Defining Defects in Myosin Structure and Function That Cause Dominant Spondylocarpotarsal Synostosis

项目总结 我们建议建立和分析第一个显性脊柱性腕关节融合症(SCT)动物模型。 由MYH3胚胎肌球蛋白重链突变引起的人类遗传病。这些 突变会导致畸形和融合的椎体，以及腕骨和颧骨的异常，这是 推测是由肌肉功能的原发缺陷引起的。我们的转基因黑腹果蝇 模型将被用来剖析其生化、生物物理、发育和生理基础。 疾病。果蝇系统将允许我们突变果蝇肌球蛋白基因，以检测 标准化遗传背景下的两个SCT等位基因。这将定义相互作用的重要性 野生型和突变型肌球蛋白分子与疾病病理学有关，并将消除导致 人类疾病的表型变异性。此外，我们将探索使用我们的转基因系统来 生产足够数量的人野生型和SCT MYH3，以确定其功能性质和 解开它们的高分辨率晶体结构。我们将测试以下假设：肌动蛋白结合，即 影响核苷酸亲和力和细丝运动，在SCT突变肌球蛋白模型中异常； 果蝇SCT肌球蛋白功能异常导致肌原纤维断裂和肌肉功能障碍 模型；人类肌球蛋白的结构-功能关系可以通过我们以果蝇为基础的 肌球蛋白表达系统。为了评估这些假设，我们将追求以下具体目标：1) 评估突变的SCT肌球蛋白的ATPase，肌动蛋白结合和肌动蛋白运动能力与野生- 类型为蛋白质。2)确定突变对肌原纤维超微结构和功能的显性影响 来自幼虫体壁和成虫胸腔的肌肉(间接飞行和跳跃肌肉)。3)探索可能性 产生、分离和测定正常人和SCT患者的结构和功能特性 MYH3蛋白利用一种独特的间接飞行肌肉表达系统。这种多方面的方法将提供 对SCT的分子和发育基础的机械论见解，并开始阐明突变是如何 在骨骼肌中，蛋白质会导致相关骨骼元素的发育缺陷。了解 导致这种疾病的潜在肌肉缺陷最终可能会产生治疗方法。

PROJECT SUMMARY We propose to build and analyze the first animal models of dominant spondylocarpotarsal synostosis (SCT), a human genetic disease that arises from mutations in the MYH3 embryonic myosin heavy chain. These mutations yield misshaped and fused vertebral bodies as well as carpal and tarsal abnormalities that are hypothesized to arise from primary defects in muscle function. Our transgenic Drosophila melanogaster models will be used to dissect the biochemical, biophysical, developmental and physiological bases of this disease. The Drosophila system will allow us to mutate the Drosophila myosin gene to examine the effects of two SCT alleles in a standardized genetic background. This will define the importance of interactions between wild-type and mutant myosin molecules to disease pathology and will obviate genetic heterogeneity that leads to phenotypic variability in the human disease. Further we will explore the use of our transgenic system to produce adequate quantities of human wild-type and SCT MYH3 to determine their functional properties and solve their high-resolution crystal structures. We will test the following hypotheses: that actin binding, which influences nucleotide affinity and filament motility, is abnormal in the SCT mutant myosin models; that functionally abnormal SCT myosin leads to myofibril disruption and muscle dysfunction in the Drosophila model; that structure-function relationships about human myosin can be discerned using our Drosophila-based myosin expression system. To evaluate these hypotheses, we will pursue the following specific aims: 1) Assess the ATPase, actin binding and actin motility capabilities of mutant SCT myosins compared to the wild- type protein. 2) Determine the dominant effects of the mutations on myofibrillar ultrastructure and function of muscles from the larval body wall and adult thorax (indirect flight and jump muscles). 3) Explore the possibility of producing, isolating and determining the structural and functional properties of normal and SCT human MYH3 protein using a unique indirect flight muscle expression system. This multifaceted approach will provide mechanistic insights into the molecular and developmental bases of SCT and begin to elucidate how mutations in a skeletal muscle protein lead to developmental defects in associated skeletal elements. Understanding the underlying muscle defects causative of the disease may ultimately yield therapeutic approaches.