Sensitivity to cyclosporin A is mediated by cyclophilin in Neurospora crassa and Saccharomyces cerevisiae

Tropschug, Maximilian; Barthelmess, Ilse B.; Neupert, Walter

doi:10.1038/342953a0

Cited by 215 publications

(141 citation statements)

References 30 publications

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“…This concentration dependence and the above analysis also strongly suggest that the immunophilin-drug complex is the biological effector. This conclusion is analogous to that reached by Tropschug et al (39) in their study of mutant strains of Neurospora crassa and Saccharomyces cerevisiae that are resistant to CsA. These mutated organisms either lost their ability to produce the CsA binding immunophilin CyP, or, if CyP was produced, it no longer bound CsA.…”

Section: Discussionsupporting

confidence: 82%

Two distinct signal transmission pathways in T lymphocytes are inhibited by complexes formed between an immunophilin and either FK506 or rapamycin.

Bierer

Mattila

Standaert

et al. 1990

Proc. Natl. Acad. Sci. U.S.A.

628

382

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Proliferation and immunologic function of T lymphocytes are initiated by signals from the antigen receptor that are inhibited by the immunosuppressant FK506 but not by its structural analog, rapamycin. On the other hand, interleukin 2 (IL-2)-induced signals are blocked by rapamycin but not by FK506. Remarkably, these two drugs inhibit each other's actions, raising the possibility that both act by means of a common immunophilin (immunosuppressant binding protein). We find that the dissociation constant of rapamycin to the FK506 binding protein FKBP (Kd = 0.2 nM) is close to the dissociation constant of FK506 to FKBP (Kd = 0.4 nM) and to their effective biologic inhibitory concentrations. However, an excess of rapamycin is needed to revert FK506-mediated inhibition of IL-2 production, apoptosis, and transcriptional activation of NF-AT, a T-cell-specific transcription factor necessary for IL-2 gene activation. Similarly, an excess of FK506 is needed to revert rapamycin-mediated inhibition of IL-2-induced proliferation. The drug concentrations required for antagonism may be explained by the relative affinity of the drugs to, and by the abundance of, the immunophilin FKBP. FKBP has been shown to catalyze the interconversion of the cisand trans-rotamers of the peptidyl-prolyl amide bond of peptide substrates; here we show that rapamycin, like FK506, is a potent inhibitor of the rotamase activity of FKBP (Ki = 0.2 nM). Neither FKBP binding nor inhibition of rotamase activity of FKBP alone is sufficient to explain the biologic actions of these drugs. Rather, these findings suggest that immunophilin bound to FK506 interferes with antigen receptor-induced signals, while rapamycin bound to the immunophilin interferes with IL-2-induced signals.Cyclosporin A (CsA) and the structurally unrelated immunosuppressant FK506 have been demonstrated to inhibit T-cell activation and to be effective in organ transplantation (1-3). CsA has been shown to bind to and inhibit the rotamase activity of a cytoplasmic enzyme, cyclophilin (CyP) (4-6). Recently, a cytoplasmic receptor for FK506, FK506 binding protein (FKBP), which is unrelated in sequence to CyP, has also been shown to have similar rotamase activity, suggesting that this common enzymatic property of immunophilin proteins (immunosuppressant binding proteins) may be related to the regulation of T-cell activation (7-10). Rapamycin is a macrolide similar in structure to FK506 and, like FK506,12) MATERIALS AND METHODSReagents, Cell Culture, Interleukin 2 (IL-2) Production, and DNA Fragmentation. The IL-2-producing murine T-cell hybridoma 16.CD2-15.20 (15) was cultured (106 cells per well) with or without a 2% (vol/vol) culture supernatant of the anti-murine CD3 monoclonal antibody (mAb) 145-2C11 (16) in the absence or presence of CsA, FK506, or rapamycin. At 24 hr, culture supernatants were dialyzed and assayed for the presence of IL-2 as described (15). DNA was isolated from the cells and assayed for induction of DNA fragmentation or apoptosis on an ethidium bromide-stained...

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Section: Discussionsupporting

confidence: 82%

Two distinct signal transmission pathways in T lymphocytes are inhibited by complexes formed between an immunophilin and either FK506 or rapamycin.

Bierer

Mattila

Standaert

et al. 1990

Proc. Natl. Acad. Sci. U.S.A.

628

382

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“…It is the most likely candidate for a cellular receptor of cyclosporin A mediating the immunosuppressive effects of the drug on T cells [2]. In accordance with this assumption, it has been shown recently that, at least in the lower eukaryotes Neurosporo crassn and Sacchuromyces cerevisiae, cyclophilin indeed mediates the cytotoxic effects of the drug on these organisms [3]. On the other hand, binding of cyclosporin A to calmodulin [4] or the prolactin receptor [5] has also been reported and the mechanism of action of cyclosporin A in T cells remains to be clearly defined.…”

mentioning

confidence: 95%

Characterization of the human cyclophilin gene and of related processed pseudogenes

Haendler¹,

Hofer²

1990

European Journal of Biochemistry

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The human cyclophilin gene was isolated from a genomic library derived from leucocyte DNA and sequenced. The gene contains five exons and four introns. The amino acid sequence deduced from the exons matches perfectly the one previously determined from the T-cell cyclophilin cDNA. A TATA box is visible in the promoter region and putative Spl binding sites are also found there as well as in the first intron. Six members of the middle repetitive Alu gene family are present in one or other orientation in the non-coding regions of the cyclophilin gene. Hybridisation of genomic DNA to probes derived from the promoter region or the first intron indicates that the cyclophilin gene is present as a single copy in the human haploid genome. Seven other cyclophilin-related DNA clones isolated from the same library were also characterized. They show a high degree of similarity to the cyclophilin cDNA and are colinear to it. However, multiple genetic lesions, often including deletion and/or insertion events which modify the reading frame, are found in these clones which are therefore likely to represent processed pseudogenes.Cyclophilin was originally isolated as the main cyclosporin-A-binding protein in the cytosol of eukaryotic cells [l]. It is the most likely candidate for a cellular receptor of cyclosporin A mediating the immunosuppressive effects of the drug on T cells [2]. In accordance with this assumption, it has been shown recently that, at least in the lower eukaryotes Neurosporo crassn and Sacchuromyces cerevisiae, cyclophilin indeed mediates the cytotoxic effects of the drug on these organisms [3]. On the other hand, binding of cyclosporin A to calmodulin [4] or the prolactin receptor [5] has also been reported and the mechanism of action of cyclosporin A in T cells remains to be clearly defined.Cyclophilin was first purified from bovine thymus [6] and subsequent cloning of the cDNA for human [7], rat [8], N . crussa [9] and yeast [lo] cyclophilin showed that the protein is highly conserved throughout evolution. It is a very abundant protein representing 0.1 -0.4% of the total cytosolic protein content of most eukaryotic cells [l I]. Furthermore, cyclophilin has recently been demonstrated to be identical to peptidylprolyl cis-trans-isomerase [12, 131, an enzyme which catalyses the isomerisation of prolyl peptide bonds and is probably essential for the correct steric folding of a number of proteins.Even though some tumour cell lines show a higher concentration of cyclophilin, most of the normal tissues and cell lines analysed so far contain similar stable levels of the protein or of its specific mRNA [8, 111. This is also the case for T lymphocytes where no modification of the high cyclophilin mRNA level was observed upon activation of these cells with phytohaemagglutinin and the tumour promoter phorbol myristate acetate [7]. These findings seem at odds with the mechanism of action of cyclosporin A, which at low concen- We recently showed that the human genome contains numerous cyclophilin-related sequences [7] an...

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“….~ focused initially on PPIase inhibition as the cause of in vivo immunosuppression, several lines of evidence now point to the CyP-CsA complex as the active immunosuppressive species (Tropschug et al, 1989;Friedman & Weissman, 1991;Liu et al, 1991b).…”

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confidence: 99%

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

et al. 1992

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Based on recent X-ray structural information, six site-directed mutants of human cyclophilin A (hCyPA) involving residues in the putative active site--54, R55, F60, Q l l l , 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, Biochemistry30, 2306-2310), H54Q, R55A, F60A, Q l l l A , 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, Q l l l A , F113A, and W121A retain 3-15% of the catalytic efficiency (kcoJKm) 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.

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Sensitivity to cyclosporin A is mediated by cyclophilin in Neurospora crassa and Saccharomyces cerevisiae

Cited by 215 publications

References 30 publications

Two distinct signal transmission pathways in T lymphocytes are inhibited by complexes formed between an immunophilin and either FK506 or rapamycin.

Two distinct signal transmission pathways in T lymphocytes are inhibited by complexes formed between an immunophilin and either FK506 or rapamycin.

Characterization of the human cyclophilin gene and of related processed pseudogenes

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

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