Regulation of Cardiac Calcium Transport

心脏钙转运的调节

• 批准号: 10670961
• 负责人: Seth L Robia
• 金额: $ 69.73万
• 依托单位: LOYOLA UNIVERSITY CHICAGO
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-07-01 至 2026-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10670961
• 关键词: ATP phosphohydrolase; Address; Adrenergic Agents; Affinity; Alberta province; Binding; Binding Sites; Biochemical; Biological Assay; Buffers; Calcium; Cardiac; Cells; Chemicals; Collaborations; Complex; Computer Simulation; Cryoelectron Microscopy; Cyclic AMP-Dependent Protein Kinases; Cytoplasm; Dependence; Disease; Docking; Enzymes; Epitopes; Exercise; Fluorescence Resonance Energy Transfer; Frequencies; Functional disorder; Funding; Future; Goals; Grant; Health; Heart; Heart Diseases; Heart Rate; Heart failure; Human; In Vitro; Kinetics; Laboratories; Mass Spectrum Analysis; Measurement; Membrane; Membrane Proteins; Methods; Microscopy; Microsomes; Modeling; Molecular; Molecular Conformation; Muscle Contraction; Mutagenesis; Mutation; Myocardial Ischemia; Myocardium; Pathogenicity; Pathologic; Peptide Fragments; Peptides; Phosphorylation; Physiological; Poison; Positioning Attribute; Predisposition; Productivity; Protein Fragment; Proteolysis; Proteome; Proteomics; Public Health; Pump; Recovery; Regulation; Research; Research Personnel; Research Project Grants; Research Support; Rest; Role; SERCA2a; Signal Transduction; Site; Structure; System; Testing; Thermodynamics; Tissues; Universities; candidate identification; cardiac pacing; computer studies; crosslink; dwarf open reading frame; experimental study; in vitro Assay; insight; molecular dynamics; multicatalytic endopeptidase complex; novel; phospholamban; physiologic model; response; simulation; structural determinants; success; therapeutic target; uptake

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.

项目摘要/摘要：拟议项目使用两个目标策略来调查监管 计划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如何被破坏。 目标 1 重点关注受磷蛋白 (PLB) 与其调节靶标 SERCA2a 的亲和力如何变化 目标 1a 实验将揭示功能差异的结构决定因素。 我们将探讨 PLB 如何稳定特异性的 SERCA 的抑制性和非抑制性调节剂。 SERCA 催化循环中的酶促中间状态，导致转运抑制。 这些实验将测试健康和疾病中监管相互作用的动态。 相互作用限制了长期钙升高后 SERCA 抑制的恢复速度。 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 运动期间心率升高，然后运动后恢复。 目标 2 将研究 PLB-SERCA 调控复合物的替代结合模式。 计算研究确定了 SERCA 上多个非规范结合位点，其中 PLB 可以在这些位点相互作用 因此，PLB-SERCA 相互作用代表了一个“模糊复合体”。 先前研究产生的假设将在拟议项目的目标 2a 中直接进行测试，使用 我们将通过光活化交联和质谱实验来确定其功能作用。 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 在目标 2b 中，我们将探讨 SERCA 模糊复合体心脏病产生的病理生理学。 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肽片段。 将通过质谱鉴定候选“毒肽”并测试其结合SERCA、抑制Ca的能力 运输，或取代本地监管合作伙伴，这些实验将为小说提供新的见解。 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。 阿尔伯塔大学将提供冷冻电镜和 SERCA 结构/功能方面的专业知识。 心脏钙处理专家，将协助杨博士和博士进行定量生理实验。 Zima 参与了之前的富有成果的高额资金周期，Jonathan Kirk 将为该项目带来大量资金。 Peter Kekenes-Huskey 的光谱测定和蛋白质组学专业知识将贡献生理模型。 SERCA 运输周期和管理运输功能的监管相互作用。 实验表明该研究团队未来成功的可能性很大，我们处于独特的地位。 在我们对心脏钙转运调节的理解方面取得了科学上的重大进展。

项目成果

Phospholamban C-terminal residues are critical determinants of the structure and function of the calcium ATPase regulatory complex.

Phospholamban C 末端残基是钙 ATP 酶调节复合物的结构和功能的关键决定因素。

• 发表时间: 2014-09-12
• 作者: Abrol, Neha;Smolin, Nikolai;Armanious, Gareth;Ceholski, Delaine K;Trieber, Catharine A;Young, Howard S;Robia, Seth L
• 通讯作者: Robia, Seth L

A structural mechanism for calcium transporter headpiece closure.

钙转运头件闭合的结构机制。

• 发表时间: 2015-01-29
• 作者: Smolin, Nikolai;Robia, Seth L
• 通讯作者: Robia, Seth L

Acute inotropic and lusitropic effects of cardiomyopathic R9C mutation of phospholamban.

磷脂班心肌病 R9C 突变的急性正性肌力和松弛作用。

• 发表时间: 2015-03-13
• 作者: Abrol, Neha;de Tombe, Pieter P;Robia, Seth L
• 通讯作者: Robia, Seth L

Dimerization of SERCA2a Enhances Transport Rate and Improves Energetic Efficiency in Living Cells.

SERCA2a 的二聚化可增强活细胞的转运速率并提高能量效率。

• 发表时间: 2020-10-06
• 影响因子: 3.4
• 作者: Bovo, Elisa;Nikolaienko, Roman;Cleary, Sean R;Seflova, Jaroslava;Kahn, Daniel;Robia, Seth L;Zima, Aleksey V
• 通讯作者: Zima, Aleksey V

Phosphomimetic mutations increase phospholamban oligomerization and alter the structure of its regulatory complex.

拟磷突变增加受磷蛋白寡聚化并改变其调节复合物的结构。

• 发表时间: 2008-10-24
• 作者: Hou, Zhanjia;Kelly, Eileen M;Robia, Seth L
• 通讯作者: Robia, Seth L