Obscurin & Myofibrils in Cardiac & Skeletal Muscle

描述(申请人提供)：近年来，我们了解了许多关于横纹肌中粗丝和细丝的组装，以及调节收缩周期中必需的钙释放和重新摄取的蛋白质。然而，我们对将粗丝和细丝整合成肌原纤维的机制以及在肌节周围如此规则地组织肌浆网(SR)和横管(T-)的蛋白质知之甚少。最近发现的暗黑蛋白为解决这些问题提供了新的方法。 Obscurin是一种约800 kDa的蛋白质，其结构与肌动蛋白具有同源性。它是Titin超家族中唯一一种至少部分集中在肌节周围的蛋白质，主要集中在M线和Z盘周围。Obscurin主要由串联的Ig结构域组成，但也有信号域，在其极端的COOH末端，有一个小的完整的膜形式的锚定蛋白1的结合位点，该结合形式集中在网络SR中。考虑到它明显能够集中在M线和Z盘周围的肌原纤维的外围，以及它对SR膜的完整蛋白的高亲和力，Oblcurin非常适合在组装和组织肌节和SR中发挥关键作用。我们建议通过四个特定的目标来检验这一一般假设：(1)了解相对于肌节内的关键结构是如何组织Oblcurin的；(2)了解Oblcurin如何与收缩装置中的元件结合；(3)评估通过siRNA和通过特定结合域的腺病毒过表达改变Oblocin的活性对肌原纤维及其相关膜的形态发生的影响；以及(4)研究Oblcurin与小骨架蛋白1的结合在SR的组装、组织和功能中的作用。 在肥厚性心肌病和肌营养不良症中，收缩装置蛋白质的突变，包括一些影响收缩元件的组装或稳定性的突变，被认为是有关联的。我们的实验结果应该会揭示一些关键的机制，这些机制涉及到将收缩蛋白组装成肌节并确定它们与SR的关联。

DESCRIPTION (provided by applicant): In recent years, we have learned a great deal about the assembly of thick and thin filaments in striated muscle, and about the proteins that regulate the release and re-uptake of Ca2+ essential for the contractile cycle. We know much less, however, about the mechanisms that integrate thick and thin filaments into myofibrils, and about the proteins that organize the sarcoplasmic reticulum (SR) and transverse (t-) tubules so regularly around sarcomere. The recent discovery of obscurin offers new ways to address these questions. Obscurin is an approximately 800 kDa protein with structural homology to titin. It is the only protein of the titin superfamily that is concentrated, at least in part, at the periphery of sarcomeres, primarily around M-lines and Z-disks. Obscurin is composed largely of tandem Ig domains but also has signaling domains and, at its extreme COOH-terminus, a binding site for a small, integral membrane form of ankyrin 1 that is concentrated in the network SR. Given its apparent ability to concentrate at the periphery of the myofibril around M-lines and Z-disks and its high affinity for an integral protein of the SR membrane, obscurin is ideally suited to play key roles in assembling and organizing both the sarcomere and the SR. We propose to test this general hypothesis through 4 specific aims: (1) to learn how obscurin is organized with respect to key structures within sarcomeres; (2) to learn how obscurin binds to elements in the contractile apparatus; (3) to assess the effect on morphogenesis of the myofibrils and associated membranes of altering the activity of obscurin with siRNA and thru adenoviral overexpression of particular binding domains; and (4) to investigate the role of obscurin's binding to small ankyrin 1 in the assembly, organization and function of the SR. Mutations in proteins of the contractile apparatus, including some that affect the assembly or stability of contractile elements, have been implicated in hypertrophic cardiomyopathies and muscular dystrophies. The results of our experiments should reveal some of the key mechanisms involved in assembling contractile proteins into sarcomeres and in determining their association with the SR.