Regulation of MyBP-C slow via phosphorylation in skeletal muscles

通过骨骼肌磷酸化缓慢调节 MyBP-C

• 批准号: 9769620
• 负责人: Aikaterini Kontrogianni-Konstantopoulos
• 金额: $ 17万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-01 至 2020-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9769620
• 关键词: ATP phosphohydrolase; Actins; Actomyosin; Adult; Affect; Aging; Alanine; Alternative Splicing; Animal Model; Arthrogryposis; Atomic Force Microscopy; Benchmarking; Binding; Biochemical; Biological; Biological Assay; Biology; Biomedical Research; C10; Cardiac; Cardiac Myosins; Complex; Cyclic AMP-Dependent Protein Kinases; Development; Distal; Embryo; Event; Exons; Exploratory/Developmental Grant; Family; Fatigue; Fibronectins; Filament; Functional disorder; Generations; Genes; Goals; Growth; Head; Heart; Immunoglobulin Domain; In Vitro; Individual; Kinetics; Knock-in; Maintenance; Mechanics; Mediating; Microfilaments; Modeling; Molecular; Morphology; Mus; Muscle; Muscle function; Mutate; Myocardium; Myopathy; Myosin ATPase; Nature; Phenotype; Phosphorylation; Phosphorylation Site; Process; Property; Protein Isoforms; Proteins; Proteomics; Protocols documentation; RNA Splicing; Regulation; Role; Sarcomeres; Sedimentation process; Side; Site; Skeletal Muscle; Slide; Striated Muscles; Structure; Testing; Text; Thick; Thick Filament; Thinness; Variant; biophysical properties; blastocyst; cell motility; citrate carrier; combinatorial; embryonic stem cell; exercise capacity; high reward; high risk; homologous recombination; in vivo; mechanical properties; mimetics; mouse model; muscle strength; mutant; myosin-binding protein C; novel; organizational structure; response; skeletal; stem; stressor; tool

ABSTRACT Myosin Binding Protein-C (MyBP-C) comprises a family of thick filament associated proteins that contributes to their assembly and maintenance, and regulates the formation of actomyosin cross-bridges during contraction. Three distinct isoforms have been characterized, including the cardiac (c), slow (s) skeletal and fast (f) skeletal. The expression of the cardiac isoform is confined in the developing and mature heart, whereas the skeletal isoforms can co-exist in the same muscle. The core structure of MyBP-C consists of seven immunoglobulin (Ig) domains and three fibronectin-III (Fn-III) domains, numbered from the NH2-terminus as C1-C10. During the last forty years, numerous studies have focused on elucidating the mechanisms that modulate the activities of cMyBP-C in the formation of actomyosin cross-bridges. On the contrary, the regulation and roles of the skeletal isoforms have remained obscure, and mainly inferred due to the structural similarity they share with cMyBP-C. Our group has been studying the slow skeletal form of MyBP-C aiming to understand its regulation and activities. Using molecular tools, we have shown that the MYBPC1 gene, encoding sMyBP-C, is heavily spliced giving rise to multiple variants that can be co-expressed in the same muscle and myofiber. These share common domains, but also differ by the inclusion or skipping of novel insertions located in the NH2-terminus, the FN-III C7 domain and the COOH-terminus. Both the NH2 and COOH termini can retain native myosin and actin and modulate the sliding velocity of actin filaments past myosin heads, though to different extents and in a variant-specific manner. Moreover, using proteomic tools, we have demonstrated that sMyBP-C undergoes phosphorylation mediated by PKA and PKC. In particular, we have identified four phosphorylation sites in the NH2-terminus of the protein, with one of them located within a unique insertion present only in select variants. We therefore hypothesize that sMyBP-C comprises a multi- faceted family of thick filament accessory proteins whose functions are regulated via complex phosphorylation of its NH2-terminus. Our goals in the current proposal are to examine how phosphorylation affects the biochemical and biophysical properties of the different sMyBP-C variants (Aim 1), and to generate the first phospho-mutant sMyBP-C animal model to assess the role of phosphorylation in vivo (Aim 2). The proposed studies will greatly advance our understanding on the regulation of the multifaceted sMyBP-C subfamily via phosphorylation, which is an outstanding biological question with important and broad implications in muscle pathophysiology.

抽象的 肌球蛋白结合蛋白-C (MyBP-C) 包含一个粗丝相关蛋白家族， 有助于其组装和维护，并调节肌动球蛋白跨桥的形成 收缩期间。三种不同亚型已被表征，包括心脏亚型 (c)、慢速亚型 (s) 骨骼亚型 和快速（f）骨骼。心脏亚型的表达仅限于发育和成熟的心脏， 而骨骼亚型可以共存于同一块肌肉中。 MyBP-C的核心结构包括 七个免疫球蛋白 (Ig) 结构域和三个纤连蛋白-III (Fn-III) 结构域，从 NH2 末端开始编号 如 C1-C10。在过去的四十年中，大量的研究集中于阐明其机制 调节 cMyBP-C 在肌动球蛋白跨桥形成中的活性。相反， 骨骼亚型的调节和作用仍然不清楚，主要是由于结构 它们与 cMyBP-C 具有相似性。我们的小组一直在研究 MyBP-C 的慢骨骼形式，旨在 了解其监管和活动。使用分子工具，我们已经证明 MYBPC1 基因， 编码 sMyBP-C，经过大量剪接，产生可以在同一序列中共表达的多个变体 肌肉和肌纤维。这些共享共同的领域，但也因包含或跳过新颖的内容而有所不同 插入位于 NH2 末端、FN-III C7 结构域和 COOH 末端。 NH2 和 COOH末端可以保留天然肌球蛋白和肌动蛋白并调节肌动蛋白丝的滑动速度 肌球蛋白头，尽管程度不同并且以特定变体的方式。此外，使用蛋白质组学工具， 我们已经证明 sMyBP-C 会经历 PKA 和 PKC 介导的磷酸化。特别是，我们 已鉴定出该蛋白质 NH2 末端的四个磷酸化位点，其中一个位于 独特的插入仅存在于选定的变体中。因此，我们假设 sMyBP-C 包含多个 粗丝辅助蛋白的多面家族，其功能通过复杂的磷酸化调节 其 NH2 末端。我们当前提案的目标是研究磷酸化如何影响 不同 sMyBP-C 变体的生化和生物物理特性（目标 1），并生成第一个 磷酸化突变体 sMyBP-C 动物模型用于评估体内磷酸化的作用（目标 2）。拟议的 研究将极大地增进我们对多方面 sMyBP-C 亚家族调控的理解 磷酸化，这是一个突出的生物学问题，对肌肉具有重要而广泛的影响 病理生理学。