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蛋白是条纹肌肉的主要肌球蛋白结合蛋白特征。为了清楚C蛋白在肌原纤维发生中的作用，我们进行了以下实验并获得了所述结果。1）小鼠C蛋白cDNA序列和C蛋白质同工型表达编码小鼠快速骨骼（F）的CDNA，并确定了整个序列。众所周知，心脏（C） - 异型类型在鸡骨骼肌发育过程中首先和瞬时表达，但是在小鼠骨骼肌中，未通过发育阶段检测到C-异型。 S-异型成型首先被检测到，其次是F-异型的出现，在退化的小鼠骨骼肌中，S-异型的表达增加了。2）2）鸡心脏C蛋白中的两种同工型，它们的表达式在表达中，我们检测到了两个变体（I型和II型）在鸡肉型C蛋白中，并确定了整个序列；它们仅因包含或排除而差异……更多的15个氨基酸残基（P-SEQ）的序列包括A型激酶的磷酸化位点。我们准备了针对P-Seq的抗体。通过与心脏C蛋白（C-315）的这种免疫细胞化学方法和其他单克隆抗体（C-315），我们发现通过发育共表达和在心室和心室的室中共定位了两种同工型，但是类型I在胚胎骨骼肌中很少表达。这些观察结果通过RT-PCR方法证实。3）C蛋白（MYBP-C）与H蛋白（MYBP-H）的竞争，在心脏肌肉细胞蛋白和H蛋白中与肌球蛋白结合，是肌球蛋白结合蛋白，是肌球蛋白结合蛋白，在肉瘤的A频段区域局部。为了阐明这两种肌球蛋白结合蛋白的功能关系，我们将编码这些蛋白质的cDNA引入了鸡肌细胞的主要培养物中，并将其组装成肌原纤维。获得的结果表明，在培养的心肌细胞中MYBP-C或MYBP-H的过表达可以分别减少MYBP-H或MYBP-C的含量。这些数据指标表明MYBP-H可以与MYBP-C竞争以与细胞中的肌球蛋白结合。较少的