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 这个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，即 Serca。 Serca在心脏周期中起着核心作用。因此，其调节对于生存和适应至关重要 不断变化的身体需求。该调节主要是由两个跨膜小肽介导的： 抑制性肽，磷脂（PLB）和刺激性肽，矮式阅读框（Dworf）。这些 监管机构竞争结合SERCA并控制其功能。我们发现驱动的细胞内Ca2+高程 心肌的收缩还会导致动态变化SERCA与PLB和Dworf的结合。 特别是，Ca2+高程只是降低了SERCA对PLB的亲和力，并增加SERCA亲和力 德沃夫。预计在Ca2+瞬态峰值期间，这将降低抑制作用并增加SERCA的刺激。 此外，我们的初步结果表明，在在 Ca2+高程在PLB五聚体中动态隔离。 PLB五聚体的缓慢解开会导致PLB 在快速心脏起搏过程中积聚在五聚会中，将PLB从SERCA隔离到运动时较低的抑制作用 心率。 AIM 1将探讨PLB五聚体的这种频率依赖性积累如何介导关键 PLB在Bowditch效应中的作用，鲍迪奇效应是一种正力频率关系，在这种关系中，更快的心率会导致更大的力量 心脏的收缩。这种现象是一种关键机制，可调节运动的心脏输出，重要的是 它缺乏心力衰竭。这是，实验将检查这种新型机制在身体上如何改变 心脏对心脏衰竭的PLB突变对肾上腺素的反应以及病理学上的反应。这些见解可能会揭示 为什么这些突变的患者更容易受到雅典/心力衰竭的影响。此外，我们的初步数据 发现PLB和DWORF亲和力的Ca2+依赖性变化是因为这些调节剂更喜欢结合不同的 SERCA酶循环的中间会议。 AIM 2将研究能量学的不同变化 SERCA-微肽在SERCA酶周期期间的结合可能在明显的抑制和刺激下 PLB和Dworf的影响。具体而言，实验将探讨PLB与ATP的紧密结合 SERCA的结合状态确定Ca2+结合介导PLB抑制。另一方面，德沃夫更喜欢与状态结合 当泵由于限速步骤而循环时，SERCA的占主导地位。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量屏障并增加SERCA酶更新。 拟议的实验将改变Serca-微肽调节的经典范例，并告知 小分子治疗心力衰竭。

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