Proteomics analysis of the cardiac myofilament subproteome reveals dynamic alterations in phosphatase subunit distribution.
Proteomics analysis of the cardiac myofilament subproteome reveals dynamic alterations in phosphatase subunit distribution.
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DOI：
10.1074/mcp.m900275-mcp200
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发表时间：
2010-03
期刊：
影响因子：
--
通讯作者：
Mayr M
中科院分区：
文献类型：
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作者：
Yin X;Cuello F;Mayr U;Hao Z;Hornshaw M;Ehler E;Avkiran M;Mayr M
肌丝蛋白负责心脏收缩。然而，肌丝亚蛋白质组迄今尚未得到全面分析。在本研究中，从啮齿动物心脏中分离心肌细胞，并用内皮素-1和异丙肾上腺素（肌丝蛋白磷酸化的强效诱导剂）刺激。随后，将心肌细胞“剥皮”，并使用配备有电子转移解离的高质量精度离子阱串联质谱仪（LTQ Orbitrap XL）分析肌丝亚蛋白质组。正如预期的那样，少量的肌丝蛋白构成了总蛋白质质量的大部分，通过电子转移解离证实了几个已知的磷酸化位点。在心肌肌丝亚蛋白质组中鉴定了超过600种额外的蛋白质，包括激酶和磷酸酶亚基。对照组和处理组心肌细胞的肌丝蛋白质组学比较表明，异丙肾上腺素处理改变了蛋白磷酸酶2A调节亚基B56α的亚细胞定位。肌细胞组分的免疫印迹分析证实，在肌球蛋白磷酸酶靶亚基亚型1和2（MYPT 1和MYPT 2）无显著变化的情况下，异丙肾上腺素的β-肾上腺素能刺激降低了肌丝组分的B56α含量。此外，免疫标记和共聚焦显微镜显示，这些蛋白质的空间重新分布与B56α从Z盘和M带区域的损失，但增加的MYPT 1/2与A带区域的肌节β-肾上腺素能刺激。总之，我们提出了第一个全面的蛋白质组学数据集的皮肤心肌细胞，并证明了蛋白质组学的潜力，解开蛋白质组成的动态变化，可能有助于神经激素调节肌丝收缩。
Myofilament proteins are responsible for cardiac contraction. The myofilament subproteome, however, has not been comprehensively analyzed thus far. In the present study, cardiomyocytes were isolated from rodent hearts and stimulated with endothelin-1 and isoproterenol, potent inducers of myofilament protein phosphorylation. Subsequently, cardiomyocytes were “skinned,” and the myofilament subproteome was analyzed using a high mass accuracy ion trap tandem mass spectrometer (LTQ Orbitrap XL) equipped with electron transfer dissociation. As expected, a small number of myofilament proteins constituted the majority of the total protein mass with several known phosphorylation sites confirmed by electron transfer dissociation. More than 600 additional proteins were identified in the cardiac myofilament subproteome, including kinases and phosphatase subunits. The proteomic comparison of myofilaments from control and treated cardiomyocytes suggested that isoproterenol treatment altered the subcellular localization of protein phosphatase 2A regulatory subunit B56α. Immunoblot analysis of myocyte fractions confirmed that β-adrenergic stimulation by isoproterenol decreased the B56α content of the myofilament fraction in the absence of significant changes for the myosin phosphatase target subunit isoforms 1 and 2 (MYPT1 and MYPT2). Furthermore, immunolabeling and confocal microscopy revealed the spatial redistribution of these proteins with a loss of B56α from Z-disc and M-band regions but increased association of MYPT1/2 with A-band regions of the sarcomere following β-adrenergic stimulation. In summary, we present the first comprehensive proteomics data set of skinned cardiomyocytes and demonstrate the potential of proteomics to unravel dynamic changes in protein composition that may contribute to the neurohormonal regulation of myofilament contraction.