Developmental Changes Affecting Cardiac Titin Function

描述(申请人提供)：Titin是一种存在于心脏和骨骼肌中的3000-4000kD的蛋白质，它被认为在控制这些组织的静息或被动张力方面发挥着重要作用。由于不同的剪接途径，心脏肌动蛋白发生了重大的发育变化。一种独特的大鼠品系被发现，它具有常染色体显性突变，导致胎儿到成人的延迟TITIN转换模式。这些动物将被用来通过超声心动图和压力-体积关系测量来测试Titin在完整动物的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关于肌动蛋白在静息张力和Frank-Starling关系中的作用的假说。我们将使用层析、圆二色谱、电泳法和表面等离子体共振技术来测试来自Titin的PEVK延伸区的带正电的PPAK多肽与带负电的多肽之间的相互作用。研究将被设计来确定PPAK和多肽的结合是否是序列中相邻区域所特有的，或者是否可能发生多种类型的相互作用。富含谷氨酸的非铁蛋白多肽也将被检查结合情况。先前提出的磷酸化位点将在表达的肌动蛋白多肽中确定，这些位点也将被检测)完整胎儿和成人的肌动蛋白的磷酸化状态。这些实验将检验这样的假设，即某些_ITE必须被磷酸化才能正确组装和/或功能。将进行基因图谱研究，以定位导致延迟发育的肌动蛋白亚型程序的突变位置。拟议的研究将提供关于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的机制提供新的见解，这是一个在理解蛋白质组方面日益重要的领域。

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.