Molecular mechanisms and regulation of the calcium pump in the heart

项目摘要 该项目的长期目标是阐明细胞外信号转导的分子机制和调控。 心脏的钙泵(肌浆网钙-ATPase，SERCA)。SERCA清除胞浆内钙离子 心肌细胞，因此在心脏的钙调节中起着中心作用。SERCA由以下人员监管 磷蛋白(PLB)，一种52个残基的磷酸化调节的膜蛋白，它抑制细胞外信号转运蛋白的活性 打气筒。心力衰竭(HF)的一个关键分子功能障碍涉及舒张期钙离子转运受损， 通常与SERCA表达不足和PLB水平未改变有关，从而产生较低的SERCA 由于PLB抑制而产生的活性。因此，迫切需要时间分辨的、原子化的表征 SERCA激活和SERCA-PLB调节，以了解钙离子失调的分子基础，并 设计适当的治疗高频的方法。这些机制很复杂，需要结构上的改变和 难以通过实验确定的域间变构通讯途径。自完成后 这些变化的实验表征可能仍然是一个棘手的问题，我们建议使用 分子模拟作为补充方法。这个项目的中心假设是分子 在适当的时空尺度上的模拟特别适合于提供时间分辨的检测 SERCA的机制和管理目前仅通过实验是无法达到的。 来自这些研究的高分辨率机制信息可以直接用于计算机辅助 发现通过特定靶向SERCA-PLB相互作用激活SERCA的命中。要验证和 综合这些假设，我们已经开发出一套强大的计算生物物理学和虚拟 SERCA和SERCA-PLB的高通量筛查方法。具体目标有三：(一) MAP配体诱导的与SERCA激活相关的结构变化。(2)确定分子 PLB对SERCA的调节机制。(3)对激活SERCA的命中执行基于结构的搜索。 在这个项目中，我们将重点放在骨架SERCA1a上，因为晶体结构仅为此而获得 但我们模拟的结构结果直接适用于心脏SERCA2a，因为 这两种异构体的动力学和功能没有显著差异，包括受PLB的调节。 模拟工作将通过合作与实验研究紧密结合； 结构和功能数据将提供验证我们的模拟和改进所需的实验测试 我们的结构模型。激活SERCA是心力衰竭广泛追求的治疗目标，而这 该项目具有巨大的潜力，可以推动我们对SERCA功能和 监管，最终实现了一种更合理的方法来解决人类健康中的一个关键问题。

Project Summary The long-term goal of this project is to elucidate the molecular mechanisms and regulation of the calcium pump (sarcoplasmic reticulum Ca2+-ATPase, SERCA) in the heart. SERCA clears cytosolic Ca2+ in cardiomyocytes, thus playing a central role in Ca2+ regulation in the heart. SERCA is regulated by phospholamban (PLB), a 52-residue phosphorylation-regulated membrane protein that inhibits the activity of the pump. A key molecular dysfunction in heart failure (HF) involves impaired Ca2+ transport during diastole, usually associated with insufficient SERCA expression and unaltered PLB levels, thus yielding lower SERCA activity due to PLB inhibition. Therefore, there is an urgent need for time-resolved, atomistic characterization of SERCA activation and SERCA-PLB regulation to understand the molecular basis of Ca2+ dysregulation, and to design appropriate approaches to HF. These mechanisms are complex, requiring structural changes and interdomain allosteric communication pathways that are difficult to determine experimentally. Since complete experimental characterization of these changes is likely to remain an intractable problem, we propose to use molecular simulations as a complementary approach. The central hypothesis of this project is that molecular simulations at appropriate spatiotemporal scales are uniquely suited to provide a time-resolved detection of SERCA mechanisms and regulation at a level of resolution currently inaccessible through experiments alone. The high-resolution mechanistic information from these studies can be directly used for computer-aided discovery of hits that activate SERCA through specifically targeting the SERCA-PLB interaction. To verify and consolidate these hypotheses, we have developed a robust battery of computational biophysics and virtual high-throughput screening approaches to SERCA and SERCA-PLB. Three Specific Aims will be pursued: (1) Map ligand-induced structural changes associated with SERCA activation. (2) Determine the molecular mechanisms for PLB regulation of SERCA. (3) Perform a structure-based search of hits that activate SERCA. For this project, we focus on skeletal SERCA1a because crystal structures have been obtained only for this isoform, but the structural results from our simulations are directly applicable to cardiac SERCA2a because there are no significant differences in the kinetics and function of both isoforms, including regulation by PLB. The simulation work will be closely coupled to experimental studies through collaborations; the combination of structural and functional data will provide the experimental tests necessary to verify our simulations and refine our structural models. Activation of SERCA is a widely pursued therapeutic goal in heart failure, and this project has great potential for pushing important frontiers in our understanding of SERCA function and regulation, ultimately enabling a more rational approach to address a critical problem in human health.