MOLECULAR STUDIES OF THE PROTEIN PHOSPHATASE CALCINEURIN

蛋白质磷酸酶钙调磷酸酶的分子研究

• 批准号: 2184347
• 负责人: FRANK M RUSNAK
• 金额: $ 16.55万
• 依托单位: MAYO CLINIC ROCHESTER
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-09-01 至 1996-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2184347
• 关键词: FK506; X ray crystallography; active sites; calcineurin; cyclosporines; enzyme activity; enzyme complex; enzyme inhibitors; enzyme mechanism; iron; manganese; metalloenzyme; molecular biology; nuclear magnetic resonance spectroscopy; peptidylprolyl isomerase; protein purification; protein sequence; recombinant proteins; tissue /cell culture; transfection /expression vector; zinc

The research in our lab has two long term objectives which, although distinct and separate, focus on a common enzyme, calcineurin. On one hand we would like to understand the enzymatic mechanism of calcineurin with emphasis on how metal ions promote phosphate ester hydrolysis. From a biomedical perspective, we are interested in the role of calcineurin in T-cells where it is known to be an important component between antigenic stimulation and transcription of the IL-2 gene. Further elucidation of the role of calcineurin in Ca2+-dependent signal transduction may provide a better understanding of the regulation of cell growth and differentiation. As a member of a family of phosphatases which hydrolyze protein serine/threonine phosphate esters calcineurin is distinguished by its Ca2+ and calmodulin dependence. In addition, calcineurin is a metalloprotein; tightly bound Fe and Zn are present in stoichiometric amounts and a divalent metal ion such as Mn2+, or Ni2+ are required for maximal activity. We are interested in understanding how these metals function int he catalytic mechanism and are using electron paramagnetic resonance spectroscopy to investigate the electronic and magnetic properties of these paramagnetic centers. These studies will provide a framework by which to understand the details of how metal ions catalyze phosphate ester hydrolysis in this class of protein phosphatases. The other objective of research in our group is a study of calcineurin in T-cell signal transduction pathways. Until recently, the role of calcineurin in vivo has been poorly understood, primarily because it is ubiquitously distributed and specific inhibitors for it that could be used to selectively study it in vivo were not available. Recently, it has been shown that calcineurin is the target of the powerful immunosuppressant drugs, cyclosporin A (CsA) and FK506, drugs given to transplant patients to prevent organ rejection. These drugs function by preventing the activation of T-cells which secrete IL-2 and cause cells of the immune system to proliferate and become activated. In the T-cell, these drugs bind to receptors and it is the complex between immunosuppressant and receptor which binds to and inhibits calcineurin. We are using both native and recombinant enzyme to understand the mechanisms by which the immunosuppressants inhibit calcineurin. Native enzyme is available in abundant amounts (20-30 mg per prep) for spectroscopic studies and we now have in hand a source of recombinant calcineurin which models the native enzyme from bovine brain in most respects. Most importantly, the recombinant enzyme is sensitive to CsA/cyclophilin and can be used to study the molecular details of the interaction between calcineurin and this drug/receptor complex. Studies are in progress using NMR spectroscopy, to map the surface of the CsA/cyclophilin complex which interacts with calcineurin. These experiments will hopefully provide information about the molecular architecture at the surface of this inhibitory complex which interacts with calcineurin and will provide a blueprint for future immunosuppressant drug design.

我们实验室的研究有两个长期目标，尽管 独特而分开的，专注于常见酶，钙调神经蛋白。 一个 我们想了解钙调神经素的酶促机制 重点是金属离子如何促进磷酸酯水解。 从 生物医学的观点，我们对钙调神经素的作用感兴趣 在T细胞中，已知它是重要组成部分 IL-2基因的抗原刺激和转录。 更远 阐明钙调蛋白在Ca2+依赖性信号中的作用 转导可能可以更好地理解细胞的调节 生长与分化。 作为水解蛋白的磷酸酶家族的成员 丝氨酸/苏氨酸磷酸酯钙调神经蛋白的区别 Ca2+和钙调蛋白依赖性。 此外，钙调蛋白是一个 金属蛋白；紧密绑定的Fe和Zn存在于化学计量中 数量和二价金属离子（例如Mn2+或Ni2+）是必需的 最大活动。 我们有兴趣了解这些金属如何 功能在催化机制中，正在使用电子顺磁性 共振光谱研究电子和磁性 这些顺磁中心的特性。 这些研究将提供 框架以了解金属离子如何催化的细节 这类蛋白质磷酸酶中的磷酸酯水解。 我们小组研究的另一个目的是钙调神经酶的研究 在T细胞信号转导途径中。 直到最近， 体内钙调神经酶的理解很少，主要是因为它是 无处不在的分布和特定抑制剂可能是 用于在体内有选择性研究的情况下不可用。 最近，它 已证明钙调神经蛋白是功能的靶标 免疫抑制剂药物，环孢菌素A（CSA）和FK506，药物 移植患者以防止器官排斥。 这些药物通过 防止分泌IL-2并引起细胞的T细胞的激活 免疫系统的增殖和激活。 在T细胞中， 这些药物与受体结合，这是 免疫抑制剂和受体结合并抑制钙调蛋白。 我们正在使用天然和重组酶来了解 免疫抑制剂抑制钙调蛋白的机制。 本国的 酶有大量（每次准备20-30毫克）可用于 光谱研究，我们现在拥有重组的来源 钙调神经酶在大多数牛大脑中对天然酶进行建模 尊重。 最重要的是，重组酶对 CSA/环磷脂，可用于研究 钙调蛋白与该药物/受体复合物之间的相互作用。 研究 使用NMR光谱进行了进行，以绘制 与钙调蛋白相互作用的CSA/环磷脂复合物。 这些 实验将有望提供有关分子的信息 这种抑制性复合物的表面建筑 使用钙调蛋白，将为未来提供蓝图 免疫抑制剂药物设计。