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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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Growth of shaking-induced fibrils with seeding is exponential.A) RENAGE gel of the time course of shaking induced conversion of recMoPrP 23–231 at 250 rpm and 37°C. B) RENAGE gel of shaking induced conversion of the same recMoPrP (same batch) under the same conditions except with seeding using 5% MoPrP 23–231 fibrils into fresh recMoPrPc. C) The chromatogram profile of each gel lane was acquired to determine the fibril content. Time dependent fibril content growth is shown plotted against time and has a sigmoidal dependence when starting with only fresh recMoPrPc23–231 (open squares, black line). Upon seeding with 5% PrP fibrils the fibril content grows logarithmically (grey circles, grey line).
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pone-0098753-g008: Growth of shaking-induced fibrils with seeding is exponential.A) RENAGE gel of the time course of shaking induced conversion of recMoPrP 23–231 at 250 rpm and 37°C. B) RENAGE gel of shaking induced conversion of the same recMoPrP (same batch) under the same conditions except with seeding using 5% MoPrP 23–231 fibrils into fresh recMoPrPc. C) The chromatogram profile of each gel lane was acquired to determine the fibril content. Time dependent fibril content growth is shown plotted against time and has a sigmoidal dependence when starting with only fresh recMoPrPc23–231 (open squares, black line). Upon seeding with 5% PrP fibrils the fibril content grows logarithmically (grey circles, grey line).

Mentions: In addition to testing the amyloid character of shaking-induced fibrils, we also tested if shaking-induced fibrils could seed and propagate fibril growth. For this we conducted a serial dilution study where small amounts of shaking-induced fibrils were added to fresh recMoPrPc 23–231. These serial dilution studies showed that if the sample is not shaken, fibril formation could not be propagated upon dilution of 5% fibril into fresh recPrPc (data not shown). However, if the sample was shaken, fibril formation occurred faster when fresh PrPc was seeded with 5% fibrils, than if no seed was added (Fig. 8A,B). The time dependence of the fibril formation as determined from RENAGE of seeded and unseeded fibril growth was fitted to exponential and sigmoidal functions, respectively (Fig. 8C). Later time points are not shown in Fig. 8C because of a loss of fibril content after the end point of the sigmoidal growth. We attribute this to loss of sample due to either fibril-fibril aggregation or adsorption of the fibrils onto the plastic container [32]. We have repeated the propagation of fibril formation by seeding fresh PrPc with the shaking-induced prion fibrils for five generations (i.e. five 1∶20 serial dilutions). During these propagation steps the kinetics seen by RENAGE did not change.


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)

Growth of shaking-induced fibrils with seeding is exponential.A) RENAGE gel of the time course of shaking induced conversion of recMoPrP 23–231 at 250 rpm and 37°C. B) RENAGE gel of shaking induced conversion of the same recMoPrP (same batch) under the same conditions except with seeding using 5% MoPrP 23–231 fibrils into fresh recMoPrPc. C) The chromatogram profile of each gel lane was acquired to determine the fibril content. Time dependent fibril content growth is shown plotted against time and has a sigmoidal dependence when starting with only fresh recMoPrPc23–231 (open squares, black line). Upon seeding with 5% PrP fibrils the fibril content grows logarithmically (grey circles, grey line).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4043794&req=5

pone-0098753-g008: Growth of shaking-induced fibrils with seeding is exponential.A) RENAGE gel of the time course of shaking induced conversion of recMoPrP 23–231 at 250 rpm and 37°C. B) RENAGE gel of shaking induced conversion of the same recMoPrP (same batch) under the same conditions except with seeding using 5% MoPrP 23–231 fibrils into fresh recMoPrPc. C) The chromatogram profile of each gel lane was acquired to determine the fibril content. Time dependent fibril content growth is shown plotted against time and has a sigmoidal dependence when starting with only fresh recMoPrPc23–231 (open squares, black line). Upon seeding with 5% PrP fibrils the fibril content grows logarithmically (grey circles, grey line).
Mentions: In addition to testing the amyloid character of shaking-induced fibrils, we also tested if shaking-induced fibrils could seed and propagate fibril growth. For this we conducted a serial dilution study where small amounts of shaking-induced fibrils were added to fresh recMoPrPc 23–231. These serial dilution studies showed that if the sample is not shaken, fibril formation could not be propagated upon dilution of 5% fibril into fresh recPrPc (data not shown). However, if the sample was shaken, fibril formation occurred faster when fresh PrPc was seeded with 5% fibrils, than if no seed was added (Fig. 8A,B). The time dependence of the fibril formation as determined from RENAGE of seeded and unseeded fibril growth was fitted to exponential and sigmoidal functions, respectively (Fig. 8C). Later time points are not shown in Fig. 8C because of a loss of fibril content after the end point of the sigmoidal growth. We attribute this to loss of sample due to either fibril-fibril aggregation or adsorption of the fibrils onto the plastic container [32]. We have repeated the propagation of fibril formation by seeding fresh PrPc with the shaking-induced prion fibrils for five generations (i.e. five 1∶20 serial dilutions). During these propagation steps the kinetics seen by RENAGE did not change.

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