Cardiac Myosin-Binding Protein C: Molecular Modulation of Actomyosin Function.

心肌肌球蛋白结合蛋白 C：肌动球蛋白功能的分子调节。

• 批准号: 8860500
• 负责人: David M Warshaw
• 金额: $ 47.18万
• 依托单位: UNIVERSITY OF VERMONT & ST AGRIC COLLEGE
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-01 至 2019-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8860500
• 关键词: Actins; Activities of Daily Living; Actomyosin; Adrenergic Agents; Affect; Affinity; Alanine; Aspartic Acid; Atomic Force Microscopy; Binding; Binding Sites; Biological Assay; Biological Models; Calcium; Calcium Binding; Cardiac; Cardiac Muscle Contraction; Cardiac Myosins; Circular Dichroism; Complex; Coupled; Cyclic AMP-Dependent Protein Kinases; DNA Sequence Alteration; Defect; Development; Diastole; Disease; Event; Fiber; Functional disorder; Generations; Genes; Head; Heart; Heart failure; Human; Hypertrophic Cardiomyopathy; Imaging Techniques; In Situ; In Vitro; Individual; Knowledge; Label; Light; Link; Literature; Measures; Microscopic; Microscopy; Molecular; Molecular Structure; Monitor; Motion; Mus; Mutagenesis; Mutate; Mutation; Myofibrils; Myosin ATPase; N-terminal; Performance; Phosphorylation; Phosphorylation Site; Proteins; Resolution; Sarcomeres; Serine; Site; Structure; Sudden Death; Systole; Testing; Thick Filament; Thin Filament; Time; Tissues; Transgenic Mice; Tropomyosin; Troponin; Uncertainty; Vertebral column; activated Protein C; adrenergic; base; biophysical techniques; cell motility; improved; in vitro Model; in vivo; innovation; light scattering; molecular mechanics; mutant; myosin-binding protein C; public health relevance; response; single-molecule FRET; targeted treatment; young adult

 DESCRIPTION (provided by applicant): Cardiac myosin-binding protein C (cMyBP-C) is a sarcomeric thick filament associated protein that is critically important to normal cardiac structure and function. The importance of cMyBP-C is emphasized by mutations to cMyBP-C being a leading cause of hypertrophic cardiomyopathy. Despite being a key regulator of cardiac contractility, the molecular mechanism by which cMyBP-C modulates actomyosin force and motion generation is far from certain. Although cMyBP-C's N-terminal domains can bind to actin and the myosin head region, it is not known which of these binding partners is physiologically relevant and whether these binding partner interactions modulate cardiac contractility by directly affecting actomyosin power generation or indirectly by altering Ca2+-dependent thin filament activation. With phosphorylation of cMyBP-C's N terminus occurring in response to ß-adrenergic stimulation, phosphorylation may offer a measure of cMyBP-C functional tunability in order to enhance cardiac contractility. We propose the following three specific aims. Aim 1 tests the hypothesis that cMyBP-C's thin filament activation and actomyosin power inhibition are independent mechanisms associated with a specific binding partner. Thus, cMyBP-C binding partner interactions will be determined using state-of-the-art molecular biophysical approaches in simplified in vitro model systems (e.g. single molecule FRET) and in situ within myofibrils (super-resolution STORM microscopy). Structural mutagenesis of cMyBP-C to ablate binding partner sites of interaction in both expressed N-terminal fragments and in mutant cMyBP-C from transgenic mice will help link cMyBP-C's functional capacities to its interaction with either the thin filament or the myosin head region. Aim 2 tests the hypothesis that cMyBP-C activates the thin filament directly through a mechanism similar to calcium activation. Thus, we have developed an in vitro single thin filament activation assay to monitor the molecular sequence of events by which expressed fluorescently-labeled N-terminal fragments of cMyBP-C initiate the cooperative recruitment of fluorescently- labeled myosin molecules to the thin filament. Aim 3 tests the hypothesis that phosphorylation of cMyBP-C tunes cMyBP-C's modulation of contractility through alterations in cMyBP-C's molecular mechanics, which in turn alters its binding partner interactions. Cardiac tissue and native thick filaments from transgenic mice expressing mutant cMyBP-C as well as expressed N-terminal fragments with one or more serines replaced by non-phosphorylatable alanines or aspartic acids (phosphomimetics) will be used in assays described in Aims 1 and 2 to characterize the effect of site-specific phosphorylation. Using atomic force microscopy, we will characterize possible mechanisms by which phosphorylation affects M-domain molecular mechanics and structure, thus modulating cMyBP-C function. With the knowledge and understanding of cMyBP-C function derived from these collective studies, targeted therapies directed at cMyBP-C binding partner interactions may be developed to help modulate and to improve cardiac performance in the failing heart.

 描述（由适用提供）：心脏肌球蛋白结合蛋白C（CMYBP-C）是一种肌肉厚的细丝相关蛋白，对正常的心脏结构和功能至关重要。 CMYBP-C的重要性通过突变对CMYBP-C是肥厚性心肌病的主要原因。尽管是心脏收缩力的关键调节剂，但CMYBP-C调节肌动蛋白力和运动产生的分子机制远非确定。尽管CMYBP-C的N末端结构域可以与肌动蛋白和肌球蛋白头部区域结合，但尚不清楚这些结合伴侣在物理上是相关的，以及这些结合伴侣是否通过直接影响Actomyosin发电或间接影响CA2+CA2+依赖性依赖性依赖性的细丝激活来与心脏收缩性相互作用。随着CMYBP-C的N末端的磷酸化响应于β-肾上腺素刺激，磷酸化可能会提供CMYBP-C功能可密义能力的量度，以增强心脏收缩力。我们提出以下三个特定目标。 AIM 1检验了CMYBP-C的细丝激活和肌动蛋白功率抑制的假设是与特定结合伴侣相关的独立机制。这是CMYBP-C结合伴侣的相互作用，将使用简化的体外模型系统（例如单分子fret）和肌纤维（超分辨率风暴显微镜）内的最先进的分子生物物理方法来确定。在表达的N末端片段和来自转基因小鼠的突变体CMYBP-C中，CMYBP-C与消融结合伴侣相互作用的结构诱变将有助于将CMYBP-C的功能能力与与薄丝或肌球蛋白头部区域的相互作用联系起来。 AIM 2检验了CMYBP-C通过类似于钙激活的机制直接激活细丝的假设。因此，我们开发了一种体外单薄细丝激活测定法，以监测事件的分子序列，通过该分子序列，CMYBP-C的表达荧光标记的N末端片段启动了荧光标记的肌球蛋白分子与细丝的合作募集。 AIM 3检验了以下假设：CMYBP-C调音通过CMYBP-C的分子力学改变CMYBP-C对收缩性的调制，从而改变其结合伴侣的相互作用。来自表达突变CMYBP-C的转基因小鼠的心脏组织和天然厚细丝，以及用一种或多种丝毫丝氨酸代替的N端片段表达的N末端片段将在AIM 1和2中描述的分析中描述的分析中描述的测定中，将使用非磷酸化的丙氨酸或天冬氨酸（磷酸性苯甲酸）（磷酸化）。使用原子力显微镜，我们将表征光谱化影响M核心分子机制和结构的可能机制，从而调节CMYBP-C功能。借助从这些集体研究得出的CMYBP-C功能的知识和理解，可以开发针对CMYBP-C结合伴侣相互作用的有针对性疗法，以帮助调节和改善失败心脏的心脏表现。