NMR Structural Analysis of Calcium Regulation in Muscle

描述（由申请人提供）：本研究的目的是阐明肌肉中钙调节的分子结构基础。肌肉的收缩和松弛是由动态的蛋白质-蛋白质复合物调节的，这些蛋白质复合物通过细胞膜循环钙。肌浆网（SR） Ca2+- atp酶（SERCA）将Ca2+从细胞质转运到SR管腔。SERCA的功能受两种膜抑制剂调控：磷蛋白（PLN）和肌磷脂（PLN），分别主要表达于心脏和骨骼肌。这两种蛋白与SERCA的膜内相互作用调节钙通量，从而调节肌肉功能。PLN的肾上腺素能磷酸化逆转SERCA抑制，而SLN抑制的逆转是由于表达水平的变化或T5磷酸化。由于几种肌病与这些钙处理蛋白有关，因此了解它们如何调节SERCA对肌肉生理学和病理生理学至关重要。我们将努力实现以下目标：目标1。定义SERCA/PLN和SERCA/SLN复合物的变构调节。目标2。确定PLN的单（T17）和双（S16/T17）磷酸化的作用。目标3。阐明SERCA/PLN和SERCA/SLN在脂质双分子层中的变构转变。目标4。测定脂质双层中PLN的寡聚结构。在项目的前五年，我们重点了解了PLN和SLN free及其与SERCA相互作用的结构动力学，为在分子水平上更深入地了解钙转运调控奠定了基础。在今后五年中，我们将在最初提出的机制的基础上，争取控制这些机制。我们的长期目标是利用我们开发的概念和模型来合理设计用于治疗目的的PLN和SLN突变体。公共卫生相关：本研究计划的重点是阐明调节肌肉钙稳态的分子机制。SERCA/PLN和SERCA/SLN是嵌入在肌浆网中的两种膜蛋白复合物，参与心肌和骨骼肌的兴奋-收缩-松弛循环。SERCA突变与Brody病有关，Brody病是一种以运动引起的肌肉痉挛、僵硬和放松障碍为特征的临床疾病。此外，PLN突变与特定的心脏疾病直接相关。例如，在扩张型心肌病患者中检测到PLN的R9C突变、R14缺失和L39截断。此外，SLN mRNA表达的下调与房颤诱导的心房重构有关。现在有直接的证据表明，通过SERCA的过表达或调节SERCA的抑制来操纵钙循环可以逆转晚期心力衰竭的许多重要临床终点。因此，了解SLN和PLN对SERCA的调控对于基因治疗等非常规药物治疗的发展至关重要。

DESCRIPTION (provided by applicant): The goal of this research is to elucidate the molecular structural basis of calcium regulation in muscle. Muscle contraction and relaxation are regulated by dynamic protein-protein complexes that cycle calcium through cell membranes. The sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA) transports Ca2+ from the cytosol into the SR lumen. SERCA's function is regulated by two membrane inhibitors: phospholamban (PLN) and sarcolipin (PLN), expressed primarily in cardiac and skeletal muscle, respectively. The intramembrane interactions of these two proteins with SERCA regulate calcium flux and thus muscle function. ¿-adrenergic phosphorylation of PLN reverses SERCA inhibition, while the reversal of SLN inhibition is due to the variation in expression levels or T5 phosphorylation. Since several myopathies are associated with these calcium handling proteins, understanding how they regulate SERCA is central to muscle physiology and pathophysiology. We will pursue the following aims: AIM 1. Defining allosteric regulation of SERCA/PLN and SERCA/SLN complexes. AIM 2. Ascertaining the role of single (T17) and double (S16/T17) phosphorylation of PLN. AIM 3. Elucidating the allosteric transitions of SERCA/PLN and SERCA/SLN in lipid bilayers. AIM 4. Determining the oligomeric structure of PLN in lipid bilayers. In the first five years of this project, we focused on understanding the structural dynamics of PLN and SLN free and in interaction with SERCA, laying the foundation for a deeper knowledge of calcium transport regulation at the molecular level. In the next five years, building on our original proposed mechanisms, we seek to gain control of these mechanisms. Our long-term goal is to harness the concepts and models we have developed for the rational design of PLN and SLN mutants for therapeutic purposes. PUBLIC HEALTH RELEVANCE: This research program focuses on the elucidation of the molecular mechanisms that regulate calcium homeostasis in muscle. SERCA/PLN and SERCA/SLN are two membrane protein complexes embedded in the sarcoplasmic reticulum that are involved in the excitation-contraction-relaxation cycle in both cardiac and skeletal muscle. SERCA mutations are linked to Brody disease, a clinical condition characterized by exercise-induced muscle cramps, stiffness, and relaxation impairments. Also, PLN mutations have been directly linked with specific heart conditions. For instance, R9C mutation, R14 deletion, and L39 truncation of PLN have been detected in patients affected by dilated cardiomyopathy. Also, down regulation of SLN mRNA expression has been correlated with atrial fibrillation-induced atrial remodeling. There is now direct evidence that manipulation of calcium cycling, either via the over-expression of SERCA or by tuning SERCA inhibition can reverse many important clinical endpoints of late stage failing heart. Therefore, understanding SERCA regulation by both SLN and PLN is central to the development of non-conventional drug therapy such as gene therapy.