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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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Related in: MedlinePlus

Shaking-induced fibrils have Proteinase K resistance.SDS-PAGE of recMoPrPc23–231 (panel A) and fibrils (panel B) without (PK-) and with PK at 1∶50, 1∶200 and 1∶400 (PK:PrP, g:g) shows that shaking-induced fibrils have 12, 13, 14 and 17 kDa resistance bands.
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pone-0098753-g009: Shaking-induced fibrils have Proteinase K resistance.SDS-PAGE of recMoPrPc23–231 (panel A) and fibrils (panel B) without (PK-) and with PK at 1∶50, 1∶200 and 1∶400 (PK:PrP, g:g) shows that shaking-induced fibrils have 12, 13, 14 and 17 kDa resistance bands.

Mentions: Naturally occurring infectious prions, as well as many in vitro converted fibril forms, are known to exhibit PK resistance [33], [34]. In fact, PK resistance is considered to be a hallmark for the presence of PrPsc. As expected, we found that shaking-induced fibrils (from recMoPrP 23–231) are PK resistant (Fig. 9A,B). As seen in this gel there are three bands of approximately 12, 13 and 14 kDa corresponding to PK resistant fragments. Interestingly, we also observed the presence of low levels of a ∼17 kDa PK resistance fragment. This may be the same, previously published, 17 kDa band that is found in PrPsc that has been PK digested after deglycosylation [15]. In fact, a 17 kDa band is also seen in recombinant PrPsc generated via PMCA and POPG/RNA that has been PK digested [18]. However in these cases, the 17 kDa fragment, from PK digested PrPsc, is often as abundant at the 12/13 kDa bands.


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)

Shaking-induced fibrils have Proteinase K resistance.SDS-PAGE of recMoPrPc23–231 (panel A) and fibrils (panel B) without (PK-) and with PK at 1∶50, 1∶200 and 1∶400 (PK:PrP, g:g) shows that shaking-induced fibrils have 12, 13, 14 and 17 kDa resistance bands.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0098753-g009: Shaking-induced fibrils have Proteinase K resistance.SDS-PAGE of recMoPrPc23–231 (panel A) and fibrils (panel B) without (PK-) and with PK at 1∶50, 1∶200 and 1∶400 (PK:PrP, g:g) shows that shaking-induced fibrils have 12, 13, 14 and 17 kDa resistance bands.
Mentions: Naturally occurring infectious prions, as well as many in vitro converted fibril forms, are known to exhibit PK resistance [33], [34]. In fact, PK resistance is considered to be a hallmark for the presence of PrPsc. As expected, we found that shaking-induced fibrils (from recMoPrP 23–231) are PK resistant (Fig. 9A,B). As seen in this gel there are three bands of approximately 12, 13 and 14 kDa corresponding to PK resistant fragments. Interestingly, we also observed the presence of low levels of a ∼17 kDa PK resistance fragment. This may be the same, previously published, 17 kDa band that is found in PrPsc that has been PK digested after deglycosylation [15]. In fact, a 17 kDa band is also seen in recombinant PrPsc generated via PMCA and POPG/RNA that has been PK digested [18]. However in these cases, the 17 kDa fragment, from PK digested PrPsc, is often as abundant at the 12/13 kDa bands.

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