Study on recombinant myosin which has high force output with high efficiency

高效率高输出力重组肌球蛋白的研究

• 批准号: 09670702
• 负责人: SUGIURA Seiryo
• 金额: $ 1.86万
• 依托单位: University of Tokyo
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1998
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09670702/
• 关键词: myosin; molecular biology; in vitro motiliy assay; optical tweezer; インビトロ アッセイ

To screen recombinant myosin which has high contractile performance. We studied the mechanical as well as biochemical characteristics of various myosin species obtained from animal model and genetically designed and expressed in cell culture. We constructed an in vitro motility assay system coupled with an optical tweezer with which we could measure the force generated by a single myosin molecule and actin filament (unitary force). We compared the unitary force generated by two cardiac myosin isoforms, V1 and V3 to find the force of two isoforms differs in duration but not in amplitude. We considered that this difference in duration of force causes the distinct energetic characteristics of these isoforms. We also evaluated the properties of mutant myosin implicated in human familial hypertrophic cardiomyopathy. We constructed recombinant myosin molecules whose mutations were located in ATPase domain, actin binding domain, proximity of reactive cysteine, and interface of light chain. We found a discrepancy between ATPase and force generating ability in these mutations. Poor prognosis of patients with these mutations suggested the significant role of these malfunctioning myosin in the pathogenesis of hypertrophic cardiomyopathy. It also showed that functional improvement of myosin by an alteration of molecular structure could be a potential treatment of heart disease.

筛选具有高收缩性能的重组肌球蛋白。我们研究了从动物模型中获得的各种肌球蛋白的机械和生化特性，并在细胞培养中进行遗传设计和表达。我们构建了一个体外运动性测定系统，结合光镊，我们可以测量单个肌球蛋白分子和肌动蛋白丝产生的力（单位力）。我们比较了两种心肌肌球蛋白亚型V1和V3产生的单位力，发现两种亚型的力在持续时间上不同，但在幅度上没有差异。我们认为，这种力的持续时间的差异导致这些异构体的不同的能量特性。我们还评估了与人类家族性肥厚型心肌病有关的突变型肌球蛋白的性质。我们构建了重组肌球蛋白分子，其突变位于ATP酶结构域、肌动蛋白结合结构域、活性半胱氨酸附近和轻链界面。我们发现在这些突变中ATP酶和力产生能力之间存在差异。这些突变的患者预后不良，提示这些肌球蛋白功能障碍在肥厚型心肌病发病机制中的重要作用。它还表明，通过改变分子结构来改善肌球蛋白的功能可能是心脏病的潜在治疗方法。

项目成果

Sugiura S.: "Comparison of unitary displacements and forces between two cardiac myosin isoforms by the optical trap technique: Molecular basis for cardiac adaptation" Circulation Research. 82. 1029-1034 (1998)

Sugiura S.：“通过光学陷阱技术比较两种心肌肌球蛋白异构体之间的单位位移和力：心脏适应的分子基础”循环研究。

Suzuki Y: "Modulation of actin filament sliding by mutations of the SH2 cysteine in Dictyostelium Myosin II" Biochemcal and Biophyscal Research Communication. 234. 701-706 (1997)

Suzuki Y：“盘基网柄菌肌球蛋白 II 中 SH2 半胱氨酸突变对肌动蛋白丝滑动的调节”生物化学和生物物理学研究通讯。

Fujita H., Sugiura S., Momomura S., Omata M., Sugi H., Sutoh K: "Characterization of mutant myosins of Dictiostelium Discoideum equinalent to human familial hypertrophic cardiomyopathy mutants" J.Clin. Invest.99. 1010-1015 (1997)

Fujita H.、Sugiura S.、Momomura S.、Omata M.、Sugi H.、Sutoh K：“相当于人类家族性肥厚性心肌病突变体的盘网菌突变体肌球蛋白的表征”J.Clin。

Aoyagi,T.: "Inhibition of carnitine synthesis protects against left ventricular dysfunction in rats with myocardial ischemia." J.Cardiovascular Pharmacology. 30. 468-474 (1997)

Aoyagi,T.：“抑制肉碱合成可防止心肌缺血大鼠的左心室功能障碍。”

Sugiura S: "Comparison of unitary displacements and forces between two cardiac myosin isoforms by the optical trap technique : Molecular basis for cardiac adaptation" Circulation Research. 82. 1029-1034 (1998)

Sugiura S：“通过光学陷阱技术比较两种心肌肌球蛋白异构体之间的单位位移和力：心脏适应的分子基础”循环研究。