Characterization of the proapoptotic intracellular mechanisms induced by a toxic conformer of the recombinant human prion protein fragment 90-231.
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 alpha-structured isoform (PrPC) to a prevalent beta-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 beta-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 beta-conformation, but not when alpha-structured, powerfully affected the survival of these cells. hPrP90-231 beta-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 beta-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 beta-sheet-rich conformation and that this effect is mediated by p38 and caspase-3 activation.
Villa
Valentina
V
Department of Oncology, Biology and Genetics, University of Genova, Viale Benedetto XV, 16132 Genova, Italy.
Corsaro
Alessandro
A
Thellung
Stefano
S
Paludi
Domenico
D
Chiovitti
Katia
K
Venezia
Valentina
V
Nizzari
Mario
M
Russo
Claudio
C
Schettini
Gennaro
G
Aceto
Antonio
A
Florio
Tullio
T
D017209:Apoptosis; D020169:Caspases / Q000378:metabolism; D002460:Cell Line; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D011328:Prions / Q000737:chemistry / Q000378:metabolism*; D020033:Protein Isoforms / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
D011328:Prions; D020033:Protein Isoforms; D011994:Recombinant Proteins; D048051:p38 Mitogen-Activated Protein Kinases; D020169:Caspases