可变剪切部位调节飞蝗横纹肌肌球蛋白马达功能的分子机制

Locust (Locusta migratoria) is not only an important agriculture pest but also a model animal for investigating insect muscle physiology. Locust has numerous types of muscle, whose contraction provides power for all kinds of movement. Muscle contraction depends on the motor function of striated myosin, which converts the energy from ATP hydrolysis into mechanical work. Unlike veterbrates, which contain multiple striated muscle myosin genes, locust has only one striated muscle myosin heavy chain (Mhc) gene, which comprises 41 exons, including 6 alternatively splicing exons. Theorily, locust Mhc gene encodes 360 muscle myosin isoforms as a result of combinations of the 6 alternative domains. Currently, we have identified 13 muscle myosin isoforms from three types of adult locust muscle. In addition, we have successfully expressed locust striated muscle myosin using insect cell expression system and obtained the purified myosin having motor function. The proposed project will investigate the roles of alternative exon-encoded regions on three aspects of locust striated muscle myosin motor function and determine the underlined molecular mechanisms, including the level of motor activity, the processitivity of the motor function, and the pH dependency of the motor function. We will use insect cell expression system to express locust striated muscle myosin isoforms and their mutants, obtain the purified recombinant myosins, and investigate their motor functions. Thereby, we will identify the key alternative domain and key residues in regulating the motor function, thus revealing the molecular mechanism of alternative domain regulation of locust striated muscle myosin motor function.

飞蝗是重要的农业害虫，也是研究昆虫肌肉生理的模式动物。飞蝗有众多类型的肌肉组织，通过肌肉收缩为各种运动提供动力。肌肉收缩依赖于横纹肌肌球蛋白的马达功能：将ATP水解产生的化学能转化为机械能。与脊椎动物不同，飞蝗基因组中只有一个横纹肌肌球蛋白重链基因，该基因由41个外显子组成，包括6个可变剪切外显子，理论上可以编码360种肌球蛋白亚型。目前我们已在飞蝗成虫的三种肌肉组织中鉴定出13种肌球蛋白亚型，并利用昆虫细胞表达系统，表达纯化得到了有马达活性的重组飞蝗肌球蛋白。本项目将从三个方面研究飞蝗肌球蛋白可变剪切部位对马达功能的影响，并阐明其分子机制：1）马达活力水平，2）马达运动的持续性，3）马达活力的pH依赖性。我们将利用昆虫细胞表达系统，表达纯化飞蝗肌球蛋白亚型及其突变体，通过分析其马达活性，确定影响马达功能的关键可变剪切部位及氨基酸残基，阐明可变剪切部位调节飞蝗肌球蛋白马达功能的分子机制。

飞蝗是重要的农业害虫，也是研究昆虫肌肉生理的模式动物。飞蝗有众多类型的肌肉组织，通过肌肉收缩为各种运动提供动力。肌肉收缩依赖于横纹肌肌球蛋白的马达功能：将ATP水解产生的化学能转化为机械能。与脊椎动物不同，飞蝗基因组中只有一个横纹肌肌球蛋白重链基因，但可以通过选择性可变剪切编码360种肌球蛋白亚型。本项目研究了飞蝗肌球蛋白可变剪切部位对马达功能的影响，分析了相关分子机制。主要成果包括：1）利用昆虫细胞表达系统，表达纯化得到了有马达活性的重组飞蝗横纹肌肌球蛋白，发现多种昆虫横纹肌肌球蛋白可以在昆虫细胞中正确折叠，而脊椎动物横纹肌不能正确折叠，表明昆虫横纹肌肌球蛋白的折叠机制与脊椎动物不同。2）发现飞蝗飞行肌肌球蛋白主要亚型的马达活性明显高于腹部节间肌的四种亚型，通过构建嵌合体方法，证明可变剪切外显子Exon-14编码的Relay区域在活性调节中起着关键作用，通过定点突变实验，证明第474位和509位残基起关键作用。3）发现飞蝗的飞行肌和跳跃肌表达不同的肌钙蛋白复合体控制肌球蛋白的马达活性，通过重组表达方法获得了完整的肌钙蛋白复合体，证明肌钙蛋白C亚基在控制肌球蛋白马达活性中起着关键作用。与跳跃肌相比，飞行肌中的肌钙蛋白在低钙条件下活性较高，在高钙条件下活性较低，暗示其他调节机制（如牵拉调节）控制飞行肌收缩舒张中起着重要作用。上述研究成果为揭示昆虫肌肉分化的分子机制奠定了基础。