Roles of Nebulin in Structure and Function of Straited Muscle

星云蛋白在狭窄肌结构和功能中的作用

• 批准号: 10231181
• 负责人: Henk L. GRANZIER
• 金额: $ 32.76万
• 依托单位: UNIVERSITY OF ARIZONA
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-07-01 至 2022-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10231181
• 关键词: Actins; Affect; Attenuated; Basic Science; Biology; C-terminal; Clinical; Complex; Data; Defect; Disease; Distal; F-Actin; Fatigue; Gene Delivery; Genes; Hypertrophy; Individual; Insulin-Like Growth Factor I; Length; Limb structure; Location; Measures; Mechanics; Modeling; Mus; Muscle; Muscle Proteins; Muscle Weakness; Muscle function; Muscular Atrophy; Mutation; Myopathy; Nemaline Myopathies; Neurons; Patients; Phenocopy; Phenotype; Pilot Projects; Play; Population; Positioning Attribute; Proteins; Regulation; Reporting; Research; Respiratory Diaphragm; Role; SH3 Domains; Sarcomeres; Serine; Signal Transduction; Skeletal Muscle; Soleus Muscle; Speed; Structure; Techniques; Testing; Therapeutic; Therapeutic Studies; Thin Filament; Width; Wiskott-Aldrich Syndrome; Work; base; cost; gene therapy; improved; insight; mouse model; muscle hypertrophy; muscular structure; nebulin; novel; response

Nebulin is an important but poorly understood sarcomeric protein found in skeletal muscle. Our previous studies with nemaline myopathy patients deficient in nebulin (NEM2 patients) revealed that nebulin protein loss results in thin filaments of reduced length, supporting that nebulin is critical for regulation of thin filament length (TFL). Reduced TFL in NEM2 patients is likely part of their severe muscle weakness because it is expected to reduce force on the descending limb of the force-sarcomere length relationship. How nebulin contributes to TFL control is controversial. To critically test nebulin’s role in TF length specification we made two novel mouse models, one in which nebulin is shortened by deleting super-repeats (SRs) 9-11 and another in which nebulin is lengthened by duplicating the same SRs. In Aim 1 we will used these contrasting and powerful models to study the role of nebulin in TFL regulation and, importantly, the functional consequence of TFL mis-specification. These studies are clinically important for multiple muscle diseases where mis-specification of TFL occurs. Nebulin is also likely to contribute to sarcomeric organization through its position in the Z-disk, where interactions with local proteins might contribute to Z-disk width regulation, myofibrillar alignment and hypertrophy signaling. It has been reported that nebulin’s C-terminal interaction with the actin assembly protein neuronal Wiskott-Aldrich syndrome protein (N-WASP) allows for a hypertrophic response to insulin-like growth factor 1 (IGF-1) stimulation. With repetitive modules forming the bulk of nebulin’s structure, the SH3 and serine-rich domains in the C-terminus stand out. To study the functions of these domains we made a mouse model which eliminates both the serine-rich and the SH3 domains and mimics mutational effects found in patients. Pilot studies show that the model has widened Z-disk and muscle atrophy. Aim 2 will investigate the effects of nebulin’s serine-rich and SH3 domains on structure and function of skeletal muscle. To test NEM2 therapeutics in Aim 3 we made a mouse in which nebulin is conditionally deleted (Neb cKO) and that phenocopies NEM2. We will use this model to test the effects of gene therapy with adeno-associated delivery of functionally distinct subdomains of nebulin. We hypothesize that expressing the full Z-disk construct improves Z-disk integrity and hypertrophy signaling and that the effects are attenuated by eliminating the serine- rich and SH3 domains. We will also test whether expressing a SR construct enhances the force generating crossbridge population and will study the effects of Z-disk and SR constructs when expressed simultaneously. With its basic science and translational foci this proposal seeks to continue our track record of cutting edge nebulin research. We have a strong research team with excellent collaborators, use state of the art techniques, created novel mouse models for this work, and have supportive pilot data. We anticipate that this proposal will provide novel insights in the complex biology of nebulin and, importantly, how to ameliorate muscle weakness in nemaline myopathy.

树云蛋白是在骨骼肌中发现的重要但知之甚少的肉瘤蛋白。我们的先前 黑甲肌病患者缺乏雾蛋白（NEM2患者）的研究表明，雾蛋白蛋白丧失 导致长度减小的细丝，支持Nebulin对于调节细丝长度至关重要 （TFL）。 NEM2患者的TFL减少可能是其严重肌肉无力的一部分 减少力量 - 晶状体长度关系的降肢上的力。树云蛋白如何促进TFL 控制是有争议的。为了批判性测试Nebulin在TF长度规范中的作用，我们制作了两只新型鼠标 模型，其中一种模型通过删除超级重复（SRS）9-11缩短了一个模型 通过复制相同的SR来延长。在AIM 1中，我们将使用这些对比和强大的模型来研究 树星蛋白在TFL调节中的作用，重要的是TFL错过指定的功能后果。 这些研究对于发生TFL错过的多种肌肉疾病至关重要。 树星蛋白也可能通过其在Z-Disk中的位置为肉瘤组织做出贡献 与局部蛋白质的相互作用可能有助于Z二型宽度调节，肌原纤维比对和肥大 信号。据报道，云蛋白与肌动蛋白总成蛋白神经元的C末端相互作用 Wiskott-Aldrich综合征蛋白（N-WASP）允许对胰岛素样生长因子1的肥厚反应1 （IGF-1）刺激。随着重复的模块形成了大部分nebulin结构，SH3和串行富含序列化的模块 C末端中的域突出。为了研究这些域的功能，我们制作了一个鼠标模型 消除了富含丝氨酸和SH3结构域以及在患者中发现的突变作用。飞行员 研究表明，该模型具有宽大的Z磁盘和肌肉萎缩。 AIM 2将研究 Nebulin在骨骼肌的结构和功能上的丝氨酸富含丝氨酸和SH3结构域。 为了在AIM 3中测试NEM2治疗，我们制作了一只小鼠，在该疗法中，有条件地删除了Nebulin（NEB CKO） 和那个表opies nem2。我们将使用该模型测试与腺相关的基因治疗的作用 在功能上不同的云母亚域的递送。我们假设表达完整的Z磁盘结构 改善Z-Disk完整性和肥大信号传导，并通过消除串行来减弱效果 Rich和SH3域。我们还将测试表达SR构建体是否增强产生的力 简单地表达时，Crossbridge的种群将研究Z-Disk和SR构建体的影响。 凭借其基础科学和翻译重点，该提案旨在继续我们的最前沿记录 树星研究。我们有一个强大的研究团队，拥有出色的合作者，使用最先进的技术， 为这项工作创建了新颖的鼠标模型，并支持了试点数据。我们预计该提议将 在云蛋白的复杂生物学中提供新颖的见解，重要的是如何改善肌肉无力 在Nemaline肌病中。