权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

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动脉粥样硬化形成的关键。我们从鸡平滑肌中分离并鉴定了一个分子量为38 kD的稳定肌球蛋白丝的蛋白。从推导的氨基酸序列来看，该基因与人p32基因同源。这种蛋白质已被鉴定为某些类型细胞中的线粒体蛋白质。当蛋白质掺入线粒体时，N-末端70个氨基酸残基被内在蛋白酶删除。我们发现该蛋白在培养的平滑肌细胞中的分布取决于分化状态。这意味着蛋白质的功能在去分化时改变。为了了解N端信号序列对定位的改变，我们尝试获得该蛋白的全长cDNA。我们构建了鸡砂囊cDNA文库并进行了筛选，但未能获得全长cDNA。主要原因是编码信号序列的核苷酸富含GC，因此cDNA合成效率不高。然后，我们获得了预先制备的cDNA文库，并筛选了全长cDNA。由于不能获得全长cDNA，我们构建了HeLa cDNA文库，目前仍在努力获得全长cDNA 2。为了了解动脉粥样硬化的分子机制，我们尝试以血管平滑肌细胞系ACO 1为模型系统。平滑肌特异性蛋白如α-肌动蛋白、高分子量钙调蛋白和结蛋白的表达水平在血清剥夺后增加。通过加入PDGF，这些蛋白的表达水平显著降低。通过观察肌原纤维蛋白的定位，PDGF的存在下，细胞骨架结构明显减少。因此，我们可以通过改变体外培养条件来控制分化水平。