NMR studies of [U-C]cyclosporin A bound to human cyclophilin B

NMR data (¹H and ¹³C chemical shifts, NOEs) on [[U-¹³C]cyclosporin A bound to cyclophilin B were compared to previously published data on the [U-¹³C]CsA/CyPA complex [Fesik et al., (1991) Biochemistry 30, 6574–6583]. Despite only 64% sequence identity between CyPA and CyPB, the conformation and active site environment of CsA when bound to CyPA and CyPB are nearly identical as judged by the similarity of the NMR data.     

Identification of two forms of cyclophilin from the hard tick Haemaphysalis longicornis

We have shown here the identification and characterization of two cyclophilin, cyclophilin A (CyPA) and B (CyPB), from the ixodid tick, Haemaphysalis longicornis. Both CyPA and CyPB contain the conserved peptidyl-prolyl isomerase (PPIase) domain, with CyPA consisting of 188 amino acids and CyPB of 216 amino acids. CyPA and CyPB share 50–67% amino acid sequence identity with the cyclophilins of other organisms, and are found in multiple organs throughout the developmental stages of ixodid ticks. In addition, recombinant CyPA and CyPB exhibited PPIase activity that could be inhibited by the cyclic peptides cyclosporin A (CsA). Silencing of CyPA through RNA interference has led to a significant reduction in the body weight of engorged ticks and their failure to lay eggs, in contrast to CyPB whose silencing did not result in any detectable phenotypic changes. Our results indicate that CyPA represents the major cyclophilin protein in H. longicornis involved in blood ingestion, viability, and oocyte development. This is the first report of cyclophilin proteins from ixodid and argasid ticks.     

1.88 A crystal structure of the C domain of hCyP33: a novel domain of peptidyl-prolyl cis-trans isomerase

Cyclophilins (CyPs) are a widespreading protein family in living organisms and possess the activity of peptidyl-prolyl cis-trans isomerase (PPIase), which is inhibited by cyclosporin A (CsA). The human nuclear cyclophilin (hCyP33) is the first protein which was found to contain two RNA binding domains at the amino-terminus and a PPIase domain at the carboxyl-terminus. We isolated the hCyP33 gene from the human hematopoietic stem/progenitor cells and expressed it in Escherichia coli, and determined the crystal structure of the C domain of hCyP33 at 1.88 A resolution. The core structure is a beta-barrel covered by two alpha-helices. Superposition of the structure of the C domain of hCyP33 with the structure of CypA suggests that the C domain contains PPIase active site which binds to CsA. Furthermore, C domain seems to be able to bind with the Gag-encoded capsid (CA) of HIV-1 and may affect the viral replication of HIV-1. A key residue of the active site is changed from Ala-103-CypA to Ser-239-hCyP33, which may affect the PPIase domain/substrates interactions.     

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.     

Cyclosporin A binding to Mycobacterium tuberculosis peptidyl-prolyl cis-trans isomerase A--investigation by CD, FTIR and fluorescence spectroscopy

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.     

A single Trp121 to Ala121 mutation in human cyclophilin alters cyclosporin A affinity and peptidyl-prolyl isomerase activity

Fluorescence and NMR spectral data have suggested an interaction between the single tryptophan in cyclophilin (CyP) and its high affinity ligand cyclosporin A (CsA). To study this interaction, a site mutation of Trp121 to Ala was introduced into human cyclophilin (CyP) and the encoded protein was expressed in E. coli. The Ala121 mutant was shown to catalyze the peptidyl-prolyl cis-trans isomerase (rotomase) reaction with several peptide substrates, albeit at less than ten percent the rate of the purified recombinant human CyP. Values for the apparent inhibition constant (Ki,app) of cyclosporin A with the human CyP and the Ala121 mutant were determined to be 1.6 +/- 0.4 nM and 640 +/- 90 nM, respectively by tight-binding inhibition analysis. The greater loss of affinity for CsA binding (400-fold) than for rotomase catalysis (20 fold) suggests that the catalytic and CsA binding properties associated with CyP can be decoupled as has been observed with an homologous protein found in E. coli (Liu, J. & Walsh, C.T. (1990) Proc. Natl. Acad. Sci. USA 87, 4028-4032).     

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.     

Anti-IL-17A blocking antibody reduces cyclosporin A-induced relapse in experimental autoimmune encephalomyelitis mice

Cyclosporin A (CsA) is effective at reducing pathogenic immune responses, but upon withdrawal of CsA the immune response often “rebounds” resulting in a relapse or exacerbation of disease. The mechanisms, cells and cytokines involved in the relapse or exacerbation after CsA withdrawal are unknown. We hypothesized that CsA withdrawal induces IL-17 production that could be responsible for relapse, and examined the effect of anti-IL-17A antibody on relapse induced after CsA withdrawal in mouse experimental autoimmune encephalomyelitis (EAE). CsA treatment markedly decreased the EAE disease score during the first episode, but augmented disease severity after CsA withdrawal, compared to untreated mice. After discontinuation of CsA the production of IL-17A was increased and the severity of relapse in EAE was reduced by treatment with anti-IL-17A antibody. These results suggest that the resumption of T cell immune responses after CsA withdrawal leads to a burst of IL-17A production that is at least partially responsible for relapse in EAE mice.     

Cyclophilin sensitivity to sanglifehrin A can be correlated to the same specific tryptophan residue as cyclosporin A

Sanglifehrin A (SFA) is a recently discovered immunosuppressant drug that shares its intracellular target with the major immunosuppressant drug cyclosporin A (CsA). Both bind to and inhibit the cyclophilins, a diverse family of proteins found throughout nature that share a conserved catalytic domain. Although they share this common protein target, the mechanism of action of the cyclophilin-SFA complex has been reported as distinct from that of the well-studied cyclophilin-CsA complex. The X-ray structure of a macrolide analogue of SFA's cyclic region complexed with cyclophilin A has recently been resolved, but this left the placement of the linear region of SFA unresolved. Using five cyclophilins from the fission yeast Schizosaccharomyces pombe, and a mutant of one of these proteins, SpCyp3-F128W, we have shown that the sensitivity of cyclophilins to SFA can be correlated to the same specific tryptophan residue that has previously been identified to correlate to CsA sensitivity, and that the tail of SFA may be responsible for mediating this sensitivity.     

Effects of lethal irradiation and cyclosporin A treatment on the growth and tumoricidal activity of a T cell clone potentially useful in cancer therapy

The TALL-104 cell line, originally derived from a patient with T cell leukemia, can be maintained indefinitely in culture in the presence of interleukin-2 (IL-2) and is endowed with a highly potent major-histocompatibilitycomplex (MHC)-non-restricted tumoricidal activity both in vitro and in animal models. The present study analyzes in detail the short- and long-term effects of irradiation and cyclosporin A (CsA) treatment on the growth and tumoricidal function of this T cell clone as compared to polyclonal lymphokine-activated killer (LAK) cell preparations from healthy donors. DNA and RNA syntheses by both TALL-104 and LAK cells were irreversibly arrested a few hours after irradiation with 40 Gy. However, 4-h⁵¹Cr-release assays, performed on different days (day 1 to day 7) after irradiation, showed that the cytotoxic efficiency of TALL-104 cells against hematopoietic and solid tumor targets was only modestly reduced, whereas that of LAK cells was severely inhibited. Moreover, the cytotoxic responses to recombinant human IL-2 and IL-12, measured 18 h after irradiation and cytokine addition, were normal in the case of TALL-104 cells but were abolished in the case of LAK cells. Co-culture of IL-2-or IL-12-preactivated TALL-104 cells with a tumor target for 5 days in the absence of cytokines resulted in a lower efficiency of lysis, as compared to the non-irradiated effectors, especially if the initial stimulus was IL-12. These findings suggest the requirement of multiple cytokine stimulation for optimal expression of tumoricidal activity by lethally irradiated TALL-104 cells. CsA, while abrogating TALL-104 cell proliferation at the low dose of 0.5 g/ml, inhibited their cytotoxic function marginally only at high doses (100 g/ml). By contrast, CsA reduced dose-dependently the cytotoxicity of LAK cells starting at very low doses (0.5 g/ml). CsA did not impair the ability of TALL-104 and LAK cells to produce interferon (IFN), tumor necrosis factor (TNF) , and granulocyte/macrophage-colony-stimulatory factor (GM-CSF) in response to IL-2, IL-12, or tumor targets. Irradiation reduced drastically IFN production by LAK, but not TALL-104 cells; release of TNF and GM-CSF by either type of effector was inhibited by 10%–50%, depending on the stimulus. The high resistance of the TALL-104 cells' tumoricidal function to irradiation and immunosuppressive drugs renders this immortal T cell clone a potentially safe and effective reagent for new adoptive-transfer approaches to cancer in MHC-incompatible recipients.     

Interferon-α-(IFN) producing CHO cell lines are resistant to the antiproliferative activity of IFN: A correlation with gene expression

CHO cell lines that constitutively produce the murine interferon-α (IFN-α) subspecies α4 and α6 were constructed. The producer cell lines were protected against viral (vesicular stomatitis virus) infection by the IFN species secreted, but were resistant to the growth inhibitory activity of the IFN species. As compared with α4, the α6 protein displayed a high antiproliferative activity when added to normal CHO cells, which correlates completely with the high antiviral activity of a6 on these cells. Three messenger ribonucleic acid (mRNA) species, which are normally induced in CHO cells by IFN treatment (1–8, 2–5A synthetase, and ISG 15) were constitutively present in CHO producer cell lines. The level of another mRNA (ISG 54), however, was very low in the producer cells as compared with its expression in short-term IFN-treated cells. These data indicate that 1–8, 2–5A synthetase and ISG 15 are not involved in the antigrowth activity of IFN in this system, but rather suggest a function of ISG 54 in this respect.