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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).

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Sonication of PrP generates oligomers.RENAGE of recMoPrP 90–231 sonicated for 8 cycles of 10 seconds each, show that oligomers are generated (lane 1). Furthermore sonication of recMoPrP 23–231 in a single PMCA-like round generates oligomers (lane 2). The formation of oligomers in a PMCA-like round is enhanced by placing the probe inside the solution (lane 3).
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pone-0098753-g010: Sonication of PrP generates oligomers.RENAGE of recMoPrP 90–231 sonicated for 8 cycles of 10 seconds each, show that oligomers are generated (lane 1). Furthermore sonication of recMoPrP 23–231 in a single PMCA-like round generates oligomers (lane 2). The formation of oligomers in a PMCA-like round is enhanced by placing the probe inside the solution (lane 3).

Mentions: Because sonication (as opposed to shaking) is commonly used for PMCA, we also tested the effect of sonication, alone, on oligomer formation. In our first experiment we investigated what sonication would do to a solution (0.5 mg/mL) of recPrP without the usual detergent additives of SDS or Triton X-100. Figure 10 shows that sonication (for 8 cycles of a 10 sec pulse) using a microprobe directly in the sample of recMoPrPc90–231 results in the formation of a mixture of large oligomers (>14-mers; 25%), 7 to 12-mers (23%) and monomers (49%). This suggests that sonication is a much more powerful and a far faster approach to prion conversion to oligomers than shaking. However, the sonication-induced conversion under these conditions does not convert all of the monomeric recPrP, even after 10 cycles of sonication (for a total sonication time of 100 sec). We also tested whether repeated sonication, using a similar scheme as in PMCA, will increase the level of prion oligomerization. We sonicated a sample of 0.5 mg/mL recMoPrPc23–231 at pH 5.5 in a 0.2 mL PCR tube for 2 min every 30 min over a 24-hour cycle. We found a small amount of oligomer (∼20%) formed when the sample was sonicated with the horn outside of the thin-walled PCR tube, and more oligomers (89%) were found when a micro tip was placed directly inside the tube, using a 24-hour cycle (Fig. 9). In this latter sample, sonication-induced conversion generated a sample of 51% large oligomers (>14-mers), 38% small oligomers (7 to 12-mers) and 1% fibrils, with 11% monomer remaining. We also tested for PK resistance in the sonicated recMoPrP 23–231 material but found that the samples were not PK resistant (data not shown). This is consistent with the very low PK resistance (in comparison to fibrils) found for β-oligomers [34]. Furthermore it indicates that the material generated from sonication, without detergents, does not generate the same prion isoform which forms spontaneously from PMCA [15].


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)

Sonication of PrP generates oligomers.RENAGE of recMoPrP 90–231 sonicated for 8 cycles of 10 seconds each, show that oligomers are generated (lane 1). Furthermore sonication of recMoPrP 23–231 in a single PMCA-like round generates oligomers (lane 2). The formation of oligomers in a PMCA-like round is enhanced by placing the probe inside the solution (lane 3).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0098753-g010: Sonication of PrP generates oligomers.RENAGE of recMoPrP 90–231 sonicated for 8 cycles of 10 seconds each, show that oligomers are generated (lane 1). Furthermore sonication of recMoPrP 23–231 in a single PMCA-like round generates oligomers (lane 2). The formation of oligomers in a PMCA-like round is enhanced by placing the probe inside the solution (lane 3).
Mentions: Because sonication (as opposed to shaking) is commonly used for PMCA, we also tested the effect of sonication, alone, on oligomer formation. In our first experiment we investigated what sonication would do to a solution (0.5 mg/mL) of recPrP without the usual detergent additives of SDS or Triton X-100. Figure 10 shows that sonication (for 8 cycles of a 10 sec pulse) using a microprobe directly in the sample of recMoPrPc90–231 results in the formation of a mixture of large oligomers (>14-mers; 25%), 7 to 12-mers (23%) and monomers (49%). This suggests that sonication is a much more powerful and a far faster approach to prion conversion to oligomers than shaking. However, the sonication-induced conversion under these conditions does not convert all of the monomeric recPrP, even after 10 cycles of sonication (for a total sonication time of 100 sec). We also tested whether repeated sonication, using a similar scheme as in PMCA, will increase the level of prion oligomerization. We sonicated a sample of 0.5 mg/mL recMoPrPc23–231 at pH 5.5 in a 0.2 mL PCR tube for 2 min every 30 min over a 24-hour cycle. We found a small amount of oligomer (∼20%) formed when the sample was sonicated with the horn outside of the thin-walled PCR tube, and more oligomers (89%) were found when a micro tip was placed directly inside the tube, using a 24-hour cycle (Fig. 9). In this latter sample, sonication-induced conversion generated a sample of 51% large oligomers (>14-mers), 38% small oligomers (7 to 12-mers) and 1% fibrils, with 11% monomer remaining. We also tested for PK resistance in the sonicated recMoPrP 23–231 material but found that the samples were not PK resistant (data not shown). This is consistent with the very low PK resistance (in comparison to fibrils) found for β-oligomers [34]. Furthermore it indicates that the material generated from sonication, without detergents, does not generate the same prion isoform which forms spontaneously from PMCA [15].

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