Abstract
Previous studies identified prion protein (PrP) mutants which act as dominant negative inhibitors of prion formation through a mechanism hypothesized to require an unidentified species-specific cofactor termed protein X. To study the mechanism of dominant negative inhibition in vitro, we used recombinant PrPC molecules expressed in Chinese hamster ovary cells as substrates in serial protein misfolding cyclic amplification (sPMCA) reactions. Bioassays confirmed that the products of these reactions are infectious. Using this system, we find that: (1) trans-dominant inhibition can be dissociated from conversion activity, (2) dominant-negative inhibition of prion formation can be reconstituted in vitro using only purified substrates, even when wild type (WT) PrPC is pre-incubated with poly(A) RNA and PrPSc template, and (3) Q172R is the only hamster PrP mutant tested that fails to convert into PrPSc and that can dominantly inhibit conversion of WT PrP at sub-stoichiometric levels. These results refute the hypothesis that protein X is required to mediate dominant inhibition of prion propagation, and suggest that PrP molecules compete for binding to a nascent seeding site on newly formed PrPSc molecules, most likely through an epitope containing residue 172.
Highlights
Prion diseases are fatal neurodegenerative diseases with inherited, sporadic, and infectious etiologies [1–3]
Over the past two decades, various investigators have observed that heterozygous animals possessing two different forms of the gene encoding the prion protein (PrP) are more difficult to infect with some strains of infectious prions than homozygous animals possessing only the most commonly occurring form of the gene encoding PrP for that species
In 1995, it was hypothesized that the inhibition of prion infection in heterozygous animals might be caused by competition between the two different types of PrP molecules for binding to a common cofactor required for prion propagation, provisionally named ‘‘protein X,’’ through a specific portion of the PrP molecule
Summary
Prion diseases are fatal neurodegenerative diseases with inherited, sporadic, and infectious etiologies [1–3]. Studies examining the transmission of prions to transgenic mice expressing human or mouse/human chimeric PrP led to the hypothesis that a species-specific cofactor, termed protein X, is required for PrPSc formation [13]. Utilizing a cell culture model of prion formation, mouse (Mo) PrP single-point mutants that could not undergo conformational conversion to form PrPSc were identified [14–16]. These MoPrP mutants acted in a dominant negative manner in that they prevented the conversion of wild type PrPC when co-expressed in scrapie-infected cells. It has been shown that other heterologous PrP molecules lacking mutations of the putative protein X binding site can interfere with conversion of MoPrPC to MoPrPSc in cell culture and biochemical assays [31– 33]. In mice, some prion strains have shorter incubation periods in mice expressing only the Prn-p(a) polymorphic allele
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