Shaking Alone Induces De Novo Conversion of Recombinant Prion Proteins to β-Sheet Rich Oligomers and Fibrils

Abstract

The formation of β-sheet rich prion oligomers and fibrils from native prion protein (PrP) is thought to be a key step in the development of prion diseases. Many methods are available to convert recombinant prion protein into β-sheet rich fibrils using various chemical denaturants (urea, SDS, GdnHCl), high temperature, phospholipids, or mildly acidic conditions (pH 4). Many of these methods also require shaking or another form of agitation to complete the conversion process. We have identified that shaking alone causes the conversion of recombinant PrP to β-sheet rich oligomers and fibrils at near physiological pH (pH 5.5 to pH 6.2) and temperature. This conversion does not require any denaturant, detergent, or any other chemical cofactor. Interestingly, this conversion does not occur when the water-air interface is eliminated in the shaken sample. We have analyzed shaking-induced conversion using circular dichroism, resolution enhanced native acidic gel electrophoresis (RENAGE), electron microscopy, Fourier transform infrared spectroscopy, thioflavin T fluorescence and proteinase K resistance. Our results show that shaking causes the formation of β-sheet rich oligomers with a population distribution ranging from octamers to dodecamers and that further shaking causes a transition to β-sheet fibrils. In addition, we show that shaking-induced conversion occurs for a wide range of full-length and truncated constructs of mouse, hamster and cervid prion proteins. We propose that this method of conversion provides a robust, reproducible and easily accessible model for scrapie-like amyloid formation, allowing the generation of milligram quantities of physiologically stable β-sheet rich oligomers and fibrils. These results may also have interesting implications regarding our understanding of prion conversion and propagation both within the brain and via techniques such as protein misfolding cyclic amplification (PMCA) and quaking induced conversion (QuIC).

The “Jekyll and Hyde” Actions of Nucleic Acids on the Prion-like Aggregation of Proteins

“…Although it became clear that an as yet unidentified negatively charged accessory molecule is important to drive the conversion process, this hypothesis was also strongly questioned because of the lack of knowledge of how and where this encounter would occur in vivo. A recent study describes the aggregation of PrP C into a ␤-sheet-rich species induced by shaking recombinant PrP under appropriate conditions without the addition of any cofactor (41). However, the infectivity of the aggregated species was not investigated in vivo.…”

Rapid chemical decontamination of infectious CJD and scrapie particles parallels treatments known to disrupt microbes and biofilms

“…This refolding parallels the relative ease of producing PrP-res forms, but not infectivity, in a test-tube. 4,[10][11][12] Dilution of the chemical denaturants here did not restore infectivity. While restoration of TSE agent infectivity has been tried for many years in various preparations, 39,55 a small amount of infectivity was recently restored by combining phenol purified 263K brain nucleic acids with recombinant PrP.…”

“…Misfolded recombinant PrP is not reproducibly infectious in animal or culture assays, even though massive amounts of PrPres amyloid are readily generated in a test tube. 4,[10][11][12][13] Huge increases in PrP-res can also coincide with a >5 log loss of FU-CJD agent from chronically infected living cell cultures. 12 This PrP-res is indistinguishable from the "infectious" PrP-res form, yet it is unable to produce any detectable agent in sensitive infectivity culture assays.…”

Endogenous Brain Lipids Inhibit Prion Amyloid Formation In Vitro

“…Investigations of the amyloid products produced by PMCA sonication have described a diverse population of conformers, including small oligomers, which may be more infectious than large aggregates (32,52). Examinations of RT-QuIC products have demonstrated amyloid fibrils; however, full characterization of the sizes and types of fibrils remains to be done (35,53,54). A second difference between the present studies and those of Deleault et al is the sequence of the recombinant protein used as the assay substrate (10,12).…”