Cardiac Myosin binding Protein-C: Molecular Mechanisms of Actomyosin Modulation

描述（由申请人提供）：心肌肌球蛋白结合蛋白c （cMyBP-C）对正常心脏功能至关重要，cMyBP-C基因突变是家族性肥厚性心肌病的主要原因之一。尽管具有重要的功能，但cMyBP-C与肌动蛋白相互作用产生力和运动时对肌球蛋白分子运动产生影响的分子机制在很大程度上仍未明确。一个尚未解决的问题是，由于cMyBP-C相对于肌凝蛋白的比例较低，并且它位于粗丝的不同区域，我们将确定cMyBP-C如何通过仅与粗丝内有限的交叉桥相互作用来调节肌动球蛋白的发电。为了在Aim #1中解决这个问题，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沿着单个天然粗丝的长度对肌动球蛋白功能的影响。在Aim #2中，我们将使用表达的cMyBP-C的n端片段来探测这些片段与肌动蛋白、受调节的细丝和/或肌凝蛋白的结合亲和力。结合运动性和激光陷阱分析，我们将确定CMyBP-C的N端是否限制了肌凝蛋白对细丝的附着率，或者是否直接影响肌凝蛋白固有的分子力学和动力学。最后，在Aim #3中，我们将描述磷酸化如何调节cMyBP-C的作用。利用现有的转基因小鼠模型，表达三种可磷酸化丝氨酸均被丙氨酸或天冬氨酸取代的cMyBP-C突变体，我们将利用含有突变体cMyBP-C的天然粗丝以及具有相同突变的n端片段来确定磷酸化的功能重要性。一旦确定了cMyBP-C的分子机制，对于与cMyBP-C基因突变相关的心力衰竭病例，可能会有新的治疗方法或临床干预。心肌肌球蛋白结合蛋白c （cMyBP-C）对正常心脏功能至关重要，cMyBP-C基因突变是家族性肥厚性心肌病的主要原因之一。尽管具有重要的功能，但cMyBP-C与肌动蛋白相互作用产生力和运动时对肌球蛋白分子运动产生影响的分子机制在很大程度上仍未明确。

DESCRIPTION (provided by applicant): Cardiac Myosin Binding Protein-C (cMyBP-C) is critical to normal cardiac performance as evidenced by genetic mutations in cMyBP-C being one of the leading causes of familial hypertrophic cardiomyopathy. Despite its functional importance, the molecular mechanism by which cMyBP-C exerts its effect on the myosin molecular motor as it interacts with actin to generate force and motion remains largely undefined. A yet unanswered question is with its low ratio relative to myosin and it being located in distinct regions of the thick filament, we will determine how cMyBP-C's modulates actomyosin's power generation by interacting with only a limited population of crossbridges within the thick filament. To address this 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. In Aim #2, we will use expressed N-terminal fragments of cMyBP-C to probe the binding affinity of these fragments for actin, the regulated thin filament, and/or myosin. In combination with motility and laser trap assays, we will determine if the N- terminus of CMyBP-C limits myosin's attachment rate to the thin filament 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 existing transgenic mouse models expressing cMyBP C mutants having alanine or aspartic acid substitutions for all three phosphorylatable serines, we will determine the functional importance of phosphorylation 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. PUBLIC HEALTH RELEVANCE Cardiac Myosin Binding Protein-C (cMyBP-C) is critical to normal cardiac performance as evidenced by genetic mutations in cMyBP-C being one of the leading causes of familial hypertrophic cardiomyopathy. Despite its functional importance, the molecular mechanism by which cMyBP-C exerts its effect on the myosin molecular motor as it interacts with actin to generate force and motion remains largely undefined.