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Trans-dominant inhibition of prion propagation in vitro is not mediated by an accessory cofactor.

Geoghegan JC, Miller MB, Kwak AH, Harris BT, Supattapone S - PLoS Pathog. (2009)

Bottom Line: 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.Bioassays confirmed that the products of these reactions are infectious.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 PrP(Sc) molecules, most likely through an epitope containing residue 172.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Dartmouth Medical School, Hanover, NH, USA.

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 PrP(C) 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) PrP(C) is pre-incubated with poly(A) RNA and PrP(Sc) template, and (3) Q172R is the only hamster PrP mutant tested that fails to convert into PrP(Sc) 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 PrP(Sc) molecules, most likely through an epitope containing residue 172.

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Inhibition of hamster PrPC conversion following hamster PrPC substrate pre-incubation with poly(A) RNA and/or Sc237.Western blots showing Sc237-seeded sPMCA propagation reactions containing wild type, Q172R HaPrP substrates, and synthetic poly(A) RNA. Reactions containing both CHO-expressed wild type HaPrPC and the Q172R mutant HaPrP substrates at ∼1∶2 (Mut∶WT) ratio (lanes 3–6 and 9–12) were subjected to three rounds of serial propagation. Wild type HaPrPC substrate was either pre-incubated (+Pre-incubation) or not (−Pre-incubation) with poly(A) RNA alone (top blot) or in combination with the Sc237 scrapie seed (bottom blot), as indicated, prior to addition of the Q172R HaPrP substrate and other components to the reaction. In all blots, samples containing recombinant wild type or mutant HaPrP substrate not subjected to proteinase K digestion are shown as a reference for comparison of electrophoretic mobility (−PK WT or Mut, respectively). All other samples were subjected to limited proteolysis with 50 µg/ml proteinase K for 1 hr at 37°C (+PK).
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ppat-1000535-g005: Inhibition of hamster PrPC conversion following hamster PrPC substrate pre-incubation with poly(A) RNA and/or Sc237.Western blots showing Sc237-seeded sPMCA propagation reactions containing wild type, Q172R HaPrP substrates, and synthetic poly(A) RNA. Reactions containing both CHO-expressed wild type HaPrPC and the Q172R mutant HaPrP substrates at ∼1∶2 (Mut∶WT) ratio (lanes 3–6 and 9–12) were subjected to three rounds of serial propagation. Wild type HaPrPC substrate was either pre-incubated (+Pre-incubation) or not (−Pre-incubation) with poly(A) RNA alone (top blot) or in combination with the Sc237 scrapie seed (bottom blot), as indicated, prior to addition of the Q172R HaPrP substrate and other components to the reaction. In all blots, samples containing recombinant wild type or mutant HaPrP substrate not subjected to proteinase K digestion are shown as a reference for comparison of electrophoretic mobility (−PK WT or Mut, respectively). All other samples were subjected to limited proteolysis with 50 µg/ml proteinase K for 1 hr at 37°C (+PK).

Mentions: As there are several possible explanations for how Q172R HaPrP exerts its dominant negative properties, we sought to examine more closely the mechanism by which Q172R HaPrP-mediated inhibition occurs. One possible mechanism is that compared to wild type HaPrPC, Q172R HaPrP has increased affinity for binding poly(A) RNA, a necessary cofactor for conversion in this in vitro assay. To test whether Q172R HaPrP might interfere with the interaction between poly(A) RNA and wild type HaPrPC, we conducted a serial propagation reaction in which CHO-expressed wild type HaPrPC substrate was allowed to pre-incubate with poly(A) RNA prior to addition of the Q172R HaPrP substrate to the reaction (Figure 5, top blot). We previously determined that our wild type HaPrPC rapidly (<15 min) binds to immobilized poly(A) RNA (data not shown) and therefore, under the tested pre-incubation conditions, the wild type HaPrPC substrate should have sufficient time to physically interact with poly(A) RNA before encountering Q172R HaPrP substrate. As previously demonstrated, when wild type and Q172R HaPrP substrates are combined simultaneously in the reaction, conversion of wild type HaPrPC substrate is inhibited (Figure 4). When wild type HaPrP substrate was allowed to incubate with poly(A) RNA before the addition of Q172R HaPrP substrate and Sc237 seed, conversion of wild type HaPrPC substrate was still inhibited (Figure 5, +Pre-incubation, lanes 10–12). Assuming that the wild type HaPrPC substrate interacted with poly(A) RNA during the pre-incubation, this finding indicates that Q172R HaPrP substrate does not block conversion of wild type HaPrPC substrate by sequestering the required cofactor, poly(A) RNA.


Trans-dominant inhibition of prion propagation in vitro is not mediated by an accessory cofactor.

Geoghegan JC, Miller MB, Kwak AH, Harris BT, Supattapone S - PLoS Pathog. (2009)

Inhibition of hamster PrPC conversion following hamster PrPC substrate pre-incubation with poly(A) RNA and/or Sc237.Western blots showing Sc237-seeded sPMCA propagation reactions containing wild type, Q172R HaPrP substrates, and synthetic poly(A) RNA. Reactions containing both CHO-expressed wild type HaPrPC and the Q172R mutant HaPrP substrates at ∼1∶2 (Mut∶WT) ratio (lanes 3–6 and 9–12) were subjected to three rounds of serial propagation. Wild type HaPrPC substrate was either pre-incubated (+Pre-incubation) or not (−Pre-incubation) with poly(A) RNA alone (top blot) or in combination with the Sc237 scrapie seed (bottom blot), as indicated, prior to addition of the Q172R HaPrP substrate and other components to the reaction. In all blots, samples containing recombinant wild type or mutant HaPrP substrate not subjected to proteinase K digestion are shown as a reference for comparison of electrophoretic mobility (−PK WT or Mut, respectively). All other samples were subjected to limited proteolysis with 50 µg/ml proteinase K for 1 hr at 37°C (+PK).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2713408&req=5

ppat-1000535-g005: Inhibition of hamster PrPC conversion following hamster PrPC substrate pre-incubation with poly(A) RNA and/or Sc237.Western blots showing Sc237-seeded sPMCA propagation reactions containing wild type, Q172R HaPrP substrates, and synthetic poly(A) RNA. Reactions containing both CHO-expressed wild type HaPrPC and the Q172R mutant HaPrP substrates at ∼1∶2 (Mut∶WT) ratio (lanes 3–6 and 9–12) were subjected to three rounds of serial propagation. Wild type HaPrPC substrate was either pre-incubated (+Pre-incubation) or not (−Pre-incubation) with poly(A) RNA alone (top blot) or in combination with the Sc237 scrapie seed (bottom blot), as indicated, prior to addition of the Q172R HaPrP substrate and other components to the reaction. In all blots, samples containing recombinant wild type or mutant HaPrP substrate not subjected to proteinase K digestion are shown as a reference for comparison of electrophoretic mobility (−PK WT or Mut, respectively). All other samples were subjected to limited proteolysis with 50 µg/ml proteinase K for 1 hr at 37°C (+PK).
Mentions: As there are several possible explanations for how Q172R HaPrP exerts its dominant negative properties, we sought to examine more closely the mechanism by which Q172R HaPrP-mediated inhibition occurs. One possible mechanism is that compared to wild type HaPrPC, Q172R HaPrP has increased affinity for binding poly(A) RNA, a necessary cofactor for conversion in this in vitro assay. To test whether Q172R HaPrP might interfere with the interaction between poly(A) RNA and wild type HaPrPC, we conducted a serial propagation reaction in which CHO-expressed wild type HaPrPC substrate was allowed to pre-incubate with poly(A) RNA prior to addition of the Q172R HaPrP substrate to the reaction (Figure 5, top blot). We previously determined that our wild type HaPrPC rapidly (<15 min) binds to immobilized poly(A) RNA (data not shown) and therefore, under the tested pre-incubation conditions, the wild type HaPrPC substrate should have sufficient time to physically interact with poly(A) RNA before encountering Q172R HaPrP substrate. As previously demonstrated, when wild type and Q172R HaPrP substrates are combined simultaneously in the reaction, conversion of wild type HaPrPC substrate is inhibited (Figure 4). When wild type HaPrP substrate was allowed to incubate with poly(A) RNA before the addition of Q172R HaPrP substrate and Sc237 seed, conversion of wild type HaPrPC substrate was still inhibited (Figure 5, +Pre-incubation, lanes 10–12). Assuming that the wild type HaPrPC substrate interacted with poly(A) RNA during the pre-incubation, this finding indicates that Q172R HaPrP substrate does not block conversion of wild type HaPrPC substrate by sequestering the required cofactor, poly(A) RNA.

Bottom Line: 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.Bioassays confirmed that the products of these reactions are infectious.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 PrP(Sc) molecules, most likely through an epitope containing residue 172.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Dartmouth Medical School, Hanover, NH, USA.

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 PrP(C) 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) PrP(C) is pre-incubated with poly(A) RNA and PrP(Sc) template, and (3) Q172R is the only hamster PrP mutant tested that fails to convert into PrP(Sc) 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 PrP(Sc) molecules, most likely through an epitope containing residue 172.

Show MeSH
Related in: MedlinePlus