STRUCTURE AND FUNCTION OF SKELETAL MUSCLE NEBULIN

骨骼肌星云蛋白的结构和功能

• 批准号: 2390552
• 负责人: KUAN WANG
• 金额: $ 28.39万
• 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/2390552
• 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.

我们提出的研究的长期目标是了解分子

项目成果

Architecture of the thin filament-Z-line junction: lessons from nebulette and nebulin homologies.

细丝-Z 线连接的结构：星云和星云蛋白同源性的教训。

• DOI: 10.1023/a:1005697226465
• 发表时间: 2000
• 期刊: Journal of muscle research and cell motility
• 影响因子: 2.7
• 作者: Moncman,CL;Wang,K
• 通讯作者: Wang,K

Molecular contacts between nebulin and actin: cross-linking of nebulin modules to the N-terminus of actin.

星云蛋白和肌动蛋白之间的分子接触：星云蛋白模块与肌动蛋白 N 末端的交联。

• DOI: 10.1021/bi961236b
• 发表时间: 1997
• 期刊: Biochemistry.
• 作者: Shih,CL;Chen,MJ;Linse,K;Wang,K
• 通讯作者: Wang,K

Human skeletal muscle nebulin sequence encodes a blueprint for thin filament architecture. Sequence motifs and affinity profiles of tandem repeats and terminal SH3.

人类骨骼肌星云蛋白序列编码细丝结构的蓝图。

• DOI: 10.1074/jbc.271.8.4304
• 发表时间: 1996
• 期刊: The Journal of biological chemistry
• 作者: Wang,K;Knipfer,M;Huang,QQ;vanHeerden,A;Hsu,LC;Gutierrez,G;Quian,XL;Stedman,H
• 通讯作者: Stedman,H