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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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Time course of the formation of prion oligomers and fibrils.A) RENAGE gel of shaking converted recMoPrP 23–231 at 250 rpm and 37°C shows a time dependent loss of monomer, formation of oligomers and subsequent formation of fibrils. The chromatogram profile of each gel lane was acquired to determine the fibril content. A representative profile after 27 hours of shaking is shown. B) Plot of the time dependent thioflavin T (ThT) fluorescence (open diamonds; black line) and RENAGE fibril peak area (purple filled squares; purple line) shows a sigmoidal growth in both ThT fluorescence enhancement and fibril formation.
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pone-0098753-g007: Time course of the formation of prion oligomers and fibrils.A) RENAGE gel of shaking converted recMoPrP 23–231 at 250 rpm and 37°C shows a time dependent loss of monomer, formation of oligomers and subsequent formation of fibrils. The chromatogram profile of each gel lane was acquired to determine the fibril content. A representative profile after 27 hours of shaking is shown. B) Plot of the time dependent thioflavin T (ThT) fluorescence (open diamonds; black line) and RENAGE fibril peak area (purple filled squares; purple line) shows a sigmoidal growth in both ThT fluorescence enhancement and fibril formation.

Mentions: The time course of shaking-induced conversion of recMoPrP 23–231 was followed using RENAGE (Fig. 7A). This allowed us to quantify the amount of monomer, oligomer and fibril throughout the conversion process, using a single technique. As seen in Fig. 7A there is a loss of monomer that is concurrent with the formation and loss of oligomers, followed by the abrupt formation of fibrils. A time course for recMoPrP 90–231 also showed a loss of monomer concurrent with the formation of oligomers and a shift to fibrils (result not shown). We also used ThT to probe for the formation of the characteristic cross-β structure found in amyloids [23], [31]. Previously we determined that shaking-induced fibrils enhance ThT fluorescence (results not shown). Consequently, we monitored the time course changes in ThT fluorescence during fibril formation, by shaking alone. Plotting the time course of ThT fluorescence over time we show a sigmoidal growth in the number of fibrils (Fig. 7B). On the same plot we also show that the growth of the fibril band in RENAGE was also sigmoidal (Fig. 7B). This suggests that the RENAGE fibril band is a suitable way to follow the kinetics of PrP fibril formation. Furthermore, the ability to overlay the growth of ThT fluorescence with the RENAGE fibril band growth indicates that it is the fibrils that are responsible for the characteristic cross-β structure of PrP amyloid fibrils. The fact that the fibrils (and not oligomers) exhibit amyloid-like structure was further confirmed when we found that PrP oligomers formed by urea conversion do not enhance ThT fluorescence (result not shown).


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)

Time course of the formation of prion oligomers and fibrils.A) RENAGE gel of shaking converted recMoPrP 23–231 at 250 rpm and 37°C shows a time dependent loss of monomer, formation of oligomers and subsequent formation of fibrils. The chromatogram profile of each gel lane was acquired to determine the fibril content. A representative profile after 27 hours of shaking is shown. B) Plot of the time dependent thioflavin T (ThT) fluorescence (open diamonds; black line) and RENAGE fibril peak area (purple filled squares; purple line) shows a sigmoidal growth in both ThT fluorescence enhancement and fibril formation.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0098753-g007: Time course of the formation of prion oligomers and fibrils.A) RENAGE gel of shaking converted recMoPrP 23–231 at 250 rpm and 37°C shows a time dependent loss of monomer, formation of oligomers and subsequent formation of fibrils. The chromatogram profile of each gel lane was acquired to determine the fibril content. A representative profile after 27 hours of shaking is shown. B) Plot of the time dependent thioflavin T (ThT) fluorescence (open diamonds; black line) and RENAGE fibril peak area (purple filled squares; purple line) shows a sigmoidal growth in both ThT fluorescence enhancement and fibril formation.
Mentions: The time course of shaking-induced conversion of recMoPrP 23–231 was followed using RENAGE (Fig. 7A). This allowed us to quantify the amount of monomer, oligomer and fibril throughout the conversion process, using a single technique. As seen in Fig. 7A there is a loss of monomer that is concurrent with the formation and loss of oligomers, followed by the abrupt formation of fibrils. A time course for recMoPrP 90–231 also showed a loss of monomer concurrent with the formation of oligomers and a shift to fibrils (result not shown). We also used ThT to probe for the formation of the characteristic cross-β structure found in amyloids [23], [31]. Previously we determined that shaking-induced fibrils enhance ThT fluorescence (results not shown). Consequently, we monitored the time course changes in ThT fluorescence during fibril formation, by shaking alone. Plotting the time course of ThT fluorescence over time we show a sigmoidal growth in the number of fibrils (Fig. 7B). On the same plot we also show that the growth of the fibril band in RENAGE was also sigmoidal (Fig. 7B). This suggests that the RENAGE fibril band is a suitable way to follow the kinetics of PrP fibril formation. Furthermore, the ability to overlay the growth of ThT fluorescence with the RENAGE fibril band growth indicates that it is the fibrils that are responsible for the characteristic cross-β structure of PrP amyloid fibrils. The fact that the fibrils (and not oligomers) exhibit amyloid-like structure was further confirmed when we found that PrP oligomers formed by urea conversion do not enhance ThT fluorescence (result not shown).

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