New mechanisms of SERCA regulation: Dimerization and Micropeptides

SERCA调控新机制：二聚化和微肽

• 批准号: 10318147
• 负责人: Seth L Robia
• 金额: $ 57.93万
• 依托单位: LOYOLA UNIVERSITY CHICAGO
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-01-01 至 2024-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10318147
• 关键词: ATP phosphohydrolase; Acute; Address; Affinity; Alberta province; Animal Model; Binding; Biological Assay; Biophysics; Ca(2+)-Transporting ATPase; Calcium; Calcium Signaling; Cardiac; Cardiac Output; Cells; Chemicals; Chicago; Collaborations; Complex; Contracts; Coupled; Coupling; Cryoelectron Microscopy; Cyclic AMP-Dependent Protein Kinases; Dimerization; Disease; Docking; Exercise; Family member; Fluorescence Microscopy; Fluorescence Spectroscopy; Heart; Heart Diseases; Heart failure; Homo; Human; Impairment; In Vitro; Ion Pumps; Ion Transport; Ions; Kinetics; Laboratories; Measurement; Measures; Membrane Proteins; Methodology; Methods; Microscopy; Minnesota; Molecular; Molecular Biology; Molecular Conformation; Muscle; Muscle relaxation phase; Myocardium; Performance; Phosphorylation; Phosphotransferases; Physiological; Play; Principal Investigator; Proteins; Protomer; Public Health; Pump; Recording of previous events; Regulation; Research Personnel; Research Project Grants; Role; Series; Site; Skeletal Muscle; Striated Muscles; Structure; Surface; Testing; Tetany; Time; Universities; dimer; experimental study; functional loss; improved; insight; mutant; novel; phospholamban; recruit; simulation; stoichiometry; therapeutic target; uptake

Project Summary/Abstract The proposed project uses a two aim strategy to explore molecular mechanisms of the regulation of ion-motive ATPases. Specifically, we will focus on regulation of calcium transporters in skeletal and cardiac muscle. While it is important in all cells, calcium transport plays a particularly critical role in striated muscle, as the uptake of calcium determines the kinetics of muscle relaxation. In the heart, the calcium transport rate also indirectly determines the strength of the cardiac contraction, since it defines the magnitude of the calcium stores and therefore the size of calcium release. Aim 1 of the present proposal focuses on regulation of SERCA by newly discovered species of micropeptides that are related to phospholamban. These new regulators include DWORF, endoregulin, myoregulin, and another-regulin. Little is known about the biophysical determinants of their functional regulation of SERCA. We will quantify the stoichiometry and binding affinity of micropeptide regulatory complexes, the dynamics of regulatory interactions, and the structural determinants of regulation. To quantify these key parameters, we will use several complementary approaches including fluorescence spectroscopy/microscopy, live cell physiological measurements, in vitro functional assays, cryoEM, and NMR. Defining the basic building blocks of micropeptide regulatory complexes is a key step in understanding their biophysical function. Aim 2 will investigate physical and functional coupling of SERCA pumps into dimeric transport complexes. We will investigate the regulation of functional coupling and determine its physiological consequences. In particular, we will test how functional coupling alters SERCA transport rate and the cooperativity of calcium- dependent ATPase activity. The experiments described in the two Aims of this application will provide new insight into fundamental mechanisms of regulation of ion-motive ATPases, and may improve our understanding of the ion transport disorders associated with heart failure. The Principal Investigator has recruited collaborating investigators to provide additional methodological expertise. Calibrated, quantitative calcium uptake measurements will be performed in live cells in the laboratory of Prof. Aleksey Zima, Loyola University Chicago. CryoEM of DWORF-SERCA complexes will be done in the lab of Prof. Howard S. Young, University of Alberta. NMR of DWORF will be done in the lab of Prof. Gianluigi Veglia, University of Minnesota. Each of the collaborators already has a history of productive collaboration with the Principal Investigator. Now they will combine their expertise as a single team to address how micropeptides and SERCA dimerization regulate calcium handling in striated muscle. Additional expertise in molecular biology and animal models of heart failure will be provided by Dr. Toni Pak and Dr. Ivana Kuo, Loyola University Chicago.

项目摘要/摘要 拟议的项目使用两个目标策略来探索离子 - 动力调节的分子机制 ATPases。具体而言，我们将重点介绍骨骼肌和心脏肌肉中钙转运蛋白的调节。尽管 在所有细胞中都很重要，钙转运在横纹肌中起着特别关键的作用，因为吸收 钙确定肌肉松弛的动力学。在心脏中，钙的传输速率也间接 确定心脏收缩的强度，因为它定义了钙存储的大小和 因此，钙释放的大小。 本提案的目的1重点介绍新发现的微肽物种对SERCA的调节 与磷团有关的。这些新调节器包括Dworf，Endoregulin，肌酸蛋白和 另一种常规蛋白。关于其SERCA功能调节的生物物理决定因素知之甚少。 我们将量化微肽调节复合物的化学计量和结合亲和力，即动力学 监管相互作用以及调节的结构决定因素。为了量化这些关键参数，我们将 使用几种互补方法，包括荧光光谱/显微镜，活细胞生理 测量，体外功能测定，冷冻和NMR。定义基本的构建块 微肽调节复合物是了解其生物物理功能的关键步骤。 AIM 2将研究SERCA泵的物理和​​功能耦合到二聚体运输复合物中。 我们将调查功能耦合的调节并确定其生理后果。在 特别是，我们将测试功能耦合如何改变Serca的运输速率和钙的合作性 依赖性ATPase活性。本应用程序的两个目标中描述的实验将提供新的 深入了解离子 - 动态ATPase调节的基本机制，并可能提高我们的理解 与心力衰竭相关的离子运输障碍。 首席调查员已招募合作调查人员，以提供其他方法论 专业知识。校准的定量钙摄取测量将在实验室的活细胞中进行 芝加哥洛约拉大学Aleksey Zima教授。 Dworf-serca综合体的冷冻将在 艾伯塔大学霍华德·杨教授的实验室。 Dworf的NMR将在教授的实验室中完成。 明尼苏达大学的吉安利吉·维格利亚（Gianluigi Veglia）。每个合作者已经有富有成效的历史 与首席研究员合作。现在，他们将把自己的专业知识结合在一起，以解决 微肽和SERCA二聚化如何调节条纹肌肉中的钙处理。其他专业知识 在分子生物学和心力衰竭的动物模型中，Toni Pak博士和Ivana Kuo博士将提供 洛约拉大学芝加哥。