Molecular and Cellular Studies of Ca2+ Transport ATPase

Ca2+ 转运 ATP 酶的分子和细胞研究

• 批准号: 6454959
• 负责人: GIUSEPPE INESI
• 金额: $ 38.9万
• 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/6454959
• 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：（由申请方提供）肌质内质网Ca 2 + ATP酶（SERCA）是主动将Ca ~（2+）转运到细胞内的操纵子 店储存的Ca 2+然后可以被释放以触发胞质Ca 2+信号传导。 Ca ~（2+）转运和Ca ~（2+）信号转导在调控细胞凋亡中起重要作用。 心脏和骨骼肌的松弛和收缩。这两种机制都是 在心力衰竭中改变。该项目的目标是：（1）澄清 利用ATP使Ca 2+逆着 （2）建立SERCA基因的表达策略 转移，并定义外源性SERCA表达的后果， 心肌细胞与目标（1）相关的研究将产生具体的 通过蛋白质化学和位点在天然和重组ATP酶中的修饰 定向诱变，并将定义这些修饰对 与Ca 2+转运偶联的连续ATP酶反应。这些发现 将与通过X射线衍射获得的晶体学数据相关， 表明各种ATP酶蛋白结构域和特定氨基酸残基 参与了能量转换。与目标（2）相关的研究将 优化SERCA cDNA递送至心肌细胞的条件， 重组腺病毒载体。这将包括对几个 启动子，以获得适当水平的外源SERCA表达 仅在心肌中，而不在其它组织如平滑肌中， 内皮、骨骼肌和肝脏。SERCA的功能后果 过表达和/或同种型对Ca 2+信号传导、收缩/舒张的开关 周期和细胞内稳态将被定义。从而优化基因 培养中心脏细胞生理学基础研究的转移策略， 并将评估其用于改善衰竭心肌的可能用途， 在原地。