New mechanisms of SERCA2a regulation: role of luminal calcium

SERCA2a调节的新机制：管腔钙的作用

基本信息

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
项目类别：
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

项目摘要

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.

项目概要/摘要 SERCA2a 钙泵在心脏功能中发挥着核心作用 SERCA2a 从心脏中清除钙的速度。细胞质是心肌舒张速率的主要决定因素，SERCA2a 也决定了心肌舒张的总量。肌浆网 (SR) 中的 Ca 决定了心脏收缩的强度，这并不奇怪，据报道，SERCA2a 功能受损在许多病理状况中，包括心力衰竭 (HF)。因此，除了激活之外，了解 SERCA 调节机制也具有重要的临床意义。在肌肉收缩过程中，SR 管腔钙 ([Ca]SR) 在调节 SR 蛋白功能中发挥着重要作用。 SERCA2a 活性控制 [Ca]SR，但人们对 [Ca]SR 的变化如何影响 SERCA2a Ca 转运知之甚少。初步结果表明 SR luminal Ca 在 SERCA2a 的调节中发挥重要作用。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 SERCA2a 的这种新机制在目标 1 中，我们将测试腔 Ca 通过增加泵的调节 SERCA2a 的假设。催化功效并通过减轻受磷蛋白 (PLB) 抑制作用进行分子动力学模拟。用于选择 SERCA2a 管腔侧参与 Ca 定点调节的特定结构域。诱变将用于识别形成管腔 Ca 结合位点的特定氨基酸并开发我们将评估管腔 Ca 调节对 Ca 转运的影响。然后，表达管腔 Ca 不敏感 SERCA2a 的肌细胞。我们将研究突变体以确定这种新机制在心脏 Ca 循环中的作用。这项工作的成果将极大地促进我们对 SERCA2a 功能的理解。这些研究将提供 SERCA2a 调控新机制的详细视图，推进我们的研究了解 Ca 泵的功能在目标 2 中，我们将检验管腔氧化还原电位调节的假设。 SERCA2a 通过稳定其腔内 Ca 结合位点，从而改善 [Ca]SR 泵的调节。动态模拟将用于预测 SERCA2a 管腔二硫键在泵催化中的作用我们将评估管腔半胱氨酸的突变是否会通过废除而导致功能丧失表型。 SERCA2a 通过管腔 Ca 调节。新开发的测量管腔氧化还原电位和 [Ca]SR 的方法。将用于定义管腔氧化还原电位和 SERCA2a 活性之间的串扰。这种新机制对 HF 中 SERCA2a 功能障碍和 Ca 处理不当的贡献。这些研究中的一个新概念可以解释 SERCA2a 结构/功能的改变如何导致缺陷 HF 中的 Ca 调节。