Structural Basis of Dilated Cardiomyopathy

描述（由申请人提供）：本研究的总体目标是阐明扩张型心肌病（DCM）的结构基础。心肌功能主要依赖于Ca-ATP酶，这是一种被受磷蛋白（PLB）抑制的膜包埋钙转运酶。在肾上腺素能刺激后，这种抑制被cAMP依赖性蛋白激酶（PKA）逆转，在S16磷酸化PLB。这一系统的破坏会导致心脏病。一种特异性PLB突变（R9 C-PLB）与遗传性DCM直接相关。R9 C-PLB不可逆地抑制PKA，阻碍wt-PLB的磷酸化，干扰Ca转运调节，并引起心力衰竭。这一发现是一项突破，已在国家媒体上广泛报道。我们将使用分子生物学、肽合成、NMR和EPR方法确定DCM的结构基础，以完成以下AIM： 目的1：研究PLB及其致死突变体R9 C的磷酸化动力学。 目的2：探索PKA/PLB和PKA/R9 C-PLB复合物的结合表面和结构。 目的3：绘制PKA及其与PLB及其R9 C突变体复合物的结构动力学。 目的4：阐明PKA/PLB复合物在脂质中的膜结构：PKA的N-肉豆蔻酰化的影响。 这项研究的直接结果将是表征R9 C-PLB/PKA复合物的高分辨率结构，并阐明R9 C突变和DCM之间的结构联系。长期目标是为涉及钙调节和心肌收缩性的新治疗方法开发分子框架。据我们所知，这些是第一个生物物理研究的蛋白质-蛋白质之间的相互作用的完整的膜蛋白和可溶性蛋白质曾经进行使用NMR光谱。

DESCRIPTION (provided by applicant): The overall goal of this research is to elucidate the structural basis of dilated cardiomyopathy (DCM). Cardiac muscle function depends critically on Ca-ATPase, a membrane-embedded Ca transport enzyme inhibited by phospholamban (PLB). Upon adrenergic stimulation, this inhibition is reversed by cAMP-dependent protein kinase (PKA), phosphorylating PLB at S16. Disruptions to this system result in heart disease. A specific PLB mutation (R9C-PLB) has been linked directly to hereditary DCM. R9C-PLB irreversibly inhibits PKA, hampering the phosphorylation of wt-PLB, interfering with Ca transport regulation, and causing heart failure. This finding represents a breakthrough that has been widely reported in the national media. We will determine the structural basis of DCM using molecular biology, peptide synthesis, NMR, and EPR methods to accomplish the following AIMs: AIM 1: Characterizing the phosphorylation kinetics of both PLB and its lethal R9C mutant. AIM 2: Probing the binding surfaces and the structures of PKA/PLB and PKA/R9C-PLB complexes. AIM 3: Mapping the structural dynamics of PKA and PKA complexes with PLB and its R9C-mutant. AIM 4: Elucidating the membrane architecture of the PKA/PLB complex in lipids: influence of N-myristoylation of PKA. The immediate outcome of this research will be to characterize the high-resolution structure of the R9C-PLB/PKA complex and to elucidate the structural links between R9C mutation and DCM. The long-term goal is to develop a molecular framework for new therapeutic approaches involving calcium regulation and heart muscle contractility. To the best of our knowledge, these are the first biophysical studies of protein-protein interactions between integral membrane proteins and soluble proteins ever carried out using NMR spectroscopy.