Investigating Novel Regulatory Mechanisms of the Cardiac Calcium Pump by Inhibitory and Stimulatory Micropeptides.

通过抑制性和刺激性微肽研究心脏钙泵的新调节机制。

• 批准号: 10537189
• 负责人: Sean Robert Cleary
• 金额: $ 2.94万
• 依托单位: LOYOLA UNIVERSITY CHICAGO
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-15 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10537189
• 关键词: Active Biological Transport; Adrenergic Agents; Affinity; Age; Binding; Biosensor; Buffers; Ca(2+)-Transporting ATPase; Calcium; Cardiac; Cardiac Output; Cardiomyopathies; Cell membrane; Chemosensitization; Clinical; Competitive Binding; Complex; Data; Development; Disease; Dissociation; Dwarfism; Enzymes; Epinephrine; Exercise; Fluorescence; Fluorescence Resonance Energy Transfer; Frequencies; Goals; Heart; Heart Rate; Heart failure; Homo; Human; Kinetics; Knock-out; Knowledge; Link; Measurement; Measures; Mediating; Mediator of activation protein; Membrane Proteins; Methods; Molecular; Molecular Conformation; Mutation; Myocardial Contraction; Myocardium; Open Reading Frames; Pathogenesis; Pathogenicity; Pathologic; Patients; Peptides; Phosphorylation; Physiological; Play; Proteins; Pump; Regulation; Relaxation; Research Personnel; Rest; Role; Sarcoplasmic Reticulum; Signal Transduction; Spectrum Analysis; TRAP Peptide; Testing; Time; Transplantation; Work; base; cardiac pacing; experimental study; extracellular; fighting; insight; monomer; novel; novel therapeutics; phospholamban; protein complex; rational design; response; small molecule; therapy design; uptake; virtual

PROJECT SUMMARY/ABSTRACT The goal of this two aim project is to explore dynamic mechanisms that regulate the cardiac calcium transporter, SERCA. SERCA plays a central role in the cardiac cycle; therefore, its regulation is critical for both survival and adapting to changing physiological demands. This regulation is primarily mediated by two transmembrane micropeptides: the inhibitory peptide, Phospholamban (PLB), and the stimulatory peptide, dwarf open reading frame (DWORF). These regulators compete to bind SERCA and control its function. We have found that intracellular Ca2+ elevations that drive contractions in cardiac muscle also cause dynamic shifts the binding equilibria of SERCA with PLB and DWORF. Specifically, Ca2+ elevations simultaneously lower the affinity of SERCA for PLB and increase SERCA affinity for DWORF. This is expected to lower inhibition and increase stimulation of SERCA during the peak of Ca2+ transients. Additionally, our preliminary results revealed that a dynamic fraction of PLB monomers that unbind from SERCA during Ca2+ elevations are dynamically sequestered in PLB pentamers. Slow unbinding of PLB pentamers causes PLB to accumulate in pentamers during rapid cardiac pacing, sequestering PLB away from SERCA to lower inhibition at exercising heart rates. Aim 1 will explore how this frequency-dependent accumulation of the PLB pentamer may mediate a critical role for PLB in the Bowditch effect, a positive force-frequency relationship in which a faster heart rate causes more forceful contractions of the heart. This phenomenon is a critical mechanism that adjusts cardiac output for exercise and, importantly, it is lacking in heart failure. Thus, experiments will examine how this novel mechanism is altered physiologically during the heart’s response to adrenaline and pathologically by PLB mutations linked to heart failure. These insights may reveal why patients with these mutations are more susceptible to arrythmias/heart failure. Additionally, our preliminary data revealed that Ca2+-dependent changes in PLB and DWORF affinity occur because these regulators prefer to bind different intermediate conformations of the SERCA enzymatic cycle. Aim 2 will investigate how distinct changes in the energetics of SERCA-micropeptide binding during the SERCA enzymatic cycle may underly the distinct inhibitory and stimulatory effects of PLB and DWORF, respectively. Specifically, experiments will explore how tight binding of PLB to the ATP- bound state of SERCA deters Ca2+ binding to mediate PLB inhibition. On the other hand, DWORF prefers to bind to states of SERCA that predominate when the pump is cycling due to rate-limiting steps. We will determine if DWORF stabilizes high energy intermediate states of the enzymatic cycle to lower an energy barrier and increase SERCA enzyme turnover. The proposed experiments will shift classic paradigms of SERCA-micropeptide regulation and inform the development of small molecules to treat heart failure.

项目总结/摘要 这两个项目的目标是探索调节心脏钙转运蛋白的动力学机制， 塞卡。SERCA在心动周期中起着核心作用;因此，它的调节对于生存和适应都至关重要 生理需求的变化。这种调节主要由两种跨膜微肽介导： 抑制肽受磷蛋白（PLB）和刺激肽矮开放阅读框（DWORF）。这些 调节剂竞争结合SERCA并控制其功能。我们发现，细胞内Ca 2+升高， 心肌收缩也引起SERCA与PLB和DWORF的结合平衡的动态变化。 具体地说，Ca 2+升高同时降低了SERCA对PLB的亲和力，并增加了SERCA对PLB的亲和力。 DWORF。预期这将降低在Ca 2+瞬变峰值期间对SERCA的抑制并增加其刺激。 此外，我们的初步结果表明，PLB单体的动态部分，从SERCA中解结合， Ca 2+升高动态隔离在PLB五聚体。PLB五聚体的缓慢解结合导致PLB 在快速心脏起搏过程中以五聚体形式积累，使PLB远离SERCA，以降低运动时的抑制作用 心率目的1将探讨PLB五聚体的这种频率依赖性积累如何介导关键的细胞凋亡。 PLB在Bowditch效应中的作用，Bowditch效应是一种积极的力量-频率关系，其中更快的心率导致更有力的力量。 心脏的收缩。这种现象是一种调节运动时心输出量的关键机制，重要的是， 它缺乏心力衰竭。因此，实验将研究这种新的机制是如何改变生理过程中， 心脏对肾上腺素的反应以及与心力衰竭有关的PLB突变的病理学。这些见解可能揭示 为什么有这些突变的患者更容易发生心律失常/心力衰竭。此外，我们的初步数据显示， 揭示了PLB和DWORF亲和力发生Ca 2+依赖性变化，因为这些调节剂更喜欢结合不同的 SERCA酶循环的中间构象。目标2将研究能量学的不同变化 在SERCA酶循环过程中的SERCA-微肽结合可能是不同的抑制和刺激作用的基础。 分别是PLB和DWORF的影响。具体来说，实验将探索PLB与ATP的紧密结合程度， SERCA的结合状态阻止Ca 2+结合以介导PLB抑制。另一方面，DWORF更倾向于绑定到状态 当泵由于速率限制步骤而循环时占主导地位的SERCA。我们将确定DWORF是否稳定 酶循环的高能量中间状态，以降低能垒并增加SERCA酶周转。 拟议的实验将改变SERCA-微肽调节的经典范例，并为开发 小分子药物来治疗心力衰竭

项目成果

Dilated cardiomyopathy variant R14del increases phospholamban pentamer stability, blunting dynamic regulation of cardiac calcium handling.

扩张型心肌病变异体 R14del 增加了受磷蛋白五聚体的稳定性，削弱了心脏钙处理的动态调节。

• DOI: 10.1101/2023.05.26.542463
• 发表时间: 2023
• 期刊: bioRxiv : the preprint server for biology
• 作者: Cleary,SeanR;Teng,AllenCT;Kongmeneck,AudreyDeyawe;Fang,Xuan;Phillips,TaylorA;Cho,EllenE;Kekenes-Huskey,Peter;Gramolini,AnthonyO;Robia,SethL
• 通讯作者: Robia,SethL