New mechanisms of SERCA2a regulation: role of luminal calcium

SERCA2a调节的新机制：管腔钙的作用

• 批准号: 10563138
• 负责人: Aleksey V Zima
• 金额: $ 43.45万
• 依托单位: LOYOLA UNIVERSITY CHICAGO
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-02-15 至 2025-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10563138
• 关键词: ATP phosphohydrolase; Affect; Affinity; Amino Acids; Arrhythmia; Attention; Binding Sites; Biological; Calcium; Cardiac; Cardiac Myocytes; Clinical; Cysteine; Cytosol; Defect; Elements; Endoplasmic Reticulum; Functional disorder; Gene Delivery; Heart Diseases; Heart failure; Impairment; Life; Measures; Molecular; Muscle Cells; Muscle Contraction; Muscle relaxation phase; Mutation; Myocardial Contraction; Myocardium; Optical Methods; Organelles; Outcome; Outcome Study; Oxidation-Reduction; Pathologic; Patients; Phenotype; Pilot Projects; Play; Proteins; Public Health; Pump; Regulation; Reporting; Research Project Grants; Research Proposals; Role; Sarcoplasmic Reticulum; Side; Site; Site-Directed Mutagenesis; Speed; Structure; Techniques; Testing; Work; biophysical techniques; design; disulfide bond; heart function; improved; in vivo; innovation; loss of function; molecular dynamics; mutant; novel; novel therapeutic intervention; novel therapeutics; phospholamban; protein function; sensor; targeted treatment; therapeutic candidate; therapeutic target; uptake

PROJECT SUMMARY/ABSTRACT SERCA2a Ca pump plays a central role in heart function. The speed at which SERCA2a removes Ca from the cytosol is the main determinant of the rate of cardiac muscle relaxation. SERCA2a also sets the total amount of Ca in the sarcoplasmic reticulum (SR), which determines the strength of cardiac contraction. It is not surprising, that impaired SERCA2a function has been reported in a number of pathological conditions, including heart failure (HF). Thus, understanding mechanisms of SERCA regulation is of great clinical importance. Besides activation of muscle contraction, SR luminal Ca ([Ca]SR) plays an important role in regulation of SR protein function. While SERCA2a activity controls [Ca]SR, less is known about how changes in [Ca]SR affect SERCA2a Ca transport. Our preliminary results suggest that SR luminal Ca plays an important role in regulation of SERCA2a. In this project we will use advanced structural analyses, innovative molecular biological techniques, new organelle-specific sensors, state-of-the-art optical methods and in vivo gene delivery to explore this new mechanism of SERCA2a regulation. In Aim 1, we will test the hypothesis that luminal Ca regulates SERCA2a by increasing the pump’s catalytic efficacy and by relieving the phospholamban (PLB) inhibitory effect. Molecular dynamic simulations will be used to select specific domains on the SERCA2a luminal side that are involved in Ca regulation. Site-directed mutagenesis will be used to identify the specific amino acids that form the luminal Ca-binding sites and to develop the luminal Ca-insensitive SERCA2a mutant. We will assess effects of the luminal Ca regulation on Ca transport, the ATPase activity and the PLB interaction. Then, myocytes expressing the luminal Ca-insensitive SERCA2a mutant will be studied to define the role of this novel mechanism in cardiac Ca cycling. We expect that the outcome of this work will greatly advance our understanding of SERCA2a function. We expect that the outcome of these studies will provide a detailed view of this new mechanism of SERCA2a regulation, advancing our understanding of the Ca pump’s function. In Aim 2, we will test the hypothesis that luminal redox potential regulates SERCA2a by stabilizing its luminal Ca binding sites, thus, improving the pump’s regulation by [Ca]SR. Molecular dynamic simulations will be used to forecast the role of the SERCA2a luminal disulfide bond in the pump catalytic cycle. We will assess whether mutation of luminal cysteines leads to a loss-of-function phenotype by abolishing SERCA2a regulation by luminal Ca. Newly developed approaches to measure luminal redox potential and [Ca]SR will be used to define the cross-talk between luminal redox potential and SERCA2a activity. We will investigate the contribution of this new mechanism to SERCA2a dysfunction and Ca mishandling in HF. The likely outcome of these studies is a new concept that can explain how alterations in SERCA2a structure/function cause defects in Ca regulation in HF.

项目总结/摘要 SERCA 2a钙泵在心脏功能中起核心作用。SERCA 2a从细胞中去除Ca的速度是恒定的。 胞质溶胶是心肌松弛速率的主要决定因素。SERCA 2a还设置了 肌浆网（SR）中的钙，决定心脏收缩的强度。这并不奇怪， SERCA 2a功能受损已被报道在许多病理状况中，包括心力衰竭， （HF）.因此，了解SERCA调节机制具有重要的临床意义。除了激活 肌浆网腔钙（[Ca]SR）在调节SR蛋白功能中起重要作用。而 SERCA 2a活性控制[Ca]SR，关于[Ca]SR的变化如何影响SERCA 2a Ca转运知之甚少。我们 初步结果表明，SR腔Ca在SERCA 2a的调节中起重要作用。在这个项目中 我们将使用先进的结构分析，创新的分子生物学技术，新的细胞器特异性， 传感器，最先进的光学方法和体内基因传递来探索SERCA 2a的这种新机制 调控在目的1中，我们将检验管腔钙通过增加泵的浓度来调节SERCA 2a的假设。 催化功效和减轻受磷蛋白（PLB）抑制作用。分子动力学模拟将 用于选择SERCA 2a管腔侧上参与Ca调节的特定结构域。定点 诱变将用于鉴定形成管腔钙结合位点的特定氨基酸，并开发 管腔Ca不敏感SERCA 2a突变体。我们将评估管腔钙调节对钙转运的影响， ATP酶活性和PLB相互作用。然后，将表达管腔钙不敏感SERCA 2a的心肌细胞 将研究突变体以确定这种新机制在心脏Ca循环中的作用。我们预计 这项工作的结果将大大推进我们对SERCA 2a功能的理解。我们预计， 这些研究将提供SERCA 2a调节的新机制的详细视图，推进我们的研究。 了解钙泵的功能。在目标2中，我们将检验管腔氧化还原电位调节 SERCA 2a通过稳定其管腔Ca结合位点，从而通过[Ca] SR改善泵的调节。 动态模拟将用于预测SERCA 2a管腔二硫键在泵催化中的作用， 周期我们将评估管腔半胱氨酸突变是否会导致功能丧失表型， 通过管腔Ca.测定管腔氧化还原电位和[Ca]SR的新方法 将用于定义管腔氧化还原电位和SERCA 2a活性之间的串扰。我们将调查 这种新机制对心力衰竭中SERCA 2a功能障碍和Ca处理不当的作用。可能的结果 这些研究中的一个新概念可以解释SERCA 2a结构/功能的改变如何导致缺陷 在HF的Ca调节中。