The NKA Interactome in Health and Disease

Na/K-ATP酶（NKA）或“钠泵”是一种转运离子的酶 穿过细胞膜细胞ATP预算的30-70%由NKA花费， 钠和钾的化学梯度。这些梯度控制细胞体积，支持电信号 （动作电位）和激励许多其他运输过程。钠处理中断与 患有心脏肥大/衰竭、帕金森病、精神分裂症和阿尔茨海默病等疾病 疾病身体不同组织中的不同细胞类型对Na/K转运有非常不同的要求， 并且转运速率必须响应生理挑战（例如锻炼）。为了使NKA适应这些不同的 在某些条件下，泵由组织特异性跨膜肽调节，所述跨膜肽增加或减少细胞内的 泵的酶活性。在本项目的目标1中，我们将重点关注正常的生理调节， FXYDs的NKA，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的光谱分析来研究不同的结构和稳定性。 NKA-FXYD调节复合物。我们将研究如何将两个泵物理耦合成一个 功能单元可以允许更快的整体循环。目标2是研究项目的平移臂。的 一个研究小组将利用蛋白质组学方法，研究FXYDs对NKA的调节在疾病中是如何变得紊乱的。 分析以比较健康和衰竭的人心脏组织中的NKA-FXYD相互作用组。我们将确定 消化的膜蛋白片段是否会破坏疾病中的NKA功能。我们还将测试 使用抑制性物质（FXYD 1）的基因递送作为改善收缩的疗法的可行性 一颗衰竭的心脏的力量最后，我们将比较激活NKA的FXYDs和抑制NKA的FXYDs。 该项目汇集了6个不同的研究人员，专门从事不同的互补方法。 项目PI的实验室，Seth Robia教授，使用荧光光谱和细胞生理学， 研究NKA-FXYD复合物在心肌细胞和异源细胞中的结构、亲和力和功能。 细胞他与两个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）。柯克将使用蛋白质组学方法，如质谱法，以发现后- 翻译修饰和新型NKA调节剂。Kekenes-Huskey教授将协助计算 蛋白质复合物的建模。Julie Bossuyt教授（MPI）将研究a-a耦合，进行 显微镜和生物化学实验来识别相互作用界面。德克萨斯州的阿蒂加斯教授（Co-I） 技术人员将对FXYD蛋白和膜蛋白片段进行详细的电生理分析。 教授Razvan Cornea（顾问）是膜蛋白生物物理学专家。他将为球队提供所有 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紊乱。

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.