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Updated: Jun 16, 2026
Isolation of Soluble and Insoluble PrP Oligomers in the Normal Human Brain
Published on: October 3, 2012
Low density subcellular fractions enhance disease-specific prion protein misfolding
James F Graham1, Sonya Agarwal1, Dominic Kurian2
1Neuropathogenesis Division, The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Alexander Robertson Building, Easter Bush Veterinary Centre, Roslin, Midlothian EH25 9RG.
Abstract:
The production of prion particles in vitro by amplification with or without exogenous seed typically results in infectivity titers less than those associated with PrP(Sc) isolated ex vivo and highlights the potential role of co-factors that can catalyze disease-specific prion protein misfolding in vivo. We used a cell-free conversion assay previously shown to replicate many aspects of transmissible spongiform encephalopathy disease to investigate the cellular location of disease-specific co-factors using fractions derived from gradient centrifugation of a scrapie-susceptible cell line. Fractions from the low density region of the gradient doubled the efficiency of conversion of recombinant PrP. These fractions contain plasma membrane and cytoplasmic proteins, and conversion enhancement can be achieved using PrP(Sc) derived from two different strains of mouse-passaged scrapie as seed. Equivalent fractions from a second scrapie-susceptible cell line also stimulate conversion. We also show that subcellular fractions enhancing disease-specific prion protein conversion prevent in vitro fibrillization of recombinant prion protein, suggesting the existence of separate, competing mechanisms of disease-specific and nonspecific misfolding in vivo.
Insights
Researchers identified cellular co-factors that enhance prion protein misfolding in vitro. These co-factors, found in plasma membrane and cytoplasmic fractions, are crucial for disease-specific prion formation in transmissible spongiform encephalopathy.
Area of Science:
- Neuroscience
- Biochemistry
- Molecular Biology
Background:
- In vitro prion production often yields lower infectivity than ex vivo PrP(Sc).
- This suggests co-factors are essential for catalyzing disease-specific prion protein misfolding in vivo.
- Transmissible spongiform encephalopathies (TSEs) involve abnormal prion protein (PrP) folding.
Purpose of the Study:
- To investigate the cellular location of disease-specific co-factors.
- To understand the role of co-factors in prion protein conversion using a cell-free assay.
Main Methods:
- Utilized a cell-free conversion assay to study prion replication.
- Employed gradient centrifugation to fractionate a scrapie-susceptible cell line.
- Tested the effect of subcellular fractions on recombinant PrP conversion efficiency.
Main Results:
- Fractions from the low-density region of the gradient doubled recombinant PrP conversion efficiency.
- These active fractions contained plasma membrane and cytoplasmic proteins.
- Conversion enhancement was observed with PrP(Sc) from two different scrapie strains.
- Equivalent fractions from a second cell line also stimulated conversion.
- Enhancing fractions inhibited non-specific in vitro fibrillization of recombinant PrP.
Conclusions:
- Disease-specific prion co-factors are located in plasma membrane and cytoplasmic fractions.
- These co-factors enhance prion conversion and may compete with non-specific misfolding pathways.
- Findings suggest distinct mechanisms govern specific and non-specific prion protein misfolding in vivo.
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