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.

项目摘要/摘要 这两个目标项目的目标是探索调节心脏钙转运蛋白的动态机制， SERCA。SERCA在心脏周期中起着中心作用；因此，它的调节对于生存和适应都至关重要。 不断变化的生理需求。这种调节主要由两种跨膜微肽介导： 抑制肽，磷蛋白(PLB)，和刺激肽，矮小开放阅读框架(DWORF)。这些 监管机构竞相约束SERCA并控制其功能。我们已经发现，推动细胞内钙离子升高的 心肌收缩也会导致SERCA与PLB和DWORF的结合平衡发生动态变化。 具体地说，钙离子升高同时降低了SERCA对PLB的亲和力，而增加了对PLB的SERCA亲和力 DWORF。这有望在钙瞬变的高峰期降低对SERCA的抑制，增加对SERCA的刺激。 此外，我们的初步结果显示，在过程中，从SERCA解离的PLB单体的动态部分 CA2+高程动态地隔离在PLB五聚体中。PLB五聚体缓慢解链导致PLB 在快速心脏起搏过程中积累在五聚体中，将PLB从SERCA隔离开来，以降低运动时的抑制 心率。目标1将探索这种依赖于频率的PLB五聚体积累如何可能调节关键的 PLB在鲍迪奇效应中的作用，这是一种正的力-频率关系，在这种关系中，较快的心率导致更强的 心脏的收缩。这种现象是调节运动的心输出量的关键机制，更重要的是， 它缺乏心力衰竭。因此，实验将检验这种新的机制是如何在 心脏对肾上腺素的反应和病理上与心力衰竭有关的原核细胞基因突变。这些见解可能会揭示 为什么具有这些突变的患者更容易发生心律失常/心力衰竭。另外，我们的初步数据 揭示了PLB和DWORF亲和力的钙依赖性变化是因为这些调节因子更喜欢结合不同的 SERCA酶循环的中间构象。目标2将调查能量学中的明显变化 SERCA-微肽在SERCA酶循环中的结合可能低于明显的抑制和刺激 PLB和DWORF的影响。具体地说，实验将探索PLB如何紧密结合到ATP- SERCA的结合状态通过抑制钙离子结合来调节原核抑制。另一方面，DWORF更喜欢绑定到状态 当泵由于限速步骤而循环时，占主导地位的SERCA。我们将确定DWORF是否稳定 酶循环的高能中间状态，以降低能量屏障，增加SERCA酶的周转。 拟议的实验将改变SERCA-微肽调节的经典范式，并为 治疗心力衰竭的小分子药物。

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