NEBULIN AS A MOLECULAR RULER OF THIN FILAMENTS

星云作为细丝的分子标尺

• 批准号: 2292317
• 负责人: KUAN WANG
• 金额: $ 2.33万
• 依托单位: UNIVERSITY OF TEXAS AT AUSTIN
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-09-01 至 1998-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2292317
• 关键词: RNase protection assay; actin binding protein; actins; calmodulin; cooperative study; gene expression; laboratory rabbit; laboratory rat; mass spectrometry; monoclonal antibody; muscle proteins; myofibrils; myosins; polymerase chain reaction; protein biosynthesis; protein isoforms; protein sequence; protein structure function; scanning transmission electron microscopy; striated muscles; tropomyosin; troponin

DESCRIPTION The long-term goal of the proposed research is to understand the physiological and developmental roles of nebulin isoforms in vertebrate skeletal muscles, which have been identified by variability in the molecular mass (600-900 kD). This giant 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 thin filament lattice during development. The specific aims are: (1) to characterize nebulin isoforms in a wide range of skeletal muscles of fetal, neonatal and adult rabbits and rats. Nebulin expression will be compared and contrasted with the transition of other myofilament proteins such as titin, myosin, C-protein, actin, tropomyosin, troponin, alpha-actin, Cap Z and tropomodulin; (2) to identify and prepare isoform-specific and pan anti-nebulin antibodies. Epitope profiles of monoclonal anti-nebulin will be characterized for each isoform. Anti-peptide antibodies will be produced to common and variable regions of nebulin sequences; (3) to correlate the size of nebulin isoforms and the length of actin filaments in selected fetal, neonatal and adult skeletal muscles of rabbit and rats. Scanning transmission electron microscopy and mass spectroscopy will be used to determine the size and contour length of nebulin isoforms. Immunolocalization of nebulin isoforms, actin, tropomyosin, tropomodulin and cap Z will be used to determine the cellular distribution and length and span of thin filaments; and (4) to characterize the molecular basis of nebulin isoform diversity. Developmentally regulated expression of nebulin exons will be determined by polymerase chain reaction and nucleotide sequencing. The proposed research would define the physiological function of nebulin and the developmental and physiological basis for its contribution to the structure, regulation, and assembly of thin filaments in skeletal muscles in healthy and diseased states.

描述 拟议研究的长期目标是了解 星云蛋白亚型在脊椎动物中的生理和发育作用 骨骼肌，已经通过分子的变异性来识别 质量(600-900kD)。这种巨大的模块化蛋白质被认为是一种 蛋白质尺调节细丝长度的蛋白质尺，如 钙-钙调蛋白介导的肌动球蛋白相互作用调节蛋白， 作为细丝格子组装的模板 发展。具体目标是：(1)表征星云蛋白亚型 在胎兔、新生兔和成年兔的广泛骨骼肌中 老鼠。星云蛋白的表达将与 其他肌丝蛋白如肌动蛋白、肌球蛋白、C蛋白、 肌动蛋白、原肌球蛋白、肌钙蛋白、α-肌动蛋白、Z帽和原调节蛋白；(2)至 鉴定和制备抗星云蛋白亚型特异性抗体和PAN抗星云蛋白抗体。 将对每个单抗星云蛋白的表位图谱进行表征 异构体。将产生的抗肽抗体分为共性和变异性 星云蛋白序列的区域；(3)关联星云蛋白亚型的大小 以及选定的胎儿、新生儿和成人的肌动蛋白细丝长度 兔和大鼠的骨骼肌。扫描透射式电子 将使用显微镜和质谱学来确定大小和 星云蛋白异构体的等高线长度。星云蛋白亚型的免疫定位， 肌动蛋白、原肌球蛋白、原肌球蛋白和Z帽将用于确定 细胞分布和细丝的长度和跨度；和(4) 描述星云蛋白异构体多样性的分子基础。 发育调节的星云蛋白外显子的表达将由 聚合酶链式反应和核苷酸测序。 这项拟议的研究将定义星云的生理功能和 它对人类健康的贡献的发育生理基础 人骨骼肌中细丝的结构、调节和组装 健康和患病的状态。