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 脊椎动物中星云蛋白亚型的生理和发育作用 骨骼肌，已通过分子变异性来识别 质量（600-900 kD）。 这种巨大的模块化蛋白质已被提议充当 蛋白质尺来调节细丝的长度，作为 钙-钙调蛋白介导的肌动球蛋白相互作用的调节蛋白， 并作为组装细丝晶格的模板 发展。 具体目标是：（1）表征星云蛋白亚型 在胎儿、新生儿和成年兔的多种骨骼肌中 老鼠。 Nebulin 表达将与 其他肌丝蛋白的转变，如肌联蛋白、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、Cap Z和原调节蛋白； (2) 至 鉴定并制备异构体特异性抗体和泛抗星云蛋白抗体。 单克隆抗nebulin的表位谱将针对每个进行表征 同工型。 抗肽抗体将产生常见的和可变的 星云蛋白序列区域； (3) 关联星云蛋白异构体的大小 以及选定胎儿、新生儿和成人肌动蛋白丝的长度 兔子和大鼠的骨骼肌。 扫描透射电子 显微镜和质谱将用于确定尺寸和 星云蛋白亚型的轮廓长度。 星云蛋白亚型的免疫定位， 肌动蛋白、原肌球蛋白、原调节蛋白和 cap Z 将用于确定 细胞分布以及细丝的长度和跨度； (4) 至 表征星云亚型多样性的分子基础。 星云蛋白外显子的发育调控表达将由以下因素决定 聚合酶链反应和核苷酸测序。 拟议的研究将定义星云蛋白的生理功能和 其贡献的发育和生理基础 骨骼肌细丝的结构、调节和组装 健康和患病状态。