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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：
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
项目类别：
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

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.

项目总结/文摘