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Role of myosin-binding poteins in myofibrillogenesis

肌球蛋白结合蛋白在肌原纤维发生中的作用

基本信息

批准号：
08044191
负责人：
OBINATA Takashi
金额：
$ 4.86万
依托单位：
DEPARTMENT OF BIOLOGY,CHIBA UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for international Scientific Research
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1998
项目状态：
已结题

项目摘要

C-protein is a major myosin-binding protein characteristic of striated muscles. In order to clarity the roles of C-protein in myofibrillogenesis, we carried out the following experiments and obtained the results as described.1) Mouse C-protein cDNA sequences and C-protein isoform expressionThe cDNAs encoding mouse fast skeletal (F) and slow skeletal (S) C-proteins were cloned and their entire sequences were determined. Cardiac (C)-isoform is known to be expressed first and transiently during development of chicken skeletal muscles, but in mouse skeletal muscle, C-isoform was not detected through developmental stages. S-isoform became detectable first, followed by appearance of F-isoform, In the degenerating mouse skeletal muscles, expression of S-isoform is increased.2) Two isoforms in chicken cardiac C-protein and their expressionWe detected two variants (type I and type II) in chicken cardiac C-protein and determined their entire sequences ; they differ only by the inclusion or exclu … More sion of a sequence of 15 amino acid residues (P-seq) which includes a phosphorylation site for A-kinase. We prepared the antibody specific for P-seq. By immunocytochemical methods with this and the other monoclonal antibody to cardiac C-protein (C-315), we found that two isoforms were coexpressed and colocalized in both atrial and ventricular muscles through development, but type I was scarcely expressed in embryonic skeletal muscle. These observations were confirmed by a RT-PCR method.3) Competition of C-protein (MyBP-C) with H-protein (MyBP-H) for binding to myosin in cardiac muscle cellsC-Protein and H-protein are myosin-binding proteins which localize in A-band regions of sarcomeres. To clarify functional relationship of these two myosin binding proteins, we introduced cDNAs encoding these proteins into primary cultures of chicken myocytes and examined their assembly into myofibrils. The results obtained indicate that overexpression of MyBP-C or MyBP-H in cultured cardiac myocytes can reduce the content of MyBP-H or MyBP-C, respectively. These data indicate that MyBP-H can compete with MyBP-C for binding to myosin in the cells. Less

c蛋白是横纹肌的一种主要肌球蛋白结合蛋白。为了明确c蛋白在肌原纤维形成中的作用，我们进行了如下实验，得到如下结果。1)小鼠c -蛋白cDNA序列及c -蛋白异构体表达克隆小鼠快骨骼(F)和慢骨骼(S) c -蛋白编码cDNA，并测定其全序列。心脏(C)-异构体在鸡骨骼肌发育过程中首先短暂表达，但在小鼠骨骼肌中，C-异构体在发育阶段均未被检测到。s -异构体首先被检测到，其次是f -异构体的出现，在退化的小鼠骨骼肌中，s -异构体的表达增加。2)鸡心脏c蛋白的两种异构体及其表达我们检测到鸡心脏c蛋白的两种变异（I型和II型），并测定了它们的全序列；它们的区别只是包含或排除了一个包含a激酶磷酸化位点的15个氨基酸残基序列（P-seq）。制备了P-seq特异性抗体。通过免疫细胞化学方法，我们发现该蛋白和另一种心脏c蛋白（C-315）单克隆抗体在发育过程中在心房肌和心室肌中共表达和共定位，但I型在胚胎骨骼肌中几乎不表达。这些观察结果被RT-PCR方法证实。3)心肌细胞中c蛋白（MyBP-C）与h蛋白（MyBP-H）结合肌球蛋白的竞争。c蛋白和h蛋白是肌球蛋白结合蛋白，定位于肌节的a带区。为了阐明这两种肌球蛋白结合蛋白的功能关系，我们将编码这两种蛋白的cdna引入鸡肌细胞的原代培养中，并检测了它们在肌原纤维中的组装。结果表明，培养心肌细胞过表达MyBP-C或MyBP-H可分别降低MyBP-H或MyBP-C的含量。这些数据表明MyBP-H可以在细胞中与MyBP-C竞争与肌球蛋白的结合。少