The NKA Interactome in Health and Disease

健康与疾病中的 NKA 相互作用组

基本信息

批准号：
10593139
负责人：
Julie B Bossuyt
金额：
$ 68.36万
依托单位：
LOYOLA UNIVERSITY CHICAGO
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-04-01 至 2026-03-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10593139
关键词：
Action Potentials Affinity Alzheimer&apos s Disease Binding Binding Proteins Biochemistry Biological Assay Biophysics Budgets Cardiac Myocytes Cell Volumes Cell membrane Cell physiology Cells Chemicals Chicago Complex Computer Models Cornea Coupled Coupling Development Dimerization Disease Electrophysiology (science)Engineering Enzymes Exercise Failure Family Fluorescence Fluorescence Resonance Energy Transfer Fluorescence Spectroscopy Foundations Functional disorder Gene Delivery Health Heart Heart Diseases Heart Hypertrophy Heart failure Human Ion Transport K ATPase Kinetics Laboratories Learning Mass Spectrum Analysis Measurement Membrane Membrane Proteins Methods Microscopy Modification Molecular Conformation Na(+)-K(+)-Exchanging ATPase Parkinson Disease Pathologic Patients Peptide Sequence Determination Peptides Pharmaceutical Preparations Phosphorylation Physiological Physiological Processes Physiology Poison Post-Translational Protein Processing Potassium Process Protein Fragment Proteins Proteomics Protomer Public Health Pump Regulation Research Research Personnel Research Project Grants Sampling Schizophrenia Signal Transduction Site-Directed Mutagenesis Sodium Structure Testing Texas Tissues Translating Translational Research Transport Process Universities Work arm cell type cofactor confocal imaging dimer experimental study feasibility testing improved insight member mutant novel novel therapeutic intervention phospholemman protein complex protein degradation protein protein interaction structural determinants

项目摘要

Project Summary/Abstract: The Na/K-ATPase (NKA), or “sodium pump”, is an enzyme that transports ions across cell membranes. Between 30-70% of the cell’s ATP budget is spent by the NKA to create strong chemical gradients for sodium and potassium. These gradients control cell volume, support electrical signaling (action potentials) and energize many other transport processes. Disruption of sodium handling is associated with diseases including cardiac hypertrophy/failure, Parkinson’s disease, schizophrenia, and Alzheimer’s disease. Various cell types in diverse tissues of the body have very different requirements for Na/K transport, and transport rates must respond to physiological challenges (e.g. exercise). To adapt NKA to these different conditions, the pump is regulated by tissue-specific transmembrane peptides that increase or decrease the pump’s enzymatic activity. In Aim 1 of the project we will focus on the normal physiological regulation of NKA by FXYDs, testing how FXYD proteins bind and stabilize specific NKA conformations to alter NKA transport kinetics. We will use novel spectroscopic assays to investigate the structure and stability of different NKA-FXYD regulatory complexes. We will investigate how physical coupling of two pumps into a single functional unit can allow faster overall cycling. Aim 2 is the translational arm of the research project. The research team will investigate how NKA regulation by FXYDs becomes disordered in disease, using proteomic analysis to compare the NKA-FXYD interactome in healthy and failing human heart tissue. We will determine whether fragments of digested membrane proteins may disrupt NKA function in disease. We will also test the feasibility of using gene delivery of an inhibitory species (FXYD1) as a therapy to improve the contraction strength of a failing heart. Finally, we will compare FXYDs that activate NKA and FXYDs that inhibit NKA. The project brings together 6 different investigators that specialize in different complementary approaches. The laboratory of the project PI, Prof. Seth Robia, uses fluorescence spectroscopy and cell physiology to investigate the structure, affinity, and function of NKA-FXYD complexes in cardiac myocytes and heterologous cells. He works closely with two Loyola University Chicago colleagues: Jonathan Kirk (Co-I) and Pete Kekenes-Huskey (Co-I). Prof. Kirk will use proteomics approaches such as mass spectrometry to discover post- translational modifications and novel NKA regulators. Prof. Kekenes-Huskey will assist with computational modeling of the protein complexes. Prof. Julie Bossuyt (MPI) will investigate a-a coupling, conducting microscopy and biochemistry experiments to identify the interaction interface. Prof. Artigas (Co-I) at Texas Tech will perform detailed electrophysiological analyses of FXYD proteins and membrane protein fragments. Prof. Razvan Cornea (Consultant) is an expert in membrane protein biophysics. He will advise the team on all aspects of the project. Together, the collaborators will uncover important new information about NKA-FXYD structure/function relationships, and learn how these mechanisms become disordered in disease.

项目摘要/摘要：Na/K-ATPase（NKA）或“钠泵”是一种运输离子的酶跨细胞膜。 NKA花费了30-70％的ATP预算来创造强大钠和钾的化学梯度。这些梯度控制细胞体积，支持电信号传导（行动电位）并为许多其他运输过程提供了能量。钠处理的破坏与疾病包括心脏肥大/衰竭，帕金森氏病，精神分裂症和阿尔茨海默氏症疾病。人体多样性时机的各种细胞类型对Na/K传输的要求截然不同，运输率必须应对身体挑战（例如锻炼）。适应NKA这些不同条件，泵受组织特异性跨膜胡椒的调节泵的酶活性。在该项目的目标1中，我们将重点介绍 NKA通过FXYD，测试FXYD蛋白如何结合和稳定特定的NKA构象以改变NKA 运输动力学。我们将使用新颖的光谱测定法研究不同的结构和稳定性 NKA-FXYD调节络合物。我们将调查两个泵的物理耦合如何功能单元可以更快地进行整体循环。 AIM 2是研究项目的转化部门。这研究小组将使用蛋白质组学研究FXYDS的NKA调节如何在疾病中变得无序分析以比较健康和失败的人类心脏组织中的NKA-FXYD相互作用。我们将确定消化的膜蛋白的碎片是否可能破坏NKA在疾病中的功能。我们还将测试使用抑制物种（FXYD1）作为改善收缩的疗法的可行性心脏失败的力量。最后，我们将比较激活抑制NKA的NKA和FXYD的FXYD。该项目汇集了6位专门研究不同完整方法的不同研究人员。 PI项目的实验室Seth Robia教授使用荧光光谱和细胞生理学研究心肌和异源的NKA-FXYD复合物的结构，亲和力和功能细胞。他与两位洛约拉大学芝加哥同事密切合作：乔纳森·柯克（Co-I）和皮特 Kekenes-Huskey（Co-I）。柯克教授将使用蛋白质组学方法（例如质谱法）来发现后发现翻译修改和新型NKA调节器。 Kekenes-Huskey教授将协助计算蛋白质复合物的建模。 Julie Bossuyt教授（MPI）将调查A-A耦合，进行显微镜和生物化学实验以识别相互作用界面。德克萨斯州Artigas（CO-I）教授技术将对FXYD蛋白和膜蛋白片段进行详细的电生理分析。 Razvan Cornea教授（顾问）是膜蛋白生物物理学的专家。他会建议团队所有人项目的各个方面。合作者将共同发现有关NKA-FXYD的重要新信息结构/功能关系，并了解这些机制如何在疾病中变得无序。