HOMEPRODUCTSCOMPANYCONTACTFAQResearchDictionaryPharmaSign Up FREE or Login

Shaking alone induces de novo conversion of recombinant prion proteins to β-sheet rich oligomers and fibrils.

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).
AuthorsCarol L Ladner-Keay, Bethany J Griffith, David S Wishart
JournalPloS one (PLoS One) Vol. 9 Issue 6 Pg. e98753 ( 2014) ISSN: 1932-6203 [Electronic] United States
PMID24892647 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
Chemical References
  • Amyloid
  • Prions
  • Recombinant Proteins
Topics
  • Amyloid (chemistry, genetics, metabolism)
  • Animals
  • Cricetinae
  • Deer
  • Hydrogen-Ion Concentration
  • Mice
  • Prions (chemistry, genetics, metabolism)
  • Protein Folding
  • Protein Structure, Secondary
  • Recombinant Proteins (chemistry, genetics, metabolism)
  • Temperature

Join CureHunter, for free Research Interface BASIC access!

Take advantage of free CureHunter research engine access to explore the best drug and treatment options for any disease. Find out why thousands of doctors, pharma researchers and patient activists around the world use CureHunter every day.
Realize the full power of the drug-disease research graph!


Choose Username:
Email:
Password:
Verify Password:
Enter Code Shown: