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: 6787161
• 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/6787161
• 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（由申请人提供）：对我们理解疾病期间心脏和骨骼肌肌丝功能或功能障碍的关键是理解肌丝组装中涉及的事件和相互作用。本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。作为本研究的第一步，将分析星云蛋白家族成员在基于肌动蛋白的细胞骨架的调节和功能中的作用。Nebulin和neblette是分别与骨骼肌和心肌的细丝相关的大模块蛋白。星云蛋白家族成员的定义元素是存在35个残基的重复序列，其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一种假设，即这些蛋白质在发育过程中充当细丝结构的蓝图。这些蛋白质还含有3个其它结构域，酸性N-末端结构域、接头结构域和Src同源3（SH 3）结构域。据推测，这些其他领域，其中许多是独特的每个家庭成员，用于调节肌动蛋白结合功能和目标相互作用的星云蛋白模块到不同的亚细胞位置。为了检验这些假设：1）三个nebulin家族成员的连接结构域（nebulin、nebulette和Lasp 1）将在体内和体外分析依赖于和不依赖于nebulin模块的肌动蛋白结合活性和靶向功能; 2）将通过免疫电子显微镜绘制nebulette的取向以及接头和N-末端结构域的位置; 3）通过检查nebulette和nebulin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查该结构域的靶向和调节功能;以及4）嵌合的星云蛋白-星云蛋白基因将被构建并在心肌细胞中表达以评估细丝尺假说。这项工作将涉及细胞和分子生物学技术，以改变心脏和骨骼肌原代培养物中的基因表达。免疫荧光和电子显微镜将用于评价体外和体内的超微结构组织，以及生物化学和生物物理技术来确定分子相互作用。这一分析的结果将有助于我们理解的作用，星云蛋白家族成员的组织和功能的细丝横纹肌和他们的作用，在细胞骨架动力学。此外，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中的作用的了解。