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We recently discovered a novel nargenicin A1 analog, 23-demethyl 8,13-deoxynargenicin (compound 9), with potential anti-cancer and anti-angiogenic activities against human gastric adenocarcinoma (AGS) cells. To identify the key molecular targets of compound 9, that are responsible for its biological activities, the changes in proteome expression in AGS cells following compound 9 treatment were analyzed using two-dimensional gel electrophoresis (2-DE), followed by MALDI/TOF/MS. Analyses using chemical proteomics and western blotting revealed that compound 9 treatment significantly suppressed the expression of cyclophilin A (CypA), a member of the immunophilin family. Furthermore, compound 9 downregulated CD147-mediated mitogen-activated protein kinase (MAPK) signaling pathway, including c-Jun N-terminal kinase (JNK) and extracellular signal-regulated protein kinase 1/2 (ERK1/2) by inhibiting the expression of CD147, the cellular receptor of CypA. Notably, the responses of AGS cells to CypA knockdown were significantly correlated with the anticancer and antiangiogenic effects of compound 9. CypA siRNAs reduced the expression of CD147 and phosphorylation of JNK and ERK1/2. In addition, the suppressive effects of CypA siRNAs on proliferation, migration, invasion, and angiogenesis induction of AGS cells were associated with G2/M cell cycle arrest, caspase-mediated apoptosis, inhibition of MMP-9 and MMP-2 expression, inactivation of PI3K/AKT/mTOR pathway, and inhibition of hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) expression. The specific interaction between compound 9 and CypA was also confirmed using the drug affinity responsive target stability (DARTS) and cellular thermal shift assay (CETSA) approaches. Moreover, in silico docking analysis revealed that the structure of compound 9 was a good fit for the cyclosporin A binding cavity of CypA. Collectively, these findings provide a novel molecular basis for compound 9-mediated suppression of gastric cancer progression through the targeting of CypA.

Background Originally discovered as receptors for immunosuppressive drugs, immunophilins consist of two major groups, FK506 binding proteins (FKBPs) and cyclosporin A binding proteins (cyclophilins, CYPs). Many members in both FKBP and CYP families are peptidyl prolyl isomerases that are involved in protein folding processes, though they share little sequence homology. It is not surprising to find immunophilins in all organisms examined so far, including viruses, bacteria, fungi, plants and animals, as protein folding represents a common process in all living systems. Scope of Review Studies on plant immunophilins have revealed new functions beyond protein folding and new structural properties beyond that of typical PPIases. This review focuses on the structural and functional diversity of plant FKBPs and CYPs. Major Conclusions The differences in sequence, structure as well as subcellular localization, have added on to the diversity of this family of molecular chaperones. In particular, the large number of immunophilins present in the thylakoid lumen of the photosynthetic organelle, promises to deliver insights into the regulation of photosynthesis, a unique feature of plant systems. However, very little structural information and functional data are available for plant immunophilins. General Significance Studies on the structure and function of plant immunophilins are important in understanding their role in plant biology. By reviewing the structural and functional properties of some immunophilins that represent the emerging area of research in plant biology, we hope to increase the interest of researchers in pursuing further research in this area. This article is part of a Special Issue entitled Proline-directed Foldases: Cell Signaling Catalysts and Drug Targets.

Cyclophilin (Cyp), a peptidyl-prolyl cis-trans isomerase (PPIase), acts as a virulence factor in many bacteria including Staphylococcus aureus. The enzymatic activity of Cyp is inhibited by cyclosporin A (CsA), an immunosuppressive drug. To precisely determine the unfolding mechanism and the domain structure of Cyp, we have investigated a chimeric S. aureus Cyp (rCyp) using various probes. Our limited proteolysis and the consequent analysis of the proteolytic fragments indicate that rCyp is composed of one domain with a short flexible tail at the C-terminal end. We also show that the urea-induced unfolding of both rCyp and rCyp-CsA is completely reversible and proceeds via the synthesis of at least one stable intermediate. The secondary structure, tertiary structure, and the hydrophobic surface area of no intermediate are fully identical to those of other intermediate or the related native protein. Further analyses reveal no loss of CsA binding activity in rCyp intermediate. The thermodynamic stability of rCyp was also significantly increased in the presence of CsA, recommending that this protein could be employed to screen new CsA derivatives in future.

Described here is a stable isotope labeling protocol that can be used with a chemical modification- and mass spectrometry-based protein-ligand binding assay for detecting and quantifying both the direct and indirect binding events that result from protein-ligand binding interactions. The protocol utilizes an H(2) (16)O(2) and H(2) (18)O(2) labeling strategy to evaluate the chemical denaturant dependence of methionine oxidation in proteins both in the presence and absence of a target ligand. The differential denaturant dependence to the oxidation reactions performed in the presence and absence of ligand provides a measure of the protein stability changes that occur as a result of direct interactions of proteins with the target ligand and/or as a result of indirect interactions involving other protein-ligand interactions that are either induced or disrupted by the ligand. The described protocol utilizes the (18)O/(16)O ratio in the oxidized protein samples to quantify the ligand-induced protein stability changes. The ratio is determined using the isotopic distributions observed for the methionine-containing peptides used for protein identification in the LC-MS-based proteomics readout. The strategy is applied to a multi-component protein mixture in this proof-of-principle experiment, which was designed to evaluate the technique's ability to detect and quantify the direct binding interaction between cyclosporin A and cyclophilin A and to detect the indirect binding interaction between cyclosporin A and calcineurin (i.e., the protein-protein interaction between cyclophilin A and calcineurin that is induced by cyclosporin A binding to cyclophilin A).

The cyclophilin of Bacillus subtilis has a moderate affinity to cyclosporin A (IC50: 120 nM) and low catalytic activity (Kcat/ Km: 1.1 microM-1 s-1) when compared to other ubiquitous peptidyl-prolyl cis-trans isomerases (PPIases). The active site residues V52, H90 and H109, which are not conserved within other peptidyl-prolyl cis-trans isomerases, were found to play an important role in cyclosporin A binding and catalytic activity. In this work we report on double mutations of these residues, which greatly improved cyclosporin A affinity and catalytic activity. The H90N/H109W mutation displayed an IC50 value of 46 nM whereas the V52M/H109F mutation exhibited over 18-fold higher catalytic activity than that detected for wild-type PPIase. The mutations H109W and H109F of the B. subtilis PPIase showed no change in cyclosporin A affinity and catalytic activity between pH 6 and 8. In contrast, wild-type PPIase (H109) showed up to 10-fold reduction below pH 7.5, both in cyclosporin A affinity and in catalytic activity. These findings clearly underline the importance of the unique H109 residue in the B. subtilis enzyme.

In order to map cyclosporin A (CsA) binding sites of cyclophilin (CyP), we synthesized the complete set of overlapping 157 octapeptides corresponding to human CyP A using the multi-pin peptide synthesis system. The pin-coupled synthetic octapeptides were examined in terms of binding ability to CsA by a modification of the enzyme-linked immunosorbent assay. Significant binding of CsA was detected with 35 synthetic N alpha-acetylated octapeptides possessing the N-terminal amino acids corresponding to the residues in positions 24-26, 42-44, 69-73, 75, 76, 89-91, 102, 116, 124-131, 144-151 and 152 in human CyP A, respectively. Other eight octapeptides showed moderate CsA binding activity. The distinct binding of octapeptides covering the C-terminal region of the CyP A was particularly significant. These data are to be compared with the information provided by X-ray and NMR studies on the CsA binding sites and furnish thus a test of the reported method. The present study also gave added insight into the CsA interaction sites of CyP.

We have recently shown that hsp56, the FK506-binding immunophilin component of both the heat shock protein (hsp90.hsp70.hsp56) heterocomplex and the untransformed glucocorticoid receptor heterocomplex, is bound directly to hsp90 (Czar, M. J., Owens-Grillo, J. K., Dittmar, K. D., Hutchison, K. A., Zacharek, A. M., Leach, K. L., Deibel, M. R., and Pratt, W. B. (1994) J. Biol. Chem. 269, 11155-11161). In this work, we show that both untransformed glucocorticoid receptor and hsp90 heterocomplexes contain CyP-40, a 40-kDa immunophilin of the cyclosporin A-binding class. CyP-40 is present in both native glucocorticoid receptor heterocomplexes and receptor heterocomplexes reconstituted with rabbit reticulocyte lysate, and the presence of CyP-40 in the receptor heterocomplex is stabilized by molybdate. Immunoadsorption of hsp90 from cell lysate yields coimmunoadsorption of both hsp56 and CyP-40, showing that both immunophilins are in native heterocomplex with hsp90. However, immunoadsorption of hsp56 does not yield coimmunoadsorption of CyP-40; thus, the two immunophilins do not exist in the same heterocomplex with hsp90. Both purified CyP-40 and hsp56 bind directly to purified hsp90, and excess CyP-40 blocks the binding of hsp56, consistent with the presence of a common immunophilin binding site on hsp90. Our data also suggest that there are at least two types of untransformed glucocorticoid receptor-hsp90 heterocomplexes, one that contains hsp56 and another that contains CyP-40. The role played by the immunophilins in steroid receptor action is unknown, but it is clear that the peptidylprolyl isomerase activity of immunophilins is not required for glucocorticoid receptor-hsp90 heterocomplex assembly and proper folding of the hormone binding domain by the hsp90-associated protein folding system of reticulocyte lysate.

The cDNA encoding a human cytosolic 40-kDa cyclophilin (CyP-40) has been inserted into a modified pGEX-3X expression vector and expressed in Escherichia coli to yield recombinant CyP-40 at levels up to 4 mg/l medium. The protein was purified to homogeneity using a cyclosporin affinity matrix and gel filtration. The recombinant CyP-40 showed peptidyl-prolyl cis-trans isomerase activity (kcat/Km = 1.12 x 10(6) M-1.s-1) comparable to that of bovine brain CyP-40. The weak affinity of CyP-40 for cyclosporin A was postulated to arise from a histidine residue that replaces a tryptophan residue critical for cyclosporin A binding and highly conserved in other cyclophilins that have high affinity for cyclosporin A. Site-directed mutagenesis to replace His141 by tryptophan yielded a protein with an approximately 20-fold greater affinity for cyclosporin A (Kdapp 11.5 +/- 2 nM as determined by tryptophan fluorescence measurements). The intrinsic isomerase activity of this mutant protein with succinyl-Ala-Ala-Pro-Phe 4-nitroanilide as substrate was about nine times greater than the value obtained for the nonmutated recombinant CyP-40 and had an activity similar to that of CyP-18. NMR difference spectroscopy and molecular modelling revealed a cyclosporin-A-binding domain that is similar to that of CyP-18.

A single amino acid substitution, Gly185-->Val, in the human P-glycoprotein (Pgp) was previously shown to cause an altered pattern of drug resistance in cell lines transfected with the MDR1 cDNA carrying this mutation. To further define the function of amino acid 185 in the Pgp, the wild-type and the mutant Val185 Pgps were expressed in Sf9 insect cells, and their biochemical properties were compared. Verapamil- and colchicine-stimulated ATPase activities were markedly increased with concomitant increase in affinity for these compounds with Gly185-->Val substitution in the Pgp. However, the vinblastine-stimulated ATPase activities of the wild-type and Val185 Pgps were nearly identical. Because transport substrate-induced ATP hydrolysis is generally thought to reflect transport function, these data suggest that colchicine and verapamil are transported at an increased rate with Gly185-->Val substitution in the Pgp. These results also indicate that amino acid 185 is involved in verapamil and colchicine, but not in vinblastine, binding/transport. Kinetic analyses indicate that cyclosporin A, an inhibitor of Pgp, binds to the verapamil and vinblastine binding/transport site(s) in the Pgp. Taken together, the results presented herein reveal that the verapamil and vinblastine binding/transport site(s) are in close proximity and that the cyclosporin A binding site spans the common region of these two drug binding/transport site(s) in the Pgp molecule.

The periplasmic Escherichia coli cyclophilin is distantly related to human cyclophilin (34% sequence identity). Peptidyl-prolyl isomerase activity, cyclosporin A binding, and inhibition of the calcium-dependent phosphatase calcineurin are compared for human and E. coli wild-type and mutant proteins. Like human cyclophilin, the E. coli protein is a cis-trans peptidyl-prolyl isomerase. However, while the human protein binds cyclosporin A tightly (Kd = 17 nM), the E. coli protein does not (Kd = 3.4 microM). The mutant F112W E. coli cyclophilin has enhanced cyclosporin binding (Kd = 170 nM). As for the human protein, the complex of the E. coli mutant with cyclosporin A inhibits calcineurin. Here we describe the structure at pH 6.2 of cyclosporin A bound to the mutant E. coli cyclophilin as solved with solution NMR methods. Despite the low overall sequence identity, the structure of the bound cyclosporin A is virtually identical in both proteins. To assess differences of the cyclosporin binding site, the solution structure of wild-type E. coli cyclophilin was compared with structures of uncomplexed human cyclophilin A and with cyclosporin bound. Despite the structural similarity of bound cyclosporin A, the architecture of the binding site in the E. coli protein is substantially different at the site most distant to tryptophan 121 (human sequence). This site is constructed by a five-residue insertion in a loop of the E. coli protein, replacing another loop in the human protein.

Malaria represents a global health, economic and social burden of enormous magnitude. Chemotherapy is at the moment a largely effective weapon against the disease, but the appearance of drug-resistant parasites is reducing the effectiveness of most drugs. Finding new drug-target candidates is one approach to the development of new drugs. The family of cyclophilins may represent a group of potential targets. They are involved in protein folding and regulation due to their peptidyl-prolyl cis – trans isomerase and/or chaperone activities. They also mediate the action of the immunosuppressive drug cyclosporin A, which additionally has strong antimalarial activity. In the genome database of the most lethal human malarial parasite Plasmodium falciparum , 11 genes apparently encoding cyclophilin or cyclophilin-like proteins were found, but most of these have not yet been characterized. Previously a pET vector conferring a C-terminal His 6 tag was used for recombinant expression and purification of one member of the P. falciparum cyclophilin family in Escherichia coli . The approach here was to use an identical method to produce all of the other members of this family and thereby allow the most consistent functional comparisons. We were successful in generating all but three of the family, plus a single amino-acid mutant, in the same recombinant form as either full-length proteins or isolated cyclophilin-like domains. The recombinant proteins were assessed by thermal melt assay for correct folding and cyclosporin A binding.

Inflammatory immune reactions occur during transplant rejections and autoimmune diseases. Such reactions are mediated by cytokines, including interleukin-8 (IL-8). Cyclosporin A (CsA) exerts immunosuppressive activities1,2 by binding to immunoregulatory proteins termed cyclophilins3. The anti-inflammatory effects of CsA are still not fully understood. Searching for novel neutrophil-activating proteins, we observed that an antiserum against human recombinant Interleukin-8 (IL-8) cross-reacted with cyclophilins in Western blots. Furthermore, native IL-8 was found to specifically bind CsA, whereas biologically inactive analogs of CsA were not bound by IL-8. Putative binding sites for CsA on IL-8 could be identified on the basis of structural similarities between IL-8 and cyclophilin. However, IL-8 lacks peptidyl-prolyl-isomerase (PPlase) enzyme activity, which is regarded as a characteristic of cyclophilins4,5,6. We conclude that the specific binding of CsA to IL-8 may explain some of the anti-inflammatory effects of CsA. IL-8 may be a novel member of the cyclophilins lacking PPlase activity.

Cyclophilins A and B (CyPA and CyPB) are cyclosporin A binding proteins that can be secreted in response to inflammatory stimuli. We recently identified CD147 as a cell-surface receptor for CyPA and demonstrated that CD147 is an essential component in the CyPA-initiated signaling cascade that culminates in ERK activation and chemotaxis. Here we demonstrate that CD147 also serves as a receptor for CyPB. CyPB induced Ca 21 flux and chemotaxis of CD147transfected, but not control, CHO cells, and the chemotactic response of primary human neutrophils to CyPB was blocked by antibodies to CD147. These results suggest that CD147 serves as a receptor for extracellular cyclophilins. © 2001 Academic Press

SUMMARY Autoantibodies against cyclophilin, a cyclosporin A binding protein, were detected in sera of 29 of 46 (63%) patients with systemic lupus erythematosus and 14 of 40 (35%) Lyme disease patients. The antibodies are directed against the denatured form of both the major and minor isoform of cyclophilin and, can be demonstrated in Western blots. Some first-degree relatives of lupus patients also express these antibodies. They are specific for cyclophilin and are not the consequence of hypergammaglobulinaetnia. Four monoclonal IgM antibodies from a patient with lepromatous leprosy also bound to cyclophilin. The generation of these antibodies may be of special interest because they are against a protein involved in the control of the immune system not known to be directly associated with DNA or RNA.

A novel family of cyclosporin A (CsA) binding proteins was identified by using the biologically active, radioiodinated photoaffinity probe [D-Lys-N epsilon-(4-azido-3-[125I]iodophenyl)propionyl)]8-CsA. In addition to cyclophilin, proteins with molecular masses of 43 kDa and approximately 50-55 kDa were labeled in Jurkat extracts and bovine calf thymus. Sequence analysis of the 43-kDa protein purified from calf thymus and subsequent Western analysis of CsA affinity-purified material from Jurkat extracts identified the 43-kDa component as actin. [D-Lys-N epsilon-(5-dimethylamino-1-naphthalenesulfonyl)]8-CsA, a fluorescent analogue of CsA, was prepared and used to measure the binding constants of cyclosporin derivatives to actin by means of a new fluorescence displacement assay. [D-Lys-N epsilon-(5-dimethylamino-1-naphthalenesulfonyl)]8-CsA and [N delta-t-butoxycarbonyl diaminobutyryl)]8-CsA bind to bovine actin at physiologically relevant concentrations, with dissociation constants of 60 +/- 33 and 570 +/- 380 nM, respectively. Because the ATPase fragment of heat shock cognate 70 (HSC 70) is structurally related to actin, the yeast homologue SSA1 was tested and found to be radiolabeled by the cyclosporin A photoaffinity reagent. The binding constant for [D-Lys-N epsilon-(5-dimethylamino-1-naphthalenesulfonyl)]8-CsA to SSA1 was determined and is 53 +/- 48 nM. These results indicate that actin and the 70-kDa heat shock protein family contain a structurally related domain for binding of cyclosporin A-related peptides.

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.

Two new peptidyl-prolyl-cis/trans-isomerases were purified to homogeneity from Fusarium sambucinum and Aspergillus niger . They belong to the class of cyclosporin A binding proteins (cyclophilins) and have molecular masses of about 18 kDa. As has been shown for other cyclophilins, the isomerase activity of the enzymes is inhibited by cyclosporin A in the nanomolar range. Furthermore binding of cyclosporin A prevents proteolytic digestion of the cyclophilin/cyclosporin complexes by the endoproteases GluC, LysC and α-chymotrypsin, in contrast to the free cyclophilins, which are readily cleaved by these proteases. We could also observe this protection for cyclophilins from sheep thymus and from the cyclosporin producing fungus Tolypocladium inflatum .

Cyclophilin, the cyclosporin A binding protein and member of the immunophilin family of proteins, demonstrates leukocyte chemotactic activity. In this study we demonstrate that FKBP, the FK506 and rapamycin binding protein, also displays leukocyte chemotactic activity. The chemotactic activity of FKBP is inhibited by FK506, however, FK506 was unable to inhibit cyclophilin-stimulated chemotactic activity. Rapamycin was unable to prevent the chemotactic activity of FKBP, similarly, the CsA analogue Me6Ala-CsA while displaying cyclophilin binding was unable to block cyclophilin-stimulated chemotactic activity. These results suggest that in addition to their intracellular role the immunophilins may also function as chemotactic agents, furthermore this activity is modulated by immunosuppressants.

1. Rat liver mitochondrial matrix peptidyl-prolyl cis-trans-isomerase (PPIase) has been purified. The major form of the enzyme has a molecular mass of 18.6 kDa, with a minor active component of 17.6 kDa. 2. The second-order rate constant for cyclosporin A binding to the enzyme was determined from the time-dependence of the inhibition of PPIase by low concentrations of cyclosporin A and found to be 0.9 microM-1.s-1 at 10 degrees C. 3. The Ki for cyclosporin A inhibition of the enzyme was 3.6 nM, and the half-life for dissociation of the enzyme-inhibitor complex was 3.6 min. 4. From the specific activity of the pure enzyme it can be calculated that isolated liver mitochondria contain approx. 45 pmol of enzyme per mg of total mitochondrial protein. Higher values estimated previously [Halestrap & Davidson (1990) Biochem. J. 268, 153-160] are explained by the use of a short (30 s) preincubation period of the enzyme with cyclosporin, which is insufficient to allow full equilibration of the binding of the inhibitor to the PPIase. 5. N-Terminal sequencing of the 18.6 and 17.5 kDa forms of PPIase show the presence of mitochondrial presequences of 13 and three amino acids respectively, with the remaining sequence having a strong sequence similarity to other cyclophilins. 6. Parallel purification and N-terminal sequencing of rat cytosolic PPIase showed the two proteins to have significant differences, implying that they are probably products of separate genes.

The cyclosporin A binding protein, cyclophilin (CyP), and the FK-506 binding protein, FKBP, catalyze the cis-to-trans isomerization of Xaa-Pro bonds in peptides. To probe the mechanism of these reactions and their nonenzymatic counterparts, we determined the following: (1) substrate specificities of CyP and FKBP; (2) dependencies of k c /K m on pH and solvent deuterium; (3) secondary deuterium isotope effects; and (4) temperature-dependencies. The results indicate that for cis-to-trans isomerization of Suc-Ala-Xaa-cis-Pro-Phe-pNA, values of k c /K m for the CyP-catalyzed reactions show little dependence on Xaa

Several autoantibodies against cytoplasmic or nuclear components of cells have been reported in autoimmune diseases. We report here a previously unrecognized autoantibody to peptidyl-prolyl cis-trans isomerase (PPIase) in patients with systemic lupus erythematosus (SLE). PPIase, which catalyzes the cis-trans isomerization of proline imidic peptide bonds in oligopeptides, has recently been found to be identical to cyclophilin, a specific binding protein of a potent immunosuppressant, cyclosporin A. IgG and IgM anti-PPIase antibodies were detected in 40 and 20% of unselected patients with SLE, respectively, by ELISA. The reactivity of these sera was confirmed by immunoblotting experiments. Sera from rheumatoid arthritis patients showed no reactivity and 1 of 8 sera from systemic sclerosis patients and 1 of 25 sera from normal controls showed only weak reactivity. Unexpectedly, the anti-PPIase antibody was unable to inhibit PPIase activity, indicating that the autoantibody recognizes an epitope of PPIase which is different from the active site of PPIase. The levels of the anti-PPIase antibody in SLE patients correlated with remissions and flares of the disease. The anti-PPIase antibody was higher in patients with active SLE than those with inactive disease. The prevalence of the active stage of the disease was significantly higher in IgG anti-PPIase antibody-positive SLE patients as compared to antibody-negative SLE patients. These data define the presence of a new autoantibody against PPIase and its association with the activity and certain clinical manifestations in SLE.

It is believed that P-glycoprotein (P-gp) is an energy-dependent drug efflux pump responsible for decreased drug accumulation in multidrug resistant (MDR) cells. In this study, we investigated whether azidopine, a photoactive dihydropyridine calcium channel blocker, is transported by P-gp in MDR Chinese hamster lung cells, DC-3F/VCRd-5L, and whether its binding site(s) on P-gp are distinct from those of Vinca alkaloids and cyclosporins. The efflux of azidopine from MDR cells was energy-dependent and inhibited by the cytotoxic agent vinblastine (VBL). Cyclosporin A (CsA), a modulator of MDR, also increased azidopine accumulation in MDR cells by decreasing the energy-dependent efflux of azidopine. P-gp in these cells was the only protein specifically bound to [3H]azidopine in photoaffinity experiments. The specific photoaffinity labeling of P-gp by [3H]azidopine was inhibited by CsA, SDZ 33-243, nonradioactive azidopine, and VBL with median concentrations (IC50) of 0.5, 0.62, 1.7, and 25 microM, respectively. The equilibrium binding of azidopine to plasma membranes of MDR variant DC-3F/VCRd-5L cells showed a single class of specific binding sites having a dissociation constant of 1.20 microM and a maximum binding capacity of 4.47 nmol/mg of protein. Kinetic analysis indicated that the inhibitory effect of VBL and CsA on azidopine binding to plasma membranes of MDR cells was noncompetitive, indicating that azidopine binds to P-gp at a binding site(s) different from the binding site(s) of these drugs.

A novel cyclosporin A binding glycoprotein of 21 kDa was isolated from human milk by several steps of cation exchange chromatography. The corresponding gene was cloned from human T cells, expressed in Escherichia coli and the recombinant protein purified. The protein shares 58% amino acid identity with the cytosolic cyclophilin and is initially synthesized with a hydrophobic leader sequence. The cyclophilin-like protein has also peptidyl-prolyl cis/trans-isomerase activity, although less efficient, that is inhibited by cyclosporin A. The existence of a secreted form of cyclophilin-like protein in addition to the previously known cytosolic cyclophilin implies that these proteins act on different in vivo targets.

We report the cloning of a neutral isoelectric form of the human peptidyl prolyl isomerase, cyclophilin, its expression in Escherichia coli, and its purification and comparison to bovine thymus cyclophilin. The cloned protein exhibited a pI of approximately 7.8 and formed a simple 1:1 complex with cyclosporin A. This cloned form had a pI similar to that observed for the neutral isoform (pI approximately 7.4) of human splenocyte cyclophilin. The bovine thymus proteins exhibited anomalous behavior on CM-cellulose chromatography but were resolved into alkaline (pI approximately 9.3) isoforms and a new neutral (pI approximately 7.8) isoform by isoelectric focusing gel electrophoresis and ultimately into at least four discrete isoforms by capillary electrophoresis. For cyclosporin A binding we observe a Kd of approximately 160 nM for an electrophoretically heterogeneous preparation of the natural bovine protein and approximately 360 nM for the more homogeneous preparation of the cloned human neutral isoform. Stopped-flow measurements of the activation energies for peptidyl-prolyl isomerase activity indicate the recombinant human protein has an activation enthalpy of 3.67 kcal/mol and an activation entropy of -47.3 cal/K-mol for cis----trans isomerization.

We report on the application of SUPREX (stability of unpurified proteins from rates of H/D exchange) to the analysis of a protein-ligand binding interaction under the ex vivo solution conditions of a human lung tumor tissue lysate. A SUPREX-derived binding free energy (i.e. DeltaDeltaG(f) value) and dissociation constant (i.e., K(d) value) were determined for the binding of cyclosporin A (CsA) to a cyclophilin A (CypA) sample in which the protein was a component of a tissue lysate derived from fresh frozen lung tumor. The DeltaDeltaG(f) and K(d) values determined by SUPREX for CsA binding to CypA in this unpurified protein sample, 4.7 +/- 0.8 kcal/mol and 77 +/- 17 nM, respectively, were comparable to the those obtained when SUPREX was used to analyze the binding of CsA to a highly purified CypA sample, 4.2 +/- 1.0 kcal/mol and 32 +/- 20 nM, respectively. Moreover, the SUPREX-derived K(d) values determined in this work were both in the range of those previously reported for the CypA-CsA complex. The results of this proof-of-principle work validate the extension of SUPREX to the thermodynamic analysis of proteins and protein-ligand binding interactions in the unpurified, ex vivo conditions of human tissue lysates,and they represent the first K(d) measurement on a protein-ligand complex under such conditions

Fructose-1,6-bisphosphatase (FBPase) is synthesized in yeast during glucose starvation but is rapidly degraded in the vacuole following the addition of glucose. FBPase trafficking to the vacuole involves two distinct steps, import into intermediate transport vesicles (Vid vesicles) and Vid vesicle trafficking to the vacuole. FBPase import into Vid vesicles requires the VID22 gene. However, VID22 affects FBPase import indirectly through a cytosolic factor. To identify the required cytosolic component, wild type cytosol was fractionated and screened for proteins that complement Deltavid22 mutant cytosol using an in vitro assay that reproduces FBPase import into Vid vesicles. Cyclophilin A (Cpr1p) was identified as a cytosolic protein that mediates Vid22p function in FBPase import. Mutants lacking Cpr1p were defective in FBPase import. Furthermore, the addition of purified Cpr1p restored FBPase import in both the Deltacpr1 and the Deltavid22 mutants. The cyclosporin A binding pocket is important for Cpr1p function, since cyclosporin A binding-deficient mutants failed to complement FBPase import in Deltacpr1 and Deltavid22 mutants. The levels of Cpr1p were reduced in the Deltavid22 mutants, implying that the expression of Cpr1p is regulated by Vid22p. Our results suggest that Cpr1p mediates Vid22p function and is directly involved in the import of FBPase into Vid vesicles.

The majority of the effects of cyclosporin A (CsA) on cells is caused by the inhibition of phosphatase activity of calcineurin (CN) by the cyclophilin A (CyPA)-CsA complex formed in the cytoplasm. Although CsA inhibits the proliferation of a large number of parasites, not all are susceptible. The presence of structurally altered CyPA with lower affinity for CsA had been suggested to be the cause of resistance. We report here the identification and cloning of a high affinity CsA-binding protein (LdCyP) from Leishmania donovani, a trypanosomatid parasite that is naturally resistant to CsA. The translated LdCyP consists of 187 amino acids with a cleavable 21-amino acid hydrophobic NH(2)-terminal extension. Modeling studies confirmed that all the residues of human CyPs responsible for interaction with CsA are sequentially and conformationally conserved in LdCyP. The purified recombinant protein displayed biochemical parameters comparable to human CyPs. Reverse transcription-polymerase chain reaction analysis confirmed that LdCyP was abundantly expressed. Immunoblot experiments and direct CsA binding studies revealed that LdCyP located in the subcellular organelles constituted the bulk of the CsA binding activity present in L. donovani, whereas the level of binding activity in the cytosol was conspicuously low. CsA selectively facilitated the secretion of LdCyP in the culture medium. Based on these results, it is concluded that the insensitivity of L. donovani to CsA is probably due to the paucity of CsA binding activity in the cytoplasm of the parasite. We suggest that LdCyP, located in the secretory pathway, may function as a chaperone by binding to membrane proteins rather than as the mediator of CN inhibition.

Full-length Entamoeba histolytica cyclophilin gene (EhCyp) was isolated, characterized and recombinantly expressed in bacterial cells. The deduced amino acid sequence of EhCyp shows 60–70% identity with cyclophilins from other organisms and has conserved the cyclophilin signature motifs and residues involved in cyclosporin A binding. Upstream of the 501 bp open reading frame of EhCyp, sequences resembling the putative consensus E. histolytica CE1, CE2 and CE3 regulatory elements were found. Northern blot assays revealed a single transcript of 0.63 kb. The transcription start was determined by primer extension at position −13 relative to the initial ATG codon. Cyclosporin A binding and peptidyl-proplyl cis–trans isomerase activities characteristic of cyclophilin were detected in soluble extracts of E. histolytica trophozoites and in the recombinant protein. In both cases, the isomerase activity was inhibited by nanomolar concentrations of cyclosporin A. Treatment of cultured trophozoites with cyclosporin A decreased their proliferation with a 50% inhibition value of 1 μg/ml and was lethal in doses over 50 μg/ml.

Cyclophilin B is a cyclosporin A-binding protein exhibiting peptidyl-prolyl cis/trans isomerase activity. We have previously shown that it interacts with two types of binding sites on T lymphocytes. The type I sites correspond to specific functional receptors and the type II sites to sulfated glycosaminoglycans. The interactions of cyclophilin B with type I and type II sites are reduced in the presence of cyclosporin A and of a synthetic peptide mimicking the N-terminal part of cyclophilin B, respectively, suggesting that the protein possesses two distinct binding regions. In this study, we intended to characterize the areas of cyclophilin B involved in the interactions with binding sites present on Jurkat cells. The use of cyclophilin B mutants modified in the N-terminal region demonstrated that the 3Lys-Lys-Lys5 and 14Tyr-Phe-Asp16 clusters are probably solely required for the interactions with the type II sites. We further engineered mutants of the conserved central core of cyclophilin B, which bears the catalytic and the cyclosporin A binding sites as an approach to localize the binding regions for the type I sites. The enzymatic activity of cyclophilin B was dramatically reduced after substitution of the Arg62 and Phe67 residues, whereas the cyclosporin A binding activity was destroyed by mutation of the Trp128 residue and strongly decreased after modification of the Phe67 residue. Only the substitution of the Trp128 residue reduced the binding of the resulting cyclophilin B mutant to type I binding sites. The catalytic site of cyclophilin B therefore did not seem to be essential for cellular binding and the cyclosporin A binding site appeared to be partially involved in the binding to type I sites.

The binding site of cyclosporin A to P-glycoprotein was characterized by using a multidrug-resistant Chinese hamster ovary cell line. P-glycoprotein photolabeled with diazirine-cyclosporin A analogue was purified by a two-step process involving continuous elution electrophoresis followed by wheat germ agglutinin-agarose precipitation. The cyclosporin A covalently bound to P-glycoprotein and to subsequent proteolytic fragments was detected by Western blot analysis using a monoclonal antibody against cyclosporin A. Proteolytic digestion of purified P-glycoprotein by V8 generated a major fragment of 15 kDa photolabeled by cyclosporin A, while proteolysis of P-glycoprotein photolabeled by [125I]-iodoaryl azidoprazosin generated a major fragment of 7 kDa. Limited proteolysis of cyclosporin A-photolabeled P-glycoprotein with trypsin indicated that the major binding site for cyclosporin A was in the C-terminal half of the protein. This cyclosporin A binding site was further characterized with chemical agents (N-chlorosuccinimide, cyanogen bromide, and 2-nitro-5-thiocyanobenzoate). These three chemical agents established a proteolytic profile of P-glycoprotein for fragments photolabeled with cyclosporin A and for fragments that contained the C494 and C219 epitopes. The smallest fragments generated by these chemical agents include the transmembrane domains (TMs) 10, 11, and 12 of P-glycoprotein. When the fragments generated by these chemical agents are aligned, the region that binds cyclosporin A is reduced to the 953-1007 residues. These combined results suggest that the major binding site of cyclosporin A occurs between the end of TM 11 and the end of TM 12.

A divergent multi-domain cyclophilin from the filarial nematodes Brugia malayi , Onchocerca volvulus and Dirofilaria immitis has a highly conserved orthologue in the free-living nematodes Caenorhabditis elegans and C. briggsae. Cyclophilins are the receptors for the immunosuppressive and anti-parasitic agent cyclosporin A and additionally these ubiquitously expressed proteins have protein folding capabilities, and exhibit proline isomerase activity. These divergent nematode cyclophilins (CYP-4 isoforms) are three domain proteins, which share 63–88% identity and have highly conserved differences present in their functionally important cyclosporin A binding and proline isomerase domains. This unusual class of nematode cyclophilins has been studied in the model nematode C. elegans , revealing a unique temporal and spatial expression pattern. The cyp- 4 transcript is most abundantly expressed in the early larval stages and is expressed exclusively in the body-wall striated muscle cells. An important functional role was established for this divergent enzyme, as specific double-stranded RNA interference experiments resulted in progeny with a phenotypically lumpy appearance. This morphological defect was predominantly expressed in the early larval stages and is consistent with an effect on body-wall muscle cell development. This study has established that this highly conserved family of nematode cyclophilins has a tissue-specific, functional role in early larval development and supports the use of C. elegans as a model for the study of orthologues in the experimentally less amenable parasitic nematodes.

When loaded with high (pathological) levels of Ca2+, mitochondria become swollen and uncoupled as the result of a large nonspecific increase in membrane permeability. This process, known as the mitochondrial permeability transition (MPT), is exacerbated by oxidative stress and adenine nucleotide depletion. These conditions match those that a heart experiences during reperfusion following a period of ischaemia. The MPT is caused by the opening of a non-specific pore that can be prevented by sub-micromolar concentrations of cyclosporin A (CsA). A variety of conditions that increase the sensitivity of pore opening to [Ca2+], such as thiol modification, oxidative stress, increased matrix volume and chaotropic agents, all enhance the binding of matrix cyclophilin (CyP) to the inner mitochondrial membrane in a CsA-sensitive manner. In contrast, ADP, membrane potential and low pH decrease the sensitivity of pore opening to [Ca2+] without affecting CyP binding. We present a model of pore opening involving CyP binding to a membrane target protein followed by Ca2+-dependent triggering of a conformational change to induce channel opening. Using the ischaemic/reperfused rat heart we have shown that the mitochondrial pore does not open during ischaemia, but does do so during reperfusion. Recovery of heart during reperfusion is improved in the presence of 0.2 µM CsA, suggesting that the MPT may be critical in the transition from reversible to irreversible reperfusion injury. (Mol Cell Biochem 174: 167–172, 1997)

Heart and liver mitochondria contain a pore in the inner membrane that is activated by Ca2+ and oxidative stress and that has been implicated in cell injury. Pore opening is blocked by cyclosporin A (CSA). Following previous indications that the interaction of CSA with the pore is inhibited by Ca2+ and promoted by ADP, we have investigated how covalent labelling of heart mitochondria by a photoactive CSA derivative is influenced by these agents. In situ photolabelling of an 11-22-kDa (approximately) membrane fraction was selectively increased in the presence of ADP and decreased in the presence of Ca2+. This fraction also accounted for all the high affinity [3H]CSA-binding capacity and contained peptidylprolyl cis-trans isomerase activity (PPIase). The membrane PPIase was extracted using Chaps as detergent, and was purified to a 22-kDa protein (SDS/PAGE). The enzyme was inhibited by CSA (Ki 5 nM). The major component of the 11-22-kDa fraction, photolabelled in an ADP/Ca(2+)-sensitive manner, also migrated at 22 kDa on SDS/PAGE; a minor 11-kDa component was also detected. On the basis of these criteria, it is suggested that the membrane PPIase may be the target for CSA when it blocks the pore. The presence of a similar PPIase in the membrane fraction of liver mitochondria was also demonstrated. The implications of these findings are discussed.

Mitochondria contain a Ca 2+ -activated pore of relevance to tissue ischaemia/reperfusion injury. The pore is blocked by cyclosporin A (CSA). A photoactive CSA derivative has been used to covalently tag CSA binding proteins together with strict criteria for selecting the relevant tagged protein(s) based on the sensitivity of CSA-pore interactions to other pore ligands. The data implicate a membrane-associated 22 kDa cyclophilin and an 11 kDa component in pore function. *

Mitochondria contain a structure which forms a large aqueous pore in the inner membrane after Ca2+ overload in the presence of Pi. In the present study, pore activation in liver mitochondria was monitored using the collapse of the inner membrane potential (delta psi). Ca(2+)-induced pore opening (delta psi collapse) was prevented by the immunosuppressant cyclosporin A, but cyclosporin A did not reverse pore opening (i.e. allow delta psi regeneration) unless ADP was also added. At concentrations that produced substantial pore blockade, [3H]cyclosporin partitioned more or less equally between membrane and soluble fractions, but the distribution was shifted slightly to the membranes in the presence of ADP. ADP also increased the binding of [3H]cyclosporin A to membranes washed free of soluble components. The indication that cyclosporin A inhibition of the pore is mediated by an ADP-sensitive membrane component was examined using a tritiated photoactivable derivative of cyclosporin A. ADP selectively increased covalent binding of this derivative to a membrane component. This component eluted from molecular-sizing columns as a 13-17-kDa-protein in the presence of 0.5% Chaps as detergent and migrated as a 10-kDa (approximately) protein in SDS/PAGE. These findings provide the first evidence that a protein of approximately 10 kDa may be part of the cyclosporin-A receptor of the Ca(2+)-activated pore. The possible implications of these findings are discussed.

A cyclophilin-related protein has recently been found to be involved in tumor recognition by natural killer cells. The N-terminal end of this 150-kDa surface molecule (NK-TR) is homologous to cyclophilin/peptidylprolyl cis-trans-isomerase. We have constructed a soluble bacterial fusion protein between the cyclophilin-homologous domain of the NK-TR molecule and glutathione S-transferase (GST) to test for the presence of peptidylprolyl cis-trans-isomerase and chaperone activities and for cyclosporin A binding. The purified NK-cyp-GST fusion protein is shown to accelerate the isomerization of the prolyl peptide bond of the substrate N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide with a kcat/KM value of 7.4 x 10(5) M-1 s-1. The isomerase activity of the NK-TR cyclophilin homolog has been determined to be relatively insensitive to inhibition by the immunosuppressive drug cyclosporin A, with an IC50 value of 770 nM as compared to 19 nM for human cyclophilin. Furthermore, the NK-cyp-GST fusion protein has been found to participate in the protein folding process as a chaperone by preventing the aggregation of early folding intermediates of carbonic anhydrase. The implications of the finding of both peptidylprolyl cis-trans-isomerase and chaperone activities within the N-terminal domain of a large, cell type-restricted, surface molecule are discussed.

The inner membrane of rat liver mitochondria contains a reversible Ca(2+)-dependent pore, opening of which is largely blocked by cyclosporin A. Analyses of [3H]cyclosporin binding to rat liver mitochondria demonstrate two classes of high-affinity binding site with capacities of less than 5 pmol and approximately 60 pmol cyclosporin.mg mitochondrial protein-1 in addition to partitioning into membrane phospholipids (0.03 pmol.mg mitochondrial protein.nM-1). Direct measurement [14C]sucrose entry into the matrix space indicates that cyclosporin A inhibits pore opening by interacting with the low-capacity sites. The same low-capacity sites (Kd cyclosporin, 8 nM) are possibly attributable to peptidylprolyl cis-trans-isomerase, although investigation of pore state interconversion from the rapid kinetics of [14C]sucrose entrapment in the matrix space does not indicate that cyclosporin-sensitive prolyl isomerization occurs at the actual step of pore opening/closure. It is suggested that the low-capacity cyclosporin-binding component may stabilize the open pore state; this is supported by the observations that Ca2+ decreases cyclosporin binding to this component and that cyclosporin brings about closure of the pre-opened pore. The implications for the possible number of functional pores in mitochondria are discussed.

The cDNA encoding human cyclophilin from the Jurkat T-cell lymphoma line has been cloned by the expression cassette polymerase chain reaction and sequenced, and an expression vector has been constructed under control of the tac promoter for efficient expression in Escherichia coli. Active cyclophilin is produced at up to 40% of soluble cell protein, facilitating a one-column purification to homogeneity. Wild-type cyclophilin was characterized for binding of the potent immunosuppressant agent cyclosporin A (Kd = 46 nM) by tryptophan fluorescence enhancement and for inhibition (IC50 = 19 nM) of cyclophilin's peptidyl-prolyl cis-trans isomerase (rotamase) activity. With N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide as the substrate, recombinant human cyclophilin has a high catalytic efficiency; kcat/Km is 1.4 X 10(7) M-1.S-1 at 10 degrees C. To test the prior suggestion that a cysteine residue may be essential for catalysis and immunosuppressant binding, the four cysteines at positions 52, 62, 115, and 161 were mutated individually to alanine and the purified mutant proteins were shown to retain full affinity for cyclosporin A and equivalent catalytic efficiency as a rotamase. Clearly the cysteines play no essential role in catalysis or cyclosporin A binding. These results rule out the recently proposed mechanism [Fischer, G., Wittmann-Liebold, B., Lang, K., Kiefhaber, T. & Schmid, F. X. (1989) Nature (London) 337, 476-478)] involving the formation of tetrahedral hemithioorthoamide. Whereas mechanisms that embody other tetrahedral intermediates may be operative, an alternative mechanism is considered that involves distortion of bound substrate with a twisted (90 degrees) peptidyl-prolyl amide bond.

The immunophilins, protein receptors for the immunosuppressing drugs cyclosporin A and FK506 and related proteins from plants, fungi, and bacteria, have been analyzed structurally and evolutionarily. The cyclosporin A binding proteins (cyclophilins) represent one ubiquitous family of homologous proteins, and the FK506- and rapamycin-binding proteins (FKBPs) constitute a second, unrelated family. Multiple sequence alignments of members of each of these two protein families define the highly conserved residues that are likely to play important structural and functional roles, and mutations in representative members of these two families that abolish or alter function have been evaluated. FKBPs have undergone greater evolutionary divergence than the cyclophilins. Evolutionary trees were constructed using two distinct programs, and these trees establish the structural relationships that allow division of each of these families into subgroups. The results lead to the suggestion that several genes encoding isozymic forms of the FKBPs and possibly also of the cyclophilins existed in prokaryotes before the emergence of eukaryotes on earth and that representatives of these genes were transmitted to both kingdoms to give rise to current subfamilies of these proteins. By contrast, compartmentalization of both classes of immunophilins appears to have arisen independently in prokaryotes and eukaryotes, late in evolutionary history.

Cyclophilin, a specific cytosolic binding protein responsible for the concentration of the immunosuppressant cyclosporin A by lymphoid cells, was purified to homogeneity from bovine thymocytes. Cation-exchange high-performance liquid chromatography resolved a major and minor cyclophilin species that bind cyclosporin A with a dissociation constant of about 2 X 10(-7) moles per liter and specific activities of 77 and 67 micrograms per milligram of protein, respectively. Both cyclophilin species have an apparent molecular weight of 15,000, an isoelectric point of 9.6, and nearly identical amino acid compositions. A portion of the NH2-terminal amino acid sequence of the major species was determined. The cyclosporin A-binding activity of cyclophilin is sulfhydryl dependent, unstable at 56 degrees C and at pH 4 or 9.5, and sensitive to trypsin but not to chymotrypsin digestion. Cyclophilin specifically binds a series of cyclosporin analogs in proportion to their activity in a mixed lymphocyte reaction. Isolation of cyclophilin from the cytosol of thymocytes suggests that the immunosuppressive activity of cyclosporin A is mediated by an intracellular mechanism, not by a membrane-associated mechanism.

Cyclosporin A, an immunosuppressive agent, inhibited the prolactin stimulated growth of rat lymphoma Nb-2 cells. In the presence of 1 ng/ml of prolactin, 50% inhibition of growth was at 5 x 10(-6) M and the inhibition was reversible. The Kd of cyclosporin A binding to the Nb-2 cells was 10(-7) M and was independent of prolactin. The Kd of prolactin binding to the Nb-2 cells was 2 x 10(-10) M. Cyclosporin A did not influence the binding of prolactin to the cells and vice versa. The inhibitory effect of cyclosporin A on the growth of Nb-2 cells is due to some step other than the binding of prolactin to the cells.

Cyclophilin (cyclosporin A-binding protein) has a dual localization in the mitochondria and in the cytosol of Neurospora crassa. The two forms are encoded by a single gene which is transcribed into mRNAs having different lengths and 5' termini (approximately 1 and 0.8 kilobases). The shorter mRNA specifies the cytosolic protein consisting of 179 amino acids. The longer mRNA is translated into a precursor polypeptide with an amino-terminal extension of 44 amino acids which is cleaved in two steps upon entry into the mitochondrial matrix. Neurospora cyclophilin shows about 60% sequence homology to human and bovine cyclophilins.

High-field 1H NMR spectroscopy has been used to study the conformation of the cytosolic cyclosporin A binding protein cyclophilin. For the drug-free form of cyclophilin, spectral editing methods in conjunction with a pH titration were used to identify all four His residues present in the protein, and two-dimensional COSY and RELAY spectroscopy was used to elucidate the scalar connectivities in the aromatic and upfield methyl regions of the spectrum. From these scalar connectivities, it was possible to distinguish between inter- and intraresidue dipolar interactions within the aromatic and upfield methyl regions of cyclophilin in the NOESY spectrum. The results of this analysis showed extensive interresidue cross-relaxation among and between these latter spectral regions indicative of the proximal relationships of several of these residues and the presence of a hydrophobic core within cyclophilin.

Circular dichroism and resolution-enhanced Fourier transform infrared reveal induction of secondary structural elements on peptidyl-prolyl cis – trans isomerase A (PpiA) from Mycobacterium tuberculosis upon binding cyclosporin A (CsA). Thermal denaturation shows aggregation of PpiA at higher temperatures (>70 °C) and CsA fails to impart stabilization in protein structure. However, CsA stabilizes PpiA structure in urea denaturation. In presence/absence of CsA, urea-induced reversible unfolding of secondary and tertiary structures follows two-state and three-state transition, respectively. The chemical unfolding results also demonstrate that loss in the tertiary structure precedes the loss in secondary structure both in presence and absence of CsA at the initial stages. Fluorescence quenching suggests presence of a positive barrier around tryptophan microenvironment of PpiA.

MUTATIONS of the Drosophila melanogaster ninaA gene affect phototransduction: ninaA mutant flies have a 10-fold reduction in the levels of rhodopsin in the R1–R6 photoreceptor cells1,2. The ninaA gene was isolated and found to encode a 237-amino-acid protein that has over 40% amino-acid sequence identity with the vertebrate cyclosporin A-binding protein, cyclophilin, a protein that seems to be involved in T-lymphocyte activation. The remarkable evolutionary conservation of cyclophilin in two phylogeneti-cally distant organisms and its involvement in diverse transduction processes suggests that this protein plays an important role in cellular metabolism. Indeed, cyclophilin has recently been shown to be a prolyl cis–trans isomerase that catalyses, in vitro, rate-limiting steps in the folding of a number of proteins3,23. Here, we present evidence for the involvement of cyclophilin-like molecules in a defined cellular process. The availability of mutations in a cyclophilin gene provides a new model system for the study of cyclophilin and cyclosporin action.

Peptidyl-prolyl cis-trans isomerase (PPIase) catalyses the cis-trans isomerization of proline imidic peptide bonds in oligopeptides and has been shown to accelerate the refolding of several proteins in vitro. Its activity has been detected in yeast, insects and Escherichia coli as well as in mammals, and it is though to be essential for protein folding during protein synthesis in the cell. We purified PPIase from pig kidney and found that its amino-acid sequence is identical to that reported for bovine cyclophilin, a protein known to bind the immunosuppressive drug, cyclosporin A (ref. 5). To investigate the functional relationship between PPIase and cyclophilin we examined the effect of cyclosporin A on PPIase activity and found that it was inhibitory. Thus we propose that the peptidyl-prolyl cis-trans isomerizing activity of PPIase may be involved in events, such as those occurring early in T-cell activation, that are suppressed by cyclosporin A.

The immunosuppressant drug [3H]cyclosporin A binds specifically and with high affinity to rat brain membrane preparations. The highest density of binding sites was observed in the hippocampus, cerebellum, cortex and basal ganglia. A similar distribution pattern was seen using a quantitative autoradiographic analysis. This distribution agrees with the localizations of cyclophilin and calcineurin reported in immunohistochemical and in situ hybridization studies. Thus inhibition of calcineurin activity following cyclosporin A binding to cyclophilin may occur in neurones, as it does in T-cells. These results suggest that the neurological side-effects of cyclosporin A may be mediated through its interaction with these proteins in neurones.

This paper reports an investigation on the regulation of the mitochondrial cyclosporin A-sensitive permeability transition pore (MTP). Energized, coupled rat liver mitochondria incubated in sucrose medium in the presence of phosphate maintain a high proton electrochemical gradient (delta microH) and a low permeability to solutes. Addition of a small (10-20 microM) Ca2+ pulse leads to a transient membrane depolarization. After Ca2+ accumulation, a high delta microH is recovered, and mitochondria remain coupled indefinitely. Yet, addition of fully uncoupling concentrations of carbonyl cyanide-p-trifluoromethoxyphenyl hydrazone (FCCP) brings about MTP opening within seconds. This finding confirms that MTP opening is the consequence rather than the cause of membrane depolarization, and allowed us to study the operation of the MTP in a synchronized population of mitochondria, since pore opening can be triggered by the addition of uncoupler under a series of experimental conditions. We find that three regulatory sites can be defined: (i) an internal Me2+ binding site: when this site is occupied by Ca2+, the pore "open" probability increases, while other Me2+ ions (Sr2+, Mn2+) have an inhibitory effect; (ii) an external Me2+ binding site: when this site is occupied by Me2+ ions, including Ca2+, the pore open probability decreases; (iii) an independent cyclosporin A binding site: when this site is occupied by cyclosporin A the pore open probability decreases. We show that at variance from the case of cyclosporin A, MTP inhibition by the phospholipase A2 inhibitors nupercaine and trifluoperazine is Ca(2+)-competitive and is presumably related to interference by these drugs with Ca2+ binding to the internal regulatory site.