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: 2184346
• 负责人: FRANK M RUSNAK
• 金额: $ 15.91万
• 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/2184346
• 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 独特且独立，重点关注一种常见的酶，钙调神经磷酸酶。 上一 我们想了解钙调神经磷酸酶的酶促机制 重点是金属离子如何促进磷酸酯水解。 从 从生物医学的角度来看，我们对钙调神经磷酸酶的作用感兴趣 在 T 细胞中，已知它是 T 细胞之间的重要组成部分 IL-2 基因的抗原刺激和转录。 更远 阐明钙调神经磷酸酶在 Ca2+ 依赖性信号中的作用 转导可以帮助我们更好地理解细胞的调控 生长和分化。 作为水解蛋白质的磷酸酶家族的成员 丝氨酸/苏氨酸磷酸酯钙调神经磷酸酶的特点是 Ca2+ 和钙调蛋白依赖。 此外，钙调磷酸酶是一种 金属蛋白；紧密结合的 Fe 和 Zn 以化学计量存在 需要一定量的二价金属离子，例如 Mn2+ 或 Ni2+ 最大活动量。 我们有兴趣了解这些金属如何 发挥催化机制并利用电子顺磁性 共振光谱研究电子和磁 这些顺磁中心的特性。 这些研究将提供 了解金属离子催化细节的框架 磷酸酯在此类蛋白磷酸酶中水解。 我们小组的另一个研究目标是钙调神经磷酸酶的研究 在 T 细胞信号转导途径中。 直到最近，角色 人们对体内钙调磷酸酶知之甚少，主要是因为它是 普遍分布的特异性抑制剂 还没有用于在体内选择性研究它的方法。 最近，它 已表明钙调神经磷酸酶是强大的目标 免疫抑制剂、环孢素 A (CsA) 和 FK506、给予以下药物 移植患者预防器官排斥反应。 这些药物的作用是通过 防止分泌 IL-2 的 T 细胞活化并引起细胞 免疫系统增殖并被激活。 在 T 细胞中， 这些药物与受体结合，它是两者之间的复合物 免疫抑制剂和结合并抑制钙调神经磷酸酶的受体。 我们正在使用天然酶和重组酶来了解 免疫抑制剂抑制钙调神经磷酸酶的机制。 本国的 酶的用量非常丰富（每次制剂 20-30 毫克） 光谱研究，我们现在手头有重组来源 钙调神经磷酸酶模拟了大多数牛脑中的天然酶 尊重。 最重要的是，重组酶对 CsA/亲环蛋白，可用于研究其分子细节 钙调神经磷酸酶和该药物/受体复合物之间的相互作用。 研究 正在使用核磁共振波谱来绘制表面 CsA/亲环蛋白复合物与钙调磷酸酶相互作用。 这些 实验有望提供有关分子的信息 该抑制复合物表面的结构相互作用 与钙调磷酸酶，将为未来提供蓝图 免疫抑制剂药物设计。