Molecular and Cellular Studies of Ca2+ Transport ATPase

Ca2+ 转运 ATP 酶的分子和细胞研究

• 批准号: 7489402
• 负责人: GIUSEPPE INESI
• 金额: $ 58.16万
• 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/7489402
• 关键词: ATP phosphohydrolase; ATPase Domain; Active Biological Transport; Adenovirus Vector; Adrenergic Agents; Amino Acids; Binding; Binding Sites; Ca(2+)-Transporting ATPase; Calcineurin; Cardiac; Cardiac Myocytes; Cell physiology; Cells; Complementary DNA; Coupled; Coupling; Cultured Cells; Data; Down-Regulation; Endoplasmic Reticulum; Enzymes; Failure; Gene Transfer; Genes; Genetic Transcription; Heart; Heart failure; Homeostasis; Hypertrophy; Kinetics; Liver; Measurement; Modification; Molecular; Myocardium; Nucleotides; Numbers; Play; Protein Chemistry; Protein Overexpression; Proteins; Range; Rate; Reaction; Recombinants; Relaxation; Research; Reverse Transcriptase Polymerase Chain Reaction; Role; Signal Pathway; Signal Transduction; Signaling Protein; Site-Directed Mutagenesis; Skeletal Muscle; Small Interfering RNA; Specificity; Tertiary Protein Structure; Transcriptional Activation; Transmembrane Domain; Up-Regulation; Work; adrenergic; base; concept; enzyme mechanism; inhibitor/antagonist; inorganic phosphate; interest; promoter; prototype; research study; 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（由申请方提供）：肌质内质网Ca 2 + ATP酶（SERCA）是将活性Ca 2+转运至细胞内储存的操纵子。储存的Ca 2+又被释放以触发胞质Ca 2+信号传导。在心肌和骨骼肌中，Ca 2+转运和Ca 2+信号传导在控制舒张和收缩以及其他功能如转录激活中起着重要作用。这些功能在心力衰竭时发生改变。本项目的目的是：（1）阐明ATP用于逆浓度梯度移动Ca 2+的分子机制;（2）建立基因转移到心肌细胞中的策略，并定义过表达或沉默SERCA和其他编码Ca 2+信号蛋白的基因的后果。与目标（1）相关的研究将通过蛋白质化学和定点诱变在天然和重组ATP酶中产生特异性修饰，并将确定这些修饰对与Ca 2+转运偶联的顺序ATP酶反应的影响。这些发现将与晶体学数据和衍射分析相关，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异性作用。这种酶作为主动转运和能量传递的原型，其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通过在特异性启动子控制下的重组腺病毒载体将外源cDNA递送至心肌细胞，从而优化基因转移和沉默策略，用于培养中的心肌细胞生理学的基础研究。将定义SERCA上调或下调对Ca 2+信号传导、收缩/舒张周期和细胞稳态的功能后果。此外，特异性沉默SERCA或其他基因（即，钙调神经磷酸酶）对其他蛋白质转录和表达的影响以及Ca 2+信号通路的重塑将被研究。这项工作将使我们能够探索和澄清一个新的和重要的概念，表明除了短期功能调节（即，肾上腺素能），Ca 2+信号传导蛋白的拷贝数、多样性和谱的长期变化是心脏重塑、肥大、衰竭和可能的治疗中的重要因素。