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，一种被磷蛋白（PLB）抑制的膜内Ca转运酶。在肾上腺素能刺激下，这种抑制被camp依赖性蛋白激酶（PKA）逆转，使PLB在S16位点磷酸化。这个系统的破坏会导致心脏病。一种特异性PLB突变（R9C-PLB）与遗传性DCM直接相关。R9C-PLB不可逆地抑制PKA，阻碍wt-PLB的磷酸化，干扰钙转运调节，导致心力衰竭。这一发现代表了一项突破，并在全国媒体上得到了广泛报道。我们将利用分子生物学、肽合成、核磁共振和EPR方法确定DCM的结构基础，以实现以下目的：

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.