New mechanisms of SERCA regulation: Dimerization and Micropeptides

SERCA调控新机制：二聚化和微肽

基本信息

批准号：
10063953
负责人：
Seth L Robia
金额：
$ 57.58万
依托单位：
LOYOLA UNIVERSITY CHICAGO
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-01-01 至 2022-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10063953
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

项目摘要

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.

项目概要/摘要该项目采用两个目标策略来探索离子动力调节的分子机制 ATP 酶。具体来说，我们将重点关注骨骼和心肌中钙转运蛋白的调节。尽管它在所有细胞中都很重要，钙运输在横纹肌中起着特别重要的作用，因为钙的吸收钙决定肌肉松弛的动力学。在心脏中，钙的转运率也间接决定心脏收缩的强度，因为它决定钙储存的大小和因此钙释放的大小。本提案的目标 1 侧重于新发现的微肽种类对 SERCA 的调节与受磷蛋白有关。这些新的调节剂包括 DWORF、内皮调节蛋白、肌调节蛋白和另一种调节蛋白。人们对 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，以及监管相互作用以及监管的结构性决定因素。为了量化这些关键参数，我们将使用多种补充方法，包括荧光光谱/显微镜、活细胞生理学测量、体外功能测定、冷冻电镜和核磁共振。定义基本构建块微肽调节复合物是了解其生物物理功能的关键一步。目标 2 将研究 SERCA 泵与二聚体运输复合物的物理和功能耦合。我们将研究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 转运速率和钙-的协同性依赖性 ATP 酶活性。本申请的两个目标中描述的实验将提供新的深入了解离子动力 ATP 酶调节的基本机制，并可能提高我们的理解与心力衰竭相关的离子转运障碍。首席研究员已招募合作研究人员来提供额外的方法论专业知识。将在实验室的活细胞中进行校准的定量钙吸收测量芝加哥洛约拉大学 Aleksey Zima 教授。 DWORF-SERCA 复合物的冷冻电镜将在艾伯塔大学 Howard S. Young 教授的实验室。 DWORF 的 NMR 将在教授的实验室进行。吉安路易吉·维利亚，明尼苏达大学。每个合作者都已经拥有富有成效的历史与首席研究员的合作。现在，他们将把他们的专业知识结合起来作为一个团队来解决微肽和 SERCA 二聚化如何调节横纹肌中的钙处理。额外的专业知识 Toni Pak 博士和 Ivana Kuo 博士将提供心力衰竭的分子生物学和动物模型方面的知识，芝加哥洛约拉大学。