Regulation of Calcineurin

钙调神经磷酸酶的调节

• 批准号: 6580833
• 负责人: PAUL M STEMMER
• 金额: $ 31.32万
• 依托单位: WAYNE STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-01-07 至 2005-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6580833
• 关键词: biological signal transduction; calcineurin; calcium; calmodulin; chemical kinetics; chimeric proteins; confocal scanning microscopy; enzyme induction /repression; enzyme inhibitors; enzyme mechanism; enzyme model; enzyme substrate complex; fluorescent dye /probe; intermolecular interaction; model design /development; molecular genetics; molecular site; oxidative stress; oxidizing agents; protein engineering; protein localization; protein structure function; radiotracer; scintillation counter; site directed mutagenesis; tissue /cell culture

DESCRIPTION (provided by applicant): Calcineurin is a Ser/Thr phosphatase and a primary regulator for several signal transduction cascades, including those involved in immune cell activation, skeletal muscle development, memory formation, and cardiac muscle hypertrophy. The hypothesis for this project is that protein-protein interactions, which determine calcineurin substrate specificity, and oxidative stress, which inactivates calcineurin in vitro, act in conjunction with Ca2+ and calmodulin to ensure proper calcineurin function in intact cells. Although Ca2+ and calmodulin are required for calcineurin activity, other regulatory mechanisms are essential for proper direction of the enzyme and to act as a brake on activity. The relative importance of these mechanisms is unknown and nothing is known about interactions among and integration of the regulatory mechanisms. The objective of this project is to determine how calcineurin targeting proteins direct the enzyme and to what extent oxidants function as an "off switch" in intact cells. To this end constructs for expression of calcineurin proteins as fusions with fluorescent calmodulin indicator proteins (CN/CaM-Ind) will be made. These CN/CaM-Ind will be used in cells with a stably integrated NFAT reporter, which measures calcineurin activity, to determine the cellular localization and calmodulin binding to calcineurin under various conditions. Aims of the project are: 1) To produce and test the CN/CaM-Ind system. 2) To determine the role of the CN-A amino terminus in enzyme activation, CN-A/CN-B interaction and NFAT substrate binding. 3) To determine if calcineurin targeting is Ca2+-dependent and if the VP/IT calcineurin binding domain directs calcineurin activity toward specific residues either by distance or direction. 4) To determine if calcineurin in intact cells is reversibly inhibited by oxidants and if targeting protein binding or calmodulin-dependent activation increase susceptibility to oxidants. Because calcineurin plays such a pivotal role in NFAT signaling and targeting is essential for calcineurin in that signaling cascade, the results of these experiments will enable us to design substrate specific calcineurin inhibitors which would be superior immunosuppressive agents. In addition, the contribution of calcineurin activity to the physiological and pathological responses to oxidative stress will be defined and interventive strategies to minimize the adverse health effects of oxidants will be found.

描述（由申请人提供）：钙调神经酶是Ser/Thr磷酸酶和A 用于几个信号转导级联的主要调节器，包括 参与免疫细胞激活，骨骼肌发育，记忆 形成和心肌肥大。这个项目的假设是 蛋白质 - 蛋白质相互作用，该蛋白质相互作用决定钙调神经酶底物 特异性和氧化应激，使钙调神经蛋白在体外失活，作用 结合Ca2+和钙调蛋白，以确保适当的钙调神经功能 在完整的细胞中。虽然钙调蛋白需要Ca2+和钙调蛋白 活动，其他调节机制对于正确的方向至关重要 酶并充当活动的制动。这些的相对重要性 机制是未知的，对之间的互动一无所知 调节机制的整合。该项目的目的是 确定钙调神经蛋白靶向蛋白如何将酶引导到什么 范围氧化剂充当完整细胞中的“关闭开关”。为此 表达钙调神经蛋白作为荧光融合的构建体 将制作钙调蛋白指示剂蛋白（CN/CAM-IND）。这些CN/CAM-IND将 在具有稳定集成的NFAT报告基因的细胞中使用，该记者测量 钙调蛋白活性，确定细胞定位和钙调蛋白 在各种条件下与钙调神经酶结合。该项目的目的是：1） 生产并测试CN/CAM-IND系统。 2）确定CN-A的作用 酶激活，CN-A/CN-B相互作用和NFAT底物中的氨基末端 结合。 3）确定钙调神经酶靶向是否为Ca2+依赖性，以及是否是否 VP/IT钙调神经蛋白结合结构域将钙调蛋白活性引向特定 残留距离或方向。 4）确定是否在 完整的细胞被氧化剂可逆抑制，如果靶向蛋白 结合或钙调蛋白依赖性激活会增加对氧化剂的敏感性。 因为钙调蛋白在NFAT信号和靶向中起着如此关键的作用 对于钙调神经酶在该信号级联反应是必不可少的，这些结果的结果 实验将使我们能够设计底物特定的钙调神经蛋白抑制剂 这将是优越的免疫抑制剂。另外，贡献 对生理和病理反应的钙调神经酶活性 将定义氧化应激和干预策略，以最大程度地减少 将发现氧化剂的不良健康影响。