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：
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
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/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

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粗（肌球蛋白）丝成分，在调节收缩中发挥关键作用，由不同的基因编码，从而产生三种不同的亚型。自从 40 年前在骨骼肌中发现以来，大多数研究都在研究快速和慢速骨骼肌 MyBP-C 亚型和第三种（心脏）亚型的表达。 MyBP-C 的研究重点关注心脏亚型，因为该亚型的突变是遗传性心肌病的主要原因。然而，最近发现慢骨骼 MyBP-C 的突变会导致骨骼肌肌病，其中一种是新生儿致命的。很明显，定义骨架 MyBP-C 同种型的分子结构和功能至关重要，因此，在这个双 PI 提案中，PI 是 Craig (UMMS) 和 Warshaw。（UVM）与 Irving（伊利诺伊州）和 Sadayappan（洛约拉）博士合作，将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以及 X 射线衍射、分子生物学和质谱来阐明分子结构骨骼 MyBP-C 的功能和功能在目标 1 中，原位和体外模型系统将有助于确定 MyBP-C 是否激活和/或机械调节在目标 2 中，通过一种新型超分辨率光学显微镜，研究了天然细丝（肌动蛋白）在来自快速和慢速大鼠骨骼纤维的天然粗丝上的钙依赖性滑动，以及收缩调节是否仅发生在粗丝中存在 MyBP-C 的地方。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作用。在补充实验中，对天然薄层和厚层进行纤维 X 射线分析和 EM 3D 重建。细丝将决定 MyBP-C 是否取代原肌球蛋白以激活细丝和/或直接影响肌球蛋白头部相互作用以调节头部功能。在目标 3 中，MyBP-C N 端结构域亚型差异在快和慢之间的结构和功能后果。 MyBP-C 将特别强调 2 个缓慢的 MyBP-C 剪接变体，这些变体被认为会影响肌动蛋白和肌球蛋白的结合，通过结构突变，N 端片段将被表达。结构域删除和缓慢的 MyBP-C 剪接插入，试图定义赋予 MyBP-C 肌动球蛋白功能调节的结构域和插入，尽管骨骼 MyBP-C 的临床影响是显而易见的，但其功能作用还远未确定，因此这种双重作用。 -PI提案紧密整合了MyBP-C结构和功能，为快速推进我们对正常状态下的快速和慢速骨骼MyBP-C亚型的理解提供了机会。