Regulation of Cardiac Calcium Transport

项目摘要/摘要：拟议的项目使用两个目标策略来调查规则 心脏钙转运。计划中的实验的目标是揭示钙的新机制。 处理并确定这些机制在疾病中是如何被破坏的。 目标1集中在磷蛋白(PLB)与其调控靶标SERCA2a的亲和力如何变化 在锻炼的时候。目标1a实验将揭示功能差异的结构性决定因素 SERCA的抑制性和非抑制性调节因子之间的关系。我们将探讨公共汽车如何稳定具体的 酶在SERCA催化循环中的中间状态，导致运输抑制。目标1b地址 健康和疾病中调控相互作用的动态。这些实验将测试如何竞争 相互作用限制了长时间钙升高后SERCA抑制的恢复速度。目标 1将揭示当心脏受到静息心脏刺激时，SERCA和PLB分子相互作用如何变化 运动期间心率加快，运动后恢复。 AIM 2将研究PLB-SERCA调控复合体的替代结合模式。我们以前的 计算研究发现PLB在SERCA上可以相互作用的多个非正则结合位点 几个不同的方向。因此，PLB-SERCA相互作用代表了一个“模糊复合体”。结构性的 先前研究产生的假设将在拟议项目的目标2a中直接测试，使用 光活化交联和质谱学实验。我们将确定 用活细胞钙摄取测量和SERCA-ATPase体外测定替代相互作用 功能。在目标2b中，我们将探索SERCA模糊复合体的病理生理学。心脏病会产生 膜蛋白的多肽片段，可能与SERCA活性或调节相互作用并破坏其活性或调节。我们 将通过质谱学鉴定候选毒肽并测试其与SERCA结合的能力，抑制Case 运输，或取代当地的监管伙伴。这些实验将为一部小说提供新的见解 心力衰竭和缺血性心脏病的病理生理机制。 赛斯·罗比亚(PI)和合作的调查人员带来了互补的专业知识和方法。霍华德·杨， 阿尔伯塔大学将提供低温EM和SERCA结构/功能方面的专业知识。阿列克西·齐马是一位 是心脏钙处理方面的专家，并将协助进行定量生理实验。杨博士和 ZZMA参与了该项目的前一个高产出供资周期。乔纳森·柯克将带来弥撒 光谱学和蛋白质组学的专业知识。Peter Kekenes-Huskey将贡献 SERCA运输周期和管理运输功能的调节相互作用。初步协作 实验表明，这个研究团队未来取得成功的可能性很高。我们处于一个独特的位置来 在我们对心脏钙转运调节的理解上取得重大科学进展。

Project Summary/Abstract: The proposed project uses a two Aim strategy to investigate the regulation of cardiac calcium transport. The goals of the planned experiments are to uncover novel mechanisms of calcium handling and determine how these mechanisms are disrupted in disease. Aim 1 is focused on how the affinity of phospholamban (PLB) for its regulatory target, SERCA2a, changes during exercise. Aim 1a experiments will reveal the structural determinants of the functional differences between inhibitory and non-inhibitory regulators of SERCA. We will explore how PLB stabilizes specific enzymatic intermediate states in the SERCA catalytic cycle, leading to transport inhibition. Aim 1b addresses the dynamics of regulatory interactions in health and disease. The experiments will test how competing interactions limit the rate of recovery of inhibition of SERCA after a prolonged period of calcium elevation. Aim 1 will reveal how SERCA and PLB molecular interactions change as the heart is stimulated from a resting heart rate to an elevated heart rate during exercise, followed by post-exercise recovery. Aim 2 will investigate alternative modes of binding for the PLB-SERCA regulatory complex. Our previous computational study identified multiple non-canonical binding sites on SERCA where PLB can interact in several alternative orientations. Thus, the PLB-SERCA interaction represents a “fuzzy complex”. The structural hypotheses generated from that previous study will be tested directly in Aim 2a of the proposed project using photoactivatable cross-linking and mass spectroscopy experiments. We will determine the functional role of the alternative interactions using live cell Ca uptake measurements and in vitro assays of SERCA ATPase function. In Aim 2b we will explore the pathophysiology of the SERCA fuzzy complex. Heart disease produces peptide fragments of membrane proteins that may interact with and disrupt SERCA activity or regulation. We will identify candidate “poison peptides” by mass spectrometry and test their ability to bind SERCA, inhibit Ca transport, or displace native regulatory partners. The experiments will provide new insight into a novel pathophysiological mechanism in heart failure and ischemic heart disease. Seth Robia (PI) and collaborating investigators bring complementary expertise and methods. Howard Young, University of Alberta, will provide expertise in cryo-EM and SERCA structure/function. Aleksey Zima is an expert in cardiac calcium handling and will assist with quantitative physiological experiments. Drs. Young and Zima participated in the previous, highly productive funding cycle for this project. Jonathan Kirk will bring mass spectrometry and proteomics expertise. Peter Kekenes-Huskey will contribute physiological models of the SERCA transport cycle and the regulatory interactions that govern transport function. Preliminary collaborative experiments suggest a high likelihood of future success for this research team. We are in a unique position to make scientifically significant advances in our understanding of the regulation of cardiac calcium transport.