title: Mechanisms of prion protein assembly into amyloid.
abstract: 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.
authors.affiliation: Institut fuer Physikalische Biologie and Biologisch-Medizinisches Forschungszentrum, Heinrich-Heine-Universitaet Duesseldorf, 40225 Duesseldorf, Germany.
authors.forename: Jan
authors.initials: J
authors.lastname: Stöhr
authors.forename: Nicole
authors.initials: N
authors.lastname: Weinmann
authors.forename: Holger
authors.initials: H
authors.lastname: Wille
authors.forename: Tina
authors.initials: T
authors.lastname: Kaimann
authors.forename: Luitgard
authors.initials: L
authors.lastname: Nagel-Steger
authors.forename: Eva
authors.initials: E
authors.lastname: Birkmann
authors.forename: Giannantonio
authors.initials: G
authors.lastname: Panza
authors.forename: Stanley B
authors.initials: SB
authors.lastname: Prusiner
authors.forename: Manfred
authors.initials: M
authors.lastname: Eigen
authors.forename: Detlev
authors.initials: D
authors.lastname: Riesner
mesh_terms: 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
chemical_list: D000682:Amyloid; D003432:Cross-Linking Reagents; D011328:Prions