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

 描述(申请人提供)：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突变强调了cMyBP-C的重要性，cMyBP-C是肥厚型心肌病的主要原因。尽管cMyBP-C是心脏收缩能力的关键调节因子，但cMyBP-C调节肌动球蛋白力量和运动产生的分子机制还远未确定。虽然cMyBP-C的N-末端结构域可以与肌动蛋白和肌球蛋白头部区域结合，但目前还不清楚这些结合伙伴中的哪一个是生理上相关的，以及这些结合伙伴的相互作用是通过直接影响肌球蛋白的发电来调节心肌收缩能力，还是通过改变依赖钙的细丝激活来间接调节心肌收缩能力。随着cMyBP-C N末端的磷酸化反应，磷酸化可能提供了一种cMyBP-C功能可调整性的措施，以增强心脏的收缩能力。我们提出了以下三个具体目标。目的1验证cMyBP-C的细丝激活和肌动球蛋白能力抑制是与特定结合伙伴相关的独立机制的假设。因此，cMyBP-C结合伙伴的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)和肌原纤维内的原位(超分辨率风暴显微镜)中确定。通过cMyBP-C的结构突变来去除转基因小鼠表达的N末端片段和突变的cMyBP-C中的结合伙伴相互作用部位，将有助于将cMyBP-C的功能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验证cMyBP-C通过与钙激活类似的机制直接激活细丝的假说。因此，我们建立了一种体外单丝激活实验来监测cMyBP-C表达荧光标记的N末端片段启动荧光标记的肌球蛋白分子协同招募到细丝的事件的分子序列。目的3验证cMyBP-C的磷酸化通过改变cMyBP-C的分子力学来调节cMyBP-C的收缩能力，进而改变其结合伙伴的相互作用的假设。在AIMS 1和2中描述的分析中，将使用来自表达突变cMyBP-C的转基因小鼠的心脏组织和天然粗丝以及用不可磷酸化的丙氨酸或天冬氨酸(拟磷酸盐)取代一个或多个丝氨酸的表达的N-末端片段来表征位点特异性磷酸化的效果。利用原子力显微镜，我们将表征磷酸化影响M-结构域分子力学和结构从而调节cMyBP-C功能的可能机制。随着这些集体研究对cMyBP-C功能的了解和理解，针对cMyBP-C结合伙伴相互作用的靶向治疗可能会被开发出来，以帮助调节和改善衰竭心脏的心功能。

 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.