U937 cell apoptosis induced by arsenite is prevented by low concentrations of mitochondrial ascorbic acid with hardly any effect mediated by the cytosolic fraction of the vitamin

partially_open6noArsenite directly triggers cytochrome c and Smac/DIABLO release in mitochondria isolated from U937 cells. These effects were not observed in mitochondria pre-exposed for 15 min to 10 µM L-ascorbic acid (AA). In other experiments, intact cells treated for 24–72 h with arsenite were found to die by apoptosis through a mechanism involving mitochondrial permeability transition. Pre-exposure (15 min) to low micromolar concentrations of AA and dehydroascorbic acid (DHA), resulting in identical cytosolic levels of the vitamin, had a diverse impact on cell survival, as cytoprotection was only observed after treatment with AA. Also the mitochondrial accumulation of the vitamin was restricted to AA exposure. An additional indication linking cytoprotection to the mitochondrial fraction of the vitamin was obtained in experiments measuring susceptibility to arsenite in parallel with loss of mitochondrial and cytosolic AA at different times after vitamin exposure. Finally, we took advantage of our recent findings that DHA potently inhibits AA transport to demonstrate that DHA abolishes all the protective effects of AA, under the same conditions in which the mitochondrial accumulation of the vitamin is prevented without affecting the overall cellular accumulation of the vitamin.embargoed_20150323Andrea Guidarelli;Mara Fiorani;Catia Azzolini;Liana Cerioni;Maddalena Scotti;Orazio CantoniGuidarelli, Andrea; Fiorani, Mara; Catia, Azzolini; Cerioni, Liana; Scotti, Maddalena; Cantoni, Orazi

Non-equivalent binding sites for Abeta1-40 on PrP determine the oligomerisation pathway

International audienceno abstrac

A seven-residue deletion in PrP leads to generation of a spontaneous prion formed from C-terminal C1 fragment of PrP

International audiencePrions result from a drastic conformational change of the host-encoded cellular prion protein (PrP), leading to the formation of β-sheet-rich, insoluble, and protease-resistant self-replicating assemblies (PrPSc). The cellular and molecular mechanisms involved in spontaneous prion formation in sporadic and inherited human prion diseases or equivalent animal diseases are poorly understood, in part because cell models of spontaneously forming prions are currently lacking. Here, extending studies on the role of the H2 α-helix C terminus of PrP, we found that deletion of the highly conserved 190HTVTTTT196 segment of ovine PrP led to spontaneous prion formation in the RK13 rabbit kidney cell model. On long-term passage, the mutant cells stably produced proteinase K (PK)-resistant, insoluble, and aggregated assemblies that were infectious for naïve cells expressing either the mutant protein or other PrPs with slightly different deletions in the same area. The electrophoretic pattern of the PK-resistant core of the spontaneous prion (ΔSpont) contained mainly C-terminal polypeptides akin to C1, the cell-surface anchored C-terminal moiety of PrP generated by natural cellular processing. RK13 cells expressing solely the Δ190-196 C1 PrP construct, in the absence of the full-length protein, were susceptible to ΔSpont prions. ΔSpont infection induced the conversion of the mutated C1 into a PK-resistant and infectious form perpetuating the biochemical characteristics of ΔSpont prion. In conclusion, this work provides a unique cell-derived system generating spontaneous prions and provides evidence that the 113 C-terminal residues of PrP are sufficient for a self-propagating prion entity

A stretch of residues within the protease-resistant core is not necessary for prion structure and infectivity

Mapping out regions of PrP influencing prion conversion remains a challenging issue complicated by the lack of prion structure. The portion of PrP associated with infectivity contains the -helical domain of the correctly folded protein and turns into a -sheet-rich insoluble core in prions. Deletions performed so far inside this segment essentially prevented the conversion. Recently we found that deletion of the last C-terminal residues of the helix H2 was fully compatible with prion conversion in the RK13-ovPrP cell culture model, using 3 different infecting strains. This was in agreement with preservation of the overall PrPC structure even after removal of up to one-third of this helix. Prions with internal deletion were infectious for cells and mice expressing the wild-type PrP and they retained prion strain-specific characteristics. We thus identified a piece of the prion domain that is neither necessary for the conformational transition of PrPC nor for the formation of a stable prion structure