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Fig. 1. Effect of AA on PTP opening in isolated mouse liver mitochondria (left) and inmitochondria of wild-type or CyP-D knock-out mouse fibroblasts (right).The CRC (Calcium Retention Capacity) assay was used to assess PTP opening followingtrains of Ca 2+ pulses and measured fluorimetrically at 25 °C in the presence of the Ca 2+indicator Calcium Green-5N. Experiments were performed either on isolated mitochondriaor on whole cells (Figure 1). The data obtained show that AA induces opening of the poreand this effect is due to its interaction with Cyp-D, as it was absent in cells derived fromCyp-D knock-out animals. All AA derivatives tested proved to be ineffective.Tests were also conducted to evaluate the effect of AA on mitochondrialdepolarization and cell death induced by clotrimazole or hexokinase II N-terminal peptide(data not shown). The results show a protective action on both events. Mitochondrialdepolarization was tested by measuring the fluorescence of TMRM, a probe thataccumulates in polarized mitochondria. Cell death was instead tested by measuring thefluorescence of propidium iodide, which is unable to cross membranes and, therefore, asignal arising from this probe means an irreversible damage of the membrane after thedeath of the cell.In conclusion, we demonstrated that AA desensitizes the PTP, a central effector of celldeath induction, by targeting the PTP regulator CyP-D. Indeed, pore inhibition by AA isnot additive with the effect of the CyP-D-binding drug CsA, and AA is ineffective onmitochondria or cells derived from CyP-D knock-out animals. Pore desensitization by AAneeds two critical residues in the peptide ring, Phe 6 and Phe 9 , and is additive withubiquinone 0 (Ub0), which acts on the PTP in a CyP-D-independent way. AA alsoabrogates mitochondrial depolarization and the resulting cell death caused by two wellcharacterizedPTP inducers, clotrimazole or a hexokinase II N-terminal peptide.Conversely, the reported AA inhibition of the toxic effects of the phallotoxin Phalloidin isindependent of PTP modulation. Our findings have implications for the comprehension ofCyP-D activity on the pore and for the development of novel PTP-targeting drugs usable ascell death inhibitors.References1. Ruzza, P., et al. J. Pept. Res. 53, 442-452 (1999).2. Nielsen, O. Acta Pharmacol. Toxicol. (Copenh) 59, 249-251 (1986).3. Welbourn, R., et al. J. Appl. Physiol. 70, 1364-1368 (1991).4. Benedetti, E., Pedone, C. J. Pept. Sci. 11, 268-272 (2005).5. Waldmeier P., et al. Curr. Med. Chem. 10, 1485-1506 (2003).6. Yang, Y.,et al. Biochem. Biophys. Res. Commun. 363, 1013-1019 (2007).329