Le Hir M, Su Q, Weber L, Woerly G, Granelli-Piperno A, Ryffel B
Institute of Toxicology, University of Zurich, Schwerzenbach/Zürich, Switzerland.
Lab Invest. 1995 Nov;73(5):727-33.
The immunosuppressant cyclosporin A (CsA) forms a trimolecular complex with cyclophilin (CPH) and calcineurins (CN) and inhibits CN phosphatase activity. Inhibition of CN phosphatase by CsA prevents the dephosphorylation of a nuclear factor in the cytosol and its nuclear translocation to the nucleus.
The intracellular distribution of CPH and CN was investigated in permeabilized Jurkat T lymphocytes and MRC fibroblasts using biochemical techniques and confocal microscopy. The site of CsA binding was identified in situ using a photoaffinity label derivative of CsA followed by immunodetection.
Cyclophilin A (CPH-A) and CN display essentially a cytosolic localization by immunofluorescence, and additional nuclear CPH-A and CN are evidenced by Western blot analysis of purified nuclei and immunofluorescence. By contrast, cyclophilin B (CPH-B) has a punctuate and reticular distribution pattern in cytoplasm, indicating an association with the endoplasmatic reticulum, but its main location is in the nuclear matrix, sparing the nucleolar region. For the in situ detection of CsA binding sites, a photolabile cyclosporine derivative (PL-CS) was used that allowed the detection of covalently bound CsA by Ab. Using the biologically active PL-CS, a punctate cytoplasmatic and nuclear immunoreactivity was obtained, which was specific and competed only with active cyclosporine derivatives. Nuclear CPH-A and -B were labeled by PL-CS, and trimolecular complexes of labeled CPH and CN were obtained by chemically cross-linking nuclear extracts.
We describe herein the accessibility of CsA to the nucleus, the presence and labeling in situ of nuclear CPH and CN. The current models of CsA action predict that CsA-CPH complexes inhibit CN activity in the cytosol. However, our present findings invite the hypothesis that CPH may capture the drug into the nucleus and target regulatory proteins or transcriptional control elements.
免疫抑制剂环孢素A（CsA）与亲环蛋白（CPH）和钙调磷酸酶（CN）形成三聚体复合物，并抑制CN磷酸酶活性。CsA对CN磷酸酶的抑制作用可防止胞质中的一种核因子去磷酸化及其向细胞核的转位。
利用生化技术和共聚焦显微镜研究了经通透处理的Jurkat T淋巴细胞和MRC成纤维细胞中CPH和CN的细胞内分布。使用CsA的光亲和标记衍生物，随后进行免疫检测，原位鉴定CsA结合位点。
通过免疫荧光法，亲环蛋白A（CPH-A）和CN主要定位于胞质，对纯化细胞核的蛋白质免疫印迹分析和免疫荧光法证明细胞核中存在额外的CPH-A和CN。相比之下，亲环蛋白B（CPH-B）在细胞质中呈点状和网状分布模式，表明其与内质网有关联，但其主要定位在核基质中，核仁区域除外。为了原位检测CsA结合位点，使用了一种光不稳定的环孢素衍生物（PL-CS），其可通过抗体检测共价结合的CsA。使用具有生物活性的PL-CS，可获得点状的细胞质和细胞核免疫反应性，该反应具有特异性，且仅与活性环孢素衍生物竞争。PL-CS标记了细胞核中的CPH-A和 -B，通过化学交联核提取物获得了标记的CPH和CN的三聚体复合物。
我们在此描述了CsA进入细胞核的可及性、细胞核CPH和CN的存在及原位标记。目前的CsA作用模型预测CsA-CPH复合物在胞质中抑制CN活性。然而，我们目前的研究结果提出了一个假设，即CPH可能将药物捕获到细胞核中，并靶向调节蛋白或转录控制元件。
