Role of UNC-89 (obscurin) in sarcomere assembly and maintenance.

UNC-89（obscurin）在肌节组装和维护中的作用。

• 批准号: 8836489
• 负责人: GUY Martin BENIAN
• 金额: $ 34.17万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-04-08 至 2019-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8836489
• 关键词: Adaptor Signaling Protein; Animal Model; Ankyrins; Antibodies; Binding; Biochemical; Biochemistry; Biological Process; Caenorhabditis elegans; Calcium; Calcium Signaling; Cardiomyopathies; Characteristics; Chronic Disease; Complex; Coupling; Cullin 1; Cullin Proteins; Development; Diabetes Mellitus; Elasticity; Elderly; Embryo; Enzymes; Family member; Filament; Genetic Models; Grant; Health; Heart; Homologous Gene; Human; Immobilization; Kidney Failure; Knockout Mice; Lead; Link; Locomotion; MEL Gene; Maintenance; Malignant Neoplasms; Mediating; Medical; Microtubules; Molecular; Molecular Genetics; Mus; Muscle; Muscle Contraction; Muscular Atrophy; Muscular Dystrophies; Myofibrils; Myopathy; Myosin ATPase; Nematoda; PH Domain; Pathogenesis; Phosphotransferases; Physiological; Play; Protein Kinase; Protein phosphatase; Proteins; Regulation; Reporting; Role; SH3 Domains; Sarcomeres; Sarcoplasmic Reticulum; Scaffolding Protein; Signal Pathway; Signal Transduction; Signaling Protein; Stretching; Striated Muscles; Structure; Tertiary Protein Structure; Ubiquitin; Ubiquitination; base; connectin; copine; cullin-3; genetic approach; human disease; insight; katanin; loss of function; mouse model; mutant; novel; obscurin; polypeptide; protein degradation; scaffold; titin kinase; ubiquitin-protein ligase

DESCRIPTION (provided by applicant): Muscle sarcomeres contain a number of giant polypeptides (0.7-4 MDa). Much is currently known about the function of the largest of these polypeptides, vertebrate titin. Much less is known about the newest vertebrate member of this family, obscurin. The pathogenesis of one form of muscular dystrophy may involve obscurin. Obscurin is the homolog of UNC-89 in C. elegans. Essential features of UNC-89 as a signaling / scaffolding protein crucial for A-band/M-line assembly were discovered before obscurin was reported. To understand how UNC-89 is localized, and how it functions, we are taking a systematic approach for identifying and studying the function of its binding partners. We discovered that the Ig1-3 segment of UNC-89 interacts with CPNA-1, a copine domain protein. Although CPNA-1 is not required for initial assembly of UNC-89 at the M-line, it is required for it retention. CPNA-1 is located at both M-lines and dense bodies, whereas UNC-89 is located only at M-lines. Therefore, we hypothesize that there are proteins which direct assembly of UNC-89 solely to the M-line. UNC-89 Ig9-11 interacts with CUL-1 (cullin 1), and UNC-89 Ig2-3 interacts with MEL-26, a substrate recognition protein for CUL-3 (cullin 3). Cullins are scaffolds for assembly of the ubiquitination machinery. One function of the CUL-3/MEL-26 complex is to promote degradation of MEI-1 (katanin). Independently, the coI of this proposal, Stephan Lange, found that in mouse heart, degradation of sAnk1.5 is promoted by a cullin 3 substrate recognition protein, KCTD6, and this is dependent upon obscurin. We hypothesize that UNC-89 (obscurin) negatively regulates the activity of cullin complexes in muscle. We further hypothesize that UNC-89 interacts with other cullins or cullin 3 adaptor proteins, and there are substrates in addition to MEI-1. In humans, muscle atrophy is associated with immobilization, chronic diseases and advanced age. Since in muscle atrophy, degradation of sarcomeric proteins is upregulated, our studies have medical relevance. Also, our collaborator and coI of this grant, Ken Norman, has found that a key function of obscurin for organization of the SR, is conserved for UNC-89. In addition, UNC-89 was found to have a physiological role in EC coupling, and this involves the conserved RacGEF, VAV-1. We hypothesize that UNC-89 and VAV-1 interact, and that VAV-1 localization and function is dependent on UNC-89. Finally, we hypothesize that the binding partners and functional mechanisms discovered for UNC-89 in C. elegans are biologically relevant for mammalian striated muscle. Specific aims are: (1) determine mechanisms which direct assembly of UNC-89 solely to the M-line; (2) determine if other cullins interact with UNC-89, if there are additional substrates for MEL-26, and determine the biochemical effects of the UNC-89 to MEL-26 interaction; (3) define the biochemistry and functional consequence of interaction between UNC-89 and myosin; (4) investigate the molecular mechanism underlying UNC-89's role in calcium regulation; and (5) determine if functional interactions and molecular mechanisms identified for nematode UNC-89 are conserved for mammalian obscurin.

描述（由申请人提供）：肌肉肌节含有许多巨型多肽（0.7-4 MDa）。目前，人们对这些多肽中最大的多肽——脊椎动物肌联蛋白的功能了解很多。人们对这个家族的最新脊椎动物成员——暗纹动物知之甚少。一种形式的肌营养不良症的发病机制可能涉及暗色蛋白。 Obscurin 是线虫中 UNC-89 的同源物。 UNC-89 作为对 A 带/M 线组装至关重要的信号/支架蛋白的基本特征在 obscurin 被报道之前就被发现了。为了了解 UNC-89 如何本地化以及它如何发挥作用，我们正在采取系统方法来识别和研究其结合伙伴的功能。我们发现 UNC-89 的 Ig1-3 片段与 CPNA-1（一种 copine 结构域蛋白）相互作用。尽管 CPNA-1 不是 UNC-89 在 M 线上的初始组装所必需的，但它的保留却是必需的。 CPNA-1 位于 M 线和致密体上，而 UNC-89 仅位于 M 线处。因此，我们假设存在将 UNC-89 仅引导至 M 线的蛋白质。 UNC-89 Ig9-11 与 CUL-1 (cullin 1) 相互作用，UNC-89 Ig2-3 与 MEL-26（CUL-3 (cullin 3) 的底物识别蛋白）相互作用。 Cullins 是用于组装泛素化机器的支架。 CUL-3/MEL-26 复合物的功能之一是促进 MEI-1（剑素）的降解。独立地，该提案的 coI Stephan Lange 发现，在小鼠心脏中，cullin 3 底物识别蛋白 KCTD6 促进了 sAnk1.5 的降解，而这依赖于 obscurin。我们假设 UNC-89（obscurin）负向调节肌肉中 cullin 复合物的活性。我们进一步假设UNC-89与其他cullin或cullin 3接头蛋白相互作用，并且除了MEI-1之外还有底物。在人类中，肌肉萎缩与不动、慢性疾病和高龄有关。由于在肌肉萎缩中，肌节蛋白的降解上调，因此我们的研究具有医学意义。此外，我们的合作者和本次资助的共同负责人 Ken Norman 发现，obscurin 用于组织 SR 的关键功能对于 UNC-89 来说是保守的。此外，UNC-89被发现在EC偶联中具有生理作用，这涉及保守的RacGEF，VAV-1。我们假设 UNC-89 和 VAV-1 相互作用，并且 VAV-1 的定位和功能依赖于 UNC-89。最后，我们假设在线虫中发现的 UNC-89 结合伴侣和功能机制与哺乳动物横纹肌具有生物学相关性。具体目标是：(1) 确定将 UNC-89 仅引导至 M 线的机制； (2) 确定其他库林是否与 UNC-89 相互作用，是否存在 MEL-26 的其他底物，并确定 UNC-89 与 MEL-26 相互作用的生化效应； (3) 定义UNC-89和肌球蛋白之间相互作用的生物化学和功能结果； (4) 研究UNC-89在钙调节中的分子机制； (5)确定线虫UNC-89的功能相互作用和分子机制对于哺乳动物暗色蛋白是否是保守的。