Developmental Changes Affecting Cardiac Titin Function

影响心脏肌联蛋白功能的发育变化

• 批准号: 6808924
• 负责人: MARION Lewis GREASER
• 金额: $ 29.1万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-07-01 至 2008-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6808924
• 关键词: cardiac myocytes; chromatography; circular dichroism; developmental genetics; echocardiography; electrophoresis; embryo /fetus protein; gene mutation; genetic mapping; glutamates; heart circulation; heart function; hemodynamics; laboratory rabbit; laboratory rat; mature animal; molecular assembly /self assembly; muscle proteins; muscle tension; peptides; phosphorylation; protein binding; protein isoforms; protein sequence; protein structure function; surface plasmon resonance

DESCRIPTION (provided by applicant): Titin is a 3000-4000 kD protein found in heart and skeletal muscle, and it has been proposed to play a major role in controlling the resting or passive tension in these tissues. Major developmental changes have been found to occur in cardiac titin as a result of alternative splicing pathways. A unique rat strain has been discovered with an autosomal dominant mutation that leads to a delayed fetal-to-adult titin transition pattern. These animals will be used to test the role of titin in cardiac hemodynamics in the intact animal using echocardiography and pressure-volume relationship measurements. Mechanical experiments on single cardiomyocytes will test hypotheses regarding titin's role in rest tension and the Frank-Starling relationship. Interactions betweer_ positively charged PPAK peptides from titin's extensible PEVK region with negatively charged polyE peptides will be tested using chromatographic, circular dichroism, electrophoretic, and surface plasmon resonance techniques. Studies will be designed to determine if binding of the PPAK and polyE peptides is specific to adjacent regions in the sequence or if multiple types of interactions can occur. Nontitin peptides rich in glutamic acid will also be examined for binding. Previously proposed phosphorylation sites will be identified in expressed titin polypeptides, and these sites will also be assayed for )hosphorylation state in intact fetal and adult titins. These experiments will test the hypothesis that certain _ites must be phosphorylated for proper assembly and/or function. Gene mapping studies will be conducted to localize the mutation site leading to the delayed developmental titin isoform program. The proposed studies will provide new information regarding the structure and function of this giant protein and its relation to human health and cardiovascular disease. The work may also provide novel insights on mechanisms of alternative splicing, an area of increasing importance in understanding the proteome.

描述（由申请人提供）：肌联蛋白是一种在心脏和骨骼肌中发现的 3000-4000 kD 蛋白质，并且已被认为在控制这些组织中的静息或被动张力方面发挥着重要作用。 已发现由于选择性剪接途径，心脏肌联蛋白发生了主要的发育变化。 已发现一种独特的大鼠品系，其具有常染色体显性突变，导致胎儿到成人肌联蛋白转变模式延迟。 这些动物将用于通过超声心动图和压力-体积关系测量来测试肌动蛋白在完整动物心脏血流动力学中的作用。 对单个心肌细胞的机械实验将检验有关肌联蛋白在静息张力和 Frank-Starling 关系中的作用的假设。 将使用色谱、圆二色性、电泳和表面等离子体共振技术来测试来自肌动蛋白可延伸PEVK区域的带正电的PPAK肽与带负电的polyE肽之间的相互作用。 研究将旨在确定 PPAK 和 PolyE 肽的结合是否对序列中的相邻区域具有特异性，或者是否可以发生多种类型的相互作用。 富含谷氨酸的非结合蛋白肽也将被检测结合。 将在表达的肌联蛋白多肽中鉴定先前提出的磷酸化位点，并且还将分析这些位点的完整胎儿和成人肌联蛋白中的磷酸化状态。 这些实验将检验以下假设：某些位点必须被磷酸化才能正确组装和/或发挥功能。 将进行基因图谱研究来定位导致发育迟缓的肌联蛋白亚型程序的突变位点。 拟议的研究将提供有关这种巨型蛋白质的结构和功能及其与人类健康和心血管疾病的关系的新信息。 这项工作还可能为选择性剪接机制提供新的见解，这是理解蛋白质组日益重要的一个领域。