New mechanisms of SERCA regulation: Dimerization and Micropeptides

项目概要/摘要 该项目采用双目标策略来探索离子动力学调节的分子机制， ATP酶具体来说，我们将集中在骨骼肌和心肌中钙转运蛋白的调节。而 在所有细胞中，钙转运都很重要，在横纹肌中起着特别关键的作用，因为 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，因为它决定了钙储存的大小， 因此钙释放的大小。 本提案的目的1集中于新发现的微肽种类对SERCA的调节 与受磷蛋白有关的基因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。很少有人知道他们的功能调节SERCA的生物物理决定因素。 我们将定量的化学计量和结合亲和力的多肽调节复合物，动力学的 监管的相互作用，以及监管的结构决定因素。为了量化这些关键参数，我们将 使用几种互补的方法，包括荧光光谱/显微镜，活细胞生理 测量、体外功能测定、cryoEM和NMR。定义的基本构建块 微肽调节复合物是理解其生物物理功能的关键步骤。 目的2将研究SERCA泵与二聚体转运复合物的物理和功能偶联。 我们将研究功能偶联的调节，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转运速率和钙离子的协同性， 依赖ATP酶活性。在本申请的两个目的中描述的实验将提供新的 深入了解离子运动ATP酶的基本调节机制，并可能提高我们的理解 与心力衰竭相关的离子转运障碍。 主要研究者招募了合作研究者，以提供额外的方法 专业知识将在实验室活细胞中进行校准的定量钙摄取测量 来自芝加哥洛约拉大学的Aleksey Zima教授。DWOF-SERCA复合物的冷冻电镜将在 霍华德S.杨，阿尔伯塔大学。DWORF的NMR将在Prof. Gianluigi Veglia，明尼苏达大学。每一个合作者都已经有了生产的历史， 与主要研究者合作。现在，他们将联合收割机将他们的专业知识作为一个单一的团队来解决 微肽和SERCA二聚化如何调节横纹肌中的钙处理。额外专门知识 在分子生物学和心力衰竭的动物模型将提供博士托尼帕克和博士郭， 洛约拉大学芝加哥。

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.