Molecular and Cellular Studies of Ca2+ Transport ATPase

Ca2+ 转运 ATP 酶的分子和细胞研究

• 批准号: 6663237
• 负责人: GIUSEPPE INESI
• 金额: $ 40.07万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 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 2006-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6663237
• 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. Stored Ca2+ can then be released to trigger cytosolic Ca2+ signaling. Both, Ca2+ transport and Ca2+ signaling play prominent roles in control of relaxation and contraction in cardiac and skeletal muscle. Both mechanisms 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, and (2) establishment of strategies for SERCA gene transfer, and definition of the consequences of exogenous SERCA expression in cardiac myocytes. 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 obtained by x-ray diffraction, to indicate how various ATPase protein domains and specific amino acid residues are involved in energy transduction. The research related to aim (2) will optimize conditions for SERCA cDNA delivery to cardiac myocytes by means of recombinant adenovirus vectors. This will include evaluation of several promoters in order to obtain appropriate levels of exogenous SERCA expression only in cardiac muscle, and not in other tissues such as smooth muscle, endothelium, skeletal muscle, and liver. The functional consequences of SERCA overexpression and/or isoform switch on Ca2+ signaling, contraction/relaxation cycle and cellular homeostasis will be defined. Thereby, we will optimize gene transfer strategies for basic studies of cardiac cell physiology in culture, and will evaluate their possible use for amelioration of failing cardiac muscle in situ.

描述：(申请人提供)肌浆网钙离子 ATPase(SERCA)是细胞内钙离子转运的操纵者 商店。然后，储存的钙离子可以被释放，以触发胞浆钙信号。 Ca~(2+)转运和Ca~(2+)信号转导在细胞周期调控中起着重要作用 心肌和骨骼肌的松弛和收缩。这两种机制都是 在心力衰竭时会发生改变。这项计划的目的是：(1)澄清 ATP被用来移动钙离子对抗钙离子的分子机制 浓度梯度；(2)SERCA基因策略的建立 外源SERCA表达在水稻中的转移和后果的定义 心肌细胞。与AIM(1)相关的研究将产生具体的 蛋白质化学和位点对天然和重组ATPase的修饰 定向突变，并将定义这些修改对 与钙离子转运相偶联的连续的ATPase反应。调查结果 将与通过x射线衍射获得的结晶学数据有关， 指出各种ATPase蛋白结构域和特定氨基酸残基是如何 都与能量传递有关。与AIM(2)相关的研究将 优化SERCA基因导入心肌细胞的条件 重组腺病毒载体。这将包括评估以下几项 启动子，以获得适当水平的外源SERCA表达 仅在心肌中，而不在其他组织中，如平滑肌， 内皮、骨骼肌和肝脏。SERCA的功能后果 钙信号的过度表达和/或异构体开关、收缩/松弛 将定义周期和细胞动态平衡。因此，我们将对基因进行优化 培养心肌细胞生理学基础研究的转移策略， 并将评估它们在改善衰竭心肌方面的可能用途 在原地。