CALCINEURIN REGULATION BY MEMBRANE PHOSPHOLIPIDS

膜磷脂对钙调磷酸酶的调节

• 批准号: 2039036
• 负责人: Brian A Perrino
• 金额: $ 9.47万
• 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/2039036
• 关键词: calcineurin; calcium flux; chemical binding; cytoplasm; 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.

该项目的广泛和长期目标是描述 膜相关蛋白的调节和生理作用 钙调神经磷酸酶在大脑中，钙调神经磷酸酶在 膜和胞质溶胶，以及几种离子通道的调节 和发动蛋白l与钙调神经磷酸酶关联 钙调神经磷酸酶的调节作用。钙调神经磷酸酶B亚基是 负责Ca 2+依赖性结合和激活 钙调磷酸酶通过膜磷脂。这个项目的主要目标 是检验钙调磷酸酶与酸性磷脂结合的假设 通过由B亚基N- 末端肉豆蔻酸和B亚基中的碱性残基簇。 将表达野生型和定点突变酶， 从细菌或Sf 9细胞中纯化。结构/功能研究 将进行Ca 2 +/磷脂结合的钙调神经磷酸酶， 调节钙调神经磷酸酶与 膜磷脂 发动蛋白I在突触囊泡循环中起关键作用。发动蛋白 I被去磷酸化并移位到质膜 在去极化诱导的Ca 2+内流期间。的主要目的 这个项目是为了验证假设，膜相关的 钙调神经磷酸酶使发动蛋白I脱磷酸化。利用重组蛋白 表达技术，钙调神经磷酸酶的体外磷酸酶活性， 在Ca 2 +/磷脂存在下， 将被定性。钙调神经磷酸酶的亚细胞区室 去磷酸化发动蛋白I将通过检查 钙调神经磷酸酶活性和分布，动力蛋白l 在Ca 2+流入纯化的突触体期间脱磷酸化。 钙调神经磷酸酶在突触可塑性中起作用， 与局灶性缺血后谷氨酸神经毒性有关。在 此外，钙调神经磷酸酶是环孢菌素的靶点， FK 5 O 6是用于预防组织排斥反应的主要药物， 器官移植，表明钙调神经磷酸酶的重要性， 免疫系统该项目的研究将阐明 通过膜靶向调节钙调神经磷酸酶，并进一步我们 了解它在学习和记忆中的生理作用， 中风和免疫功能