cMyBP-C: Phosphorylation-Dependent Regulation In Vivo

我们的目的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性心肌病的主要原因之一，这证明cMyBP-C对正常心脏功能至关重要。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。此外，蛋白质磷酸化模式的变化密切相关

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.