Genetics and Molecular Biology of Myosin

肌球蛋白的遗传学和分子生物学

基本信息

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
项目类别：
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

项目摘要

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）：我们将使用一种综合和多学科的方法来研究肌球蛋白重链（MHC）蛋白的S1头域如何驱动肌肉功能。肌球蛋白是肌肉的分子运动，也是肌原纤维厚细丝的主要成分。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。我们的研究使用模型有机体果蝇melanogaster，该果蝇具有单个肌肉MHC基因，但通过替代RNA剪接产生多种形式的蛋白质（同工型）。使用MHC无效突变体与种系转化结合使用，我们表达蛋白质的工程版本，并利用它们来测试基本和新颖的假设，这些假设可以预测特定肌球蛋白域和氨基酸残基所赋予的结构，生化，纤维机械，生理和运动特性。我们系统的一个创新方面是，将在体外，骨骼和心脏肌肉以及完整生物体中测试功能。因此，我们可以直接确定特定的生化特性定义生理或运动特征的程度。为此，我们将利用一系列体外和体内测定的电池：ATPase，肌动蛋白和核苷酸亲和力，体外运动，X射线晶体学，分子建模，电子显微镜，孤立的光纤力学，基于视频的心脏成像和有机体激动。我们的第一个目的是阐明肌球蛋白电动机的关键通信元素的作用，称为中继域。我们将确定我们假设在机械化学循环期间与转换器域或与SH1-SH2螺旋区相互作用的继电器关键氨基酸残基的特定瞬态相互作用的重要性。为此，我们将将转基因方法与经典遗传学结合起来，以引入和抑制突变。我们的第二个目标将在机械化学周期期间测试预测的同工型特异性相互作用。为此，我们将利用果蝇系统表达飞行和胚胎肌肉肌球蛋白同工型，这些肌球蛋白同工型将在多种核苷酸结合态中结晶并进行比较。这种方法还将用于人类β-心肌球蛋白的结构分析，这将是原子分辨率分析的第一个哺乳动物条纹肌肉肌球蛋白。我们的第三个目标将检验我们关于肌球蛋白突变影响的假设，该突变已知会导致限制性心肌病。我们将通过在肌球蛋白头杆连接处突变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产生的生化，生物物理，机械和运动缺陷。我们还将检查突变是否影响肌球蛋白头的柔韧性，并确定其如何影响果蝇心脏（背血管）结构和功能。总体而言，我们的新型综合分析将允许测试模型，以将化学能转移到运动中，并会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：我们研究了肌球蛋白运动蛋白的替代形式之间的结构和功能差异，以阐明在正常运动期间这些“同工型”如何差异调节各种肌肉类型的收缩。我们还将通过开发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开始和进展中的作用。这与人类健康有关，因为肌球蛋白的突变会导致心脏病，例如扩张，限制性和肥厚性心肌病，以及骨骼肌疾病，例如纳入人体肌病，中心核心疾病，早期发作远端肌病和肌球蛋白储存。