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

定义导致显性腕跗骨骨联结的肌球蛋白结构和功能缺陷

• 批准号: 9899926
• 负责人: Sanford I Bernstein
• 金额: $ 19.87万
• 依托单位: SAN DIEGO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-04-01 至 2022-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9899926
• 关键词: ATP phosphohydrolase; Abnormal Cell; Actin-Binding Protein; Actins; Adult; Affect; Affinity; Alleles; Animal Model; Binding; Biochemical; Biological Models; Biophysics; Birth; Cells; Chest; Congenital abnormal Synostosis; Crystallization; Cytoskeleton; Defect; Development; Disease; Dominant Genetic Conditions; Drosophila genus; Drosophila melanogaster; Elements; Embryo; Filament; Foot Deformities; Functional disorder; Genes; Genetic; Genetic Diseases; Genetic Heterogeneity; Goals; Hand; Hand deformities; Heterotopic Ossification; Human; Human Genetics; Intervertebral disc structure; Joints; Lead; Link; Maps; Methods; Modeling; Molecular; Molecular Structure; Mus; Muscle; Muscle Proteins; Muscle function; Mutate; Mutation; Myofibrils; Myosin ATPase; Myosin Heavy Chains; Nucleotides; Pathology; Phenotype; Physiological; Plant Roots; Production; Property; Proteins; Research; Resolution; Signal Transduction; Skeletal Muscle; Skeletal Muscle Myosins; Standardization; Structural defect; Structure; Structure-Activity Relationship; System; Testing; Therapeutic; Tissues; Transgenic Model; Transgenic Organisms; Vertebral column; alpha Actin; base; beta Actin; bone; causal variant; cell motility; cell type; cost efficient; design; filamin; foot; genome sequencing; human disease; insight; mechanical pressure; muscular structure; mutant; postnatal; relating to nervous system; skeletal; skeletal disorder; spine bone structure; vertebra body; whole genome

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.

项目摘要 我们建议构建和分析主要的链球链球杆骨突发症（SCT）的动物模型，A 人类遗传疾病是由MYH3胚胎肌球蛋白重链中的突变引起的。这些 突变产生畸形和融合的椎体以及腕骨和tarsal异常 假设是由肌肉功能的主要缺陷引起的。我们的转基因果蝇Melanogaster 模型将用于剖析此模型的生化，生物物理，发育和生理基础 疾病。果蝇系统将使我们能够突变果蝇肌球蛋白基因，以检查 标准化遗传背景中的两个SCT等位基因。这将定义相互作用的重要性 野生型和突变体肌球蛋白分子到疾病病理学，将消除导致的遗传异质性 在人类疾病中的表型变异性。此外，我们将探索转基因系统的使用 产生足够数量的人类野生型和SCT MYH3来确定其功能特性，并 解决其高分辨率晶体结构。我们将检验以下假设：肌动蛋白结合， 在SCT突变体肌球蛋白模型中，影响核苷酸的亲和力和细丝运动性异常。那 在功能上异常的SCT肌球蛋白导致果蝇中的肌原纤维破坏和肌肉功能障碍 模型;可以使用我们的基于果蝇的结构功能关系可以辨别 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） 评估与野生的ATPase，肌动蛋白结合和肌动蛋白运动能力 型蛋白质。 2）确定突变对肌原纤维超微结构和功能的主要影响 幼体体壁和成年胸腔（间接飞行和跳跃肌肉）的肌肉。 3）探索可能性 产生，隔离和确定正常和SCT人类的结构和功能特性 MYH3蛋白使用独特的间接飞行肌肉表达系统。这种多方面的方法将提供 机械洞察SCT的分子和发展碱基，并开始阐明突变 在骨骼肌蛋白中，导致相关骨骼元素的发育缺陷。了解 疾病的基本肌肉缺陷可能最终产生治疗方法。