Mechanisms of prion protein assembly into amyloid.
The conversion of the alpha-helical, cellular isoform of the prion protein (PrP(C)) to the insoluble, beta-sheet-rich, infectious, disease-causing isoform (PrP(Sc)) is the key event in prion diseases. In an earlier study, several forms of PrP were converted into a fibrillar state by using an in vitro conversion system consisting of low concentrations of SDS and 250 mM NaCl. Here, we characterize the structure of the fibril precursor state, that is, the soluble state under fibrillization conditions. CD spectroscopy, analytical ultracentrifugation, and chemical cross-linking indicate that the precursor state exists in a monomer-dimer equilibrium of partially denatured, alpha-helical PrP, with a well defined contact site of the subunits in the dimer. Using fluorescence with thioflavin T, we monitored and quantitatively described the kinetics of seeded fibril formation, including dependence of the reaction on substrate and seed concentrations. Exponential, seed-enhanced growth can be achieved in homogeneous solution, which can be enhanced by sonication. From these data, we propose a mechanistic model of fibrillization, including the presence of several intermediate structures. These studies also provide a simplified amplification system for prions.
Stöhr
Jan
J
Institut fuer Physikalische Biologie and Biologisch-Medizinisches Forschungszentrum, Heinrich-Heine-Universitaet Duesseldorf, 40225 Duesseldorf, Germany.
Weinmann
Nicole
N
Wille
Holger
H
Kaimann
Tina
T
Nagel-Steger
Luitgard
L
Birkmann
Eva
E
Panza
Giannantonio
G
Prusiner
Stanley B
SB
Eigen
Manfred
M
Riesner
Detlev
D
D000682:Amyloid / Q000737:chemistry* / Q000378:metabolism* / Q000648:ultrastructure; D002942:Circular Dichroism; D003432:Cross-Linking Reagents / Q000737:chemistry; D019281:Dimerization; D008854:Microscopy, Electron; D011328:Prions / Q000737:chemistry* / Q000378:metabolism* / Q000648:ultrastructure; D014461:Ultracentrifugation
D000682:Amyloid; D003432:Cross-Linking Reagents; D011328:Prions