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（由申请人提供）：扩张型心肌病（DCM）是最常见的心肌病形式，可由收缩蛋白（例如肌球蛋白）的突变引起，然而，基于肌球蛋白的 DCM 的结构、分子和生理学起源导致心脏扩张。我们将利用果蝇中强大的遗传工具来生成第一个基于肌球蛋白的 DCM 果蝇模型，并确定疾病的多学科基础。将采用方法来确定肌球蛋白中的单个氨基酸变化如何破坏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，我们将为含有已知导致 DCM 的突变的肌球蛋白生成第一个 X ra 晶体结构。预测在人类中调节肌动蛋白结合的突变 His 标记的肌球蛋白将在间接飞行肌 (IFM) 中表达、纯化并用于晶体学研究。我们将测试以下假设：DCM 突变。破坏肌动蛋白结合位点附近或内部的分子内相互作用的肌球蛋白会重新定向对肌动蛋白结合重要的关键残基，Aim 2 将采用多种方法来更好地了解与人肌球蛋白 DCM 突变相关的生化、细胞生物学和功能缺陷。我们将从 IFM 中表达和纯化突变肌球蛋白，用于生化/生物物理测定（肌动蛋白共沉淀、ATP 酶、体外运动），以确定肌球蛋白引起的 DCM 的分子基础IFM 的超微结构分析将深入了解突变引起的肌原纤维组装和维护缺陷。此外，我们将使用飞行和跳跃测试来确定此类突变的表达是否会导致骨骼肌功能障碍。肌球蛋白中的肌球蛋白可以削弱肌动蛋白的亲和力，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，我们将评估由于表达而在果蝇心脏中发生的重塑事件。尽管已知果蝇心脏可以重塑为扩张表型，但尚不清楚它是否会响应已知导致人类 DCM 的肌球蛋白突变而扩张。我们将对显微解剖心脏进行心脏生理和超微结构分析。准备检验以下假设：DCM 相关肌球蛋白突变的表达会导致心肌收缩力缺陷，并导致类似于人类状况的病理重塑，即心脏扩张，总体而言，我们的项目将提供详细而全面的分析，以更好地了解肌球蛋白功能障碍如何导致 DCM，并确定使用果蝇作为人类 DCM 评估工具的可行性。这些研究将为结构生物学、生物化学、细胞和分子 生物学旨在研究与心脏病相关的蛋白质功能障碍，为申请人在学术界从事相关职业做好准备。