No abstract

The immunosuppressive effects of the fungal metabolite cyclosporin A (CsA) are mediated primarily by binding to cyclophilins (Cyps). The resulting CsA-Cyp complex inhibits the Ca2+-regulated protein phosphatase calcineurin and down-regulates signal transduction events. Previously we reported that CsA is a potent inhibitor of infections transmitted by the human pathogenic protozoan parasite Leishmania major in vitro and in vivo, but does not effect the extracellular growth of L. major itself. It is unknown how L. major exerts this resistance to CsA. Here we report that a major Cyp, besides additional isoforms with the same N-terminal amino acid sequence, was expressed in L. major. The cloned and sequenced gene encodes a putative 174-residue protein called L. major Cyp 19 (LmCyp19). The recombinant LmCyp19 exhibits peptidyl-prolyl cis/trans isomerase activity with a substrate specificity and an inhibition by CsA that are characteristic of other eukaryotic Cyps. To determine whether calcineurin is involved in the discrimination of the effects of CsA we also examined the presence of a parasitic calcineurin and tested the interaction with Cyps. Despite the expression of functionally active calcineurin by L. major, neither LmCyp19 nor other L. major Cyps bound to its own or mammalian calcineurin. The amino acid sequence of most Cyps includes an essential arginine residue around the calcineurin-docking side. In LmCyp19 this is replaced by an asparagine residue. This exchange and additional charged residues are apparently responsible for the lack of LmCyp19 interaction with calcineurin. These observations indicate that resistance of L. major to CsA in vitro is mediated by the lack of complex formation with calcineurin despite CsA binding by parasitic Cyp.

Tissue factor (TF), the primary initiator of blood coagulation with structural homology to the cytokine receptor family, has been implicated in various vascular processes including metastasis, angiogenesis, and atherosclerosis. Within the vasculature, monocytes and endothelial cells (EC) can be activated to synthesize TF depending on the induction of NF-κB. Despite the undisputed value of cyclosporin A (CsA) as an immunosuppressant, problems have emerged due to induction of vascular changes by a poorly understood mechanism. We demonstrate that CsA has opposite effects on TF gene expression, inhibiting NF-κB-mediated TF gene transcription in monocytes but enhancing it in EC. To test whether CsA binding proteins (cyclophilins) can mediate these CsA effects we used a nonimmunosuppressant analog of CsA that binds to cyclophilins but does not inhibit the Ca2+/calmodulin-dependent phosphatase calcineurin (Cn). This drug lacked regulatory function for NF-κB and TF expression suggesting that Cn is responsible for the inverse gene regulation. The key function of Cn was supported by experiments demonstrating that other phosphatase inhibitors also either positively or negatively regulated NF-κB in monocytes and EC. Calcineurin was demonstrated to regulate NF-κB activation at the level of IκBα degradation, because agonist-induced phosphorylation and subsequent degradation of IκBα is prevented by Cn inhibitors in monocytes but enhanced in EC. These data identify Cn as an opposite regulator in generating transcriptionally active NF-κB, and they confirm the presumption that the ability of Cn to participate in NF-κB transactivation is not T cell specific.

A screening procedure for protein-protein interactions in cellular extracts using a green fluorescent protein (GFP) and affinity capillary electrophoresis (ACE) was established. GFP was fused as a fluorescent indicator to the C-terminus of a cyclophilin (rDmCyp20) from Drosophila melanogaster. Cyclophilins (Cyps) belong to the ubiquitously distributed enzyme family of peptidyl-prolyl cis/trans isomerases (PPlases) and are well known as cellular targets of the immunosuppressive drug cyclosporin A (CsA). The PPlase activity of the GFP fused rDmCyp20 as well as the high affinity to CsA remain intact. Using native gel electrophoresis and ACE mobility-shift assays, it was demonstrated that the known moderate affinity of Cyp20 to the capsid protein p24 of HIV-1 was detectable in the case of rDmCyp20 fused to the fluorescent tag. For the p24 / rDmCyp20-GFP binding an ACE method was established which allowed to determine a dissociation constant of Kd = 20+/-1.5 x 10(-6) M. This result was verified by size-exclusion chromatography and is in good agreement with published data for the nonfused protein. Moreover the fusion protein was utilized to screen rDmCyp20-protein interactions by capillary electrophoresis in biological matrices. A putative ligand of rDmCyp20 in crude extracts of embryonic D. melanogaster was discovered by mobility-shift assays using native gel electrophoresis with fluorescence imaging and ACE with laser-induced fluorescence detection. The approach seems applicable to a wide range of proteins and offers new opportunities to screen for moderate protein-protein interactions in biological samples.