STRUCTURE AND FUNCTION OF SKELETAL MUSCLE NEBULIN

骨骼肌星云蛋白的结构和功能

基本信息

批准号：
2083212
负责人：
KUAN WANG
金额：
$ 26.2万
依托单位：
UNIVERSITY OF TEXAS AT AUSTIN
依托单位国家：
美国
项目类别：
财政年份：
1995
资助国家：
美国
起止时间：
1995-04-15 至 2000-03-31
项目状态：
已结题

项目摘要

The long term goal of our proposed research is to understand the molecular structure of nebulin and to define how nebulin contributes to the mechanical properties and developmental programs of skeletal muscle cells. Nebulin is a giant actin binding protein that comprises nearly 2% of myofibrillar proteins. This modular protein has been proposed to act as a protein ruler to regulate the length of thin filaments, as a calcium- calmodulin-mediated regulatory protein of actomyosin interaction, and as a template for the assembly of the thin filament lattice during development. We propose to evaluate these hypotheses critically using a combination of biochemical, biophysical, structural and cellular approaches. The specific aims are: (l) to correlate the size of nebulin isoforms and the length of actin filaments in a wide range of skeletal muscles. Scanning transmission electron microscopy and mass spectrometry will be used to help determine the size and contour length of nebulin isoforms of mature and developing skeletal muscles of rabbit, mouse, and chicken. lmmunolocalization of actin and tropomyosin will be used to help define the length of actin filaments. (2) to prepare nebulin-containing thin filaments and study the arrangement of nebulin on the thin filaments by high resolution cryoelectron microscopy. (3) to investigate the mechanisms of nebulin-actin interaction and nebulin's inhibition of actomyosin interaction, its reversal by calcium and calmodulin and its potential regulation by phosphorylation and phospholipids. Cosedimentation, solid phase binding, ATPase assays, in vitro motility assays, and fluorescence spectroscopy will be used to reveal molecular interactions. (4) to define molecular interfaces of nebulin with actin, myosin, and calmodulin. Chemical crosslinking, protein sequencing and mutant nebulin fragments will he used to identify important amino acids at the interface. (5) to complete the coding sequence of human skeletal muscle nebulin, to define the mechanisms with which nebulin isoforms are generated and to catalog functional domains. (6) to elucidate the role of nebulin in myofibrillogenesis in developing mouse skeletal muscle in culture. The synthesis and assembly of nebulin and its transcripts in normal cells and in cells that overexpress nebulin fragments will be monitored, and thin filament length will be determined by immunoelectron microscopy. The presence of nebulin in the contractile machinery of skeletal muscles poses new challenges and elicits renewed interest in the understanding of thin filaments. The proposed research would define the physiological function of nebulin and the molecular basis for its contribution to the structure, regulation, and assembly of thin filaments in skeletal muscles in healthy and diseased states.

我们提出的研究的长期目标是了解分子星云蛋白的结构并定义星云蛋白如何促进骨骼肌细胞的机械特性和发育程序。 Nebulin 是一种巨大的肌动蛋白结合蛋白，包含近 2% 肌原纤维蛋白。这种模块化蛋白质已被提议充当蛋白质尺子调节细丝的长度，作为钙- 钙调蛋白介导的肌动球蛋白相互作用的调节蛋白，以及用于组装细丝晶格的模板发展。我们建议使用以下方法批判性地评估这些假设：生物化学、生物物理、结构和细胞的结合接近。具体目标是： (l) 将星云蛋白亚型的大小与肌动蛋白的长度相关联广泛骨骼肌中的细丝。扫描传输电子显微镜和质谱分析将用于帮助确定成熟和发育的星云蛋白亚型的大小和轮廓长度兔子、小鼠和鸡的骨骼肌。免疫定位肌动蛋白和原肌球蛋白将用于帮助定义肌动蛋白的长度细丝。 (2) 制备含星云细丝并研究其排列通过高分辨率冷冻电子在细丝上观察星云显微镜。 (3) 研究nebulin-actin相互作用的机制星云蛋白对肌动球蛋白相互作用的抑制作用，其被钙逆转和钙调蛋白及其通过磷酸化和的潜在调节磷脂。共沉降、固相结合、ATP 酶测定体外运动测定和荧光光谱将用于揭示分子相互作用。 (4) 定义星云蛋白与肌动蛋白、肌球蛋白和肌球蛋白的分子界面钙调蛋白。化学交联、蛋白质测序和突变星云蛋白片段将用于识别界面处的重要氨基酸。 (5) 完成人骨骼肌星云蛋白的编码序列，定义星云蛋白亚型的生成机制并目录功能域。 (6) 阐明nebulin在肌原纤维形成中的作用培养中的小鼠骨骼肌。星云蛋白的合成与组装及其在正常细胞和过度表达 nebulin 的细胞中的转录本将监测碎片，并确定细丝长度通过免疫电子显微镜。骨骼肌收缩机制中存在星云蛋白提出了新的挑战并引起了人们对理解的新兴趣细丝。拟议的研究将定义生理学星云蛋白的功能及其贡献的分子基础骨骼肌细丝的结构、调节和组装在健康和患病状态下。