Hsp90 modifies the conformation of recombinant mouse prion protein in vitro
Yuji Sakasegawa, Naomi S. Hachiya and Kiyotoshi Kaneko
Dept. Cortical Func. Disorders, Natl. Inst. Neurosci., Natl. Center Neurol.
Psychiat., CREST, JST, 4-1-1 Ogawa-Higashi, Kodaira, Tokyo 187-8502 Japan <e-mail> [email protected]
Abstract
In mammals, prions reproduce by recruiting the normal cellular isoform of the prion protein (PrP^) and by stimulating its conversion into the patho- logical conformational isoform (PrP^^). Since the conversion process is thought to require unfolding of PrP^ to cause prion infection, to identify such activities, we have tried to construct an in vitro protein-unfolding as- say using Trypsin-digestion susceptibility of E. coli-expressed recombi- nant mouse prion protein (recPrP). The highly purified recPrP was re- folded as a soluble monomeric protein in 1 M arginine and a glutathione redox system-containing solution. We explored protein-unfolding activi- ties of a mouse neuroblastoma cell line, Neuro-2a, which was widely used for prion infection experiments, and found that the protein unfolding ac- tivities existed in the cell extracts, particularly, in the Cytosolic fraction (100,000g sup). From the Cytosolic fraction we purified the activity to homogeneity by three steps of chromatography. The active polypeptide has a molecular weight of 90 kDa in denature condition (SDS-PAGE) and 200 kDa in native condition (Native-PAGE). The MALDI-TOF-MS/MS analysis revealed that the purified polypeptide was identical to Hsp90.
The protein unfolding activity of the purified Hsp90 was inhibited by treatment of ATP-hydrolyzing enzyme, Apyrase. These results suggested that Hsp90 can modify the recombinant prion protein's conformation in the presence of ATP and might be required for conformation change of PrP^ to PrP^^ in prion propagation.
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