Genetics and Molecular Biology of Myosin

肌球蛋白的遗传学和分子生物学

• 批准号: 7999955
• 负责人: Sanford I Bernstein
• 金额: $ 11.92万
• 依托单位: SAN DIEGO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-01-25 至 2011-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7999955
• 关键词: ATP phosphohydrolase; Actins; Address; Affect; Affinity; Alternative Splicing; Amino Acids; Animal Model; Binding; Binding Sites; Biochemical; Biological Assay; Cardiac; Cardiomyopathies; Central Core Myopathy; Characteristics; Chemicals; Communication; Crystallization; Crystallography; Defect; Development; Distal Muscular Dystrophies; Dorsal; Drosophila genus; Drosophila melanogaster; Electron Microscopy; Elements; Embryo; Engineering; Fiber; Frequencies; Genes; Genetic; Genetic Models; Goals; Head; Health; Heart; Heart Diseases; Human; Human Development; Hypertrophic Cardiomyopathy; Image; In Vitro; Inclusion Bodies; Investigation; Kinetics; Lead; Locomotion; Mechanics; Mediating; Microfilaments; Modeling; Molecular; Molecular Biology; Molecular Models; Molecular Motors; Motor; Movement; Muscle; Muscle Contraction; Muscle Fibers; Muscle function; Mutate; Mutation; Myocardium; Myopathy; Myosin ATPase; Myosin Heavy Chains; Nucleotides; Organism; Output; Physiological; Physiology; Production; Proline; Property; Protein Isoforms; Proteins; Resolution; Restrictive Cardiomyopathy; Role; Site-Directed Mutagenesis; Skeletal Muscle; Staging; Striated Muscles; Structure; Structure-Activity Relationship; Suppressor Mutations; System; Systems Analysis; Testing; Therapeutic Intervention; Thick Filament; Thin Filament; Transgenic Organisms; Variant; Video Microscopy; Whole Organism; Wing; Work; analog; base; beta-Myosin; cell motility; early onset; flexibility; human disease; in vitro activity; in vitro testing; in vivo; innovation; insight; interdisciplinary approach; molecular modeling; muscular structure; mutant; myosin storage myopathy; novel; public health relevance; retinal rods; skeletal

DESCRIPTION (provided by applicant): We will use an integrative and multidisciplinary approach to investigate how the S1 head domain of the myosin heavy chain (MHC) protein drives muscle function. Myosin is the molecular motor of muscle and the major component of myofibrillar thick filaments. Its ATP-dependent interaction with actin-containing thin filaments powers muscle contraction. Our studies use the model organism Drosophila melanogaster, which has a single muscle MHC gene but produces multiple forms of the protein (isoforms) by alternative RNA splicing. Using MHC null mutants in conjunction with germline transformation, we express engineered versions of the protein and employ them to test basic and novel hypotheses that predict structural, biochemical, fiber mechanical, physiological and locomotory properties imparted by specific myosin domains and amino acid residues. An innovative aspect of our system is that functions will be tested in vitro, in skeletal and cardiac muscle and in intact organisms. Therefore, we can determine directly and to what degree a specific biochemical property defines a physiological or locomotory characteristic. To this end, we will utilize a battery of in vitro and in vivo assays: ATPase, actin and nucleotide affinity, in vitro motility, x-ray crystallography, molecular modeling, electron microscopy, isolated fiber mechanics, video-based cardiac imaging and organismal locomotion. Our first aim is to elucidate the role of a critical communication element of the myosin motor called the relay domain. We will determine the importance of specific transient interactions of key amino acid residues of the relay that we hypothesize to interact with the converter domain or with the SH1-SH2 helix region during the mechanochemical cycle. For this, we will combine the transgenic approach with classical genetics to introduce and suppress mutations. Our second aim will test predicted isoform-specific interactions during the mechanochemical cycle. To this end, we will exploit the Drosophila system to express flight and embryonic muscle myosin isoforms that will be crystallized and compared in multiple nucleotide binding states. This approach will also be used for structural analysis of human beta-cardiac myosin, which will be the first mammalian striated muscle myosin analyzed at atomic resolution. Our third aim will test our hypotheses about the effects of a mutation in myosin that is known to cause restrictive cardiomyopathy. We will create a Drosophila model of this human disease by mutating the invariant proline at the myosin head-rod junction. We will define the biochemical, biophysical, mechanical and locomotory defects engendered by the myosin mutation. We will also examine whether the mutation affects the flexibility of the myosin head and determine how it influences Drosophila heart (dorsal vessel) structure and function. Overall, our novel integrative analyses will permit testing of models for the transduction of chemical energy into movement and will yield insight into how myosin functions in muscle. Further, we will directly address the role of myosin in human muscle disease, by defining the molecular basis of a restrictive cardiomyopathy. PUBLIC HEALTH RELEVANCE: We study the structural and functional differences among alternative forms of the myosin motor protein in order to elucidate how these "isoforms" differentially regulate contraction of various muscle types during normal locomotion. We will also examine the role of myosin mutation in muscle disease initiation and progression by developing a genetic model for myosin-based restrictive cardiomyopathy. This is relevant to human health in that mutations in myosin cause heart diseases such as dilated, restrictive and hypertrophic cardiomyopathy, as well as skeletal muscle diseases such as inclusion body myopathy, central core disease, early onset distal myopathy and myosin storage myopathy.

描述（由申请人提供）：我们将使用综合和多学科的方法来研究肌球蛋白重链（MHC）蛋白的S1头部结构域如何驱动肌肉功能。肌球蛋白是肌肉的分子马达，是肌原纤维粗丝的主要成分。它依赖于ATP与肌动蛋白的相互作用，为肌肉收缩提供动力。我们的研究使用的模式生物果蝇，它有一个单一的肌肉MHC基因，但产生多种形式的蛋白质（异构体）的选择性RNA剪接。使用MHC无效突变体与种系转化相结合，我们表达了蛋白质的工程版本，并利用它们来测试基本和新的假设，这些假设预测了特定肌球蛋白结构域和氨基酸残基赋予的结构、生化、纤维机械、生理和运动特性。我们的系统的一个创新方面是，功能将在体外测试，在骨骼肌和心肌和完整的生物体。因此，我们可以直接确定一个特定的生化特性在多大程度上定义了一个生理或运动特征。为此，我们将利用一系列体外和体内测定：ATP酶、肌动蛋白和核苷酸亲和力、体外运动性、X射线晶体学、分子建模、电子显微镜、分离纤维力学、基于视频的心脏成像和生物体运动。我们的第一个目标是阐明一个关键的通信元件的肌球蛋白马达称为中继域的作用。我们将确定我们假设在机械化学循环期间与转换器结构域或与SH 1-SH 2螺旋区域相互作用的继电器的关键氨基酸残基的特定瞬时相互作用的重要性。为此，我们将结合联合收割机的转基因方法与经典遗传学引入和抑制突变。我们的第二个目标是在机械化学循环过程中测试预测的异构体特异性相互作用。为此，我们将利用果蝇系统来表达飞行和胚胎肌肉肌球蛋白亚型，这些亚型将在多个核苷酸结合状态下结晶和比较。这种方法也将用于人类β-心肌肌球蛋白的结构分析，这将是第一个在原子分辨率下分析的哺乳动物横纹肌肌球蛋白。我们的第三个目标是检验我们关于肌球蛋白突变的影响的假设，这种突变已知会引起限制性心肌病。我们将通过突变肌球蛋白头杆连接处不变的脯氨酸来创建这种人类疾病的果蝇模型。我们将定义由肌球蛋白突变引起的生化、生物物理、机械和运动缺陷。我们还将研究突变是否影响肌球蛋白头部的灵活性，并确定它如何影响果蝇心脏（背血管）的结构和功能。总的来说，我们的新的综合分析将允许测试模型的化学能转换成运动，并将产生洞察肌球蛋白在肌肉中的功能。此外，我们将通过定义限制性心肌病的分子基础，直接讨论肌球蛋白在人类肌肉疾病中的作用。公共卫生关系：我们研究肌球蛋白马达蛋白的替代形式之间的结构和功能差异，以阐明这些“异构体”在正常运动过程中如何差异调节各种肌肉类型的收缩。我们也将通过建立肌球蛋白为基础的限制性心肌病的遗传模型来研究肌球蛋白突变在肌肉疾病的发生和发展中的作用。这与人类健康有关，因为肌球蛋白的突变引起心脏病，如扩张性、限制性和肥厚性心肌病，以及骨骼肌疾病，如包涵体肌病、中央核心病、早发性远端肌病和肌球蛋白沉积肌病。