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。肌球蛋白是肌肉的运动分子，是肌原纤维粗丝的主要成分。它与含有肌动蛋白的细丝的atp依赖性相互作用为肌肉收缩提供动力。我们的研究使用模式生物黑腹果蝇，它有一个单一的肌肉MHC基因，但通过选择性RNA剪接产生多种形式的蛋白质（同种异构体）。利用MHC零突变体与种系转化相结合，我们表达了该蛋白的工程版本，并利用它们来测试基本的和新的假设，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、纤维力学、生理和运动特性。我们系统的一个创新之处在于，其功能将在体外、骨骼肌和心肌以及完整的生物体中进行测试。因此，我们可以直接确定特定的生化特性在多大程度上定义生理或运动特性。为此，我们将利用一系列体外和体内分析：atp酶、肌动蛋白和核苷酸亲和力、体外运动性、x射线晶体学、分子模型、电子显微镜、分离纤维力学、基于视频的心脏成像和有机体运动。我们的第一个目标是阐明肌凝蛋白马达的一个关键通信元件的作用，称为中继域。我们将确定接力的关键氨基酸残基的特定瞬态相互作用的重要性，我们假设在机械化学循环中与转换结构域或与SH1-SH2螺旋区域相互作用。为此，我们将把转基因方法与经典遗传学相结合，引入和抑制突变。我们的第二个目标是在机械化学循环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球蛋白同型异构体，这些异构体将被结晶并在多个核苷酸结合状态下进行比较。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上分析的哺乳动物横纹肌肌球蛋白。我们的第三个目标将测试我们关于肌球蛋白突变影响的假设，这种突变已知会导致限制性心肌病。我们将通过突变肌凝蛋白头杆连接处的不变性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。我们还将研究这种突变是否会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，我们新颖的综合分析将允许测试化学能转化为运动的模型，并将深入了解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，直接解决肌球蛋白在人类肌肉疾病中的作用。公共卫生相关性：我们研究了不同形式的肌球蛋白运动蛋白之间的结构和功能差异，以阐明这些“同种异构体”如何在正常运动过程中差异调节各种肌肉类型的收缩。我们还将通过建立基于肌球蛋白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进展中的作用。这与人类健康有关，因为肌球蛋白突变可引起扩张性、限制性和肥厚性心肌病等心脏病，以及包涵体肌病、中央核心病、早发性远端肌病和肌球蛋白储存性肌病等骨骼肌疾病。