ABSTRACT
Transmissible spongiform encephalopathies are a group of neurodegenerative disorders caused by a posttranslational, conformational change in the cellular isoform of the prion protein (PrP(C)) into an infectious, disease-associated form (PrP(Sc)). Increasing evidence supports a role for PrP(C) in the cellular response to oxidative stress. We investigated the effect of oxidative stress mediated by paraquat exposure on SH-SY5Y neuroblastoma cells. A loss of mitochondrial membrane potential and subsequent reduction in ATP production were demonstrated in untransfected SH-SY5Y cells, an effect that was ameliorated by the expression of PrP(C). Cells expressing either PrP-DeltaOct, which lacks the octapeptide repeats, or PrP-DA, in which the N-terminus is tethered to the membrane, showed increased sensitivity to paraquat compared with cells expressing wild-type PrP(C) as shown by reduced viability, loss of their membrane integrity, and reduced mitochondrial bioenergetic measurements. Exposure of prion-infected mouse SMB15S cells to paraquat resulted in a reduction in viability to levels similar to those seen in the untransfected SH-SY5Y cells. However, "curing" the cells with pentosan sulfate restored the viability to the level observed in the SH-SY5Y cells expressing PrP(C). These data would indicate that the molecular mechanism promoting cellular resistance to oxidative stress had been compromised in the infected SMB15S cells, which could be reinstated upon curing. Our study supports the hypothesis that PrP(C) expression protects cells against paraquat-induced oxidative injury, demonstrates the significance of the N-terminal region of the protein in mediating this protective effect, and also shows that the biochemical consequences of prion infection may be reversed with therapeutic intervention.
Subject(s)
Neurons/drug effects , Neuroprotective Agents/pharmacology , Oxidants/poisoning , Oxidative Stress , Paraquat/poisoning , Prions/pharmacology , Adenosine Triphosphate/antagonists & inhibitors , Animals , Cell Line , Cell Survival/drug effects , Drug Resistance , Energy Metabolism/drug effects , Humans , Membrane Potential, Mitochondrial/drug effects , Mice , Mitochondria/drug effects , Mitochondria/metabolism , Neurons/metabolism , Pentosan Sulfuric Polyester/pharmacology , Prions/genetics , Protein Isoforms/genetics , Protein Isoforms/pharmacology , Scrapie/metabolism , Scrapie/pathology , Scrapie/physiopathology , TransfectionABSTRACT
We are confronted daily to unknown microorganisms that have yet to be characterized, detected, and/or analyzed. We propose, in this study, a multidimensional strategy using polyclonal antibodies, consisting of a novel proteomic tool, the ProteomeLab PF2D, coupled to immunological techniques and mass spectrometry (i-PF2D-MS/MS). To evaluate this strategy, we have applied it to Bacillus subtilis, considered here as our unknown bacterial model.
Subject(s)
Antigens, Bacterial/analysis , Bacillus subtilis/immunology , Mass Spectrometry/methods , Proteomics/methods , Amino Acid Sequence , Antibodies, Bacterial/immunology , Antibodies, Bacterial/ultrastructure , Bacillus subtilis/ultrastructure , Blotting, Western , Hydrophobic and Hydrophilic Interactions , Immune Sera , Isoelectric Point , Microscopy, Electron , Molecular Sequence DataABSTRACT
We propose a multi-dimensional strategy, associating immunodetection to a protein fractionating two-dimensional liquid chromatography tool, for serological characterization of microbial antigens. The originality of such immunoproteomic approaches resides in their application in large-scale studies for rapid serotyping of micro-organisms, evaluation of immunomes and could be proposed in the development and monitoring of vaccines.
Subject(s)
Antigens, Bacterial/analysis , Antigens, Bacterial/immunology , Bacillus subtilis/immunology , Bacterial Proteins/analysis , Bacterial Proteins/immunology , Proteomics , Blotting, Western , Chromatography, Liquid , Electrophoresis, Polyacrylamide Gel , Immunologic Techniques , Microscopy/methods , Proteome/analysis , Serotyping , Spectrum Analysis , Tandem Mass SpectrometryABSTRACT
A synthetic peptide corresponding to the 106-126 amyloidogenic region of the cellular human prion protein (PrP(c)) is useful for in vitro study of prion-induced neuronal cell death. The aim of the present work was to examine the implication of the cellular prion protein in the toxicity mechanism induced by PrP 106-126. The effect of PrP 106-126 was investigated both on human neuroblastoma SH-SY5Y cells and on SH-SY5Y overexpressing murine cellular prions (wtPrP). We show by metabolic assay tests and ATP assays that PrP(c) expression does not modulate the toxicity of the prion peptide. Moreover, we investigated the effect of this peptide on an established non neuronal model, rabbit kidney epithelial A74 cells that express a doxycycline-inducible murine PrP(c) gene. We show for the first time that the prion peptide 106-126 does not exert any toxic effect on this cell line in the presence or absence of doxycycline. Our results show that the PrP 106-126-induced cell alteration is independent of PrP(c) expression. Rather, it seems to act via an interaction with lipidic components of the plasma membrane as strengthened by our results showing the differential susceptibility of neuronal and non neuronal cell lines that significantly differ by their membrane fatty acid composition.
