M-Line Proteins and A-Band Assembly in Skeletal Muscle

描述（由申请人提供）：肌原纤维形成是一个高度复杂的过程，依赖于许多收缩蛋白、细胞骨架蛋白和信号蛋白的协调组装和整合，形成规则的阵列，即肌瘤。肌球蛋白结合蛋白C （MyBP-C）是一种与粗纤维相关的蛋白质，被认为在肌肉细胞内具有结构和调节作用，有助于粗纤维的正常组装和稳定，并调节可参与收缩周期的肌球蛋白头的数量。暗隐蛋白是一种巨大的肌原纤维蛋白，紧密围绕在z盘和m线周围，与MyBP-C慢速（MyBP-C（+）慢速）的一种新型异构体特异性地直接相互作用，这种异构体似乎选择性地集中在m线。因此，我们假设，通过形成可能作为支架结构的原始m线，obscurin与MyBP-C（+）的结合缓慢地促进了肌球蛋白在规则a带中的组装、稳定和维持。我们提出通过三个具体目标来验证这一假设：(1)利用广泛的分子、细胞和免疫学方法，表征分化和成年骨骼肌纤维中的新型MyBP-C（+）慢亚型；（II）通过体外和体内结合实验定性地研究了暗色蛋白与MyBP-C（+）缓慢的相互作用，并通过表面等离子体共振技术定量地研究了暗色蛋白与MyBP-C（+）缓慢的相互作用。（III）利用腺病毒介导的基因转移和小抑制RNA技术研究了暗色蛋白与MyBP-C（+）缓慢结合在肌球蛋白粗丝组装和组织成周期性a带中的生理意义。从拟议的研究中获得的知识将为将粗丝整合到正常骨骼纤维的肌节以及它们如何在人类肌肉疾病中受损的分子机制提供新的见解。

DESCRIPTION (provided by applicant): Myofibrillogenesis is a highly complex process that depends on the coordinated assembly and integration of a number of contractile, cytoskeletal and signaling proteins into regular arrays, the sarcomeres. Myosin Binding Protein C (MyBP-C), a protein associated with thick filaments, is believed to have both a structural and a regulatory role within the muscle cell, by contributing to the normal assembly and stabilization of thick filaments and modulating the number of myosin heads available for involvement in the contractile cycle. Obscurin, a giant myofibrillar protein, that closely surrounds Z-disks and M-lines, interacts specifically and directly with a novel isoform of MyBP-C slow, MyBP-C(+) slow, that appears to selectively concentrate at the M-line. Consequently, we hypothesize that the binding of obscurin with MyBP-C(+) slow contributes to the assembly, stabilization and maintenance of sarcomeric myosin into regular A-bands, through formation of primitive M-lines that may serve as scaffolding structures. We propose to test this hypothesis through three specific aims: (I) to characterize the novel MyBP-C(+) slow isoform in differentiating and adult skeletal muscle fibers, using a wide array of molecular, cellular and immunological approaches; (II) to study the interaction between obscurin and MyBP-C(+) slow qualitatively, by in vitro and in vivo binding assays, and quantitatively, by surface plasmon resonance technology and (III) to investigate the physiological significance of the binding of obscurin to MyBP-C(+) slow in the assembly and organization of myosin thick filaments into periodic A-bands, using adenovirally-mediated gene transfer and small inhibitory RNA technology. Knowledge gained from the proposed studies will provide new insights into the molecular mechanisms that integrate thick filaments into sarcomeres of normal skeletal fibers and how they are compromised in human muscle disease.