodeling in response to specific pathological or physiological conditions. In the former, myocardial growth is a risk factor for cardiac failure and faster protein synthesis is a major factor driving cardiomyocyte growth. Our goal was to quantify the rapid effects of different pro-hypertrophic stimuli on the synthesis of specific proteins in ARVC and to determine whether such effects are caused by alterations on mRNA abundance or the translation of specific mRNAs. Cardiomyocytes have very low rates of protein synthesis, posing a challenging problem in terms of studying changes in the synthesis of specific proteins, which also applies to other nondividing primary cells. To study the rates of accumulation of specific proteins in these cells, we developed an optimized version of the Quantitative Noncanonical Amino acid Tagging LC/MS proteomic method to label and selectively enrich newly synthesized proteins in these primary cells while eliminating the suppressive effects of pre-existing and highly abundant nonisotope-tagged polypeptides. Our data revealed that a classical pathologic (phenylephrine; PE) and the recently identified insulin stimulus that also contributes to the development of pathological cardiac hypertrophy (insulin), both increased the synthesis of proteins involved in, e.g. glycolysis, the Krebs cycle and beta-oxidation, and sarcomeric components. However, insulin increased synthesis of many metabolic enzymes to a greater extent than PE. Using a novel validation method, we confirmed that synthesis of selected candidates is indeed up-regulated by PE and insulin. Synthesis of all proteins studied was up-regulated by signaling through mammalian target of rapamycin complex 1 without changes in their mRNA levels, showing the key importance of translational control in the rapid effects of hypertrophic stimuli. Expression of PKM2 was up-regulated in rat hearts following TAC. This isoform possesses specific regulatory properties, so this finding indicates it may be involved in metabolic remodeling and also serve as a novel candidate biomarker. Levels of translation factor eEF1 also increased during TAC, likely contributing to faster cell mass accumulation. Interestingly those two candidates were not up-regulated in pregnancy or exercise induced CH, indicating PKM2 and eEF1 were pathological CH specific markers. We anticipate that the methodologies described here will be valuable for other researchers studying protein synthesis in primary cells.