Skeletal myosin-binding protein C (MyBP-C): molecular structure and function

骨骼肌球蛋白结合蛋白 C (MyBP-C)：分子结构和功能

基本信息

批准号：
9116778
负责人：
ROGER W CRAIG
金额：
$ 47.2万
依托单位：
UNIV OF MASSACHUSETTS MED SCH WORCESTER
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-08-01 至 2020-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9116778
关键词：
Actins Actomyosin Address Affect Affinity Alanine Alternative Splicing Antibodies Attention Binding Binding Sites Biological Assay Biological Models Biophysics Calcium Cardiac Cardiac Myosins Clinical Collaborations Congenital cardiomyopathy Coupled Data Electrons Fiber Filament Generations Genes Head Health Heart Hypertrophic Cardiomyopathy Illinois Image Imaging Techniques In Situ In Vitro Individual Lasers Lead Light Link Literature Mass Spectrum Analysis Measurement Microfilaments Microscopic Microscopy Molecular Molecular Biology Molecular Conformation Molecular Structure Motion Muscle Muscle Contraction Muscle Fibers Mutagenesis Mutation Myopathy Myosin ATPase N-terminal Physiology Play Proline Protein Isoforms Proteins RNA Splicing Rattus Resolution Roentgen Rays Role Skeletal Muscle Skeletal Muscle Myosins Slide Striated Muscles Structure Techniques Testing Thick Thick Filament Thin Filament Total Internal Reflection Fluorescent Tropomyosin Variant Vertebral column X ray diffraction analysis X-Ray Diffraction base cell motility in vitro Model in vivo insight molecular mass myosin-binding protein C novel reconstruction research study single molecule skeletal stoichiometry

项目摘要

DESCRIPTION (provided by applicant): Myosin-binding protein C (MyBP-C) is a thick (myosin) filament component of vertebrate striated muscle that plays a key role in modulating contraction. Three distinct isoforms are encoded by different genes, resulting in the expression of fast and slow skeletal muscle MyBP-C isoforms and a third (cardiac) isoform. Since its discovery in skeletal muscle 40 years ago, most studies of MyBP-C have focused on the cardiac isoform, because mutations in this isoform are a prime cause of inherited cardiomyopathies. However, the recent discovery that mutations in slow skeletal MyBP-C cause skeletal muscle myopathies, one of which is neonatally lethal, makes it clear that defining the molecular structure and function of the skeletal MyBP-C isoforms is critically important. Therefore, in this dual-PI proposal, PIs Craig (UMMS) and Warshaw (UVM), in collaboration with Drs. Irving (Illinois) and Sadayappan (Loyola), will combine their labs' expertise in high resolution imaging and single molecule biophysics coupled with X-ray diffraction, molecular biology and mass spectrometry to elucidate the molecular structure and function of skeletal MyBP-C. In Aim 1, in situ and in vitro model systems will help determine if MyBP-C activates and/or mechanically modulates the calcium- dependent sliding of native thin (actin) filaments over native thick filaments from fast and slow rat skeletal fibers and whether contractile modulation occurs only where MyBP-C exists in the thick filament. In Aim 2, through a novel super-resolution light microscopic technique, we will determine whether the MyBP-C N terminus functions by binding to actin and/or myosin. In complementary experiments, fiber X-ray analysis and EM 3D reconstruction of native thin and thick filaments will determine if MyBP-C displaces tropomyosin to activate the thin filament and/or directly influences myosin head interactions to modulate head function. In Aim 3, the structural and functional consequences of MyBP-C N-terminal domain isoform differences between fast and slow MyBP-C will be characterized with special emphasis on 2 slow MyBP-C splice variants thought to affect actin and myosin binding. Through structural mutagenesis, N-terminal fragments will be expressed with domain deletions and slow MyBP-C splice inserts in an effort to define the domains and inserts that confer MyBP-C's modulation of actomyosin function. Although skeletal MyBP-C's clinical impact is apparent, its functional role is far from certain and thus this dual-PI proposal, tightly integrating MyBP-C structure and function, offers an opportunity to rapidly advance our understanding of both fast and slow skeletal MyBP-C isoforms in their normal state.

描述（由适用提供）：肌球蛋白结合蛋白C（MYBP-C）是脊椎动物策略的厚（肌球蛋白）细丝成分，在调节收缩中起着关键作用。三种不同的同工型由不同的基因编码，从而导致骨骼肌肉MYBP-C同工型和第三个（心脏）同工型的表达。自从40年前发现其在骨骼肌中发现，MYBP-C的大多数研究都集中在心脏同工型上，因为该同工型的突变是遗传性心肌病的主要原因。然而，最近的发现是，慢速骨骼MYBP-C中的突变引起骨骼肌肌病，其中一种是新生儿致命的，这清楚地表明，定义骨骼MYBP-C同工型的分子结构和功能很重要。因此，在此双PI提案中，Pis Craig（UMMS）和Warshaw（UVM）与Drs合作。 Irving（Illinois）和Sadayappan（Loyola）将结合其实验室在高分辨率成像和单个分子生物物理学方面的专业知识，再加上X射线衍射，分子生物学和质谱法，以阐明骨骼MyBP-C的分子结构和功能。在AIM 1中，原位和体外模型系统将有助于确定MYBP-C是否激活和/或机械地调节天然薄（肌动蛋白）丝（肌动蛋白）丝（依赖性钙）纤维的钙依赖性滑动，而天然厚的细丝和慢速大鼠骨骼骨骼纤维以及收缩调制是否仅在厚丝中存在MYBP-C才出现。在AIM 2中，通过一种新颖的超分辨率光显微镜技术，我们将通过与肌动蛋白和/或肌球蛋白结合来确定MYBP-C N末端是否功能。在完整的实验中，天然细丝和厚细丝的纤维X射线分析和EM 3D重建将确定MYBP-C移位是否会激活细丝和/或直接影响肌球蛋白头部相互作用以调节头部功能。在AIM 3中，快速和慢的MYBP-C之间MYBP-C N末端域同工型的结构和功能后果将以2慢的MyBP-C剪接变体特别强调被认为会影响肌动蛋白和肌球蛋白结合。通过结构诱变，N末端碎片将用域缺失和减慢MYBP-C剪接插入片段表示，以努力定义域并插入该域MYBP-C对Actomyosin功能的调节。尽管骨骼MYBP-C的临床影响显而易见，但其功能作用远非确定，因此，该双PI提案紧密整合了MYBP-C结构和功能，它提供了一个机会，可以快速促进我们对正常状态下快速和缓慢的骨骼MYBP-C同工型的理解。