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Isolation of Soluble and Insoluble PrP Oligomers in the Normal Human Brain
Published on: October 3, 2012
In vitro prion protein conversion in detergent-solubilized membranes
Koren Nishina1, Nathan R Deleault, Ralf W Lucassen
1Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755, USA.
Abstract:
A fundamental event in the pathogenesis of prion disease is the conversion of PrP(C), a normal glycophosphatidyl-anchored glycoprotein, into an infectious isoform designated PrP(Sc). In a modified version of the protein misfolding cyclic amplification (PMCA) technique [Saborio et al. (2001) Nature 411, 810-813], protease-resistant PrP(Sc)-like molecules (PrPres) can be amplified in vitro in a species- and strain-specific manner from crude brain homogenates, providing a biochemical model of the prion conversion reaction [Lucassen et al. (2003) Biochemistry 42, 4127-4135]. In this study, we investigated the ability of enriched membrane subsets and detergent-solubilized membrane preparations to support PrPres amplification. Membrane fractionation experiments showed that purified synaptic plasma membrane preparations enriched in PrP(C) but largely depleted of late endosomal and lysosomal markers were sufficient to support PrPres amplification. Detergent solubilization experiments showed that a small group of select detergents could be used to produce soluble preparations that contain PrP(C) and fully support PrPres amplification. The stability of PrPres amplification ability in detergent-solubilized supernatants was dependent on detergent concentration. These results lead to the surprising conclusion that membrane attachment is not required for PrP(C) to convert efficiently into PrPres in vitro and also indicate that biochemical purification of PrPres amplification factors from brain homogenates is a feasible approach.
Insights
Prion disease pathogenesis involves converting normal PrP(C) into infectious PrP(Sc). This study shows membrane attachment is not required for PrP(C) conversion in vitro, suggesting purification of amplification factors is possible.
Area of Science:
- Neuroscience
- Biochemistry
- Molecular Biology
Background:
- Prion diseases are linked to the misfolding of cellular prion protein (PrP(C)) into infectious PrP(Sc).
- Protein Misfolding Cyclic Amplification (PMCA) is an in vitro method to amplify PrP(Sc)-like molecules (PrPres).
- Understanding the requirements for PrP(C) conversion is crucial for modeling prion pathogenesis.
Purpose of the Study:
- To investigate whether membrane association is necessary for PrP(C) conversion into PrPres in vitro.
- To determine if detergent-solubilized membrane preparations can support PrPres amplification.
- To assess the feasibility of purifying factors involved in prion conversion.
Main Methods:
- Utilized a modified Protein Misfolding Cyclic Amplification (PMCA) technique.
- Employed membrane fractionation to isolate synaptic plasma membrane preparations enriched in PrP(C).
- Performed detergent solubilization experiments on membrane preparations.
Main Results:
- Purified synaptic plasma membranes, depleted of endosomal/lysosomal markers, supported PrPres amplification.
- Specific detergents allowed for the creation of soluble PrP(C) preparations that fully supported PrPres amplification.
- The ability of detergent-solubilized preparations to amplify PrPres was dependent on detergent concentration.
Conclusions:
- Membrane attachment is not essential for efficient in vitro conversion of PrP(C) to PrPres.
- Detergent-solubilized membrane preparations containing PrP(C) can serve as a model for prion conversion.
- These findings suggest that biochemical purification of prion conversion factors from brain homogenates is a viable strategy.
