Substitution in Position 3 of Cyclosporin A Abolishes the Cyclophilin-mediated Gain-of-function Mechanism but Not Immunosuppression

Abstract

Both immunosuppressive drugs cyclosporin A (CsA) 1 and tacrolimus (FK506) are believed to inhibit antigen-specific activation of lymphocytes by blocking T cell receptor (TCR) signaling cascades through targeting the Ca 2ϩ /calmodulindependent Ser/Thr phosphatase calcineurin (1, 2), which represents a specific bottleneck of antigen-receptor signaling. The drugs alone do not inhibit the enzymatic activity of calcineurin. CsA and FK506 bind to and inhibit calcineurin only after interaction with their respective peptidyl-prolyl cis/trans isomerases (PPIases) (EC 5.2.1.8), cyclophilins (Cyps), and FK506-binding proteins (FKBPs) through a gain-of-function mechanism. Therefore, these particular PPIases were also termed immunophilins. Among the many known human PPIases, the most abundant cytosolic members cyclophilin 18 (Cyp18) and FKBP12 were found as major receptor proteins for CsA and FK506, respectively (2, 3). Based on the structures of FKBP12-FK506-calcineurin (4, 5) and Cyp18-CsA-calcineurin complexes (6, 7), it is proposed that the inhibition of calcineurin results from blockage of access for protein substrates to the active site by the drug-immunophilin complexes, but does not involve a direct contact to the catalytic center of the protein phosphatase.

Inhibition of calcineurin‐NFAT signaling by the pyrazolopyrimidine compound NCI3

“…Comparison of the NCI3-mediated suppression of the NFAT-and NF-jB-luciferase reporter gene expression with the CsA-mediated effects indicated that the pathways inducing the activity of these two transcription factors exhibited different sensitivity to NCI3 and CsA. Whereas CsA inhibited the expression of both reporter gene constructs with similar efficiencies (IC 50 of 2.0 and 4.0 nM) [18], NCI3 exhibited IC 50 values of 2 AE 0.1 lM for the NFAT and 7 AE 1 lM for the NF-jB-driven construct. Interestingly, the TNF-amediated stimulation of the NF-jB reporter plasmid in Jurkat cells remained unaffected by CsA and NCI3 ( Table 2), suggesting that in this signaling pathway calcineurin is not involved.…”

“…The biotinylated and non-biotinylated VIVIT peptide (MAGPHPVI-VITGPHEE) [18], the reversed VIVIT peptide (EEHPGTIVIVPH-PGAM), and the biotinylated RII peptide (DLDVPIPGRFDRRV-SVAAE-OH) were synthesized by solid-phase peptide synthesis (Quartett, Berlin, Germany).…”

“…The phosphatase activity of the four major Ser/Thr protein phosphatases PP1, PP2A, PP2B, and PP2C against protein substrates and their inhibition was assayed with biotinylated 33 P-labeled phospho-casein or, in the case of PP1, with biotinylated 33 P-labeled phosphorylase a, using a scintillation proximity assay as described previously [15,18]. The protein phosphatase concentrations were adjusted to an activity level of approximately 80% dephosphorylation of the substrate within 20 min at 30 C.…”

“…PBMC were stimulated with 10 ng/mL PMA and 1 lg/mL ionomycin (both Sigma) for 5 h. Brefeldin A (5 lg/mL) was added for the last 3 h of stimulation. Cells were then fixed in 2% paraformaldehyde for 20 min, permeabilized by washing in PBS supplemented with 0.5% saponin and 1% FCS, incubated with an anti-IL-2 PE-conjugated antibody and measured by flow cytometry [18].…”

Dephosphorylation of Bcl‐10 by calcineurin is essential for canonical NF‐κB activation in Th cells

“…Bcl-10 phosphorylation events can both positively and negatively regulate NF-kB activation (reviewed in [12]). Several studies have reported that not only NFAT, but also NF-kB activation by antigen-specific T-cell stimulation is dependent on the phosphatase activity of CaN, identified by using the immunosuppressive drug cyclosporine A (CsA) [13][14][15][16]. CsA binds to its matchmaker protein cyclophilin A and, subsequently, this complex inhibits the Ser/Thr phosphatase activity of CaN [17][18][19][20].…”