MECHANISMS OF IMMUNOSUPPRESSION BY FK506

新型免疫抑制剂FK 506及其结构类似物 雷帕霉素抑制不同的信号转导途径， 与称为FK 506结合的细胞内受体家族结合 蛋白质或FKBP。 FKBP 12（以前称为FKBP）是主要的 T细胞中的细胞受体。 FK 506-FKBP 12复合物，如CsA-CyP 复合物，已被证明结合并抑制，在体外， 钙调神经磷酸酶（Cn），一种Ca+2-钙调蛋白依赖性丝氨酸/苏氨酸 磷酸酶。 我们已经证实FK 506和CsA，而不是雷帕霉素， 抑制体内钙调磷酸酶活性，现在建议研究其作用 钙调神经磷酸酶在T细胞活化中的作用 此外，我们还克隆了 表征了新型FK 506和雷帕霉素受体。 我们建议： 1.分析钙调神经磷酸酶在T细胞活化中的作用。 钙调 蛋白质水平将与生物活性和能力相关， 用药物抑制 2.表征FKBP多基因家族。 我们已经确定 FKBP 12表达缺陷的功能活性细胞系 和FKBP 13。 这些细胞系将用野生型转染， 突变的FKBP 12和FKBP 13，并研究它们的功能。 的 还将研究FK 506和雷帕霉素抑制功能的能力。 FKBP 12是否是T细胞抑制的相关受体将被确定。 解决，并将进行有限数量的信息突变， 确认预测的药物结合口袋。 亚细胞定位 将研究每种克隆的FKBP的生物学作用， 的受体。 3.设计实验以使用以下方法分离雷帕霉素-FKBP靶蛋白： FKBP 12和FKBP 25，蛋白质-蛋白质存在的测定 FKBPs和其他细胞蛋白之间的相互作用， 细胞内的推定靶蛋白。 我们将尝试 免疫沉淀结合到细胞表面的内源性蛋白质或肽。 免疫亲和素 雷帕霉素在上游调控中的作用 将研究p70 S6激酶的激活剂。 对这些蛋白质及其机制有更深入的生物学了解 FK 506和雷帕霉素的抑制作用可能允许合理设计 具有免疫抑制活性但毒性有限的药剂，或药剂 调节特定的信号通路。 这种方法最终可能 导致设计用于同种异体移植物的激动剂或抑制剂 移植，也可能用于自身免疫性疾病。

A novel immunosuppressive agent, FK506 and its structural analog rapamycin inhibit different signal transduction pathways yet both agents bind to the same family of intracellular receptors termed FK506-binding proteins, or FKBPs. FKBP12 (previously termed FKBP) is the predominant cellular receptor in T cells. The FK506-FKBP12 complex, like the CsA-CyP complex, has been shown to bind to and inhibit, in vitro, the activity of calcineurin (Cn), a Ca+2-calmodulin dependent serine/threonine phosphatase. We have confirmed that FK506 and CsA, but not rapamycin, inhibit calcineurin activity in vivo, and now propose to examine the role of calcineurin in T cell activation. In addition, we have cloned and characterized novel FK506 and rapamycin receptors. We propose to: 1. Analyze the role of calcineurin in T cell activation. Calcineurin protein levels will be correlated with biologic activity and with ability to inhibit with drug. 2. Characterize the FKBP multigene family. We have identified functionally active cell lines deficient in their expression of FKBP12 and of FKBP13. These cell lines will be transfected with wild type and mutated FKBP12 and FKBP13, and their function will be studied. The ability of FK506 and rapamycin to inhibit function will also be studied. Whether FKBP12 is the relevant receptor for T cell inhibition will be addressed, and a limited number of informative mutations will be made to confirm the predicted drug binding pocket. The subcellular localization of each of the cloned FKBPs will be studied, as will the biological role of the receptors. 3. Design experiments to isolate the rapamycin-FKBP target protein using both FKBP12 and FKBP25, assay for the presence of protein-protein interactions between the FKBPs and other cellular proteins, and isolate putative target proteins within the cell. We will attempt to immunoprecipitate endogenous proteins or peptides bound to the immunophilins. The role of rapamycin in regulating the upstream activators of p70 S6 kinases will be studied. A greater biological understanding of these proteins and of the mechanism of inhibition by FK506 and by rapamycin may allow rational design of agents with immunosuppressive activity but limited toxicity, or of agents that modulate specific signalling pathways. This approach may ultimately lead to the design of agonists or inhibitors for use in allograft transplantation, and possibly in autoimmune disorders.