cMyBP-C: Molecular Mechanisms of Actomyosin Modulation

CMyBP-C对肌动球蛋白力发电系统的作用机制在很大程度上还不清楚。由于cMyBP-C相对肌球蛋白的比例较低，并且位于粗丝的不同区域，我们将在这个项目中确定cMyBP-C是如何通过与有限的交叉桥相互作用来调节肌球蛋白的发电，或者是否协同影响粗丝内的所有交叉桥。该项目作为动物(项目3，Robbins)、整个心脏(核心B，Kass)和纤维(核心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(例如激光陷阱分析)来探讨cMyBP-C对肌动球蛋白功能的影响，该纤维是从项目#3(Robbins)中设计并在Core C(Robbins)中生产的转基因小鼠中分离出来的单个天然粗丝。在目标#2中，我们将使用在核心C(Robbins)中产生的cMyBP-C的N端表达片段来探索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)和核心B(Palmer)中获得的数据将有助于定义cMyBP-C的特定结合伙伴(即肌球蛋白和/或肌动蛋白)，从而有助于确定其 生理作用部位。结合运动性和激光陷阱分析，我们将确定cMyBP-C的N端是否限制了肌球蛋白对肌动蛋白的附着率，或者它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)，其中三个可磷酸化的丝氨酸中的一个或多个具有丙氨酸或天冬氨酸替代，我们将确定磷酸化的功能重要性以及使用天然Thick的每个位点的等级重要性 含有突变的cMyBP-C以及具有相同突变的N-末端片段的细丝。一旦cMyBP-C的分子机制被确定，在与cMyBP-C基因突变相关的心力衰竭病例中，新的治疗或临床干预的潜力可能成为可能。

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.