cMyBP-C: Phosphorylation-Dependent Regulation In Vivo

cMyBP-C：体内磷酸化依赖性调节

• 批准号: 8215310
• 负责人: Jeffrey Robbins
• 金额: $ 25.56万
• 依托单位: UNIVERSITY OF VERMONT & ST AGRIC COLLEGE
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-02-01 至 2015-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8215310
• 关键词: Actins; Actomyosin; Affect; Alanine; Animals; Aspartate; Binding; Binding Sites; Biochemical Genetics; Calmodulin; Cardiac; Cardiac Myocytes; Cardiac Myosins; Charge; Collaborations; Cyclic AMP-Dependent Protein Kinases; Data; Development; Dissection; Failure; Familial Hypertrophic Cardiomyopathy; Fiber; Gene Mutation; Generations; Genetic Engineering; Heart; Heart Diseases; Heart failure; Human; Hypertrophy; In Vitro; Individual; Injury; Ischemia; Kinetics; Mechanics; Mediating; Modeling; Molecular; Molecular Motors; Motion; Mus; Mutate; Mutation; Myosin ATPase; Organ; Outcome; Pathologic; Pattern; Performance; Phenotype; Phosphorylation; Phosphorylation Site; Phosphotransferases; Physiological; Population; Post-Translational Protein Processing; Preparation; Protein Isoforms; Protein Kinase C; Proteins; Regulation; Role; Sarcomeres; Serine; Site; Stress; Structure; Structure-Activity Relationship; Techniques; Testing; Thick Filament; Thin Filament; Tissues; Transgenic Mice; Transgenic Organisms; human data; in vivo; mimetics; myosin-binding protein C; novel therapeutics; response; skeletal; therapeutic target

Our objective is to understand the structure-function relationships and the functional consequences of posttranslational modifications of cardiac myosin binding protein C (cMyBP-C). cMyBP-C is critical to normal cardiac performance as evidenced by genetic mutations in cMyBP-C being one of the leading causes of familial hypertrophic cardiomyopathy. Despite its functional importance, the molecular mechanism by which cMyBP-C exerts its effect on the myosin molecular motor as it interacts with actin to generate force and motion remains largely undefined. Additionally, changes in the protein's phosphorylation patterns are tightly coordinated with the heart's response to stress and failure but the consequences of these changes in terms of the interactions with myosin and actin remain largely unexplored and are critical for the control of contractility. Aim 1 will test the hypothesis that the 3 phosphorylated serines in the cardiac-specific insertion of cMyBP-C (Ser-273, Ser-282 and Ser-302) are not functionally equivalent and that hierarchal patterns of phosphorylation exist for cMyBP-C. A corollary is that these patterns are functionally important. In order to understand the role of each phosphorylation site, Ser-273, Ser-282 and Ser-302 will be mutated to either a nonphosphorylatable residue (alanine) or a charged phosphorylation mimetic (aspartate) singly and in combination. Aim 2 will test the hypothesis that cMyBP-C has a defined binding site or sites for actin and show, in collaboration with Core B, that this interaction provides an elastic and viscous load on the sarcomere. Although the interactions between cMyBP-C and myosin itself are well defined, cMyBP-C binds to actin but the physiological significance of this binding is obscure and the cMyBP-C residues/regions responsible for this interaction is/are undefined. Using a combination of biochemical and genetic approaches we will first define the regions of cMyBP-C that are responsible for actin binding, mutate them and express those mutations in the cardiomyocyte population via transgenic replacement in order to determine their physiological significance.

我们的目标是了解心肌肌球蛋白结合蛋白 C (cMyBP-C) 的结构与功能关系以及翻译后修饰的功能后果。 cMyBP-C 对正常心脏功能至关重要，cMyBP-C 的基因突变是家族性肥厚型心肌病的主要原因之一。尽管其功能很重要，但 cMyBP-C 在与肌动蛋白相互作用产生力和运动时对肌球蛋白分子马达发挥作用的分子机制在很大程度上仍不清楚。此外，蛋白质磷酸化模式的变化与 与心脏对压力和衰竭的反应相协调，但这些变化在与肌球蛋白和肌动蛋白相互作用方面的后果在很大程度上仍未被探索，并且对于收缩力的控制至关重要。目标 1 将检验以下假设：cMyBP-C 心脏特异性插入中的 3 个磷酸化丝氨酸（Ser-273、Ser-282 和 Ser-302）在功能上并不等效，并且 cMyBP-C 存在磷酸化的分层模式。推论是这些模式在功能上很重要。为了了解每个磷酸化位点的作用，Ser-273、Ser-282 和 Ser-302 将单独或组合突变为不可磷酸化残基（丙氨酸）或带电磷酸化模拟物（天冬氨酸）。目标 2 将进行测试 假设 cMyBP-C 具有明确的肌动蛋白结合位点，并与 Core B 合作表明这种相互作用为肌节提供了弹性和粘性负载。尽管 cMyBP-C 和肌球蛋白本身之间的相互作用已明确定义，但 cMyBP-C 与肌动蛋白结合，但这种结合的生理意义尚不清楚，并且负责这种相互作用的 cMyBP-C 残基/区域未定义。通过结合生化和遗传学方法，我们将首先定义 cMyBP-C 中负责肌动蛋白结合的区域，对其进行突变并通过转基因替换在心肌细胞群中表达这些突变，以确定其生理意义。