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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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RENAGE of shaking-converted prions under various conditions.A) ShPrP 90–232 oligomers are formed by shaking in pH 5.5, 6.2 and 7.4 buffers, at 350 rpm and 37°C. B) RENAGE after shaking at 350 rpm for 1 day with full length recMoPrP 23–231 (lane 1), truncated recMoPrP 90–231 (lane 2) and C-terminal domain recMoPrP 120–231 (lane 3) and after 2 days with recMoPrP 23–231 (lane 4), truncated recMoPrP 90–231 (lane 5) and C-terminal domain recMoPrP 120–231 (lane 6). Samples in panel B were shaken at 350 rpm and 37°C in pH 6.2 buffer. C) Shaking a 0.6 mL solution of recShPrP90–232 in a 0.6 mL centrifuge tube without any air or bubbles for two weeks (lane 1) as compared to the same sample of 0.4 mL in a 1.5 mL centrifuge tube (i.e. with air), shaken for one week (lane 2).
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pone-0098753-g003: RENAGE of shaking-converted prions under various conditions.A) ShPrP 90–232 oligomers are formed by shaking in pH 5.5, 6.2 and 7.4 buffers, at 350 rpm and 37°C. B) RENAGE after shaking at 350 rpm for 1 day with full length recMoPrP 23–231 (lane 1), truncated recMoPrP 90–231 (lane 2) and C-terminal domain recMoPrP 120–231 (lane 3) and after 2 days with recMoPrP 23–231 (lane 4), truncated recMoPrP 90–231 (lane 5) and C-terminal domain recMoPrP 120–231 (lane 6). Samples in panel B were shaken at 350 rpm and 37°C in pH 6.2 buffer. C) Shaking a 0.6 mL solution of recShPrP90–232 in a 0.6 mL centrifuge tube without any air or bubbles for two weeks (lane 1) as compared to the same sample of 0.4 mL in a 1.5 mL centrifuge tube (i.e. with air), shaken for one week (lane 2).

Mentions: Shaking-induced oligomers were also found at pH 6.2 using recShPrP 90–232 and recMoPrP 90–231 (result shown for recShPrP 90–232 in Fig. 3A). The distribution of oligomers formed at pH 6.2 is similar to that formed at pH 5.5. However at pH 6.2 there is an increase in high molecular oligomers (16 to 20-mers) relative to oligomers at 8 to 12-mers (Fig. 3A). Furthermore shaking-induced conversion occurred when recShPrP 90–232 and recMoPrP 90–231 were shaken at room temperature. In contrast when shaking-induced conversion was tested at pH 7.4 the oligomers formed are predominantly large oligomers (>16-mers) and included a large oligomer band referred to as a fibril band on the RENAGE gels. Prion protein constructs of different lengths for MoPrP were assessed for their ability to convert to oligomers after 24 and 48 hrs shaking at pH 6.2, using RENAGE. Shaking-induced conversion occurs for full length recMoPrP 23–231 in a manner similar to truncated recMoPrP 90–231 although apparently with different kinetics (Fig. 3B). In contrast, shaking the C-terminal domain (recMoPrP 120–231) causes faster conversion as seen at 24 hrs and then after 48 hrs only large oligomers are visible by RENAGE (Fig. 3B). In this recMoPrP 120–321 sample which was shaken for 48 hours, there is a loss in the total amount of protein deposited on the gel in addition to the formation of a visible precipitate in the sample tube. This could indicate that after (C-terminal domain) oligomers form they preferentially progress to aggregates rather than fibrils. Conversion of these three different lengths of MoPrP occurred similarly at pH 5.5, except the C-terminal (recMoPrP 120–231) low molecular weight oligomers (ie. 8-mers) were not as distinct. We proceeded to characterize shaking-induced conversion at pH 5.5, because of the efficiency of forming oligomers for recMoPrP 90–231 and recMoPrP 23–231 at this pH and because the sodium acetate buffer was more amenable to CD analysis than the buffer containing MES. It is also notable that shaking-induced conversion occurs irrespective of the presence of the His6x purification tag (Fig. S1), with oligomers of the same size formed with or without the His6x tag. We also converted cervid PrP 94–233 to oligomers and fibrils, as seen by RENAGE (results 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)

RENAGE of shaking-converted prions under various conditions.A) ShPrP 90–232 oligomers are formed by shaking in pH 5.5, 6.2 and 7.4 buffers, at 350 rpm and 37°C. B) RENAGE after shaking at 350 rpm for 1 day with full length recMoPrP 23–231 (lane 1), truncated recMoPrP 90–231 (lane 2) and C-terminal domain recMoPrP 120–231 (lane 3) and after 2 days with recMoPrP 23–231 (lane 4), truncated recMoPrP 90–231 (lane 5) and C-terminal domain recMoPrP 120–231 (lane 6). Samples in panel B were shaken at 350 rpm and 37°C in pH 6.2 buffer. C) Shaking a 0.6 mL solution of recShPrP90–232 in a 0.6 mL centrifuge tube without any air or bubbles for two weeks (lane 1) as compared to the same sample of 0.4 mL in a 1.5 mL centrifuge tube (i.e. with air), shaken for one week (lane 2).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4043794&req=5

pone-0098753-g003: RENAGE of shaking-converted prions under various conditions.A) ShPrP 90–232 oligomers are formed by shaking in pH 5.5, 6.2 and 7.4 buffers, at 350 rpm and 37°C. B) RENAGE after shaking at 350 rpm for 1 day with full length recMoPrP 23–231 (lane 1), truncated recMoPrP 90–231 (lane 2) and C-terminal domain recMoPrP 120–231 (lane 3) and after 2 days with recMoPrP 23–231 (lane 4), truncated recMoPrP 90–231 (lane 5) and C-terminal domain recMoPrP 120–231 (lane 6). Samples in panel B were shaken at 350 rpm and 37°C in pH 6.2 buffer. C) Shaking a 0.6 mL solution of recShPrP90–232 in a 0.6 mL centrifuge tube without any air or bubbles for two weeks (lane 1) as compared to the same sample of 0.4 mL in a 1.5 mL centrifuge tube (i.e. with air), shaken for one week (lane 2).
Mentions: Shaking-induced oligomers were also found at pH 6.2 using recShPrP 90–232 and recMoPrP 90–231 (result shown for recShPrP 90–232 in Fig. 3A). The distribution of oligomers formed at pH 6.2 is similar to that formed at pH 5.5. However at pH 6.2 there is an increase in high molecular oligomers (16 to 20-mers) relative to oligomers at 8 to 12-mers (Fig. 3A). Furthermore shaking-induced conversion occurred when recShPrP 90–232 and recMoPrP 90–231 were shaken at room temperature. In contrast when shaking-induced conversion was tested at pH 7.4 the oligomers formed are predominantly large oligomers (>16-mers) and included a large oligomer band referred to as a fibril band on the RENAGE gels. Prion protein constructs of different lengths for MoPrP were assessed for their ability to convert to oligomers after 24 and 48 hrs shaking at pH 6.2, using RENAGE. Shaking-induced conversion occurs for full length recMoPrP 23–231 in a manner similar to truncated recMoPrP 90–231 although apparently with different kinetics (Fig. 3B). In contrast, shaking the C-terminal domain (recMoPrP 120–231) causes faster conversion as seen at 24 hrs and then after 48 hrs only large oligomers are visible by RENAGE (Fig. 3B). In this recMoPrP 120–321 sample which was shaken for 48 hours, there is a loss in the total amount of protein deposited on the gel in addition to the formation of a visible precipitate in the sample tube. This could indicate that after (C-terminal domain) oligomers form they preferentially progress to aggregates rather than fibrils. Conversion of these three different lengths of MoPrP occurred similarly at pH 5.5, except the C-terminal (recMoPrP 120–231) low molecular weight oligomers (ie. 8-mers) were not as distinct. We proceeded to characterize shaking-induced conversion at pH 5.5, because of the efficiency of forming oligomers for recMoPrP 90–231 and recMoPrP 23–231 at this pH and because the sodium acetate buffer was more amenable to CD analysis than the buffer containing MES. It is also notable that shaking-induced conversion occurs irrespective of the presence of the His6x purification tag (Fig. S1), with oligomers of the same size formed with or without the His6x tag. We also converted cervid PrP 94–233 to oligomers and fibrils, as seen by RENAGE (results 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