New mechanisms of SERCA regulation: Dimerization and Micropeptides

项目摘要/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。 ATPase。具体地说，我们将重点研究骨骼肌和心肌中钙转运蛋白的调节。而当 它在所有细胞中都很重要，钙转运在横纹肌中起着特别关键的作用，因为摄取 钙决定肌肉松弛的动力学。在心脏，钙的转运率也间接 决定心脏收缩的强度，因为它定义了钙储存和 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 与磷脂蛋白有关的物质。这些新的调节剂包括DWORF、内皮调节素、肌调节素和 另一种--调节素。关于它们对SERCA的功能调控的生物物理决定因素知之甚少。 我们将量化微肽调节复合体的化学计量和结合亲和力，动力学 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 使用几种互补的方法，包括荧光光谱/显微镜、活细胞生理学 测量、体外功能分析、低温电子显微镜和核磁共振。定义的基本构建块 微肽调节复合体是了解其生物物理功能的关键一步。 目标2将研究SERCA泵与二聚体传输复合体的物理和功能耦合。 我们将研究功能偶联的调节，并确定其生理后果。在……里面 特别是，我们将测试功能偶联如何改变SERCA转运速率和钙的协同性- 依赖于ATPase活性。本应用程序的两个目标中描述的实验将提供新的 洞察离子动力ATPase调节的基本机制，可能会提高我们的理解 与心力衰竭相关的离子转运障碍。 首席调查员已经招募了合作调查人员，以提供更多的方法 专业知识。校准后的定量钙摄取测量将在实验室的活细胞中进行。 来自芝加哥洛约拉大学的Aleksey Zima教授。DWORF-SERCA复合体的低温EM将在#年完成 艾伯塔大学霍华德·S·杨教授的实验室。DWORF的核磁共振研究将在他的实验室进行。 吉安路易吉·维格利亚，明尼苏达大学。每一位合作者都有一段多产的历史 与首席调查员合作。现在他们将结合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的钙转运。其他专业知识 在分子生物学和心力衰竭的动物模型方面，将由Toni Pak博士和Ilana Kuo博士提供， 芝加哥洛约拉大学。

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.