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: 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
• 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

 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（由申请人提供）：肌球蛋白结合蛋白C（MyBP-C）是脊椎动物横纹肌的一种粗（肌球蛋白）丝成分，在调节收缩中起关键作用。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，在缓慢的骨骼肌MyBP-C突变导致骨骼肌肌病，其中之一是致命的，明确了骨骼肌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提案中，PI克雷格（UMMS）和Warshaw（UVM）与欧文（伊利诺伊州）和Sadayappan（Loyola）博士合作，将联合收割机结合他们实验室在高分辨率成像和单分子生物物理学方面的专业知识，结合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。在目的1中，原位和体外模型系统将有助于确定MyBP-C是否激活和/或机械调节来自快和慢大鼠骨骼纤维的天然细（肌动蛋白）丝在天然粗丝上的钙依赖性滑动，以及是否仅在MyBP-C存在于粗丝中的情况下发生收缩调节。在目标2中，通过一种新的超分辨率光学显微镜技术，我们将确定MyBP-C N端是否通过与肌动蛋白和/或肌球蛋白结合来发挥功能。在补充实验中，纤维X射线分析和EM 3D重建的天然薄和厚的细丝将确定是否MyBP-C取代原肌球蛋白激活细丝和/或直接影响肌球蛋白头相互作用，以调节头部功能。在目标3中，快速和慢速MyBP-C之间MyBP-C N-末端结构域同种型差异的结构和功能后果将被表征，特别强调被认为影响肌动蛋白和肌球蛋白结合的2种慢速MyBP-C剪接变体。通过结构诱变，N-末端片段将与结构域缺失和缓慢MyBP-C剪接插入物一起表达，以努力定义赋予MyBP-C对肌动球蛋白功能的调节的结构域和插入物。虽然骨骼MyBP-C的临床影响是显而易见的，但其功能作用远未确定，因此这种紧密整合MyBP-C结构和功能的双PI提案提供了一个机会，以快速推进我们对正常状态下快速和慢速骨骼MyBP-C亚型的理解。