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(申请人提供)：肌肉肌节含有许多巨型多肽(0.7-4丙二醛)。目前，人们对这些多肽中最大的一种--脊椎动物肌动蛋白的功能知之甚少。关于这个家族中最新的脊椎动物成员--暗黑龙，人们所知的要少得多。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-的同源物。UNC-89作为信号/支架蛋白在A-带/M-线组装中起关键作用的基本特征在Oblcurin被报道之前就已被发现。为了了解UNC-89是如何本地化的，以及它是如何发挥作用的，我们正在采取一种系统的方法来确定和研究其结合伙伴的功能。我们发现UNC-89的IG1-3片段与Copine结构域蛋白CPNA-1相互作用。虽然CPNA-1不是UNC-89在M线的初始组装所必需的，但它是保持其稳定性所必需的。CPNA-1位于M线和致密体上，而UNC-89仅位于M线上。因此，我们假设存在将UNC-的组装仅定向到M-线的蛋白质。UNC-89 IG9-11与Cul-1(Cullin 1)相互作用，UNC-89 IG2-3与Cul-3(Cullin 3)底物识别蛋白MEL-26相互作用。库林是组装泛素化机器的支架。CuL-3/MEL-26复合体的一个功能是促进Mei-1(Katanin)的降解。独立地，该提案的COI Stephan Lange发现，在小鼠心脏中，sAnk1.5的降解是由cullin 3底物识别蛋白KCTD6促进的，这依赖于Oblcurin。我们假设UNC-89(Oblcurin)对肌肉中cullin复合体的活性具有负性调节作用。我们进一步假设，UNC-与其他Cullins或cullin3接头蛋白相互作用，除MeI-1外，还存在底物。在人类中，肌肉萎缩与制动、慢性病和高龄有关。由于在肌肉萎缩中，肌节蛋白的降解被上调，我们的研究具有医学意义。此外，我们的合作者和这笔赠款的COI，Ken Norman，已经发现组织SR的一个关键功能是为UNC-89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的定位和功能取决于UNC-89。最后，我们假设在线虫中发现的UNC-的结合伙伴和功能机制与哺乳动物横纹肌具有生物学意义。具体目标包括：(1)确定仅将UNC-89组装到M线的机制；(2)确定其他库林细胞是否与UNC-89相互作用，以及是否有额外的底物用于MEL-26；以及确定UNC-89与MEL-26相互作用的生化效应；(3)确定UNC-89与肌球蛋白相互作用的生化和功能后果；(4)研究UNC-89与肌球蛋白相互作用的分子机制；(5)确定已确定的线虫UNC-89的功能相互作用和分子机制是否在哺乳动物暗蛋白中是保守的。