The Role of Nebulin Isoforms in Thin Filament Assembly

星云蛋白亚型在细丝组装中的作用

• 批准号: 6600873
• 负责人: Carole L. Moncman
• 金额: $ 29.4万
• 依托单位: UNIVERSITY OF KENTUCKY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-08-04 至 2007-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6600873
• 关键词: SDS polyacrylamide gel electrophoresis; actin binding protein; binding sites; cardiac myocytes; complementary DNA; gene expression; genetically modified animals; green fluorescent proteins; immunoelectron microscopy; laboratory mouse; laboratory rabbit; microfilaments; muscle proteins; myocardium; polymerase chain reaction; protein isoforms; protein protein interaction; protein structure function; striated muscles; tissue /cell culture; western blottings

DESCRIPTION (provided by applicant): Crucial to our understanding of cardiac and skeletal muscle myofilament function or dysfunction during disease is an understanding of the events and interactions involved in myofilament assembly. The long term goal of this project is to elucidate these interactions. Essential to this work is defining key proteins and assessing their role in the structure. As an initial step in this research, the roles of nebulin family members in the regulation and function of the actin based cytoskeleton will be analyzed. Nebulin and nebulette are large modular proteins associated with the thin filaments of skeletal muscle and cardiac muscle, respectively. The defining element of the nebulin family members is the presence of a 35 residue repeat that serves as the basic actin binding motif for the family members. The organization of the repeat domain has led to the postulation that these proteins serve as blueprints for the architecture of the thin filaments during development. These proteins also contain 3 other domains, an acidic N-terminal domain, a linker domain and a Src homology 3 (SH3) domain. It is hypothesized that these other domains, many of which are unique to each family member, serve to regulate actin binding functions and target interactions of the nebulin modules to distinct subcellular locations. To test these hypotheses: 1) the linker domains of three nebulin family members (nebulin, nebulette and Lasp 1) will be analyzed both in vivo and in vitro for actin binding activity and targeting functions dependent and independent of the nebulin modules; 2) the orientation of nebulette and the position of the linker and N-terminal domains will be mapped by immuno-electron microscopy; 3) the N-terminal domains of nebulette and nebulin will be examined for targeting and regulatory functions by examining the ability of this domain to interact with thin filaments in vivo and in vitro; and 4) chimeric nebulin-nebulette genes will be constructed and expressed in cardiomyocytes to evaluate the thin filament ruler hypothesis. This work will involve cellular and molecular biological techniques to alter gene expression in primary cultures of cardiac and skeletal muscle. Immunofluorescence and electron microscopy will be used to evaluate ultrastructural organization both in vitro and in vivo and biochemical and biophysical techniques to define molecular interactions. The results of this analysis will aid in our understanding of the roles of nebulin family members in the organization and functions of the thin filaments of striated muscle and to their roles in cytoskeletal dynamics. In addition, information gained here will add to our knowledge of the roles of these proteins in normal and diseased states.

描述(申请人提供)：对于我们理解疾病期间心肌和骨骼肌肌丝功能或功能障碍的关键是对肌丝组装过程中所涉及的事件和相互作用的理解。这个项目的长期目标是阐明这些相互作用。这项工作的关键是定义关键蛋白质并评估它们在结构中的作用。作为这项研究的第一步，我们将分析星云蛋白家族成员在肌动蛋白细胞骨架调控和功能中的作用。星云蛋白和星云蛋白是分别与骨骼肌和心肌细丝相关的大型模块化蛋白。星云蛋白家族成员的决定因素是存在一个35个残基的重复序列，作为家族成员的基本肌动蛋白结合基序。重复结构域的组织导致了这样的假设，即这些蛋白质在发育过程中作为细丝结构的蓝图。这些蛋白还含有另外3个结构域，即酸性N末端结构域、连接器域和Src同源3(SH3)结构域。据推测，这些其他结构域，其中许多是每个家庭成员独一无二的，用于调节肌动蛋白结合功能，并针对不同的亚细胞位置的星云蛋白模块的相互作用。为了验证这些假设：1)将在体内和体外分析三个星云蛋白家族成员(星云蛋白、星云蛋白和LASP1)的连接域的肌动蛋白结合活性和依赖和独立于星云蛋白模块的靶向功能；2)免疫电子显微镜将绘制星云蛋白的方向、连接体和N-末端结构域的位置；3)通过检测星云蛋白和星云蛋白的N-末端结构域在体内和体外与细丝相互作用的能力来检测该结构域的靶向和调节功能；4)构建星云-星云嵌合基因并在心肌细胞中表达，以验证细丝标尺假说。这项工作将涉及改变心肌和骨骼肌原代培养中基因表达的细胞和分子生物学技术。免疫荧光和电子显微镜将被用来评估体外和体内的超微结构组织，生物化学和生物物理技术将被用来定义分子相互作用。这一分析结果将有助于我们理解星云蛋白家族成员在横纹肌细丝的组织和功能中的作用以及它们在细胞骨架动力学中的作用。此外，在这里获得的信息将增加我们对这些蛋白质在正常和疾病状态下的作用的了解。