CALCINEURIN REGULATION BY MEMBRANE PHOSPHOLIPIDS

膜磷脂对钙调磷酸酶的调节

• 批准号: 6187982
• 负责人: Brian A Perrino
• 金额: $ 10.43万
• 依托单位: UNIVERSITY OF NEVADA RENO
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-04-01 至 2002-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6187982
• 关键词: calcineurin; calcium flux; chemical binding; cytoplasm; dynamin; gene induction /repression; guanosinetriphosphatases; ionic bond; membrane lipids; myristates; phospholipids; phosphoproteins; protein structure function; recombinant proteins; site directed mutagenesis; synaptosomes; tissue /cell culture

The broad, long-term objectives of this project are to characterize the regulation and physiological roles of membrane-associated calcineurin. In brain, the equal distribution of calcineurin between the membrane and cytosol, and regulation of several ion channels and dynamin l by calcineurin implicates membrane-associated calcineurin in their regulation. The calcineurin B subunit is responsible for the Ca2+-dependent binding and activation of calcineurin by membrane phospholipids. A major goal of this project is to test the hypothesis that calcineurin binds to acidic phospholipids by a myristoyl-electrostatic switch composed of the B subunit N- terminal myristic acid and a basic residue cluster in the B subunit. Wild-type and site-directed mutant enzymes will be expressed and purified from bacteria or Sf9 cells. Structure/function studies of Ca2+/phospholipid-bound calcineurin will be carried out to identify the mechanism regulating the interaction between calcineurin and membrane phospholipids. Dynamin I plays a key role in synaptic vesicle recycling. Dynamin I is dephosphorylated and translocates to the plasma membrane during depolarization-induced Ca2+ influx. A primary objective of this project is to test the hypothesis that membrane-associated calcineurin dephosphorylates dynamin I. Using recombinant protein expression techniques, the in vitro phosphatase activity of calcineurin toward purified dynamin l in the presence of Ca2+/phospholipids will be characterized. The subcellular compartment where calcineurin dephosphorylates dynamin I will be identified by,examining calcineurin activity and distribution, and dynamin l dephosphorylation during Ca2+ influx into purified synaptosomes. Calcineurin plays a role in synaptic plasticity, and has been implicated in glutamate neurotoxicity following focal ischemia. In addition, the finding that calcineurin is the target of cyclosporin and FK5O6, the major drugs used to prevent tissue rejection following organ transplantation, indicates the importance of calcineurin in the immune system. The studies from this project will elucidate calcineurin regulation by membrane targeting, and further our understanding of its physiological roles in learning and memory, stroke, and immune function.

该项目的广泛、长期目标是描述 膜相关蛋白的调节及其生理作用 钙调神经磷酸酶。在大脑中，钙调神经磷酸酶在脑内的平均分布 膜和胞浆与几种离子通道的调节 而动力蛋白L通过钙调神经磷酸酶牵连膜相关蛋白 钙调神经磷酸酶在它们的调节中。钙调神经磷酸酶B亚单位是 负责钙离子依赖的结合和激活 钙调神经磷酸酶由膜磷脂组成。这个项目的一个主要目标 是为了检验钙调神经磷酸酶与酸性磷脂结合的假设 通过由B亚基N-组成的肉豆蔻基-静电开关 末端肉豆蔻酸和B亚基中的碱性残基簇。 野生型和定点突变酶将被表达并 从细菌或Sf9细胞中提纯。的结构/功能研究 将进行钙离子/磷脂结合的钙调神经磷酸酶鉴定 钙调神经磷酸酶与钙调神经磷酸酶相互作用的调控机制 膜磷脂。 Dynamin I在突触小泡循环中起着关键作用。动感 I被去磷酸化并转移到质膜上 在去极化诱导的钙内流过程中。的主要目标是 这个项目是为了验证与膜相关的假说 钙调神经磷酸酶利用重组蛋白对动力蛋白I进行脱磷酸化 钙调神经磷酸酶体外表达技术及活性研究 钙/磷脂存在下对纯化动力蛋白L的研究 将被描述为。钙调神经磷酸酶所在的亚细胞室 去磷酸化的动力素I将通过检查鉴定 钙调神经磷酸酶的活性和分布，以及动力蛋白L 钙离子内流到纯化的突触体内时的去磷酸化。 钙调神经磷酸酶在突触可塑性中发挥作用，并一直 与局部缺血后谷氨酸神经毒性有关。在……里面 此外，发现钙调神经磷酸酶是环孢素A和环孢素的靶标 FK5O6，预防术后组织排斥反应的主要药物 表明钙调神经磷酸酶在器官移植中的重要性 免疫系统。这个项目的研究将阐明 钙调神经磷酸酶通过膜靶向调节，并进一步我们的 了解它在学习和记忆中的生理作用， 中风和免疫功能。