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首页> 外文期刊>Molecular & cellular proteomics: MCP >Proteomics analysis of the cardiac myofilament subproteome reveals dynamic alterations in phosphatase subunit distribution.
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Proteomics analysis of the cardiac myofilament subproteome reveals dynamic alterations in phosphatase subunit distribution.

机译：心脏肌丝亚蛋白质组的蛋白质组学分析揭示了磷酸酶亚基分布的动态变化。

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摘要

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 B56alpha. Immunoblot analysis of myocyte fractions confirmed that beta-adrenergic stimulation by isoproterenol decreased the B56alpha 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 B56alpha from Z-disc and M-band regions but increased association of MYPT1/2 with A-band regions of the sarcomere following beta-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.
机译：肌丝蛋白负责心脏收缩。然而，迄今为止尚未对肌丝亚蛋白质组进行全面分析。在本研究中，从啮齿动物的心脏中分离出心肌细胞，并用内皮素-1和异丙肾上腺素（肌丝蛋白磷酸化的强力诱导剂）刺激心肌细胞。随后，将心肌细胞“剥皮”，并使用配备电子转移解离的高精度离子阱串联质谱仪（LTQ Orbitrap XL）分析肌丝亚蛋白质组。如预期的那样，少量的肌丝蛋白构成了总蛋白质量的大部分，并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出600多种其他蛋白质，包括激酶和磷酸酶亚基。对照和处理过的心肌细胞的肌丝蛋白质组学比较表明，异丙肾上腺素治疗改变了蛋白磷酸酶2A调节亚基B56alpha的亚细胞定位。对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（MYPT1和MYPT2）没有明显变化的情况下，异丙肾上腺素对β-肾上腺素能的刺激降低了肌丝组分的B56alpha含量。此外，免疫标记和共聚焦显微镜揭示了这些蛋白质的空间再分布，其中Z-disc和M-band区域的B56alpha丢失，但在β-肾上腺素刺激后，MYPT1 / 2与肌小节的A-带区域的结合增加。总而言之，我们介绍了皮肤心肌细胞的第一个综合蛋白质组学数据集，并证明了蛋白质组学揭示蛋白质成分动态变化的潜力，该动态变化可能有助于肌丝收缩的神经激素调节。

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