STRUCTURE AND FUNCTION OF SKELETAL MUSCLE NEBULIN

骨骼肌星云蛋白的结构和功能

• 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
• 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
• 来源: https://reporter.nih.gov/project-details/2083212
• 关键词: actin binding protein; actins; calmodulin; chickens; epitope mapping; immunoelectron microscopy; intermolecular interaction; laboratory mouse; laboratory rabbit; mass spectrometry; microfilaments; molecular cloning; molecular size; myofibrils; myogenesis; myosins; phosphorylation; protein isoforms; protein sequence; protein structure function; sarcomeres; scanning transmission electron microscopy; striated muscles; structural biology; tissue /cell culture

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.

我们拟议研究的长期目标是了解分子 星云蛋白的结构，并确定如何星云蛋白有助于 骨骼肌细胞的机械性能和发育程序。 Nebulin是一种巨大的肌动蛋白结合蛋白， 肌原纤维蛋白这种模块化蛋白被认为是一种 蛋白质的统治者，以调节细丝的长度，作为钙- 钙调蛋白介导的肌动球蛋白相互作用的调节蛋白，以及作为 用于组装细丝网格的模板， 发展我们建议用一个 生物化学、生物物理、结构和细胞的组合 接近。具体目标是： (l)将星云蛋白亚型的大小和肌动蛋白的长度联系起来， 骨骼肌中的纤维扫描透射 电子显微镜和质谱仪将被用来帮助确定 成熟期和发育期星云蛋白亚型的大小和轮廓长度 兔、小鼠和鸡骨骼肌。免疫定位 肌动蛋白和原肌球蛋白将被用来帮助确定肌动蛋白的长度 细丝。 (2)制备含有星云蛋白的细丝， 用高分辨冷电子显微镜观察了星云蛋白在细丝上的分布 显微镜 (3)研究星云蛋白-肌动蛋白相互作用的机制， 星云蛋白对肌动球蛋白相互作用的抑制，钙离子的逆转 和钙调蛋白及其通过磷酸化的潜在调节， 磷脂共沉淀，固相结合，ATP酶测定， 体外运动性测定和荧光光谱将用于 揭示分子间的相互作用 (4)定义星云蛋白与肌动蛋白、肌球蛋白和 钙调素化学交联、蛋白质测序和突变型星云蛋白 片段将用于鉴定界面处的重要氨基酸。 (5)完成人类骨骼肌星云蛋白的编码序列， 定义星云蛋白异构体产生的机制， 目录功能域。 (6)为了阐明星云蛋白在发育中肌原纤维发生中的作用， 培养的小鼠骨骼肌。星云蛋白的合成与组装 在正常细胞和过度表达星云蛋白的细胞中 将监测碎片，并确定细丝长度 通过免疫电镜。 星云蛋白在骨骼肌收缩机制中的存在 提出了新的挑战，并激发了人们对理解 细丝拟议的研究将定义生理 星云蛋白的功能和它的贡献的分子基础 骨骼肌中细丝的结构、调节和装配 在健康和疾病状态下。