Abstract
The most unorthodox feature of the prion disease is the existence of an abnormal infectious isoform of the prion protein, PrP(Sc). According to the "protein-only" hypothesis, PrP(Sc) propagates its abnormal conformation in an autocatalytic manner using the normal isoform, PrP(C), as a substrate. Because autocatalytic conversion is considered a key element of prion replication, in this study I tested whether in vitro conversion of recombinant PrP into abnormal isoform displays specific features of an autocatalytic process. I found that recombinant human PrP formed two distinct beta-sheet rich isoforms, the beta-oligomer and the amyloid fibrils. The kinetics of the fibrils formation measured at different pH values were consistent with a model in which the beta-oligomer was not on the kinetic pathway to the fibrillar form. As judged by electron microscopy, an acidic pH favored to the long fibrils, whereas short fibrils morphologically similar to "prion rods" were formed at neutral pH. At neutral pH the conversion to the fibrils can be seeded with small aliquots of preformed fibrils. As small as 0.001% aliquot displayed seeding activity. The conversion of human PrP was seeded with high efficacy only with the preformed fibrils of human but not mouse PrP and vice versa. These studies illustrate that in vitro conversion of recombinant PrP displays specific features of an autocatalytic process and mimics the transmission barrier of prion propagation observed in vivo. I speculate that this model can be used as a rapid assay for assessing the intrinsic propensities of prion transmission between different species.
Highlights
Spontaneous conversion of the normal cellular isoform of the prion protein, PrPC, into the abnormal pathological isoform, PrPSc, underlies sporadic forms of prion disease, including Creutzfeldt-Jakob disease in humans [1]
Formation of the -Oligomer versus the Amyloid Fibrils— Typical kinetics of spontaneous in vitro fibril formation of Hu recPrP at 37 °C showed a substantial lag-phase followed by a rapid increase of amyloid fibrils accumulation as measured by thioflavin T (ThT) fluorescence (Fig. 1a)
In a previous study, using mouse and Syrian hamster recPrPs, we showed that acidic pH favored formation of the -oligomer, which is off the kinetic pathway to the amyloid fibrils [24]
Summary
Spontaneous conversion of the normal cellular isoform of the prion protein, PrPC, into the abnormal pathological isoform, PrPSc, underlies sporadic forms of prion disease, including Creutzfeldt-Jakob disease in humans [1]. I found that recombinant human PrP formed two distinct -sheet rich isoforms, the -oligomer and the amyloid fibrils. These studies illustrate that in vitro conversion of recombinant PrP displays specific features of an autocatalytic process and mimics the transmission barrier of prion propagation observed in vivo.
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