Molecular and Cellular Studies of Ca2+ Transport ATPase

描述（由申请人提供）：肌内质网Ca2+ atp酶（SERCA）是活跃的Ca2+转运到细胞内储存的操作员。储存的Ca2+反过来释放触发细胞质Ca2“1”信号。在心脏和骨骼肌中，Ca2+转运和Ca2+信号在松弛和收缩的控制以及转录激活等其他功能中发挥着重要作用。这些功能在心力衰竭时发生改变。这个项目的目的是：(1)澄清分子机制，即ATP被用来移动Ca2+对浓度梯度；(2)建立基因转移到心肌细胞的策略，并定义SERCA和其他编码Ca2+信号蛋白的基因过表达或沉默的后果。与目标(1)相关的研究将通过蛋白质化学和位点定向诱变对天然和重组atp酶进行特异性修饰，并将确定这些修饰对与Ca2+转运相关的顺序atp酶反应的影响。这些发现将与晶体学数据和衍射分析有关，以表明各种atp酶蛋白结构域和特定氨基酸残基如何参与能量转导。将定义抑制剂的结合位点和特定作用。作为主动转运和能量转导的原型，该酶的机制有望在分子和原子水平上得到解决。与目标(2)相关的研究将主要基于在特定启动子的控制下，通过重组腺病毒载体将外源cDNA传递到心肌细胞，从而优化基因转移和沉默策略，用于培养心肌细胞生理的基础研究。SERCA上调或下调对Ca2+信号、收缩/松弛周期和细胞稳态的功能后果将被定义。此外，将研究沉默特异性SERCA或其他基因（即钙调磷酸酶）对其他蛋白质的转录和表达以及Ca2+信号通路重塑的影响。这项工作将使我们能够探索和阐明一个新的重要概念，即除了短期功能调节（即肾上腺素能）外，Ca2+信号蛋白拷贝数、多样性和谱的长期变化是心脏重塑、肥厚、衰竭和可能治疗的重要因素。

DESCRIPTION (provided by applicant): The Sarco-Endoplasmic Reticulum Ca2+ ATPase (SERCA) is the operator of active Ca2+ transport into intracellular stores. The stored Ca2+ is in turn released to trigger cytosolic Ca2"1" signaling. In cardiac and skeletal muscle Ca2+ transport and Ca2+ signaling play prominent roles in control of relaxation and contraction, as well as other functions such as transcriptional activation. These functions are altered in cardiac failure. The aims of this project are: (1) clarification of the molecular mechanism whereby ATP is utilized to move Ca2+ against a concentration gradient; (2) establishment of strategies for gene transfer into cardiac myocytes, and definition of the consequences of overexpression or silencing SERCA and other genes encoding Ca2+ signaling proteins. The research related to aim (1) will produce specific modifications in native and recombinant ATPase by protein chemistry and site directed mutagenesis, and will define the effects of these modifications on the sequential ATPase reactions that are coupled to Ca2+ transport. The findings will be related to crystallographic data and diffraction analysis, to indicate how various ATPase protein domains and specific amino acid residues are involved in energy transduction. Binding sites and specific effects of inhibitors will be defined. It is expected that the mechanism of this enzyme, as a prototype of active transport and energy transduction, will be solved at the molecular and atomic level. The research related to aim (2) will be mostly based on exogenous cDNA delivery to cardiac myocytes by means of recombinant adenovirus vectors under control of specific promoters, thereby optimizing gene transfer and silencing strategies for basic studies of cardiac cell physiology in culture. The functional consequences of SERCA up- or downregulation on Ca2+ signaling, contraction/relaxation cycle and cellular homeostasis will be defined. In addition the effects of silencing specifically SERCA or other genes (i.e., calcineurin) on transcription and expression of other proteins and remodeling of the Ca2+ signaling pathways will be studied. This work will allow us to explore and clarify a new and important concept indicating that in addition to short term functional modulation (i.e., adrenergic), long term changes in copy number, diversity and profile of Ca2+ signaling proteins are important factors in cardiac remodeling, hypertrophy, failure, and possible treatment.