Ralf W Lucassen | PubMed Health

PubMed2004

In vitro prion protein conversion in detergent-solubilized membranes.

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.

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PubMed2003

RNA molecules stimulate prion protein conversion.

Much evidence supports the hypothesis that the infectious agents of prion diseases are devoid of nucleic acid, and instead are composed of a specific infectious protein. This protein, PrP(Sc), seems to be generated by template-induced conformational change of a normally expressed glycoprotein, PrP(C) (ref. 2). Although numerous studies have established the conversion of PrP(C) to PrP(Sc) as the central pathogenic event of prion disease, it is unknown whether cellular factors other than PrP(C) might be required to stimulate efficient PrP(Sc) production. We investigated the biochemical amplification of protease-resistant PrP(Sc)-like protein (PrPres) using a modified version of the protein-misfolding cyclic amplification method. Here we report that stoichiometric transformation of PrP(C) to PrPres in vitro requires specific RNA molecules. Notably, whereas mammalian RNA preparations stimulate in vitro amplification of PrPres, RNA preparations from invertebrate species do not. Our findings suggest that host-encoded stimulatory RNA molecules may have a role in the pathogenesis of prion disease. They also provide a practical approach to improve the sensitivity of diagnostic techniques based on PrPres amplification.

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