Regulation of Calcineurin

说明(申请人提供)：钙调神经磷酸酶是一种丝氨酸/苏氨酸磷酸酶，是一种 几个信号转换级联的初级调节器，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 决定钙调神经磷酸酶底物的蛋白质-蛋白质相互作用 特异性和氧化应激，在体外使钙调神经磷酸酶失活 结合钙离子和钙调蛋白确保正常的钙调神经磷酸酶功能 在完好无损的细胞中。尽管钙调神经磷酸酶需要钙离子和钙调素 除了活动之外，其他监管机制对于正确引导 酶，并对活动起到刹车作用。这些因素的相对重要性 机制未知，对和之间的相互作用也一无所知 整合监管机制。这个项目的目标是 确定钙调神经磷酸酶靶向蛋白如何引导酶以及导向什么 在完好的细胞中，范围氧化剂起到“关闭开关”的作用。为此， 钙调神经磷酸酶融合荧光蛋白表达载体的构建 将制备钙调素指示蛋白(CN/CaM-Ind)。这些Cn/Cam-Ind将 用于具有稳定集成的NFAT报告程序的单元中，该报告程序可测量 钙调神经磷酸酶的活性，以确定细胞定位和钙调素 在不同条件下与钙调神经磷酸酶结合。该项目的目标是：1) 制作并测试CN/Cam-IND系统。2)确定CN-A的作用 酶激活中的氨基末端、CN-A/CN-B相互作用和NFAT底物 有约束力的。3)确定钙调神经磷酸酶靶向是否依赖于钙离子，以及 VP/IT钙调神经磷酸酶结合结构域将钙调神经磷酸酶活性导向特定的 残留物由距离或方向决定。4)确定钙调神经磷酸酶在 完整的细胞被氧化剂和靶向蛋白可逆地抑制 结合或钙调蛋白依赖的激活增加了对氧化剂的敏感性。 因为钙调神经磷酸酶在NFAT信号转导和靶向中起着至关重要的作用 是钙调神经磷酸酶在信号级联中所必需的，这些结果 实验将使我们能够设计底物特异性钙调神经磷酸酶抑制剂 这将是更好的免疫抑制药。此外，贡献 钙调神经磷酸酶的活性对生理和病理反应的影响 将定义氧化应激，并制定干预策略，以将 将会发现氧化剂对健康的不利影响。

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.