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.

项目摘要/摘要 SERCA2A CA泵在心脏功能中起着核心作用。 SERCA2A删除CA从 细胞质是心肌松弛率的主要确定。 SERCA2A还设置了总数 肌质网（SR）中的Ca，它决定了心脏收缩的强度。这不足为奇， 在许多病理状况中，已经报道了SERCA2A功能受损的功能，包括心力衰竭 （HF）。这是理解SERCA调节机制的临床重要性。除了激活 肌肉收缩，SR Luminal Ca（[CA] SR）在调节SR蛋白功能中起重要作用。尽管 SERCA2A活性控制[CA] SR，对[CA] SR的变化如何影响SERCA2A CA传输的了解少。我们的 初步结果表明，SR Luminal CA在SERCA2A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递送，以探索SERCA2A的这种新机制 规定。在AIM 1中，我们将检验以下假设，即腔内CA通过增加泵的调节SERCA2A 催化效率并通过缓解磷团（PLB）抑制作用。分子动态模拟将 用于选择与CA调节有关的SERCA2A腔侧的特定域。站点定向 诱变将用于识别形成腔内ca结合位点的特定氨基酸，并发展 腔内ca不敏感的SERCA2A突变体。我们将评估管腔CA调节对CA转运的影响， ATPase活性和PLB相互作用。然后，表达腔内CA不敏感的SERCA2A的心肌细胞 突变体将研究以定义这种新机制在心脏CA循环中的作用。我们期望 这项工作的结果将大大提高我们对SERCA2A功能的理解。我们期望结果 这些研究将为SERCA2A调节的这种新机制提供详细的看法，从而推进了我们 了解CA泵的功能。在AIM 2中，我们将测试腔氧化电势调节的假设 SERCA2A通过稳定其腔内Ca结合位点，从而通过[CA] SR改善泵的调节。分子 动态模拟将用于预测SERCA2A腔二硫键在泵催化中的作用 循环。我们将评估腔半胱氨酸的突变是否通过废除功能丧失表型导致功能丧失表型 SERCA2A管腔调节。新开发的方法来测量发光氧化还原电位和[CA] SR 将用于定义腔氧化还原电位和SERCA2A活性之间的串扰。我们将调查 这种新机制对HF中SERCA2A功能障碍和CA不当的贡献。可能的结果 这些研究是一个新概念，可以解释SERCA2A结构/功能的变化如何导致缺陷 在HF中的CA调节中。