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。

基本信息

批准号：
9282730
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
项目类别：
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/9282730
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 Microscopy Molecular Molecular Structure Monitor Motion Mus Mutagenesis Mutate Mutation Myofibrils Myosin ATPase N-terminal Performance Phosphorylation Phosphorylation Site Physiological Proteins Recruitment Activity Resolution Sarcomeres Serine Site Structure Sudden Death Systole Testing Thick Filament Thin Filament Time Tissues Transgenic Mice Tropomyosin Troponin Uncertainty Vertebral column base biophysical techniques cell motility improved in vitro Model in vivo innovation light scattering microscopic imaging molecular domain molecular mechanics mutant myosin-binding protein C public health relevance response single-molecule FRET targeted treatment young adult

项目摘要

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（由申请人提供）：心肌肌球蛋白结合蛋白 C (cMyBP-C) 是一种肌节粗丝相关蛋白，对正常心脏结构和功能至关重要 cMyBP-C 的突变强调了 cMyBP-C 的重要性。尽管 cMyBP-C 是心肌收缩力的关键调节因子，但其调节肌动球蛋白力量和运动产生的分子机制却远非如此。虽然 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伙伴中的哪一个具有生理相关性，也不知道这些结合伙伴相互作用是否通过直接影响肌动球蛋白发电或间接影响心肌收缩力。改变 Ca2+ 依赖性细丝激活。cMyBP-C 的 N 末端因 β-肾上腺素刺激而发生磷酸化，磷酸化可提供一种测量cMyBP-C 功能可调性，以增强心脏收缩力。目标 1 测试 cMyBP-C 的细丝激活和肌动球蛋白功率抑制是与特定结合伙伴相关的独立机制的假设。 C 结合配偶体相互作用将使用最先进的分子生物物理方法在简化的体外模型系统（例如单分子 FRET）和肌原纤维内进行原位测定（超分辨率风暴显微镜）cMyBP-C 的结构突变消除了表达的 N 末端片段和转基因小鼠突变 cMyBP-C 中相互作用的结合伙伴位点，这将有助于将 cMyBP-C 的功能能力与其相互作用联系起来。目标 2 测试了 cMyBP-C 通过类似于钙激活的机制直接激活细丝的假设。我们开发了一种体外单细丝激活试验来监测事件的分子序列，通过该试验，表达的 cMyBP-C 荧光标记 N 末端片段启动荧光标记肌球蛋白分子向细丝 Aim 3 测试的协同招募。假设 cMyBP-C 的磷酸化通过改变 cMyBP-C 的分子力学来调节 cMyBP-C 的收缩性调节，从而改变其与心脏的结合伙伴相互作用。来自表达突变体 cMyBP-C 的转基因小鼠的组织和天然粗丝以及用不可磷酸化的丙氨酸或天冬氨酸（磷酸模拟物）取代的一个或多个丝氨酸表达的 N 末端片段将用于目标 1 和 2 中描述的测定中使用原子力显微镜表征位点特异性磷酸化的影响，我们将表征磷酸化影响 M 结构域分子力学和结构的可能机制，从而通过这些集体研究对 cMyBP-C 功能的了解和理解，可以开发针对 cMyBP-C 结合伴侣相互作用的靶向疗法，以帮助调节和改善衰竭心脏的心脏功能。