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

1.平滑肌细胞去分化是诱发动脉粥样硬化的关键事件。我们从鸡的平滑肌中分离并鉴定了一种38kD的稳定肌球蛋白细丝的蛋白质。从推导的氨基酸序列来看，它是人p32的同源物。这种蛋白在某些类型的细胞中被鉴定为线粒体蛋白。当蛋白质进入线粒体后，N-末端70个氨基酸残基被固有的蛋白酶删除。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发生了变化。为了了解N-末端信号序列的变化对定位的影响，我们试图获得该蛋白的全长cDNA。我们构建了鸡肌胃的cDNA文库，并对其进行了筛选，但未能获得全长的cDNA。主要原因是编码信号序列的核苷酸富含GC，导致合成效率不高。接下来，我们获得了预先构建的cDNA文库，并对其全长进行了筛选。由于未能获得全长cDNA2，我们构建了HeLa的cDNA2文库，目前仍在努力获得全长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ACO1建立模型系统。去血清后，α-肌动蛋白、高分子钙调蛋白和结蛋白的表达水平增加。加入PDGF后，这些蛋白的表达水平显著降低。通过对肌原纤维蛋白定位的观察，发现PDGF的存在使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。

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.