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a Department of Oncology, Biology and Genetics, University of Genova, 16132 Genova, Italy b Department of Scienze degli Alimenti, Veterinary School, University of Teramo, Teramo, Italy c Department of Biomedical Sciences, University G. D'Annunzio, Chieti, Italy d Department of Health Sciences, University of Molise, Campobasso, Italy
Key Words: prion protein • apoptosis • p38 MAP kinase • caspases
Address for correspondence: Prof. Tullio Florio, Department of Oncology, Biology and Genetics, University of Genova, Viale Benedetto XV, 16132 Genova, Italy. Voice and fax: +39-010-3538806. e-mail: tullio.florio{at}unige.it
Prion diseases comprise a group of fatal neurodegenerative disorders that affect both animals and humans. The transition of the prion protein (PrP) from a mainly
 -structured isoform (PrPC) to a prevalent  -sheet-containing protein (PrPSc) is believed to represent a major pathogenetic mechanism in prion diseases. To investigate the linkage between PrP neurotoxicity and its conformation, we used a recombinant prion protein fragment corresponding to the amino acidic sequence 90–231 of human prion protein (hPrP90–231). Using thermal denaturation, we set up an experimental model to induce the process of conversion from PrPC to PrPSc. We report that partial thermal denaturation converts hPrP90–231 into a -sheet-rich isoform, displaying a temperature- and time-dependent conversion into oligomeric structures that share some physico-chemical characteristics with brain PrPSc. SH-SY5Y cells were chosen to characterize the potential neurotoxic effect of hPrP90–231 in its different structural conformations. We demonstrated that hPrP90–231 in -conformation, but not when -structured, powerfully affected the survival of these cells. hPrP90–231 -structured caused DNA fragmentation and a significant increase in caspase-3 proteolytic activity (maximal effects + 170%), suggesting the occurrence of apoptotic cell death. Finally, we investigated the involvement of MAP kinases in the regulation of -hPrP90–231-dependent apoptosis. We observed that the p38 MAP kinase blocker SB203580 prevented the apoptotic cell death evoked by hPrP90–231, and Western blot analysis revealed that the exposure of the cells to the peptide induced p38 phosphorylation. In conclusion, we demonstrate that the hPrP90–231 elicits proapoptotic activity when in -sheet-rich conformation and that this effect is mediated by p38 and caspase-3 activation.
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