Structural Intermediates in the Putative Pathway from the Cellular Prion Protein to the Pathogenic Form

Abstract

The conversion of the alpha-helical, protease sensitive and noninfectious form of the prion protein (PrP(C)) into an insoluble, protease resistant, predominantly beta-sheeted and infectious form (PrP(Sc)) is the fundamental event in prion formation. In the present work, two soluble and stable intermediate structural states are newly identified for recombinant Syrian hamster PrP(90-231) (recPrP), a dimeric alpha-helical state and a tetra- or oligomeric, beta-sheet rich state. In 0.2% SDS at room temperature, recPrP is soluble and exhibits alpha-helical and random coil secondary structure as determined by circular dichroism. Reduction of the SDS concentration to 0.06% leads first to a small increase in alpha-helical content, whereas further dilution to 0.02% results in the aquisition of beta-sheet structure. The reversible transition curve is sigmoidal within a narrow range of SDS concentrations (0.04 to 0.02%). Size exclusion chromatography and chemical crosslinking revealed that the alpha-helical form is dimeric, while the beta-sheet rich form is tetra- or oligomeric. Both the alpha-helical and beta-sheet rich intermediates are soluble and stable. Thus, they should be accessible to further structural and mechanistic studies. At 0.01% SDS, the oligomeric intermediates aggregated into large, insoluble structures as observed by fluorescence correlation spectroscopy. Our results are discussed with respect to the mechanism of PrP(Sc) formation and the propagation of prions.

Structural changes of membrane-anchored native PrP C

“…Consequently, dimers or oligomers of PrP C are formed on the membrane. Indeed, dimers of PrP C and recPrP have been observed in solution in several studies (7,29).…”

“…It leads to an increase in ␤-sheet structure, insolubility, and partial resistance against digestion with proteinase K (3)(4)(5)(6). This conversion has been investigated in vitro, predominantly by using recombinant PrP (recPrP), expressed in Escherichia coli, (7)(8)(9)(10). However, the eucaryotic PrP C is posttranslationally modified, carrying two N-glycosylations and a GPI anchor.…”

Mechanisms of prion protein assembly into amyloid

“…A very similar SDS-conversion system was also used successfully by other groups (24). Lowering the SDS concentration from 0.2% to 0.02%, recPrP is transformed from a monomeric, partially denatured and otherwise mainly ␣-helical structure through a dimer with an ␣-helical content similar to that of PrP C and a soluble oligomeric, ␤-sheetrich state to polymorphic, ␤-sheet-rich aggregates (25).…”

“…The truncated form of recPrP of Syrian hamster residues 90 -231 was used. It was expressed, purified, and refolded as described (25,35). PrP Sc was isolated to high purity by using a modified protocol of the NaPTA precipitation method (22,36).…”

Misfolding of the prion protein: linking biophysical and biological approaches

“…The synaptic loss is a prominent feature of prion diseases, which is observed at the early pre-clinical stages [25,40,52] while neuronal death is typically observed at late clinical stages [16]. Other authors underlined the difficulties to establish the identification of the early molecular events associated with neuronal degeneration by a valuable biological model system [50,84].…”