Mechanisms of prion protein assembly into amyloid

Abstract

The conversion of the ␣-helical, cellular isoform of the prion protein (PrP C ) to the insoluble, ␤-sheet-rich, infectious, diseasecausing 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, ␣-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.dimer ͉ seeding ͉ fibril ͉ precursor state

Homodimerization as a molecular switch between low and high efficiency PrPCcell surface delivery and neuroprotective activity

“…[7][8][9][10] Also, the connection between dimerization and toxicity has been supported for several years by a model suggesting in vivo noxiousness of cross-linked PrP C at neurons surface.…”

Oligomeric-Induced Activity by Thienyl Pyrimidine Compounds Traps Prion Infectivity

“…PrP Sc dimers and trimers could be transitory and unstable species (DeMarco et al, 2006;Silveira et al, 2005), possibly referring to pre-amyloid states (Stöhr et al, 2008). Indeed, from electron crystallographic studies established using PrP Sc purified from infected brain homogenates, two models of prion proto-fibril propagation are currently emerging: the ␤-helix model and the spiral model.…”

“…According to the prion hypothesis, PrP Sc can trigger the autocatalytic conversion of PrP C into PrP Sc , involving several PrP Sc pre-amyloid intermediates, generated through a complex mechanism of oligomerization (Silveira et al, 2005;DeMarco et al, 2006;Stöhr et al, 2008). The replication cycle proceeds with the self-assembling of PrP Sc oligomers into proto-fibrils, which in turn grow into amyloid fibrils.…”

Distinct Structures of Scrapie Prion Protein (PrPSc)-seeded Versus Spontaneous Recombinant Prion Protein Fibrils Revealed by Hydrogen/Deuterium Exchange

“…In contrast to brain-derived PrP C , large scale purification can be readily accomplished for bacterially expressed rPrP, a form of PrP lacking glycosylation and the glycophosphatidylinositol anchor. The latter protein can spontaneously polymerize into amyloid fibrils, and much insight has been gained into mechanistic and structural aspects of this reaction (22)(23)(24)(25)(26)(27)(28). However, although rPrP fibrils were shown to cause or accelerate a transmissible neurodegenerative disorder in transgenic mice overexpressing a PrP C variant encompassing residues 89 -231, the infectivity titer of these "synthetic prions" was extremely low (29) or absent altogether (4).…”