Autumnalamide targeted proteins of the immunophilin family

Abstract

Previous works with autumnalamide reported that Store Operated Calcium (SOC) channels were blocked through mitochondrial modulation. In the present paper we studied the effect of autumnalamide on ionomycin Ca²⁺ fluxes. Thus, autumnalamide did not modify ionomycin-sensitive intracellular pools while the ionomycin-induced Ca²⁺ influx was blocked with similar potency whether the incubation was done before or after ionomycin-sensitive pools depletion. Nevertheless, autumnalamide was not able to inhibit ionomycin-induced Ca²⁺ influx once the membrane channels were activated. Moreover, the compound efficiently inhibited flufenamic acid (FFA) Ca²⁺ release induced in this organelle but no the next influx. Since in previous work the effect of autumnalamide was inhibited by cyclosporine A (CsA), structures that target this drug were studied. Therefore, the affinity of autumnalamide for cyclophilin D (Cyp D) was examined. The K_D obtained for Cyp D- autumnalamide was 1.51±1.399. Moreover, the K_D for Cyp A- autumnalamide was calculated. The peptide had a similar order of Cyp A binding affinity than CsA (8.08±1.23 and 6.85±1.1μM respectively). After testing autumnalamide-binding capacity for Cyp A, the activity of this compound on Cyp A pathway was tested. Thus, the effect on interleukin (IL)-2 release on activated T-lymphocytes was checked. Autumnalamide was able to reduce IL-2 levels near to T cells in resting conditions. Next, the effect over calcineurin and NFATc1 was also evaluated. While CsA inhibits both calcineurin and NFATc1, autumnalamide did not produce any effect. From these results we can conclude that, autumnalamide targeted mitochondrion and prevent T-cells from IL-2 production through the modulation of SOC Ca²⁺ channels.

Keywords: Ca(2+); Cyclophilin; Mitochondria; Phormidium autumnale; SOC channels.