骨架蛋白Cypher作为PKA的锚定蛋白参与扩张型心肌病发病机制的研究

Dilated cardiomyopathy(DCM) has complicated etiology and it's treatment is undesirable. Dysfunction on cytoskeleton proteins due to mutations is one of important mechanisms, which brings potential targets for therapy. It has been well demonstrated that Cypher is a DCM associated gene. Our previous study has investigated that Cypher is a novel A kinase anchoring protein (AKAP). Moreover, one of cardiomyopathy associated Cypher mutation T206I, enhanced the interaction between Cypher and PKA. AKAP is a scaffold protein for protein kinases and phosphatases to compartamentate their activities, therefore to fine regulate protein phosphorylation. Using Cypher deficient and T206I mutation knockin mouse models, together with phosphoproteomic and mass spectrometry, we will find new substrates for AKAP of Cypher and the molecular mechanisms how T206I causes dilated cardiomyopathy. Our results bring new molecular events to understand dilated cardiomyopathy, finally for best clilical treatments.

扩张型心肌病（DCM）病因复杂，疗效差。骨架蛋白基因突变致功能失衡是引起DCM的重要机制，这为寻找合适的干预靶点提供了机会。目前明确骨架蛋白Cypher缺失可导致DCM。前期实验我们发现Cypher是一个蛋白激酶A（cyclic-AMP dependent protein kinase A，PKA）的锚定蛋白（A kinase anchoring protein, AKAP），模拟结构和体外实验证实其中与心肌病相关的T206I突变可增强Cypher与PKA的结合。AKAP可调控PKA和蛋白磷酸酶的活性，影响下游蛋白的磷酸化。本课题用Cypher基因敲除和T206I敲入小鼠，用分子生物学技术，磷酸化蛋白组学方法、酵母双杂交以及质谱分析，探索Cypher-PKA复合物如何调控下游蛋白的磷酸化及T206I突变后致病的分子机理，从而揭示Cypher与DCM发病的关系，为寻找DCM干预靶点提供依据。

扩张型心肌病（DCM）病因复杂，可导致严重心力衰竭和心律失常引起猝死。研究发现一些编码心肌细胞骨架蛋白的基因突变与 DCM相关。目前已明确骨架蛋白 Cypher 缺失可使心肌细胞肌小节 Z 线有序结构的破坏，从而导致 DCM 的发生。前期实验我们发现 Cypher 是一个AKAP，即PKA的锚定蛋白，AKAP 可调控 PKA 和蛋白磷酸酶的活性，从而影响下游蛋白的磷酸化参与信号传导。所以本研究将继续深入探讨Cypher作为信号传导或者中继的重要节点发挥的未知功能。.在本项目研究中，我们基于Cypher 基因敲除小鼠，采用磷酸化蛋白组学和质谱分析方法，筛选了以Cypher为AKAP的受PKA调控的底物蛋白及可能的磷酸化位点, 包括Troponin I, cardiac muscle ser-23/24，beta catenin ser-552/675，Junction plakoglobin ser-665，Vimentin ser-72等；并在体内体外验证了筛选出的底物蛋白（Vimentin、β-catenin、γ-catenin）与Cypher的相互作用；最后在细胞和组织水平验证Cypher对相关底物蛋白磷酸化水平的调控。实验结果表明Cypher可与Vimentin、β-catenin、γ-catenin等相互作用，并与之形成复合物从而调控Vimentin 72位点及β-catenin675位点的磷酸化水平。本研究从信号传导的角度揭示了 Cypher 与 DCM 发病的可能关系，为寻找 DCM 干预靶点提供理论依据。