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: 8632004
• 负责人: GUY Martin BENIAN
• 金额: $ 36.14万
• 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/8632004
• 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; 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; human disease; insight; katanin; loss of function; mouse model; mutant; novel; obscurin; polypeptide; protein degradation; public health relevance; scaffold; titin kinase; ubiquitin-protein ligase

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 its 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.

肌肉肌节含有许多巨型多肽(0.7-4个丙二醛)。目前人们对此已有很多了解。 这些多肽中最大的一种是脊椎动物Titin的功能。人们对这种最新的脊椎动物知之甚少 这个家庭的一员，暗黑。一种类型的肌营养不良的发病机制可能涉及遮盖素。 Obscurin是线虫中UNC-89的同源物。UNC-89作为信令/脚手架的基本特征 在Oblcurin被报道之前，对A-带/M-线组装至关重要的蛋白质就被发现了。要了解如何 UNC-89是本地化的，以及它的功能如何，我们正在采取系统的方法来识别和研究 其结合伙伴的功能。我们发现UNC-89的IG1-3片段与CPNA-1、a Copine结构域蛋白。尽管UNC-89在M线的初始组装不需要CPNA-1，但它是 需要它的保留。CPNA-1位于M线和致密体上，而UNC-89位于 仅限于M-Line。因此，我们假设存在引导UNC-89组装的蛋白质单独作用于 M线。UNC-89 IG9-11与CUL-1(Cullin 1)相互作用，UNC-89 IG2-3与底物MEL-26相互作用 Cul-3识别蛋白(Cullin 3)。库林是组装泛素化机器的支架。一 CuL-3/MEL-26复合体的功能是促进MEI-1(Katanin)的降解。独立地，的COI 这项名为Stephan Lange的提议发现，在小鼠的心脏中，sAnk1.5的降解是由cullin 3促进的 底物识别蛋白，KCTD6，这是依赖于暗蛋白。我们假设UNC-89 (Darcurin)负向调节肌肉中cullin复合体的活性。我们进一步假设UNC-89 与其他cullins或cullin 3接头蛋白相互作用，除Mei-1外，还有底物。在……里面 在人类中，肌肉萎缩与制动、慢性病和高龄有关。自年起 肌肉萎缩，肌节蛋白降解上调，我们的研究具有医学意义。另外， 我们的合作者和这笔赠款的COI肯·诺曼发现，Oblcurin对于组织 SR，是为北卡罗来纳大学-保留的。此外，UNC-89被发现在EC偶联中具有生理作用， 这涉及到保守的racgef，VAV-1。我们假设UNC-89和VAV-1相互作用， VAV-1的定位和功能依赖于UNC-。最后，我们假设结合伙伴和 在线虫中发现的UNC-89的作用机制与哺乳动物条纹动物的生物学相关 肌肉。具体目标是：(1)确定将UNC-89的组装只定向到M线的机制； (2)确定是否有其他库林斯与UNC-89相互作用，是否有额外的MEL-26底物，并确定 UNC-89与MEL-26相互作用的生化效应；(3)生物化学和功能 UNC-89与肌球蛋白相互作用的结果；(4)探讨其可能的分子机制 S在钙调节中的作用；以及(5)确定功能相互作用和分子机制 已鉴定为线虫的UNC-89对哺乳动物黑点蛋白保守。