Thioredoxin converts the Syrian hamster (29-231) recombinant prion protein to an insoluble form.
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.
Requena
J R
JR
Laboratory of Biochemistry, NHLBI, National Institutes of Health, Bethesda, MD 20892, USA. requena@nih.gov
Levine
R L
RL
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*
D004220:Disulfides; D011328:Prions; D011994:Recombinant Proteins; D013879:Thioredoxins; D009249:NADP; D013880:Thioredoxin-Disulfide Reductase; D019286:Endopeptidase K; D004229:Dithiothreitol