Molecular and Cellular Studies of Ca2+ Transport ATPase

Ca2+ 转运 ATP 酶的分子和细胞研究

• 批准号: 7073010
• 负责人: GIUSEPPE INESI
• 金额: $ 58.35万
• 依托单位: CALIFORNIA PACIFIC MED CTR RES INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-09-01 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7073010
• 关键词: X ray crystallography; adenosine triphosphate; binding sites; biological signal transduction; calcium transporting ATPase; cardiac myocytes; complementary DNA; enzyme activity; gene expression; gene mutation; laboratory mouse; laboratory rabbit; laboratory rat; muscle contraction; newborn animals; nucleotides; phosphorylation; sarcoplasmic reticulum; site directed mutagenesis; tissue /cell culture; transfection; transfection /expression vector

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.

描述(申请人提供)：肌浆网钙ATPase(SERCA)是活性钙离子转运到细胞内储存库的操作者。储存的钙离子随后被释放，以触发胞浆中的钙“1”信号。在心肌和骨骼肌中，Ca~(2+)转运和Ca~(2+)信号在松弛和收缩以及转录激活等其他功能中起着重要的作用。这些功能在心力衰竭时会发生改变。该项目的目标是：(1)阐明利用ATP来逆浓度梯度移动钙的分子机制；(2)建立基因转移到心肌细胞的策略，并定义过度表达或沉默SERCA和其他编码钙信号蛋白的基因的后果。与AIM(1)相关的研究将通过蛋白质化学和定点突变在天然和重组ATPase中产生特定的修饰，并将确定这些修饰对与钙离子转运耦合的连续ATPase反应的影响。这些发现将与结晶学数据和衍射分析相关联，以表明不同的ATPase蛋白结构域和特定氨基酸残基是如何参与能量传递的。将定义抑制剂的结合位置和特定效果。该酶作为主动转运和能量转导的原型，其作用机制有望在分子和原子水平上得到解决。与AIM(2)相关的研究将主要基于特定启动子控制下的重组腺病毒载体将外源基因导入心肌细胞，从而优化基因转移和沉默策略，以用于培养的心肌细胞生理学基础研究。SERCA上调或下调对Ca~(2+)信号、收缩/松弛周期和细胞内稳态的功能后果将被定义。此外，还将研究沉默SERCA或其他基因(即钙调神经磷酸酶)对其他蛋白质的转录和表达以及钙信号通路重塑的影响。这项工作将使我们能够探索和澄清一个新的重要概念，即除了短期的功能调节(即肾上腺素能)外，拷贝数、钙信号蛋白的多样性和分布的长期变化是心脏重构、肥厚、衰竭和可能的治疗的重要因素。