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

描述（由申请方提供）：心肌肌球蛋白结合蛋白-C（cMyBP-C）对正常心脏功能至关重要，这一点通过cMyBP-C的基因突变得到证明，cMyBP-C是家族性肥厚型心肌病的主要原因之一。尽管cMyBP-C在功能上很重要，但当它与肌动蛋白相互作用以产生力和运动时，cMyBP-C对肌球蛋白分子马达发挥作用的分子机制在很大程度上仍然不确定。一个尚未回答的问题是其相对于肌球蛋白的低比例，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。为了解决目标#1中的这一问题，我们将使用最先进的单分子生物物理学技术（例如激光陷阱测定）来探测cMyBP-C沿单个天然粗丝长度对肌动球蛋白功能沿着发挥的作用。在目标#2中，我们将使用表达的cMyBP-C的N-末端片段来探测这些片段对肌动蛋白、受调节的细丝和/或肌球蛋白的结合亲和力。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对细丝的附着率，或者是否直接影响肌球蛋白的固有分子力学和动力学。最后，在目标#3中，我们将描述磷酸化如何调节cMyBP-C作用。使用现有的转基因小鼠模型表达cMyBP C突变体具有丙氨酸或天冬氨酸取代的所有三个磷酸化丝氨酸，我们将确定磷酸化的功能的重要性，使用天然粗丝含有突变cMyBP-C以及具有相同的突变的N-末端片段。一旦确定了cMyBP-C的分子机制，在与cMyBP-C基因突变相关的心力衰竭病例中，可能有可能进行新的治疗或临床干预。心脏肌球蛋白结合蛋白-C（cMyBP-C）对正常心脏功能至关重要，cMyBP-C的基因突变是家族性肥厚型心肌病的主要原因之一。尽管cMyBP-C在功能上很重要，但当它与肌动蛋白相互作用以产生力和运动时，cMyBP-C对肌球蛋白分子马达发挥作用的分子机制在很大程度上仍然不确定。

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.