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：
7789874
负责人：
David M Warshaw
金额：
$ 43.21万
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：
2010
资助国家：
美国
起止时间：
2010-02-01 至 2015-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7789874
关键词：
Actins Activities of Daily Living Actomyosin Adrenergic Agents Affect Affinity Alanine Animals Arts 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 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Actomyosin Force Estragating System的影响的分子机制在很大程度上不确定。由于其相对于肌球蛋白的比例较低，并且位于厚细丝的不同区域，我们将在该项目中确定CMYBP-C的CMYBP-C如何通过与有限的Crossbridges群体相互作用来调节Actomyosin的发电，或者它是否合作地影响了厚细丝中的所有Crossbridges。该项目是动物（项目＃3，Robbins），全心（Core B，Kass）和纤维（Core B，帕尔默（Palmer，Maughan））研究之间的生理桥梁。在AIM＃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沿长度沿长度上的肌动蛋白功能施加的作用，该长度沿长度施加了从项目＃3（Robbins）中设计的单基因厚细丝，并在Core C（Robbins）中产生。在AIM＃2中，我们将使用在Core C（Robbins）中产生的CMYBP-C的N末端片段来探测这些片段对肌动蛋白和/或肌球蛋白的结合亲和力。因此，这些数据以及项目＃1（CRAIG），项目＃3（Robbins）和Core B（Palmer）中获得的数据将有助于定义CMYBP-C的特定绑定伙伴（即肌球蛋白和/或肌动蛋白），从而有助于它生理作用部位。结合运动性和激光陷阱测定，我们将确定CMYBP-C的N末端是否限制了肌球蛋白对肌动蛋白的附着率，还是直接影响肌球蛋白固有的分子力学和动力学。最后，在AIM＃3中，我们将表征磷酸化如何调节CMYBP-C的作用。使用表达具有丙氨酸或天冬氨酸取代的CMYBP C突变体的转基因小鼠模型（核心C，Robbins），用于三种可磷酸化的丝氨酸中的一个或多个，我们将确定磷酸化的功能重要性和使用本地厚度的层次重要性的功能重要性包含突变CMYBP-C以及具有相同突变的N末端片段的细丝。一旦定义了CMYBP-C的分子机制，在与CMYBP-C中的遗传突变有关的心力衰竭的情况下，新型治疗或临床干预的潜力可能会发生。