Abstract
During prion infection, the normal, protease-sensitive conformation of prion protein (PrPC) is converted via seeded polymerization to an abnormal, infectious conformation with greatly increased protease-resistance (PrPSc). In vitro, protein misfolding cyclic amplification (PMCA) uses PrPSc in prion-infected brain homogenates as an initiating seed to convert PrPC and trigger the self-propagation of PrPSc over many cycles of amplification. While PMCA reactions produce high levels of protease-resistant PrP, the infectious titer is often lower than that of brain-derived PrPSc. More recently, PMCA techniques using bacterially derived recombinant PrP (rPrP) in the presence of lipid and RNA but in the absence of any starting PrPSc seed have been used to generate infectious prions that cause disease in wild-type mice with relatively short incubation times. These data suggest that lipid and/or RNA act as cofactors to facilitate the de novo formation of high levels of prion infectivity. Using rPrP purified by two different techniques, we generated a self-propagating protease-resistant rPrP molecule that, regardless of the amount of RNA and lipid used, had a molecular mass, protease resistance and insolubility similar to that of PrPSc. However, we were unable to detect prion infectivity in any of our reactions using either cell-culture or animal bioassays. These results demonstrate that the ability to self-propagate into a protease-resistant insoluble conformer is not unique to infectious PrP molecules. They suggest that the presence of RNA and lipid cofactors may facilitate the spontaneous refolding of PrP into an infectious form while also allowing the de novo formation of self-propagating, but non-infectious, rPrP-res.
Highlights
The fatal, transmissible neurological diseases scrapie in sheep, Creutzfeldt-Jakob Disease in humans and bovine spongiform encephalopathy in cattle are known as transmissible spongiform encephalopathies (TSEs)
They are known as prion diseases because they are associated with abnormal proteins known as prions that are derived from the mammalian prion protein (PrP)
By comparing the intensity of the 16 kDa band to a standard dilution series of undigested recombinant PrP (rPrP) (Figure 1A, lanes 1–4), we estimated that approximately 0.3–3% of the total rPrP in the reaction was converted to rPrP-res
Summary
The fatal, transmissible neurological diseases scrapie in sheep, Creutzfeldt-Jakob Disease in humans and bovine spongiform encephalopathy in cattle are known as transmissible spongiform encephalopathies (TSEs). They are known as prion diseases because they are associated with abnormal proteins known as prions that are derived from the mammalian prion protein (PrP). The normal protease-sensitive and soluble host prion protein (PrPC) is converted to an infectious, proteaseresistant and insoluble form (PrPSc) which has a different tertiary structure. Detergent insolubility, increased resistance to degradation by proteases, an increased amount of beta-sheet structure relative to PrPC, and especially the ability to self-propagate are all considered hallmarks of infectious prions. The mechanisms which underlie the refolding of PrPC into PrPSc remain poorly understood
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