Human and Escherichia coli cyclophilins: sensitivity to inhibition by the immunosuppressant cyclosporin A correlates with a specific tryptophan residue

The human T-cell protein cyclophilin shows high affinity for and is the proposed target of the major immunosuppressant drug cyclosporin A (CsA). Cyclophilin also has peptidyl prolyl cis-trans isomerase activity that is inhibited by CsA with an IC50 of 6 nM, while by contrast a homologous PPIase from Escherichia coli has been found to be much less sensitive to CsA, shown here to be 500-fold less potent at an IC50 of 3000 nM. This E. coli rotamase lacks the single highly conserved tryptophan residue of eukaryotic cyclophilins, and we show here that mutation of the natural F112 to W112 enhances E. coli rotamase susceptibility to CsA inhibition by 23-fold. Correspondingly, the human W121 mutations to F121 or A121 yield cyclophilins with 75- and 200-fold decreased sensitivity to CsA, while kcat/Km values of rotamase activity in a tetrapeptide assay drop only 2- and 13-fold, respectively. This complementary gain and loss of CsA sensitivity to mutation to or from tryptophan validate the indole side chain as a major determinant in immunosuppressant drug recognition and the separation of PPIase catalytic efficiency from CsA affinity.     

A fluorescence polarization-based assay for peptidyl prolyl cis/trans isomerase cyclophilin A

Peptidyl prolyl cis/trans isomerase cyclophilin A (CypA) serves as a cellular receptor for the important immunosuppressant drug, cyclosporin A. In addition, CypA and its enzyme family have been found to play critical roles in a variety of biological processes, including protein trafficking, HIV and HCV infection/replication, and Ca(2+)-mediated intracellular signaling. For these reasons, cyclophilins have emerged as potential drug targets for several diseases. Therefore, it is extremely important to screen for novel small molecule cyclophilin inhibitors. Unfortunately, the biochemical assays reported so far are not adaptable to a high-throughput screening format. Here, we report a fluorescence polarization-based assay for human CypA that can be adapted to high-throughput screening for drug discovery. The technique is based on competition and uses a fluorescein-labeled cyclosporin A analog and purified human CypA to quantitatively measure the binding capacity of unlabeled inhibitors. Detection by fluorescence polarization allows real-time measurement of binding ratios without separation steps. The results obtained demonstrated significant correlation among assay procedures, suggesting that the application of fluorescence polarization in combination with CypA is highly advantageous for the accurate assessment of inhibitor binding.     

Parasite cyclophilins and antiparasite activity of cyclosporin A

Cyclosporin A (CsA) was initially developed as an immunosuppressive drug. In the past several years, it has been shown to possess antiparasite activity independent of the immune system. It is not known how the drug exerts these antiparasite effects, or why it is stage and/or species specific. The answers may lie in the enzymatic function of cyclophilins. The cyclophilins are a growing family of proteins that exhibit peptidyl-prolyl cis-trans isomerase (PPiase) activity and bid CsA to varying degrees. PPiases have been shown to play a role in the folding of many essential proteins. Antony Page, Sanjay Kumar and Clotilde Carlow here review parasite cyclophilins and their association with CsA. The possible biological function of parasite cyclophilins and their potential role in future drug discovery are also discussed.     

Crystal structure of the complex of brugia malayi cyclophilin and cyclosporin A

The resistance of the human parasite Brugia malayi to the antiparasitic activity of cyclosporin A (CsA) may arise from the presence of cyclophilins with relatively low affinity for the drug. The structure of the complex of B. malayi cyclophilin (BmCYP-1) and CsA, with eight independent copies in the asymmetric unit, has been determined at a resolution of 2.7 A. The low affinity of BmCYP-1 for CsA arises from incomplete preorganization of the binding site so that the formation of a hydrogen bond between His132 of BmCYP-1 and N-methylleucine 9 of CsA is associated with a shift in the backbone of approximately 1 A in this region.     

Structure of human cyclophilin A in complex with the novel immunosuppressant sanglifehrin A at 1.6 A resolution

Sanglifehrin A (SFA) is a novel immunosuppressant isolated from Streptomyces sp. that binds strongly to the human immunophilin cyclophilin A (CypA). SFA exerts its immunosuppressive activity through a mode of action different from that of all other known immunophilin-binding substances, namely cyclosporine A (CsA), FK506, and rapamycin. We have determined the crystal structure of human CypA in complex with SFA at 1.6 A resolution. The high resolution of the structure revealed the absolute configuration at all 17 chiral centers of SFA as well as the details of the CypA/SFA interactions. In particular, it was shown that the 22-membered macrocycle of SFA is deeply embedded in the same binding site as CsA and forms six direct hydrogen bonds with CypA. The effector domain of SFA, on the other hand, has a chemical and three-dimensional structure very different from CsA, already strongly suggesting different immunosuppressive mechanisms. Furthermore, two CypA.SFA complexes form a dimer in the crystal as well as in solution as shown by light scattering and size exclusion chromatography experiments. This observation raises the possibility that the dimer of CypA.SFA complexes is the molecular species mediating the immunosuppressive effect.     

A proposed molecular model for the interaction of calcineurin with the cyclosporin A-cyclophilin A complex.

Cyclosporin A (CsA) and FK506 are potent natural product immunosuppressants that induce their biological effects by forming an initial complex with cytosolic proteins termed immunophilins. These drug immunophilin complexes then bind to and inhibit the serine/threonine protein phosphatase calcineurin (CN). Two classes of immunophilin have been identified with cyclophilins (CyP's) being proteins specifically binding CsA and FKBPs specifically binding FK506. Solution and crystal structures of various CsA-CyP and FK506-FKBP complexes have been determined and show no apparent structural similarity between the two classes of drug protein complexes. These findings raise the question as to how, given their structural differences, these two complexes can both inhibit CN. While the crystal structure of the FK506-FKBP12-CN complex has been reported, no structure for a CsA-CyP CN complex has been determined. Here are reported studies that use various modelling strategies to construct a model for the interaction of the cyclosporin A- cyclophilin A complex with calcineurin. The first stage of constructing this model consisted of using conformational comparison of CsA and FK506, GRID and GROUP analysis and restrained molecular dynamics to dock CsA into the FK506 binding site of the FK506-FKBP12-CN structure. An initial model for the CsA-CyPA-CN complex was then constructed by superimposing the structure of the CsA-CyPA complex onto the docked CsA molecule. This model was then optimised with molecular dynamics simulations run on sterically clashing regions. The validity of the model for the CsA-CyPA-CN complex was then examined with respect to the effect of chemical modifications to CsA and amino acid substitutions within CyPA on the ability of the drug-immunophilin complex to inhibit calcineurin.     

Overexpression, purification, and characterization of yeast cyclophilins A and B.

Two isoforms of yeast cyclophilins, yCyPA and yCyPB, have been subcloned, expressed in Escherichia coli, and purified to homogeneity. The full-length (163-amino acid) yeast CyPA was easily expressed and purified; however, only a genetically truncated, 186-residue form of yCyPB lacking a putative 20-amino acid signal sequence could be purified. Each yeast cyclophilin isoform is a peptidyl-prolyl isomerase, inhibitable by the immunosuppressive drug CsA (IC50's of 40 +/- 8 nM and 101 +/- 14 nM at 18 nM concentrations of yCyPA and yCyPB, respectively). Polyclonal antibodies raised against recombinant yCyPA detected native yCyPA in yeast cell extracts by both immunoprecipitation and Western blot analysis. However, polyclonal antibodies raised against recombinant yCyPB detected no native yCyPB in yeast cell extracts by Western blot analysis; small amounts of yCyPB were found in the culture broth, suggesting secretion extracellularly of this isoform. Northern analysis indicated that both yCyPA mRNA and yCYPB mRNA (at a much lower level) were detectable in cell-free extracts. Characterization of the yeast cyclophilin proteins demonstrated that their catalytic properties and sensitivity to CsA parallel those of the human cyclophilins.     

Cloning and characterization of Giardia intestinalis cyclophilin

The cyclophilins (Cyps) are family members of proteins that exhibit peptidylprolyl cis-trans isomerase (PPIase, EC 5.2.1.8) activity and bind the immunosuppressive agent cyclosprin A (CsA) in varying degrees. During the process of random sequencing of a cDNA library made from Giardia intestinalis WB strain, the cyclophilin gene (gicyp 1) was isolated. An open reading frame of gicyp 1 gene was 576 nucleotides, which corresponded to a translation product of 176 amino acids (Gicyp 1). The identity with other Cyps was about 58-71%. The 13 residues that constituted the CsA binding site of human cyclophilin were also detected in the amino acid sequence of Gicyp 1, including tryptophan residue essential for the drug binding. The single copy of the gicyp 1 gene was detected in the G. intestinalis chromosome by southern hybridization analysis. Recombinant Gicyp 1 protein clearly accelerated the rate of cis-->trans isomerization of the peptide substrate and the catalysis was completely inhibited by the addition of 0.5 microM CsA.     

Cyclosporin A, the cyclophilin class of peptidylprolyl isomerases, and blockade of T cell signal transduction.

Cyclosporin A, the major immunosuppressive drug in transplantation, and the more potent therapeutic drug candidate, FK506, have led to the discovery of two superfamilies of immunosuppressant binding proteins, the cyclophilins and the FK binding proteins. These proteins, enzymes with high kcat values for isomerization of X-Pro bonds in peptides and protein substrates, are distributed in all cell compartments where protein folding normally occurs. It is likely that they play major roles in the protein folding and protein trafficking in the cell. It is also likely that they have been suborned in T cells by the immunosuppressant drugs that are potent pseudosubstrate ligands that selectively block the signal transduction cascade. The discovery of the inhibition of protein phosphatase 2B (calcineurin) by the drug-immunophilin complex (CsA-CyP or FK506-FKBP) provides evidence for a specific downstream target of the drug-immunophilin complexes and may prompt a search for endogenous ligands of cyclophilin and FKBP that may effect signal transduction regulation. The molecular insights gained over a short time in this area have been remarkable; they promise to elucidate the steps in T cell activation and delineate new targets for immunosuppressive therapy.     

Functional analysis of Leishmania major cyclophilin

A potent immunosuppressive drug cyclosporin A (CsA) is known to inhibit human cell infection by the pathogenic protozoan parasite Leishmania major both in vitro and in vivo. The proposed mechanism of action involves CsA binding to Leishmania major-expressed cyclophilin and subsequent down-regulation of signaling events necessary for establishing productive infection. Recently, we identified a ubiquitously expressed membrane protein, CD147, as a signaling receptor for extracellular cyclophilins in mammalian cells. Here we demonstrate that, while being enzymatically active, the Leishmania cyclophilin, unlike its human homologue, does not interact with CD147 on the cell surface of target cells. CD147 facilitates neither Leishmania binding nor infection. Primary structure and biochemical analyses revealed that the parasite's cyclophilin is defective in heparan binding, an event required for signaling interaction between CD147 and human cyclophilin. When the heparan-binding motif was reconstituted in Leishmania cyclophilin, it regained the CD147-dependent signaling activity. These results underscore a critical role of cyclophilin-heparan interactions in CD147-mediated signaling events and argue against the role of Leishmania cyclophilin in parasite binding to target cells.     

A cell-impermeable cyclosporine A derivative reduces pathology in a mouse model of allergic lung inflammation

Although the main regulators of leukocyte trafficking are chemokines, another family of chemotactic agents is cyclophilins. Intracellular cyclophilins function as peptidyl-prolyl cis-trans isomerases and are targets of the immunosuppressive drug cyclosporine A (CsA). Cyclophilins can also be secreted in response to stress factors, with elevated levels of extracellular cyclophilins detected in several inflammatory diseases. Extracellular cyclophilins are known to have potent chemotactic properties, suggesting that they might contribute to inflammatory responses by recruiting leukocytes into tissues. The objective of the present study was to determine the impact of blocking cyclophilin activity using a cell-impermeable derivative of CsA to specifically target extracellular pools of cyclophilins. In this study, we show that treatment with this compound in a mouse model of allergic lung inflammation demonstrates up to 80% reduction in inflammation, directly inhibits the recruitment of Ag-specific CD4(+) T cells, and works equally well when delivered at 100-fold lower doses directly to the airways. Our findings suggest that cell-impermeable analogs of CsA can effectively reduce inflammatory responses by targeting leukocyte recruitment mediated by extracellular cyclophilins. Specifically blocking the extracellular functions of cyclophilins may provide an approach for inhibiting the recruitment of one of the principal immune regulators of allergic lung inflammation, Ag-specific CD4(+) T cells, into inflamed airways and lungs.     

Transgenic mice overexpressing cyclophilin A are resistant to cyclosporin A-induced nephrotoxicity via peptidyl-prolyl cis-trans isomerase activity

Cyclosporin A (CsA) suppresses immune reaction by inhibiting calcineurin activity after forming complex with cyclophilins and is currently widely used as an immunosuppressive drug. Cyclophilin A (CypA) is the most abundantly and ubiquitously expressed family member of cyclophilins. We previously showed that CsA toxicity is mediated by ROS generation as well as by inhibition of peptidyl-prolyl cis-trans isomerase (PPIase) activity of CypA in CsA-treated myoblasts [FASEB J. 16 (2002) 1633]. Since CsA-induced nephrotoxicity is the most significant adverse effect in its clinical utilization, we here investigated the role of CsA inhibition of CypA PPIase activity in its nephrotoxicity using transgenic mouse models. Transgenic mice of either wild type (CypA/wt) or R55A PPIase mutant type (CypA/R55A), a dominant negative mutant of CypA PPIase activity, showed normal growth without any apparent abnormalities. However, CsA-induced nephrotoxicity was virtually suppressed in CypA/wt mice, but exacerbated in CypA/R55A mice, compared to that of littermates. Also, life expectancy was extended in CypA/wt mice and shortened in CypA/R55A mice during CsA administration. Besides, CsA-induced nephrotoxicity was inversely related to the levels of catalase expression and activity. In conclusion, our data provide in vivo evidence that supplement of CypA PPIase activity allows animal's resistance toward CsA-induced nephrotoxicity.     

A Magnaporthe grisea cyclophilin acts as a virulence determinant during plant infection

Cyclophilins are peptidyl prolyl cis-trans isomerases that are highly conserved throughout eukaryotes and that are best known for being the cellular target of the immunosuppressive drug cyclosporin A (CsA). The activity of CsA is caused by the drug forming a complex with cyclophilin A and inhibiting the calmodulin-dependent phosphoprotein phosphatase calcineurin. We have investigated the role of CYP1, a cyclophilin-encoding gene in the phytopathogenic fungus Magnaporthe grisea, which is the causal agent of rice blast disease. CYP1 putatively encodes a mitochondrial and cytosolic form of cyclophilin, and targeted gene replacement has shown that CYP1 acts as a virulence determinant in rice blast. Cyp1 mutants show reduced virulence and are impaired in associated functions, such as penetration peg formation and appressorium turgor generation. CYP1 cyclophilin also is the cellular target for CsA in Magnaporthe, and CsA was found to inhibit appressorium development and hyphal growth in a CYP1-dependent manner. These data implicate cyclophilins as virulence factors in phytopathogenic fungi and also provide evidence that calcineurin signaling is required for infection structure formation by Magnaporthe.     

Active site mutants of human cyclophilin A separate peptidyl-prolyl isomerase activity from cyclosporin A binding and calcineurin inhibition.

Based on recent X-ray structural information, six site-directed mutants of human cyclophilin A (hCyPA) involving residues in the putative active site--H54, R55, F60, Q111, F113, and H126--have been constructed, overexpressed, and purified from Escherichia coli to homogeneity. The proteins W121A (Liu, J., Chen, C.-M., & Walsh, C.T., 1991a, Biochemistry 30, 2306-2310), H54Q, R55A, F60A, Q111A, F113A, and H126Q were assayed for cis-trans peptidyl-prolyl isomerase (PPIase) activity, their ability to bind the immunosuppressive drug cyclosporin A (CsA), and protein phosphatase 2B (calcineurin) inhibition in the presence of CsA. Results indicate that H54Q, Q111A, F113A, and W121A retain 3-15% of the catalytic efficiency (kcat/Km) of wild-type recombinant hCyPA. The remaining three mutants (R55A, F60A, and H126Q) each retain less than 1% of the wild-type catalytic efficiency, indicating participation by these residues in PPIase catalysis. Each of the mutants bound to a CsA affinity matrix. The mutants R55A, F60A, F113A, and H126Q inhibited calcineurin in the presence of CsA, whereas W121A did not. Although CsA is a competitive inhibitor of PPIase activity, it can complex with enzymatically inactive cyclophilins and inhibit the phosphatase activity of calcineurin.     

Characterisation of a cyclophilin isoform in Trypanosoma cruzi

The immunosuppressive drug cyclosporin A (CsA) has shown antiparasitic activity against several protozoans and helminths, when complexed to proteins called cyclophilins (CyPs). In this paper, the molecular characterisation of one member of the CyP family in Trypanosoma cruzi is reported. TcCyP19 gene proved to be highly conserved compared to CyPs from other organisms and was highly homologous to a Trypanosoma brucei brucei CyPA. This gene was expressed in Escherichia coli and the purified recombinant protein exhibited a peptidyl prolyl cis-trans isomerase activity that was inhibited by CsA (IC(50) = 18.4 + /-0.8 nM). The TcCyP19 gene was located on two chromosomal bands in T. cruzi CL Brener clone.     

Delineation of the calcineurin-interacting region of cyclophilin B

The immunosuppressant drug cyclosporin A (CsA) inhibits T-cell function by blocking the phosphatase activity of calcineurin. This effect is mediated by formation of a complex between the drug and cyclophilin (CyP), which creates a composite surface able to make high-affinity contacts with calcineurin. In vitro, the CyPB/CsA complex is more effective in inhibiting calcineurin than the CyPA/CsA and CyPC/CsA complexes, pointing to fine structural differences in the calcineurin-binding region. To delineate the calcineurin-binding region of CyPB, we mutated several amino acids, located in two loops corresponding to CyPA regions known to be involved, as follows: R76A, G77H, D155R, and D158R. Compared to wild-type CyPB, the G77H, D155R, and D158R mutants had intact isomerase and CsA-binding activities, indicating that no major conformational changes had taken place. When complexed to CsA, they all displayed only reduced affinity for calcineurin and much decreased inhibition of calcineurin phosphatase activity. These results strongly suggest that the three amino acids G77, D155, and D158 are directly involved in the interaction of CyPB/CsA with calcineurin, in agreement with their exposed position. The G77, D155, and D158 residues are not maintained in CyPA and might therefore account for the higher affinity of the CyPB/CsA complex for calcineurin.