Shaking alone induces de novo conversion of recombinant prion proteins to β-sheet rich oligomers and fibrils.

Carol L Ladner-Keay,David S Wishart,Bethany J Griffith,Human Rezaei

doi:10.1371/journal.pone.0098753

Carol L Ladner-Keay, David S Wishart + Show 2 more

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https://doi.org/10.1371/journal.pone.0098753

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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).

Highlights

Prion protein (PrP) is a highly conserved membrane-bound protein that is abundant in the neuronal cells of vertebrates
Our results clearly show that shaking-alone can convert recombinant PrPc to b-sheet rich oligomers and fibrils
This is the first demonstration that the conversion of native recombinant PrP to b-sheet oligomers and fibrils can occur under physiological conditions

Summary

Introduction

Prion protein (PrP) is a highly conserved membrane-bound protein that is abundant in the neuronal cells of vertebrates. An abundance of misfolded PrPsc proteins on the neuronal cell surface or within endosomes leads to the accumulation of extracellular amyloid protein deposits that eventually lead to cell death and the manifestation of neuronal disease To better understand this physiological process, a number of cell-free, de novo methods have been developed that allow recombinant (rec) PrPc to be converted to a b-sheet rich isoform. These include protein misfolding cyclic amplification (PMCA) [13,14,15] and quaking-induced conversion (QuIC) [16] In these prion amplification methods, small amounts of prions (PrPsc) are added to large amounts of native PrPc (recombinant or brain-derived) and the mixed samples are shaken or sonicated for days.

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