Analysis of the function of myosin-stabilizing protein in living smooth muscle cell

活体平滑肌细胞中肌球蛋白稳定蛋白的功能分析

基本信息

批准号：
12680691
负责人：
OKAGAKI Tsuyoshi
金额：
$ 2.3万
依托单位：
Mie University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12680691/
关键词：
vascular cell smooth muscle cell myosin myosin filament

项目摘要

1. Dedifferentiation of smooth muscle cell is key event for induction of atherosclerosis. We isolated and characterized a protein with 38kD that stabilize myosin filament from chicken smooth muscle. From deduced amino acid sequence of cDNA, it was homologue of human p32. This protein has been identified as a mitochondrial protein in some types of cell. Upon incorporation of the protein into mitochondria, N-terminal 70 amino acid residue are deleted by intrinsic protease. We found that the protein is distributed in cultured smooth muscle cell depending on the state of differentiation. This means that the function of the protein is changed upon dedifferentiation. To understand the N-terminal signal sequence on the change of localization, we tried to obtain full length cDNA of the protein. We made cDNA library of chicken gizzard and screened the cDNA, but could not obtained the full length cDNA. The main reason was that the nucleotide coding the signal sequence is quite GC-rich, so that cDNA synthesis was not efficient. Next we obtained pre-made cDNA library and also screened the full length cDNA. Since we could not obtained the full length cDNA, we made cDNA library of HeLa, now we are still doing effort to obtain the full length cDNA2. To understand molecular mechanism of atherosclerosis, we tried to make model system by means of vascular smooth muscle cell line ACO1. The expression level of smooth muscle specific protein such as α-actin, high molecular weight caldesmon, and desmin was increased upon serum deprivation. By addition of PDGF, expression level of these protein was remarkably reduced. By observation of the localization of myofibrillar protein, cytoskeletal structure was clearly diminished in the presence of PDGF. Thus we could control the level of differentiation by changing the culture condition in vitro.

1。平滑肌细胞的去分化是诱导动脉粥样硬化的关键事件。我们用38KD隔离并表征了一种蛋白质，该蛋白质稳定了鸡平滑肌的肌球蛋白细丝。从推导的cDNA氨基酸序列中，它是人p32的同源物。在某些类型的细胞中，该蛋白已被鉴定为线粒体蛋白。将蛋白质掺入线粒体中后，N-末端70氨基酸的保留将被内在蛋白酶删除。我们发现该蛋白质根据分化状态分布在培养的平滑肌细胞中。这意味着蛋白质的功能在去分化后会改变。为了理解N端信号序列在定位的变化上，我们试图获得蛋白质的全长cDNA。我们制作了鸡皮bizz的cDNA库并筛选了cDNA，但无法获得全长cDNA。主要原因是编码信号序列的核苷酸非常富含GC，因此cDNA合成不高。接下来，我们获得了预制的cDNA库，还筛选了全长cDNA。由于我们无法获得全长cDNA，因此我们制造了HeLa的cDNA库，现在我们仍在努力获得全长的cDNA2。为了了解动脉粥样硬化的分子机制，我们试图通过血管平滑肌细胞系ACO制造模型系统。血清剥夺时，平滑肌特异性蛋白（例如α-肌动蛋白，高分子量Caldesmon和Desmin）的表达水平增加。通过添加PDGF，这些蛋白质的表达水平明显降低。通过观察肌原纤维蛋白的定位，在存在PDGF的情况下，细胞骨架结构明显减少。我们可以通过在体外改变培养条件来控制分化的水平。