title: Thioredoxin converts the Syrian hamster (29-231) recombinant prion protein to an insoluble form.
abstract: The prion protein (PrP) is an essential, and probably the only, component of the infectious agent responsible for the transmissible spongiform encephalopathies. In its cellular (PrP(C)) form, it is a soluble, alpha-helix-rich protein of yet unknown function attached to the outer membrane of neurons through a glycosylphosphatidyl inositol anchor. In its pathogenic, "scrapie" form (PrP(Sc)), it appears as an aggregate showing no detectable covalent modifications but displaying a profoundly altered conformation enriched in beta-sheet structure. Reduction of the single disulfide bridge in the prion protein with millimolar concentrations of dithiothreitol results in transformation of the alpha-helix-rich to the beta-sheet-rich conformation, with concomitant decrease in solubility. We report here that thioredoxin coupled with thioredoxin reductase and NADPH efficiently reduces recombinant Syrian hamster (29-231) prion protein under physiologically relevant conditions. The reduced prion protein immediately becomes insoluble and precipitates, although it does not gain significant resistance to proteinase K. The thioredoxin/thioredoxin reductase system is approximately 7000 times more efficient than dithiothreitol.
authors.affiliation: Laboratory of Biochemistry, NHLBI, National Institutes of Health, Bethesda, MD 20892, USA. requena@nih.gov
authors.forename: J R
authors.initials: JR
authors.lastname: Requena
authors.forename: R L
authors.initials: RL
authors.lastname: Levine
mesh_terms: D000818:Animals; D011232:Chemical Precipitation; D002851:Chromatography, High Pressure Liquid; D006224:Cricetinae; D004220:Disulfides / Q000378:metabolism; D004229:Dithiothreitol / Q000378:metabolism; D019286:Endopeptidase K / Q000378:metabolism; D007700:Kinetics; D013058:Mass Spectrometry; D008647:Mesocricetus; D009249:NADP / Q000378:metabolism; D011328:Prions / Q000378:metabolism*; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000378:metabolism; D012995:Solubility; D013880:Thioredoxin-Disulfide Reductase / Q000378:metabolism; D013879:Thioredoxins / Q000378:metabolism*
chemical_list: D004220:Disulfides; D011328:Prions; D011994:Recombinant Proteins; D013879:Thioredoxins; D009249:NADP; D013880:Thioredoxin-Disulfide Reductase; D019286:Endopeptidase K; D004229:Dithiothreitol