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

Ladner-Keay CL, Griffith BJ, Wishart DS - PLoS ONE (2014)

Bottom Line: 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.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).

View Article: PubMed Central - PubMed

Affiliation: Department of Computing Science, University of Alberta, Edmonton, Alberta, Canada; Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada; National Institute for Nanotechnology, Edmonton, Alberta, Canada.

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).

Show MeSH

Related in: MedlinePlus

Electron microscopy confirms the formation of oligomers and fibrils seen in RENAGE.Negative stain EM of shaking-induced prion oligomers (panel A) and fibrils (panel B). The oligomers shown here were formed from shaking recMoPrP 90–231 at 350 rpm at room temperature for 1 day. The fibril sample was formed by shaking recShPrP 90–232 at 350 rpm at 37°C for 5 days. The corresponding RENAGE analysis of the same sample is shown alongside the micrograph. The indicated scale bar = 100 nm.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4043794&req=5

pone-0098753-g005: Electron microscopy confirms the formation of oligomers and fibrils seen in RENAGE.Negative stain EM of shaking-induced prion oligomers (panel A) and fibrils (panel B). The oligomers shown here were formed from shaking recMoPrP 90–231 at 350 rpm at room temperature for 1 day. The fibril sample was formed by shaking recShPrP 90–232 at 350 rpm at 37°C for 5 days. The corresponding RENAGE analysis of the same sample is shown alongside the micrograph. The indicated scale bar = 100 nm.

Mentions: To characterize the progression of PrP monomers to prion fibrils throughout the shaking period, we analyzed the shaking-converted isoforms by negative stain electron microscopy (EM). These samples were generated using different shaking conditions and different time points to provide an oligomer sample free of fibrils and a fibril sample free of oligomers. A sample of prion oligomers was generated by shaking recMoPrPc 90–231 monomers at 350 rpm, at room temperature for 1 day. The sample was shown by RENAGE to contain only oligomer bands and no fibril band. The sample was shaken at room temperature to enrich for oligomers and avoid the formation of fibrils, which was routinely found when shaking recPrP at room temperature, rather than 37°C. EM analysis of this sample showed that the oligomers were ∼20 nm disc-like structures (Fig. 5A). It should be noted that there is an enrichment of high molecular weight oligomers (∼20-mers) in this sample that likely aided in visualizing the oligomers by EM. EM characterization also confirmed what the RENAGE analysis initially showed: that the sample contained PrP oligomers only and no detectable fibrils. In contrast, PrPc that was shaken for 5 days at 350 rpm at 37°C, showed only a fibril band on RENAGE and contained abundant rod-like fibrils as seen by EM (Fig. 5B). The dominant species seen on the grid were these rod-like fibrils with no significant patches of the oligomeric structures that are seen in panel A. EM was also performed for recMoPrP 90–231 and recMoPrP 23–231 fibril samples (based on RENAGE) and showed the formation of similar rod-like fibrils (results not shown). However EM of shaking-induced conversion of MoPrP 120–231 did not show any rod-like fibrils, but rather only showed round clusters consistent with amorphous aggregates. However EM cannot rule out that fibrils are formed by shaking this C-terminal construct. This is because the fibrils may have been stuck to the tube and were at low abundance.


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

Ladner-Keay CL, Griffith BJ, Wishart DS - PLoS ONE (2014)

Electron microscopy confirms the formation of oligomers and fibrils seen in RENAGE.Negative stain EM of shaking-induced prion oligomers (panel A) and fibrils (panel B). The oligomers shown here were formed from shaking recMoPrP 90–231 at 350 rpm at room temperature for 1 day. The fibril sample was formed by shaking recShPrP 90–232 at 350 rpm at 37°C for 5 days. The corresponding RENAGE analysis of the same sample is shown alongside the micrograph. The indicated scale bar = 100 nm.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4043794&req=5

pone-0098753-g005: Electron microscopy confirms the formation of oligomers and fibrils seen in RENAGE.Negative stain EM of shaking-induced prion oligomers (panel A) and fibrils (panel B). The oligomers shown here were formed from shaking recMoPrP 90–231 at 350 rpm at room temperature for 1 day. The fibril sample was formed by shaking recShPrP 90–232 at 350 rpm at 37°C for 5 days. The corresponding RENAGE analysis of the same sample is shown alongside the micrograph. The indicated scale bar = 100 nm.
Mentions: To characterize the progression of PrP monomers to prion fibrils throughout the shaking period, we analyzed the shaking-converted isoforms by negative stain electron microscopy (EM). These samples were generated using different shaking conditions and different time points to provide an oligomer sample free of fibrils and a fibril sample free of oligomers. A sample of prion oligomers was generated by shaking recMoPrPc 90–231 monomers at 350 rpm, at room temperature for 1 day. The sample was shown by RENAGE to contain only oligomer bands and no fibril band. The sample was shaken at room temperature to enrich for oligomers and avoid the formation of fibrils, which was routinely found when shaking recPrP at room temperature, rather than 37°C. EM analysis of this sample showed that the oligomers were ∼20 nm disc-like structures (Fig. 5A). It should be noted that there is an enrichment of high molecular weight oligomers (∼20-mers) in this sample that likely aided in visualizing the oligomers by EM. EM characterization also confirmed what the RENAGE analysis initially showed: that the sample contained PrP oligomers only and no detectable fibrils. In contrast, PrPc that was shaken for 5 days at 350 rpm at 37°C, showed only a fibril band on RENAGE and contained abundant rod-like fibrils as seen by EM (Fig. 5B). The dominant species seen on the grid were these rod-like fibrils with no significant patches of the oligomeric structures that are seen in panel A. EM was also performed for recMoPrP 90–231 and recMoPrP 23–231 fibril samples (based on RENAGE) and showed the formation of similar rod-like fibrils (results not shown). However EM of shaking-induced conversion of MoPrP 120–231 did not show any rod-like fibrils, but rather only showed round clusters consistent with amorphous aggregates. However EM cannot rule out that fibrils are formed by shaking this C-terminal construct. This is because the fibrils may have been stuck to the tube and were at low abundance.

Bottom Line: 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.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).

View Article: PubMed Central - PubMed

Affiliation: Department of Computing Science, University of Alberta, Edmonton, Alberta, Canada; Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada; National Institute for Nanotechnology, Edmonton, Alberta, Canada.

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).

Show MeSH
Related in: MedlinePlus