TOWARD A PHYSICAL DESCRIPTION OF IMMUNOSUPPRESSION

免疫抑制的物理描述

• 批准号: 3309007
• 负责人: IAN M ARMITAGE
• 金额: $ 22.87万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-08-01 至 1997-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3309007
• 关键词: artificial immunosuppression; biophysics; calcineurin; calcium; calmodulin; chemical kinetics; conformation; crosslink; cyclosporines; enzyme activity; enzyme complex; enzyme mechanism; enzyme structure; enzyme substrate analog; fluorescence; immunosuppressive; molecular cloning; nuclear magnetic resonance spectroscopy; peptidylprolyl isomerase; phosphatase inhibitor; posttranslational modifications; protein isoforms; protein purification; protein structure function; site directed mutagenesis; stop flow technique; thermodynamics

This application, an extension of research derived from a program project grant, has as its focus the structural and functional role of the cyclophilin (CyP) family of proteins. In this rapidly evolving field, we will continue our structural and kinetic characterization of the interaction between the Cyclosporin A (CsA) related ligands as they associate with the CyP family members and calcineurin, currently considered a major target within various cell types. A central focus will be NMR methods coordinated with an assessment of kinetic and thermodynamic parameters of these interactions with stopped-flow fluorescence studies. Human CyP-18, the predominant cytosolic receptor, as well as new CyP-40c species will be provided by isolation from tissues and in recombinant form from E. Coli. We will also prepare and study site-directed mutant forms of these species. In addition, two new membrane bound CyP species, CyP-22m, richly associated with the endoplasmic reticulum and CyP-20, will be available in their native glycosylated state. Recently, it has been shown that the ligand- immunophilin complex associates with calcineurin in a Ca++-dependent reaction, thus a variety of multidimensional metallo-NMR as well as 1H, 13C and 15N techniques are possible. Specifically, 113Cd NMR methods will be used to provide an isomorphic, magnetically active probe of the Ca++ sites as they are involved in the structure and dynamics of these multimeric immunophilin complexes. Additionally, multidimensional NMR methods utilizing 1H, 13C and 19F isotopically labeled CsA derivatives will aid in the identification of specific amino acids located at sites of interaction in the multimeric complex. Independent assessment of kinetic aspects of these interactions will be accomplished by transient state fluorescence measurements using the single tryptophan in CyP which has been shown to sensitively reflect CyP's interaction with ligands such as CsA. This interaction will be assessed by stopped-flow fluorescence studies to develop a complete kinetic and thermodynamic understanding of the CsA-CyP interaction as well as provide a basis for characterizing new CyP proteins and site-directed mutant forms. These studies will also be extended to examine the interaction of calcineurin with the immunophilin complex. Preliminary information obtained by chemical crosslinking has revealed an unexpected topological relationship of the components in the CyP-calcineurin complex. These results will be extended to the substrate site and potential new cellular targets for the CyP-CsA complex may also be examined with these physical techniques. These unique opportunities for integrating different talents are expected not only to define specific amino acid loci of interaction with dimensional information but also serve as a guide for new concepts in drug synthesis and an understanding of the mechanism of action of this new class of immunosuppressive agent.

该应用程序是从计划项目中得出的研究的扩展 格兰特（Grant）的重点是结构和功能的作用 蛋白质家族（CYP）家族。 在这个快速发展的领域， 我们将继续我们的结构和动力学表征 环孢菌素A（CSA）相关的配体之间的相互作用 目前与CYP家族成员和钙调神经酶合作 被认为是各种细胞类型中的主要目标。 中心重点 将是NMR方法，并评估动力学和 与停止流相互作用的热力学参数 荧光研究。 人CYP-18，主要的胞质受体， 以及新的CYP-40C物种将通过与组织分离提供 并以大肠杆菌的重组形式。 我们还将准备和学习 这些物种的位置突变形式。 此外，两个新 膜结合的CYP物种，CYP-22M，与 内质网和CYP-20，将在其本地使用 糖基化状态。 最近，已经表明配体 - 免疫光蛋白复合物与钙调神经蛋白酶在Ca ++中的相关性 反应，因此多种多维金属-NMR以及1H 13C和15N技术是可能的。 具体而言，113CD NMR方法 将用于提供同构的，磁性活性的探针 Ca ++站点参与这些结构和动态 多聚体免疫蛋白复合物。 另外，多维NMR 利用1H，13C和19F同位素标记的CSA衍生物的方法 将有助于识别位于位置的特定氨基酸 多聚体复合物中的相互作用。 独立评估 这些相互作用的动力学方面将通过瞬态完成 使用CYP中的单调的荧光测量 已被证明可以敏感地反映CYP与配体的相互作用 作为CSA。 这种相互作用将通过停止流量荧光进行评估 研究以对对的完全动力学和热力学理解 CSA-CYP互动以及为新的表征提供基础 CYP蛋白质和位置突变体形式。 这些研究也将是 扩展以检查钙调蛋白与免疫蛋白的相互作用 复杂的。 化学交联获得的初步信息已有 揭示了组件中的意外拓扑关系 CYP-钙调蛋白络合物。 这些结果将扩展到基板 CYP-CSA复合物的位点和潜在的新细胞靶 通过这些物理技术进行检查。 预计这些独特的融合人才的机会 不仅要定义特定的氨基酸基因座与 维度信息，但也可以作为新概念的指南 药物合成和对此作用机理的理解 新的免疫抑制剂。