Structural and biochemical mechanisms of myosin-induced dilated cardiomyopathy

肌球蛋白诱导的扩张型心肌病的结构和生化机制

• 批准号: 8911613
• 负责人: Adriana Trujillo
• 金额: $ 3.03万
• 依托单位: SAN DIEGO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-08-19 至 2019-08-18
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8911613
• 关键词: ATP Hydrolysis; ATP phosphohydrolase; Academia; Actins; Actomyosin; Affinity; Alternative Splicing; Amino Acids; Arrhythmia; Binding; Binding Sites; Biochemical; Biochemistry; Biological; Biological Assay; Cardiac; Cardiomyopathies; Cells; Cellular biology; Complex; Contractile Proteins; Crystallography; Defect; Dilated Cardiomyopathy; Disease; Disease Progression; Drosophila genus; Drosophila melanogaster; Etiology; Event; Exhibits; Functional disorder; Gene Family; Generations; Genes; Genetic; Genome; Haploidy; Heart; Heart Diseases; Human; In Vitro; Induced Mutation; Lead; Location; Maintenance; Modeling; Molecular; Molecular Biology; Molecular Motors; Muscle; Muscle function; Mutation; Myosin ATPase; Myosin Type II; Organism; Patients; Phenotype; Physiological; Preparation; Protein Isoforms; Proteins; Sedimentation process; Skeletal Muscle; Striated Muscles; Structure; System; Testing; Thin Filament; Training; X-Ray Crystallography; base; career; cell motility; fly; in vivo; insight; interdisciplinary approach; muscular structure; muscular system; mutant; novel; public health relevance; response; screening; structural biology; tool

 DESCRIPTION (provided by applicant): Dilated cardiomyopathy (DCM), the most common cardiomyopathy form, can result from mutations in contractile proteins (e.g. myosin). However, the structural, molecular, and physiological origins leading to cardiac dilation in myosin-based DCM are not well understood. We will take advantage of the powerful genetic tools available in Drosophila to generate the first fly models of myosin-based DCM and determine the mechanistic basis of disease. Multidisciplinary approaches will be implemented to determine how single amino acid changes in myosin disrupt intramolecular interactions and cause biochemical, structural and physiological defects in striated muscles. In Aim 1, we will generate the first X-ra crystal structures for myosin harboring mutations known to cause DCM in humans and predicted to modulate actin binding. Mutant His-tagged myosin will be expressed in indirect flight muscles (IFMs), purified, and use for crystallography. We will test the hypothesis that: DCM mutations in myosin that disrupt intramolecular interactions near or within the actin- binding site re-orient key residues important for actin binding. Aim 2 will implement a variety of approaches to better understand the biochemical, cell biological, and functional defects associated with human myosin DCM mutations. We will express and purify mutant myosin from IFMs for biochemical/biophysical assays (actin co- sedimentation, ATPase, in vitro motility) to determine the molecular basis of DCM due to myosin mutations. Ultrastructural analyses of IFMs will provide insight into the defects in myofibrillar assembly and maintenance induced by the mutations. Furthermore, we will determine if expression of such mutations cause skeletal muscle dysfunction using flight and jump tests. We will test the hypothesis that: mutations in myosin can weaken actin affinity, reduce enzymatic activity of myosin, and cause structural and functional defects in indirect flight muscles. In Aim 3, we will assess remodeling events that occur in the Drosophila heart due to expression of myosin DCM mutations. Although it is known that the Drosophila heart can remodel into a dilated phenotype, it is unknown if it dilates in response to myosin mutations known to cause DCM in humans. We will perform cardiac physiological and ultrastructural analyses of micro-dissected heart preparations to test the hypothesis that: expression of DCM-associated myosin mutations causes defects in cardiac contractility and leads to pathological remodeling akin to the human condition, i.e. cardiac dilation, arrhythmias, and ultrastructural defects. Overall, our project will provide detailed and comprehensive analyses to better understand how myosin dysfunction causes DCM and to determine the feasibility of using Drosophila as an assessment tool for human DCM. These studies will offer outstanding training in structural biology, biochemistry, and cell and molecular biology aimed at studying protein dysfunction related to heart disease to prepare the applicant for a related career in academia.

 描述(申请人提供)：扩张型心肌病(DCM)是最常见的心肌病类型，可由收缩蛋白(如肌球蛋白)突变引起。然而，在肌球蛋白为基础的扩张型心肌病中，导致心脏扩张的结构、分子和生理来源还不是很清楚。我们将利用果蝇可用的强大遗传工具来产生基于肌球蛋白的DCM的第一个果蝇模型，并确定疾病的机制基础。将采用多学科方法来确定肌球蛋白中单一氨基酸的变化如何扰乱分子内相互作用，并导致横纹肌的生化、结构和生理缺陷。在目标1中，我们将为肌球蛋白产生第一个X-ra晶体结构，该晶体结构含有已知的导致人类DCM的突变，并被预测为调节肌动蛋白结合。突变的His标记的肌球蛋白将在间接飞行肌肉(IFMS)中表达、纯化并用于结晶学。我们将测试这样的假设：肌球蛋白中的DCM突变破坏了肌动蛋白结合位点附近或内部的分子内相互作用，从而重新定位了对肌动蛋白结合重要的关键残基。AIM 2将实施多种方法，以更好地了解与人类肌球蛋白DCM突变相关的生化、细胞生物学和功能缺陷。我们将从IFMS中表达和纯化突变的肌球蛋白，用于生化/生物物理分析(肌动蛋白共沉淀，ATPase，体外运动性)，以确定肌球蛋白突变引起的扩张型心肌病的分子基础。IFMS的超微结构分析将深入了解突变导致的肌原纤维组装和维护方面的缺陷。此外，我们将通过飞行和跳跃测试来确定这种突变的表达是否会导致骨骼肌功能障碍。我们将验证这样的假设：肌球蛋白突变可以削弱肌动蛋白亲和力，降低肌球蛋白的酶活性，并导致间接飞行肌肉的结构和功能缺陷。在目标3中，我们将评估由于肌球蛋白DCM突变表达而在果蝇心脏中发生的重塑事件。虽然已知果蝇的心脏可以重塑为扩张的表型，但尚不清楚它是否会因肌球蛋白突变而扩张，已知的肌球蛋白突变会导致人类DCM。我们将对显微解剖的心脏标本进行心脏生理和超微结构分析，以检验以下假设：DCM相关肌球蛋白突变的表达会导致心脏收缩能力的缺陷，并导致类似于人类情况的病理性重构，即心脏扩张、心律失常和超微结构缺陷。总体而言，我们的项目将提供详细和全面的分析，以更好地了解肌球蛋白功能障碍是如何导致DCM的，并确定使用果蝇作为人类DCM评估工具的可行性。这些研究将提供结构生物学、生物化学以及细胞和分子方面的卓越培训。 生物学旨在研究与心脏病相关的蛋白质功能障碍，为申请者在学术界从事相关职业做准备。