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: 2518977
• 负责人: FRANK M RUSNAK
• 金额: $ 18.17万
• 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 2000-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2518977
• 关键词: Escherichia coli; FK506; Mossbauer spectrometry; X ray crystallography; active sites; calcineurin; calcium binding protein; cyclosporines; electron nuclear double resonance spectroscopy; electron spin resonance spectroscopy; enzyme activity; enzyme mechanism; metalloenzyme; nuclear magnetic resonance spectroscopy; peptidylprolyl isomerase; phosphatase inhibitor; protein purification; recombinant proteins; tissue /cell culture

DESCRIPTION: The objectives of the study are to understand the molecular mechanisms of catalysis, regulation, and inhibition of the serine/threonine protein phosphatases calcineurin and protein phosphatase 1 (PP1). Since little is known regarding the mechanism of phosphate ester hydrolysis of these enzymes, the long term goals of this study will be to arrive at a model for the catalytic mechanism which can be tested by biochemical and spectroscopic approaches. The broad objectives are: 1) to characterize the binuclear metal centers of calcineurin and PP1 and determine their role in catalysis; 2) to understand the structural basis for divalent metal ion activation of these phosphatases; 3) to examine the role(s) of specific amino acid residues surrounding the binuclear metal centers; 4) to investigate the inactivation of calcineurin by mechanism-based inhibitors; and 5) to understand the structural basis for calcineurin inhibition by cyclosporin A (CsA)-cyclophilin complexes. The above objectives will be accomplished by using a variety of techniques including EPR, Mossbauer, NMR, ENDOR, and ESEEM spectroscopies. In addition, metal analyses and determinations of enzyme activity of calcineurin and PP1 in various states will be carried out in order to determine whether a correlation exists between enzyme activity and redox state of the bound metal ions. These spectroscopic techniques should also be able to resolve discrepancies between current active site models of calcineurin and PP1, and will provide additional handles for uncovering aspects regarding the mechanism of catalysis. X-ray crystallography will be applied to understand the similarities and differences of calcineurin inhibition by CsA-cyclophilin and FK506-FKBP complexes. Since calcineurin is the target of the immunosuppressant drugs cyclosporin A and FK506, a knowledge of the mechanism of CsA-cyclophilin inhibition and details relating to the mechanism of catalysis may assist in the future design of novel immunosuppressive agents with decreased toxicity. The continued study of calcineurin inactivation by mechanism-based inactivators will provide information about the role of active site residues and can assist in the design of additional structural elements for increased inhibitor potency.

描述：研究的目标是了解分子 丝氨酸/苏氨酸的催化，调节和抑制机制 蛋白质磷酸酶钙调蛋白和蛋白质磷酸酶1（PP1）。 自从 关于磷酸酯水解的机理知之甚少 这些酶，这项研究的长期目标将是到达 催化机制的模型，可以通过生化和 光谱法。 广泛的目标是：1）表征 钙调神经酶和PP1的双核金属中心，并确定它们在 催化; 2）了解二价金属离子的结构基础 这些磷酸酶的激活； 3）检查特定的作用 双核金属中心周围的氨基酸残基； 4）到 通过基于机制的抑制剂研究钙调神经酶的失活； 5）了解通过 环孢菌素A（CSA） - 环酚蛋白复合物。 以上目标将是 通过使用包括EPR，Mossbauer，NMR等各种技术来完成 Endor和ESEEM光谱。 此外，金属分析和 确定钙调神经酶和PP1在各种状态的酶活性 将进行以确定是否存在相关性 酶活性和结合金属离子的氧化还原状态之间。 这些 光谱技术也应该能够解决差异 在当前的钙调神经酶和PP1的活动现场模型之间，将提供 其他方面，以揭示有关机制的方面 催化。 X射线晶体学将应用于了解 CSA-周期性蛋白的钙调神经素抑制作用的相似性和差异 和FK506-FKBP复合物。 由于钙调蛋白是 免疫抑制药物环孢菌素A和FK506，对 CSA-环素抑制的机制以及与 催化机制可以帮助未来的新颖设计 免疫抑制剂毒性降低。 继续研究 通过基于机制的灭活剂将钙调神经酶灭活将提供 有关活动现场残留作用的信息，可以协助 设计其他结构元素，以增加抑制剂效力。