Deciphering the Role of Skeletal Muscle Protein STAC3 in Calcium Release

描述(由申请人提供)：骨骼肌疾病是全世界成千上万人的疼痛和虚弱的来源(2)。这些疾病可由一系列外因(3)或涉及肌肉结构(4，5)、代谢(6)或信号成分(7)的基因突变引起。该项目的目标将是了解最近发现的致病基因Stac3(8，9)的功能。在小鼠中，由于缺乏肌肉收缩，整体消融Stac3会导致胎儿完全瘫痪和围产儿死亡(9)。出生时Stac3缺失的致命性表明了该蛋白对正常肌肉发育和功能的重要性；然而，围产期的致死性排除了对幼年和成年小鼠Stac3功能的分析，这两个生命阶段与理解人类疾病最相关。由于钙处理缺陷会导致青少年和成年人的疾病，这项研究的第一个目标将是使用可诱导的cre-lox模型来消除出生后小鼠的Stac3。这一策略将消除大部分但不是全部Stac3，并有望准确地模拟影响蛋白质表达或稳定性的突变。这些突变体的特征将为深入了解Stac3在出生后肌肉功能中的作用提供更深入的了解，并将为理解Stac3突变导致的人类表型提供合理的基础。拟议项目的第二个目标将是确定Stac3的互动伙伴。Stac3很可能与表型相似的复合体相互作用，即二氢吡啶受体复合体或兰诺定受体复合体，因此将使用培养的肌肉细胞的蛋白质下拉来系统地评估这些复合体的组成成分的相互作用。除了个人候选人分析，质谱学将用于交叉验证积极互动的候选人，并提供一种公正的方法来发现意想不到的互动。该项目的第三个目标将是了解Stac3蛋白中的哪些残基是其功能所必需的。为了实现这一目标，将使用转录激活因子样效应子核酸酶(广为人知的TALEN)来建立C2C12成肌细胞系。然后，这些细胞可以很容易地被操纵，通过逆转录病毒转导表达突变结构，然后评估对刺激的钙释放。这一策略将为快速鉴定突变结构提供一个低成本、可重复性的系统，并将通过消除对原代敲除成肌细胞的需求来减少完成这项研究所需的动物数量。不能挽救钙释放的突变体将被评估与目标2中确定的蛋白质失去相互作用。完成这些目标将提高对Stac3功能和兴奋-收缩偶联的理解，这可能成为各种肌肉疾病的新治疗策略的基础。

DESCRIPTION (provided by applicant): Skeletal muscle disease is a source of pain and debilitation for many thousands of people worldwide (2). These diseases can result from a range of extrinsic causes (3) or genetic mutations involving structural (4, 5), metabolic (6) or signaling components of the muscle (7). The goal of this project will be to understand the function of the recently identified disease-causing gene, STAC3 (8, 9). Global ablation of STAC3 in mice causes complete fetal paralysis and perinatal death due to lack of muscle contraction (9). The lethality of STAC3 deletion at birth demonstrates the importance of this protein for normal muscle development and function; however, perinatal lethality precludes analysis of STAC3 function in juvenile and adult mice, the life stages most relevant for understanding human disease. Because defects in calcium handling cause disease in juvenile and adult humans, the first aim of this study will be to ablate STAC3 in postnatal mice using an inducible cre-lox model. This strategy will eliminate most but not all STAC3 and is expected to accurately model mutations that affect protein expression or stability. Characterization of these mutants will provide deeper insight into the role of STAC3 in postnatal muscle function and will provide a rational basis for understanding human phenotypes resulting from STAC3 mutations. The second aim of the proposed project will be to identify interaction partners of STAC3. It is likely that STAC3 interacts with complexes associated with similar phenotypes, namely the dihydropyridine receptor complex or the ryanodine receptor, so components of these complexes will be systematically evaluated for interaction using protein pull-down from cultured muscle cells. In addition to individual candidate analysis, mass spectrometry will be used to cross validate positively interacting candidates and to provide an unbiased method to discover unexpected interactions. The third aim of this project will be to understand which residues in the STAC3 protein are required for its function. To accomplish this aim, a knockout C2C12 myoblast cell line will be generated using Transcription Activator-Like Effector Nucleases (widely known as TALENs). These cells can then be easily manipulated to express mutant constructs using retroviral transduction followed by assessment of calcium release in response to excitation. This strategy will provide a low cost, reproducible system for rapidly characterizing mutant constructs and will reduce the number of animals needed to complete this study by eliminating the need for primary knockout myoblasts. Mutants that are unable to rescue calcium release will then be assessed for loss of interaction with proteins identified in aim 2. Completion of these aims will improve understanding of STAC3 function and excitation-contraction coupling, which may serve as a basis for new treatment strategies for a variety of muscle maladies.