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

• 批准号: 9301480
• 负责人: ROGER W CRAIG
• 金额: $ 45.69万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-08-01 至 2020-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9301480
• 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; Heart; Hypertrophic Cardiomyopathy; Illinois; Imaging Techniques; In Situ; In Vitro; Individual; Lasers; Lead; Light; Link; Literature; Mass Spectrum Analysis; Measurement; Mechanics; Microfilaments; Microscopic; Microscopy; Molecular; Molecular Biology; Molecular Conformation; Molecular Structure; Motion; Muscle; Muscle Contraction; Muscle Fibers; Mutagenesis; Mutation; Myopathy; Myosin ATPase; N-terminal; Neonatal; Physiological; 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; Thinness; Total Internal Reflection Fluorescent; Tropomyosin; Variant; Vertebral column; X ray diffraction analysis; X-Ray Diffraction; base; cell motility; experimental study; high resolution imaging; in vitro Model; in vivo; insight; molecular mass; myosin-binding protein C; novel; public health relevance; reconstruction; single molecule; skeletal; stoichiometry

 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(申请人提供)：肌球蛋白结合蛋白C(MyBP-C)是脊椎动物横纹肌中一种粗大的(肌球蛋白)细丝成分，在调节收缩方面发挥关键作用。三种不同的亚型由不同的基因编码，导致快速和慢速骨骼肌MyBP-C亚型和第三种(心脏)亚型的表达。自从40年前在骨骼肌中发现MyBP-C以来，大多数对MyBP-C的研究都集中在心脏亚型上，因为这种亚型的突变是遗传性心肌病的主要原因。然而，最近发现缓慢的骨骼MyBP-C突变会导致骨骼肌肌病，其中一种是新生儿致命的，这清楚地表明，确定骨骼MyBP-C亚型的分子结构和功能是至关重要的。因此，在这个双PI方案中，PIS Craig(UMMS)和Warshaw(UVM)将与伊利诺伊州的Irving博士和Loyola的Sadayappan博士合作，将他们实验室在高分辨率成像和单分子生物物理方面的专业知识与X射线衍射、分子生物学和质谱学相结合，以阐明骨骼MyBP-C的分子结构和功能。在目标1中，原位和体外模型系统将有助于确定MyBP-C是否激活和/或机械地调节天然细纤维(肌动蛋白)在天然粗纤维上的滑动，以及收缩调制是否仅发生在粗纤维中存在MyBP-C的地方。在目标2中，通过一种新的超分辨光学显微镜技术，我们将确定MyBP-C N末端是否通过与肌动蛋白和/或肌球蛋白结合而发挥功能。在互补性实验中，天然细丝和粗丝的纤维X射线分析和EM 3D重建将确定MyBP-C是否取代原肌球蛋白激活细丝和/或直接影响肌球蛋白头部相互作用来调节头部功能。在目标3中，将特别强调影响肌动蛋白和肌球蛋白结合的两个慢速MyBP-C剪接变异体，以表征快MyBP-C和慢MyBP-C之间的MyBP-C N末端结构和功能上的差异。通过结构突变，N-末端片段将通过结构域的缺失和缓慢的MyBP-C剪接插入来表达，以努力定义MyBP-C对肌动球蛋白功能的调控的结构域和插入。尽管骨骼MyBP-C的临床影响是显而易见的，但它的功能作用还远未确定，因此，这种紧密结合MyBP-C结构和功能的双PI方案为我们提供了一个迅速提高我们对正常状态下快、慢骨骼MyBP-C亚型的理解的机会。