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疼痛和衰弱的根源(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的功能（8,9）。在小鼠中，由于缺乏肌肉收缩，全盘消融STAC3会导致胎儿完全瘫痪和围产期死亡(9)。出生时STAC3缺失的致命性证明了该蛋白对正常肌肉发育和功能的重要性；然而，围产期致死率阻碍了对幼年和成年小鼠（与了解人类疾病最相关的生命阶段）中STAC3功能的分析。由于钙处理缺陷在青少年和成年人中都会引起疾病，因此本研究的第一个目的将是使用可诱导的cre-lox模型在出生后小鼠中切除STAC3。该策略将消除大部分但不是全部的STAC3，并有望准确地模拟影响蛋白质表达或稳定性的突变。这些突变的特征将提供对STAC3在出生后肌肉功能中的作用的更深入的了解，并将为理解由STAC3突变引起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的相互作用伙伴。很可能STAC3与具有相似表型的复合物相互作用，即二氢吡啶受体复合物或ryanodine受体，因此这些复合物的组分将通过从培养肌肉细胞中提取蛋白质来系统地评估其相互作用。除了单个候选分析外，质谱法还将用于交叉验证积极相互作用的候选物质，并提供一种无偏倚的方法来发现意想不到的相互作用。该项目的第三个目标是了解STAC3蛋白中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核酸酶（俗称TALENs）生成敲除C2C12成肌细胞系。这些细胞可以很容易地通过逆转录病毒转导来表达突变体，然后评估钙释放对兴奋的反应。该策略将为快速表征突变构建体提供低成本、可重复的系统，并通过消除对原发性敲除成肌细胞的需求，减少完成本研究所需的动物数量。无法恢复钙释放的突变体将被评估与目的2中鉴定的蛋白质相互作用的丧失。这些目标的完成将提高对STAC3功能和兴奋-收缩耦合的理解，这可能为各种肌肉疾病的新治疗策略提供基础。

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.