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

RENAGE indicates that shaking recPrPc generates oligomers.A PrP PICUP ladder (lane 1) is used to size the oligomers formed by shaking recMoPrP 90–231 in pH 5.5 buffer at 350 rpm and 37°C for 1 day (lane 2). Shaking-induced oligomers are predominantly a distribution of 8-mers to 13-mers. In comparison oligomers formed in urea and salt exhibit a bimodal size distribution of 7-mers to 12-mers (lane 3). Longer periods of shaking recMoPrP 90–231 (shaking at 350 rpm, 37°C for 2 days) will also generate a fibril band and bimodal distribution of 8-mers to 12-mers and larger oligomers (>16-mers) (lane 4).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0098753-g001: RENAGE indicates that shaking recPrPc generates oligomers.A PrP PICUP ladder (lane 1) is used to size the oligomers formed by shaking recMoPrP 90–231 in pH 5.5 buffer at 350 rpm and 37°C for 1 day (lane 2). Shaking-induced oligomers are predominantly a distribution of 8-mers to 13-mers. In comparison oligomers formed in urea and salt exhibit a bimodal size distribution of 7-mers to 12-mers (lane 3). Longer periods of shaking recMoPrP 90–231 (shaking at 350 rpm, 37°C for 2 days) will also generate a fibril band and bimodal distribution of 8-mers to 12-mers and larger oligomers (>16-mers) (lane 4).

Mentions: RENAGE [22] was initially used to discover the formation of oligomers induced by shaking recShPrP 90–232 and recMoPrP 90–231 at 37°C and 350 rpm, in buffer at pH 5.5. The size of these recMoPrP 90–231 oligomers follows a Gaussian or near-Gaussian distribution ranging from octamers to dodecamers as determined using a specially prepared PrP PICUP ladder (Fig. 1). This PrP PICUP ladder is generated by non-specifically cross-linking MoPrP 90–231 using the PICUP reagent [22]. The same size and distribution of shaking-induced oligomers was produced from shaking recShPrP 90–232 (results not shown). Interestingly, this size distribution is distinct from what is seen when oligomers of heptamers and larger are formed using more conventional techniques: 3 M urea and 200 mM NaCl at pH 4 [22] (Fig. 1). Shaking the PrP oligomer samples for longer times caused the formation of larger oligomers (>16 mers) and a protein band that barely enters the stacking gel as seen for recMoPrP 90–231 (marked as the fibril band, Fig. 1, lane 4). This result is also seen for all recPrP constructs that we studied (results not shown). Formation of these shaking-induced oligomers occurs with or without the addition of 150 mM NaCl (results not shown). This is in contrast to urea induced conversion, where conversion to oligomers is dependent on the presence of NaCl [28].


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)

RENAGE indicates that shaking recPrPc generates oligomers.A PrP PICUP ladder (lane 1) is used to size the oligomers formed by shaking recMoPrP 90–231 in pH 5.5 buffer at 350 rpm and 37°C for 1 day (lane 2). Shaking-induced oligomers are predominantly a distribution of 8-mers to 13-mers. In comparison oligomers formed in urea and salt exhibit a bimodal size distribution of 7-mers to 12-mers (lane 3). Longer periods of shaking recMoPrP 90–231 (shaking at 350 rpm, 37°C for 2 days) will also generate a fibril band and bimodal distribution of 8-mers to 12-mers and larger oligomers (>16-mers) (lane 4).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0098753-g001: RENAGE indicates that shaking recPrPc generates oligomers.A PrP PICUP ladder (lane 1) is used to size the oligomers formed by shaking recMoPrP 90–231 in pH 5.5 buffer at 350 rpm and 37°C for 1 day (lane 2). Shaking-induced oligomers are predominantly a distribution of 8-mers to 13-mers. In comparison oligomers formed in urea and salt exhibit a bimodal size distribution of 7-mers to 12-mers (lane 3). Longer periods of shaking recMoPrP 90–231 (shaking at 350 rpm, 37°C for 2 days) will also generate a fibril band and bimodal distribution of 8-mers to 12-mers and larger oligomers (>16-mers) (lane 4).
Mentions: RENAGE [22] was initially used to discover the formation of oligomers induced by shaking recShPrP 90–232 and recMoPrP 90–231 at 37°C and 350 rpm, in buffer at pH 5.5. The size of these recMoPrP 90–231 oligomers follows a Gaussian or near-Gaussian distribution ranging from octamers to dodecamers as determined using a specially prepared PrP PICUP ladder (Fig. 1). This PrP PICUP ladder is generated by non-specifically cross-linking MoPrP 90–231 using the PICUP reagent [22]. The same size and distribution of shaking-induced oligomers was produced from shaking recShPrP 90–232 (results not shown). Interestingly, this size distribution is distinct from what is seen when oligomers of heptamers and larger are formed using more conventional techniques: 3 M urea and 200 mM NaCl at pH 4 [22] (Fig. 1). Shaking the PrP oligomer samples for longer times caused the formation of larger oligomers (>16 mers) and a protein band that barely enters the stacking gel as seen for recMoPrP 90–231 (marked as the fibril band, Fig. 1, lane 4). This result is also seen for all recPrP constructs that we studied (results not shown). Formation of these shaking-induced oligomers occurs with or without the addition of 150 mM NaCl (results not shown). This is in contrast to urea induced conversion, where conversion to oligomers is dependent on the presence of NaCl [28].

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