Synthetic miniprion PrP106.
Elucidation of structure and biological properties of the prion protein scrapie (PrP(Sc)) is fundamental to an understanding of the mechanism of conformational transition of cellular (PrP(C)) into disease-specific isoforms and the pathogenesis of prion diseases. Unfortunately, the insolubility and heterogeneity of PrP(Sc) have limited these studies. The observation that a construct of 106 amino acids (termed PrP106 or miniprion), derived from mouse PrP and containing two deletions (Delta 23-88, Delta 141-176), becomes protease-resistant when expressed in scrapie-infected neuroblastoma cells and sustains prion replication when expressed in PrP(0/0) mice prompted us to generate a corresponding synthetic peptide (sPrP106) to be used for biochemical and cell culture studies. sPrP106 was obtained successfully with a straightforward procedure, which combines classical stepwise solid phase synthesis with a purification strategy based on transient labeling with a lipophilic chromatographic probe. sPrP106 readily adopted a beta-sheet structure, aggregated into branched filamentous structures without ultrastructural and tinctorial properties of amyloid, exhibited a proteinase K-resistant domain spanning residues 134-217, was highly toxic to primary neuronal cultures, and induced a remarkable increase in membrane microviscosity. These features are central properties of PrP(Sc) and make sPrP106 an excellent tool for investigating the molecular basis of the conformational conversion of PrP(C) into PrP(Sc) and prion disease pathogenesis.
Bonetto
Valentina
V
Dulbecco Telethon Institute, Department of Molecular Biochemistry and Pharmacology, Istituto di Ricerche Farmacologiche Mario Negri, via Eritrea 62, Milan 20157, Italy. bonetto@marionegri.it
Massignan
Tania
T
Chiesa
Roberto
R
Morbin
Michela
M
Mazzoleni
Giulia
G
Diomede
Luisa
L
Angeretti
Nadia
N
Colombo
Laura
L
Forloni
Gianluigi
G
Tagliavini
Fabrizio
F
Salmona
Mario
M
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D017096:Prion Diseases / Q000503:physiopathology; D011328:Prions / Q000138:chemical synthesis / Q000737:chemistry* / Q000648:ultrastructure; D011487:Protein Conformation; D011994:Recombinant Proteins; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D010446:Peptide Fragments; D011328:Prions; D011994:Recombinant Proteins; C466227:miniprion protein PrP106, mouse