Analysis of stabilization and degradation mechanism of muscle fiber of vascular smooth muscle cell upon transformatioon

血管平滑肌细胞转化后肌纤维的稳定和降解机制分析

• 批准号: 15590224
• 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: 2003
• 资助国家: 日本
• 起止时间: 2003 至 2004
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15590224/
• 关键词: protein; pharmacology; myosin; cell, tissue; atherosclerosis; hypertension; brain blood vessel; 血管平滑筋; 形質変換; ミオシン線維

Cultured vascular smooth muscle cell line AC01 become to be in transformed state in the presence of PDGF, whereas it to be in well differentiated state in the presence of sodium butyrate (NaB). mRNA was isolated from AC01 cells cultured under these two types of conditions to make cDNA. We examined expression of several marker protein such as α-actin, high molecular weight caldesmon. By RT-PCR analysis, expression level of these two proteins was down regulated upon addition of PDGF, and up regulated in the presence of NaB. We further made subtraction library from these two types of cDNA as described below.To examine the nature of p32, a myosin regulatory protein, in living smooth muscle cell, we raised antibody against N-terminal signal sequence of the protein. By indirect immunofluorescence microscopy the antibody stained cytoskeletal filaments, suggesting the localization on myosin filaments. And western blotting using antibody against p32mat, that stain both p32mat and p32FL, showed that most of p32 existing in cytoskeleton of AC01 cell is p32FL (42 kDa) but not p32mat (38 kDa). These observations suggest that p32 is binding to myosin filament as p32FL containing signal sequence. Although p32mat bind to myosin and stimulate its assembly, p32FL expressed in E. coli did not interact with myosin. The discrepancy indicates that p32FL should be post-translationaly modified to interact with myosin in vivo.We further tried to make subtraction library to screen specific gene that is up regulated upon transformation. Using two types of cDNA that are obtained from PDGF treated and NaB treated cells, PCR-select subtraction have been performed to hybridize common gene in these two groups of cDNA. Subtracted cDNA was subcloned to plasmid to construct library.

在PDGF存在下，培养的血管平滑肌细胞系AC01处于转化状态，而在丁酸钠（NAB）存在下，它处于分化良好的状态。将mRNA从在这两种类型的条件下培养的AC01细胞中分离出来，以制成cDNA。我们检查了几种标记蛋白的表达，例如α-肌动蛋白，高分子量Caldesmon。通过RT-PCR分析，加入PDGF后，调节这两种蛋白质的表达水平，并在NAB存在下进行调节。如下所述，我们进一步从这两种类型的cDNA中减去了库。要检查p32（一种肌球蛋白调节蛋白的性质，在活的平滑肌细胞中），我们提出了针对蛋白质N端信号序列的抗体。通过间接免疫荧光显微镜，抗体染色的细胞骨骼丝，表明在肌球蛋白丝上定位。使用针对p32mat的抗体染色的蛋白质印迹，染色p32mat和p32fl，表明在AC01细胞的细胞骨架中存在的大多数p32是p32fl（42 kDa），但没有p32mat（38 kDa）。这些观察结果表明，p32与肌球蛋白细丝作为含有信号序列的p32fl结合。尽管p32mat与肌球蛋白结合并刺激其组装，但在大肠杆菌中表达的p32FL与肌球蛋白没有相互作用。差异表明p32fl应该在翻译后修改以与体内与肌球蛋白相互作用。我们进一步试图使减法库以筛选在转化后受到调节的特定基因。使用两种类型的cDNA，这些cDNA从经过PDGF处理的细胞和NAB处理的细胞中获得，已经进行了PCR选择的减法，以杂交这两组cDNA中的共同基因。将减去的cDNA取代至质粒以构建文库。

项目成果

A calmodulin-dependent protein kinase from lower eukaryote Physarum polycephalum.

来自低等真核生物多头绒泡菌的钙调蛋白依赖性蛋白激酶。

• 发表时间: 2005
• 期刊: Biochem.Biophys.Res.Commun. 328
• 作者: Nakmaura;A.et al.
• 通讯作者: A.et al.

Gao, Y., K.Kawano, S.Yoshiyama, H.Kawamichi, X.Wang, A.Nakamura, K.Kohama: "Myosin light chain kinase stimulate smooth muscle myosin ATPase activity by binding to the myosin heads without phosphyorylating the myosin light chain"Biochem.Biophys.Res.Commun.

高，Y.，K.Kawano，S.Yoshiyama，H.Kawamichi，X.Wang，A.Nakamura，K.Kohama：“肌球蛋白轻链激酶通过与肌球蛋白头结合而不磷酸化肌球蛋白来刺激平滑肌肌球蛋白 ATP 酶活性

Metal-free and Ca2+-bound structures of a multidomain EF-hand protein, CBP40, from the lower eukaryote Physarum polycephalum.

来自低等真核生物多头绒泡菌的多域 EF 手蛋白 CBP40 的无金属和 Ca2 结合结构。

• 发表时间: 2003
• 期刊: Structure 11
• 作者: Iwasaki W;Sasaki H;Nakamura A;Kohama K;Tanokura M.
• 通讯作者: Tanokura M.

Biochemical properties of ordinary and dark muscle myosin from carp skeletal muscle.

• DOI: 10.1093/jb/mvi121
• 发表时间: 2005-09
• 期刊: Journal of biochemistry
• 影响因子: 2.7
• 作者: T. Okagaki;Masaki Takami;K. Hosokawa;M. Yano;S. Higashi-Fujime;A. Ooi

Partial specific volume and adiabatic compressibility of G-Actin depend on the bound nucleotide.

G-肌动蛋白的部分比容和绝热压缩性取决于结合的核苷酸。

• 发表时间: 2003
• 期刊: J.Biochem. 133
• 作者: Kikumoto;M.;Tamura;Y.;Ooi;A.;Mihashi;K.
• 通讯作者: K.