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TOWARD A PHYSICAL DESCRIPTION OF IMMUNOSUPPRESSION

免疫抑制的物理描述

基本信息

批准号：
2187413
负责人：
IAN M ARMITAGE
金额：
$ 23.3万
依托单位：
UNIVERSITY OF MINNESOTA
依托单位国家：
美国
项目类别：
财政年份：
1993
资助国家：
美国
起止时间：
1993-08-01 至 2000-02-29
项目状态：
已结题

项目摘要

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.

该应用程序是一个项目研究的延伸赠款的重点是结构和功能作用亲环蛋白 (CyP) 家族蛋白质。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) 相关配体之间的相互作用与 CyP 家族成员和钙调磷酸酶相关，目前被认为是各种细胞类型中的主要靶标。一个中心焦点将核磁共振方法与动力学评估相协调这些与停流相互作用的热力学参数荧光研究。人类 CyP-18，主要的胞质受体，以及新的 CyP-40c 物种将从组织中分离出来以及来自大肠杆菌的重组形式。我们也会准备和学习这些物种的定点突变形式。此外，还有两个新膜结合的 CyP 物种，CyP-22m，与内质网和 CyP-20，将以它们的天然形式提供糖基化状态。最近，研究表明配体亲免素复合物与 Ca++ 依赖的钙调神经磷酸酶相关反应，因此有多种多维金属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活性探针 Ca++ 位点，因为它们参与这些结构和动态多聚亲免素复合物。此外，多维核磁共振利用 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点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来完成使用 CyP 中的单一色氨酸进行状态荧光测量，已被证明能够灵敏地反映 CyP 与配体的相互作用，例如作为环孢素A。这种相互作用将通过停流荧光进行评估研究以形成完整的动力学和热力学理解 CsA-CyP 相互作用，并为表征新的 CyP 蛋白和定点突变形式。这些研究也将扩展到检查钙调神经磷酸酶与亲免素的相互作用复杂的。通过化学交联获得的初步信息揭示了组件中意想不到的拓扑关系 CyP-钙调磷酸酶复合物。这些结果将扩展到基材 CyP-CsA 复合物的位点和潜在的新细胞靶标也可能用这些物理技术进行检查。预计这些独特的机会可以整合不同的人才不仅要定义与相互作用的特定氨基酸位点维度信息也可以作为新概念的指南药物合成及其作用机制的理解新型免疫抑制剂。

项目成果

期刊论文数量（8）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Impact of angiotensin-converting enzyme substrate conformation on fractional hydrolysis in lung.

血管紧张素转换酶底物构象对肺中部分水解的影响。

DOI：
10.1152/ajplung.1996.270.2.l251
发表时间：
1996
期刊：
The American journal of physiology
影响因子：
0
作者：
Merker,MP;Armitage,IM;Audi,SH;Kakalis,LT;Linehan,JH;Maehl,JR;Roerig,DL;Dawson,CA
通讯作者：
Dawson,CA

Isolation, cDNA sequences, and biochemical characterization of the major cyclosporin-binding proteins of Toxoplasma gondii.

弓形虫主要环孢菌素结合蛋白的分离、cDNA 序列和生化特征。