cMyBP-C: Phosphorylation-Dependent Regulation In Vivo

我们的目标是了解心肌肌球蛋白结合蛋白C(cMyBP-C)翻译后修饰的结构-功能关系和功能后果。CMyBP-C基因突变是家族性肥厚型心肌病的主要原因之一，cMyBP-C对正常心功能至关重要。尽管cMyBP-C具有重要的功能，但cMyBP-C在肌球蛋白与肌动蛋白相互作用产生力和运动时对肌球蛋白分子马达产生影响的分子机制在很大程度上仍不清楚。此外，蛋白质的磷酸化模式的变化是紧密的 与心脏对应激和衰竭的反应相协调，但这些变化在与肌球蛋白和肌动蛋白相互作用方面的后果仍在很大程度上尚不清楚，并且对控制收缩能力至关重要。目的1验证心脏特异插入cMyBP-C的3个磷酸化丝氨酸(Ser-273、Ser-282和Ser-302)在功能上不相同的假设，以及cMyBP-C存在磷酸化的层级模式的假设。推论是，这些模式在功能上很重要。为了了解每个磷酸化位点的作用，将Ser-273、Ser-282和Ser-302单独和组合突变为非磷酸化残基(丙氨酸)或带电磷酸化模拟物(天冬氨酸)。AIM 2将测试 假设cMyBP-C有一个或多个已定义的肌动蛋白结合位点，并与核心B合作表明，这种相互作用在肌节上提供了弹性和粘性负载。虽然cMyBP-C与肌球蛋白本身之间的相互作用已有很好的定义，但cMyBP-C与肌动蛋白结合，但这种结合的生理意义尚不清楚，负责这种相互作用的cMyBP-C残基/区域也不确定。我们将结合生物化学和遗传学的方法，首先定义负责肌动蛋白结合的cMyBP-C区域，突变它们，并通过转基因替换在心肌细胞中表达这些突变，以确定它们的生理意义。

Our objective is to understand the structure-function relationships and the functional consequences of posttranslational modifications of cardiac myosin binding protein C (cMyBP-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. Additionally, changes in the protein's phosphorylation patterns are tightly coordinated with the heart's response to stress and failure but the consequences of these changes in terms of the interactions with myosin and actin remain largely unexplored and are critical for the control of contractility. Aim 1 will test the hypothesis that the 3 phosphorylated serines in the cardiac-specific insertion of cMyBP-C (Ser-273, Ser-282 and Ser-302) are not functionally equivalent and that hierarchal patterns of phosphorylation exist for cMyBP-C. A corollary is that these patterns are functionally important. In order to understand the role of each phosphorylation site, Ser-273, Ser-282 and Ser-302 will be mutated to either a nonphosphorylatable residue (alanine) or a charged phosphorylation mimetic (aspartate) singly and in combination. Aim 2 will test the hypothesis that cMyBP-C has a defined binding site or sites for actin and show, in collaboration with Core B, that this interaction provides an elastic and viscous load on the sarcomere. Although the interactions between cMyBP-C and myosin itself are well defined, cMyBP-C binds to actin but the physiological significance of this binding is obscure and the cMyBP-C residues/regions responsible for this interaction is/are undefined. Using a combination of biochemical and genetic approaches we will first define the regions of cMyBP-C that are responsible for actin binding, mutate them and express those mutations in the cardiomyocyte population via transgenic replacement in order to determine their physiological significance.