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

基本信息

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
项目类别：
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

项目摘要

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.

项目概要我们建议建立并分析第一个显性椎骨跗骨连接（SCT）的动物模型，这是一种由 MYH3 胚胎肌球蛋白重链突变引起的人类遗传病。这些突变会产生畸形和融合的椎体以及腕骨和跗骨异常推测是由肌肉功能的主要缺陷引起的。我们的转基因黑腹果蝇模型将用于剖析其生物化学、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，以检查以下因素的影响：标准化遗传背景中的两个 SCT 等位基因。这将定义之间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的关系，并将消除导致的遗传异质性人类疾病的表型变异。此外，我们将探索使用我们的转基因系统产生足够数量的人类野生型和 SCT MYH3 以确定其功能特性和解析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，影响核苷酸亲和力和丝运动性，在 SCT 突变肌球蛋白模型中异常；那功能异常的 SCT 肌球蛋白导致果蝇肌原纤维破坏和肌肉功能障碍模型;可以使用我们基于果蝇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）与野生型相比，评估突变型 SCT 肌球蛋白的 ATP 酶、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显着影响来自幼虫体壁和成虫胸部的肌肉（间接飞行和跳跃肌肉）。 3）探索可能性产生、分离和确定正常和 SCT 人类的结构和功能特性 MYH3蛋白采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对 SCT 的分子和发育基础的机制见解，并开始阐明突变是如何发生的骨骼肌蛋白中的缺陷会导致相关骨骼元素的发育缺陷。了解导致该疾病的潜在肌肉缺陷最终可能会产生治疗方法。