Cardiac Myosin-Binding Protein C: Molecular Modulation of Actomyosin Function.

 描述（由申请方提供）：心肌肌球蛋白结合蛋白C（cMyBP-C）是一种肌节粗丝相关蛋白，对正常心脏结构和功能至关重要。cMyBP-C的突变是肥厚型心肌病的主要原因，这强调了cMyBP-C的重要性。尽管cMyBP-C是心肌收缩力的关键调节因子，但其调节肌动球蛋白力和运动产生的分子机制远未确定。虽然cMyBP-C的N-末端结构域可以结合肌动蛋白和肌球蛋白头部区域，但尚不清楚这些结合伴侣中的哪一个是生理相关的，以及这些结合伴侣的相互作用是否通过直接影响肌动球蛋白发电或间接通过改变Ca 2+依赖性细丝激活来调节心脏收缩力。随着cMyBP-C的N末端的磷酸化响应于β-肾上腺素能刺激而发生，磷酸化可以提供cMyBP-C功能可调性的量度，以增强心脏收缩力。我们提出以下三个具体目标。目的1检验cMyBP-C的细丝激活和肌动球蛋白功率抑制是与特异性结合伴侣相关的独立机制的假设。因此，cMyBP-C结合配偶体相互作用将使用最先进的分子生物物理方法在简化的体外模型系统（例如单分子FRET）和肌原纤维内原位（超分辨率STORM显微镜）进行测定。对cMyBP-C进行结构诱变，以消除表达的N-末端片段和转基因小鼠突变cMyBP-C中相互作用的结合伴侣位点，将有助于将cMyBP-C的功能能力与其与细丝或肌球蛋白头部区域的相互作用联系起来。目的2测试cMyBP-C通过类似于钙激活的机制直接激活细丝的假设。因此，我们已经开发了一种体外单细丝激活测定来监测事件的分子序列，通过所述事件，表达的cMyBP-C的荧光标记的N-末端片段启动荧光标记的肌球蛋白分子向细丝的协同募集。目的3检验cMyBP-C的磷酸化通过改变cMyBP-C的分子力学来调节cMyBP-C对收缩性的调节的假设，这反过来又改变了其结合伴侣的相互作用。在目的1和2所述的试验中，将使用表达突变型cMyBP-C的转基因小鼠的心脏组织和天然粗肌丝以及表达的一个或多个丝氨酸被不可磷酸化的丙氨酸或天冬氨酸（磷酸模拟物）替代的N-末端片段，以表征位点特异性磷酸化的影响。使用原子力显微镜，我们将表征磷酸化影响M结构域分子力学和结构的可能机制，从而调节cMyBP-C功能。随着对cMyBP-C功能的了解和理解，可以开发针对cMyBP-C结合伴侣相互作用的靶向治疗，以帮助调节和改善衰竭心脏的心脏性能。

 DESCRIPTION (provided by applicant): Cardiac myosin-binding protein C (cMyBP-C) is a sarcomeric thick filament associated protein that is critically important to normal cardiac structure and function. The importance of cMyBP-C is emphasized by mutations to cMyBP-C being a leading cause of hypertrophic cardiomyopathy. Despite being a key regulator of cardiac contractility, the molecular mechanism by which cMyBP-C modulates actomyosin force and motion generation is far from certain. Although cMyBP-C's N-terminal domains can bind to actin and the myosin head region, it is not known which of these binding partners is physiologically relevant and whether these binding partner interactions modulate cardiac contractility by directly affecting actomyosin power generation or indirectly by altering Ca2+-dependent thin filament activation. With phosphorylation of cMyBP-C's N terminus occurring in response to ß-adrenergic stimulation, phosphorylation may offer a measure of cMyBP-C functional tunability in order to enhance cardiac contractility. We propose the following three specific aims. Aim 1 tests the hypothesis that cMyBP-C's thin filament activation and actomyosin power inhibition are independent mechanisms associated with a specific binding partner. Thus, cMyBP-C binding partner interactions will be determined using state-of-the-art molecular biophysical approaches in simplified in vitro model systems (e.g. single molecule FRET) and in situ within myofibrils (super-resolution STORM microscopy). Structural mutagenesis of cMyBP-C to ablate binding partner sites of interaction in both expressed N-terminal fragments and in mutant cMyBP-C from transgenic mice will help link cMyBP-C's functional capacities to its interaction with either the thin filament or the myosin head region. Aim 2 tests the hypothesis that cMyBP-C activates the thin filament directly through a mechanism similar to calcium activation. Thus, we have developed an in vitro single thin filament activation assay to monitor the molecular sequence of events by which expressed fluorescently-labeled N-terminal fragments of cMyBP-C initiate the cooperative recruitment of fluorescently- labeled myosin molecules to the thin filament. Aim 3 tests the hypothesis that phosphorylation of cMyBP-C tunes cMyBP-C's modulation of contractility through alterations in cMyBP-C's molecular mechanics, which in turn alters its binding partner interactions. Cardiac tissue and native thick filaments from transgenic mice expressing mutant cMyBP-C as well as expressed N-terminal fragments with one or more serines replaced by non-phosphorylatable alanines or aspartic acids (phosphomimetics) will be used in assays described in Aims 1 and 2 to characterize the effect of site-specific phosphorylation. Using atomic force microscopy, we will characterize possible mechanisms by which phosphorylation affects M-domain molecular mechanics and structure, thus modulating cMyBP-C function. With the knowledge and understanding of cMyBP-C function derived from these collective studies, targeted therapies directed at cMyBP-C binding partner interactions may be developed to help modulate and to improve cardiac performance in the failing heart.