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

描述（由申请人提供）：肌肉肌节含有大量巨多肽（0.7-4 MDa）。目前，人们对这些多肽中最大的一种——脊椎动物titin的功能了解甚多。人们对这个家族中最新的脊椎动物成员——盲鳗知之甚少。一种形式的肌营养不良的发病机制可能与暗蛋白有关。隐孔蛋白是秀丽隐杆线虫中UNC-89的同源物。UNC-89作为a波段/ m线组装至关重要的信号/脚手架蛋白的基本特征在obscurin报道之前就被发现了。为了了解UNC-89是如何定位的，以及它是如何起作用的，我们正在采取系统的方法来识别和研究它的结合伙伴的功能。我们发现UNC-89的Ig1-3片段与copine结构域蛋白CPNA-1相互作用。虽然在m线初始装配UNC-89时不需要CPNA-1，但在保留UNC-89时需要CPNA-1。CPNA-1位于m -线和致密体，而UNC-89仅位于m -线。因此，我们假设存在将UNC-89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的底物识别蛋白MEL-26 （cullin 3）相互作用。Cullins是组装泛素化机器的支架。CUL-3/MEL-26复合物的一个功能是促进MEI-1 （katanin）的降解。独立地，该提案的发起人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cullins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，肌肉萎缩与固定不动、慢性疾病和老年有关。由于在肌肉萎缩中，肌肉合成蛋白的降解是上调的，因此我们的研究具有医学意义。此外，我们的合作者和这项资助的coI Ken Norman发现，UNC-89保留了暗色蛋白组织SR的一个关键功能。此外，发现UNC-89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，我们假设秀丽隐杆线虫中发现的UNC-89的结合伙伴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直接组装到m线的机制；(2)确定其他cullins是否与UNC-89相互作用，MEL-26是否有其他底物，并确定UNC-89与MEL-26相互作用的生化效应；(3)明确UNC-89与肌球蛋白相互作用的生物化学和功能后果；(4)研究UNC-89参与钙调控的分子机制；(5)确定线虫UNC-89的功能相互作用和分子机制是否保守于哺乳动物的obscurin。

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.