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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 brain PrPC conversion by dominant negative mutant PrP.Reactions containing either purified brain-derived HaPrPC alone (−Mutant, lanes 2–5) or in combination with either Q172R, immunopurified Q172R (Q172R Pure), T215K, or different concentrations of Q219K HaPrP mutant substrates (+Mutant, lanes 8–11), as indicated, were subjected to three rounds of serial propagation. Q172R Pure was tested at ∼1∶2 (Mut∶WT) ratio; Q172R Low was tested at ∼1∶5 ratio; and all other conditions were tested at ∼5∶1 ratio. In each blot, an arrowhead demarks the expected mobility of the ∼27–30 kDa PK-resistant brain-derived PrPSc species. All reactions were supplemented with synthetic poly(A) RNA. In all blots, a sample containing wild type or mutant HaPrP substrate not subjected to proteinase K digestion is shown in the lane(s) preceding the corresponding PK-digested samples as a reference for comparison of electrophoretic mobility (−PK WT or Mut). 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-g004: Inhibition of hamster brain PrPC conversion by dominant negative mutant PrP.Reactions containing either purified brain-derived HaPrPC alone (−Mutant, lanes 2–5) or in combination with either Q172R, immunopurified Q172R (Q172R Pure), T215K, or different concentrations of Q219K HaPrP mutant substrates (+Mutant, lanes 8–11), as indicated, were subjected to three rounds of serial propagation. Q172R Pure was tested at ∼1∶2 (Mut∶WT) ratio; Q172R Low was tested at ∼1∶5 ratio; and all other conditions were tested at ∼5∶1 ratio. In each blot, an arrowhead demarks the expected mobility of the ∼27–30 kDa PK-resistant brain-derived PrPSc species. All reactions were supplemented with synthetic poly(A) RNA. In all blots, a sample containing wild type or mutant HaPrP substrate not subjected to proteinase K digestion is shown in the lane(s) preceding the corresponding PK-digested samples as a reference for comparison of electrophoretic mobility (−PK WT or Mut). All other samples were subjected to limited proteolysis with 50 µg/ml proteinase K for 1 hr at 37°C (+PK).

Mentions: While it was clear the Q172R HaPrP substrate could inhibit the conversion of wild type HaPrPC substrate, we still could not conclude whether T215K and Q219K HaPrP substrates possess the same dominant negative properties. Our HaPrP substrate expressed in CHO cells has a higher relative molecular weight as compared to brain-derived HaPrPC; thus, the molecular weight of protease-resistant PrP derived from the conversion of these different PrP substrates should also be distinguishable by SDS-PAGE. We decided to exploit this biochemical difference between HaPrP substrates to determine if T215K and Q219K HaPrP substrate act as dominant negatives. We conducted seeded sPMCA propagation reactions containing both immunopurified brain-derived wild type HaPrPC and mutant HaPrP substrates supplemented with poly(A) RNA (Figure 4). Consistent with previous studies, brain-derived wild type HaPrPC substrate was converted into autocatalytic HaPrPSc in serial propagation reactions supplemented with poly(A) RNA (Figure 4, lanes 3–5, all blots). When Q172R HaPrP substrate was added to the reaction, conversion of brain-derived wild type HaPrPC substrate was blocked (Figure 4, lanes 9–11, top blot).


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 brain PrPC conversion by dominant negative mutant PrP.Reactions containing either purified brain-derived HaPrPC alone (−Mutant, lanes 2–5) or in combination with either Q172R, immunopurified Q172R (Q172R Pure), T215K, or different concentrations of Q219K HaPrP mutant substrates (+Mutant, lanes 8–11), as indicated, were subjected to three rounds of serial propagation. Q172R Pure was tested at ∼1∶2 (Mut∶WT) ratio; Q172R Low was tested at ∼1∶5 ratio; and all other conditions were tested at ∼5∶1 ratio. In each blot, an arrowhead demarks the expected mobility of the ∼27–30 kDa PK-resistant brain-derived PrPSc species. All reactions were supplemented with synthetic poly(A) RNA. In all blots, a sample containing wild type or mutant HaPrP substrate not subjected to proteinase K digestion is shown in the lane(s) preceding the corresponding PK-digested samples as a reference for comparison of electrophoretic mobility (−PK WT or Mut). All other samples were subjected to limited proteolysis with 50 µg/ml proteinase K for 1 hr at 37°C (+PK).
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1000535-g004: Inhibition of hamster brain PrPC conversion by dominant negative mutant PrP.Reactions containing either purified brain-derived HaPrPC alone (−Mutant, lanes 2–5) or in combination with either Q172R, immunopurified Q172R (Q172R Pure), T215K, or different concentrations of Q219K HaPrP mutant substrates (+Mutant, lanes 8–11), as indicated, were subjected to three rounds of serial propagation. Q172R Pure was tested at ∼1∶2 (Mut∶WT) ratio; Q172R Low was tested at ∼1∶5 ratio; and all other conditions were tested at ∼5∶1 ratio. In each blot, an arrowhead demarks the expected mobility of the ∼27–30 kDa PK-resistant brain-derived PrPSc species. All reactions were supplemented with synthetic poly(A) RNA. In all blots, a sample containing wild type or mutant HaPrP substrate not subjected to proteinase K digestion is shown in the lane(s) preceding the corresponding PK-digested samples as a reference for comparison of electrophoretic mobility (−PK WT or Mut). All other samples were subjected to limited proteolysis with 50 µg/ml proteinase K for 1 hr at 37°C (+PK).
Mentions: While it was clear the Q172R HaPrP substrate could inhibit the conversion of wild type HaPrPC substrate, we still could not conclude whether T215K and Q219K HaPrP substrates possess the same dominant negative properties. Our HaPrP substrate expressed in CHO cells has a higher relative molecular weight as compared to brain-derived HaPrPC; thus, the molecular weight of protease-resistant PrP derived from the conversion of these different PrP substrates should also be distinguishable by SDS-PAGE. We decided to exploit this biochemical difference between HaPrP substrates to determine if T215K and Q219K HaPrP substrate act as dominant negatives. We conducted seeded sPMCA propagation reactions containing both immunopurified brain-derived wild type HaPrPC and mutant HaPrP substrates supplemented with poly(A) RNA (Figure 4). Consistent with previous studies, brain-derived wild type HaPrPC substrate was converted into autocatalytic HaPrPSc in serial propagation reactions supplemented with poly(A) RNA (Figure 4, lanes 3–5, all blots). When Q172R HaPrP substrate was added to the reaction, conversion of brain-derived wild type HaPrPC substrate was blocked (Figure 4, lanes 9–11, top blot).

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