cMyBP-C: Molecular Mechanisms of Actomyosin Modulation

cMyBP-C：肌动球蛋白调节的分子机制

基本信息

批准号：
8215309
负责人：
David M Warshaw
金额：
$ 39.62万
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：
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/8215309
关键词：
Actins Activities of Daily Living Actomyosin Adrenergic Agents Affect Affinity Alanine Animals Aspartic Acid Binding Biological Assay Biomechanics Cardiac Cardiac Myosins Clinical Complement Cyclic AMP-Dependent Protein Kinases Data Equilibrium Familial Hypertrophic Cardiomyopathy Fiber Gene Mutation Generations Head Heart Heart failure In Vitro Intervention Kinetics Knockout Mice Label Lasers Length Literature Location Mechanics Molecular Molecular Structure Motion Motor Mus Mutation Myocardium Myosin ATPase Myosin Subfragments N-terminal Pattern Performance Phosphorylation Phosphorylation Site Physiological Population Positioning Attribute Property Regulation Relative (related person)Serine Site Structure System Techniques Thick Thick Filament Thin Filament Transgenic Mice adrenergic base cell motility design molecular mechanics mouse model mutant myosin-binding protein C novel novel therapeutics programs research study single molecule stoichiometry

项目摘要

The molecular mechanism by which cMyBP-C exerts its effect on actomyosin force power generating system remains largely undefined. With its low ratio relative to myosin and it being located in distinct regions of the thick filament, we will determine in this Project how cMyBP-C's modulates actomyosin's power generation by either interacting with a limited population of crossbridges or whether it cooperatively affects all crossbridges within the thick filament. This project serves as a physiological bridge between the animal (Project #3, Robbins), whole heart (Core B, Kass) and fiber (Core B, Palmer, Maughan) studies. In Aim #1, we will use state-of-the-art single molecule biophysical techniques (e.g. laser trap assay) to probe the effect that cMyBP-C exerts on actomyosin function along the length a single native thick filament isolated from transgenic mice designed in Project #3 (Robbins) and produced in Core C (Robbins). In Aim #2, we will use expressed N-terminal fragments of cMyBP-C produced in Core C (Robbins) to probe the binding affinity of these fragments for actin and/or myosin. Thus, these data and that obtained in Project #1 (Craig), Project #3 (Robbins) and Core B (Palmer) will help define cMyBP-C's specific binding partners (i.e. myosin and/or actin) and thus its physiological site of action. In combination with motility and laser trap assays, we will determine if the N-terminus of CMyBP-C limits myosin's attachment rate to actin or if it directly affects myosin's inherent molecular mechanics and kinetics. Finally, in Aim #3 we will characterize how phosphorylation regulates cMyBP-C action. Using transgenic mouse models (Core C, Robbins) expressing cMyBP C mutants having alanine or aspartic acid substitutions for one or more of the three phosphorylatable serines, we will determine the functional importance of phosphorylation and the hierarchical importance of each site using native thick filaments containing mutant cMyBP-C as well as N-terminal fragments having the same mutations. Once the molecular mechanism of cMyBP-C is defined, the potential for novel therapeutics or clinical intervention may be possible in cases of heart failure associated with genetic mutations in cMyBP-C.

cMyBP-C 对肌动球蛋白力发电系统发挥作用的分子机制在很大程度上仍不清楚。由于其相对于肌球蛋白的比率较低且位于粗丝的不同区域，我们将在本项目中确定 cMyBP-C 如何通过与有限数量的横桥相互作用或是否协同影响内部的所有横桥来调节肌动球蛋白的发电。粗丝。该项目充当动物（项目#3，Robbins）、整个心脏（Core B，Kass）和纤维（Core B，Palmer，Maughan）研究之间的生理桥梁。在目标#1中，我们将使用最先进的单分子生物物理技术（例如激光陷阱测定）来探测cMyBP-C对从设计的转基因小鼠中分离出的单个天然粗丝的长度对肌动球蛋白功能的影响。在项目 #3 (Robbins) 中并在 Core C (Robbins) 中制作。在目标 #2 中，我们将使用 Core C (Robbins) 中产生的 cMyBP-C 表达的 N 端片段来探测这些片段与肌动蛋白和/或肌球蛋白的结合亲和力。因此，这些数据以及在项目 #1 (Craig)、项目 #3 (Robbins) 和 Core B (Palmer) 中获得的数据将有助于定义 cMyBP-C 的特定结合伴侣（即肌球蛋白和/或肌动蛋白），从而确定其生理作用部位。结合运动性和激光陷阱测定，我们将确定 CMyBP-C 的 N 末端是否限制肌球蛋白与肌动蛋白的附着率，或者是否直接影响肌球蛋白固有的分子力学和动力学。最后，在目标 3 中，我们将描述磷酸化如何调节 cMyBP-C 的作用。使用表达 cMyBP C 突变体的转基因小鼠模型（Core C、Robbins），该突变体具有丙氨酸或天冬氨酸取代三种可磷酸化丝氨酸中的一个或多个，我们将使用天然粗蛋白确定磷酸化的功能重要性和每个位点的层次重要性含有突变体cMyBP-C的丝以及具有相同突变的N端片段。一旦 cMyBP-C 的分子机制被明确，对于与 cMyBP-C 基因突变相关的心力衰竭病例，可能有可能出现新的治疗方法或临床干预。