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）：钙调神经磷酸酶是一种 Ser/Thr 磷酸酶和 多种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。该项目的假设是 蛋白质-蛋白质相互作用，决定钙调神经磷酸酶底物 特异性和氧化应激（可在体外使钙调神经磷酸酶失活）发挥作用 与 Ca2+ 和钙调蛋白结合，确保适当的钙调神经磷酸酶功能 在完整的细胞中。尽管钙调神经磷酸酶需要 Ca2+ 和钙调蛋白 活动，其他监管机制对于适当指导至关重要 酶并作为活动的制动器。这些的相对重要性 机制是未知的，并且对于 和 之间的相互作用一无所知 整合监管机制。该项目的目标是 确定钙调神经磷酸酶靶向蛋白如何引导酶以及作用于什么 在某种程度上，氧化剂在完整细胞中起到“关闭开关”的作用。为此 用于表达钙调磷酸酶蛋白与荧光融合蛋白的构建体 将制备钙调蛋白指示蛋白（CN/CaM-Ind）。这些 CN/CaM-Ind 将 可用于具有稳定集成的 NFAT 报告基因的细胞，该报告基因可测量 钙调神经磷酸酶活性，以确定细胞定位和钙调蛋白 在各种条件下与钙调神经磷酸酶结合。该项目的目标是： 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 绑定。 3) 确定钙调磷酸酶靶向是否依赖于 Ca2+，以及是否 VP/IT 钙调磷酸酶结合域将钙调磷酸酶活性导向特定 按距离或方向排列的残基。 4) 确定钙调神经磷酸酶是否在 完整的细胞会被氧化剂可逆地抑制，并且如果靶向蛋白质 结合或钙调蛋白依赖性激活会增加对氧化剂的敏感性。 因为钙调神经磷酸酶在 NFAT 信号传导和靶向中起着至关重要的作用 对于该信号级联中的钙调磷酸酶至关重要，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剂。此外，贡献 钙调神经磷酸酶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尽量减少氧化应激 人们会发现氧化剂对健康有不良影响。