Regulation of Cardiac Calcium Transport

项目概要/摘要：拟议的项目使用两个目标的战略，以调查的监管， 心脏钙转运计划实验的目标是揭示钙的新机制 处理和确定这些机制是如何在疾病中被破坏的。 目的1关注受磷蛋白（PLB）对其调节靶点SERCA 2a的亲和力如何变化 在运动中。目的1a实验将揭示功能差异的结构决定因素 之间的抑制性和非抑制性调节剂的SERCA。我们将探讨PLB如何稳定特定的 在SERCA催化循环中的酶中间状态，导致转运抑制。目标1b涉及 健康和疾病中的调节相互作用的动力学。这些实验将测试如何竞争 在长时间的钙升高后，这种相互作用限制了SERCA抑制的恢复速率。目的 1将揭示SERCA和PLB分子相互作用如何随着心脏从静息心脏受到刺激而变化 运动期间心率升高，然后运动后恢复。 目的2将研究PLB-SERCA调节复合物的替代结合模式。我们以前的 计算研究确定了SERCA上的多个非典型结合位点，其中PLB可以在 几种不同的方向。因此，PLB-SERCA相互作用代表了“模糊复合物”。结构性 从以前研究中得出的假设将在拟议项目的目标2a中直接进行测试， 光活化交联和质谱实验。我们将确定 使用活细胞Ca摄取测量和SERCA ATP酶体外测定的替代相互作用 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 可能与SERCA活性或调节相互作用并破坏SERCA活性或调节的膜蛋白的肽片段。我们 将通过质谱法鉴定候选的“毒肽”，并测试它们结合SERCA、抑制Ca 转移或取代本地监管合作伙伴。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。 Seth Robia（PI）和合作研究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冷冻EM和SERCA结构/功能方面的专业知识。Aleksey Zima是一名 心脏钙处理专家，将协助定量生理实验。杨博士和 Zima参与了该项目的上一个高生产力的融资周期。乔纳森·柯克将带来弥撒 光谱学和蛋白质组学专业知识。彼得Kekenes-Huskey将贡献的生理模型， SERCA转运循环和控制转运功能的调节相互作用。初步合作 实验表明这个研究小组将来很有可能取得成功。我们处于一个独特的位置， 在我们对心脏钙转运调节的理解方面取得科学上的重大进展。

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.