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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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sPMCA propagation with CHO-expressed PrP substrates and Prnp0/0 brain homogenate.(A) Reactions containing either wild type or mutant mouse (Mo) PrP substrate alone were originally seeded with RML scrapie brain homogenate and propagated for three rounds of sPMCA. (B) Reactions containing either wild type MoPrPC substrate alone (−Mutant, lanes 2–5) or in combination with either Q171R, V214I, V214K, or Q218K MoPrP mutant substrates at approximately equimolar concentrations (+Mutant, lanes 8–11), as indicated, were subjected to three rounds of serial propagation. (C) Reactions containing either wild type or mutant hamster (Ha) PrP substrate alone were originally seeded with Sc237 scrapie brain homogenate and propagated for three rounds of sPMCA reconstituted with Prnp0/0 brain homogenate. (D) Reactions containing either wild type HaPrP substrates alone (−Mutant, lanes 2–5) or in combination with either Q172R, T215K, or Q219K HaPrP mutant substrates at approximately equimolar concentrations (+Mutant, lanes 8–11), as indicated, were subjected to three rounds of serial propagation. An arrowhead demarks the ∼25 kDa PK-resistant T215K HaPrP species. All reactions were reconstituted with Prnp0/0 brain homogenate. In all blots, a sample containing wild type or mutant PrP substrate not subjected to proteinase K digestion is shown in the lanes preceding the corresponding PK-digested samples as a reference for comparison of electrophoretic mobility (−PK PrP, WT, or Mut). All other samples were subjected to limited proteolysis with proteinase K (25 µg/ml for 30 min. for mouse, 50 µg/ml for 1 hr. for hamster) at 37°C (+PK).
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ppat-1000535-g001: sPMCA propagation with CHO-expressed PrP substrates and Prnp0/0 brain homogenate.(A) Reactions containing either wild type or mutant mouse (Mo) PrP substrate alone were originally seeded with RML scrapie brain homogenate and propagated for three rounds of sPMCA. (B) Reactions containing either wild type MoPrPC substrate alone (−Mutant, lanes 2–5) or in combination with either Q171R, V214I, V214K, or Q218K MoPrP mutant substrates at approximately equimolar concentrations (+Mutant, lanes 8–11), as indicated, were subjected to three rounds of serial propagation. (C) Reactions containing either wild type or mutant hamster (Ha) PrP substrate alone were originally seeded with Sc237 scrapie brain homogenate and propagated for three rounds of sPMCA reconstituted with Prnp0/0 brain homogenate. (D) Reactions containing either wild type HaPrP substrates alone (−Mutant, lanes 2–5) or in combination with either Q172R, T215K, or Q219K HaPrP mutant substrates at approximately equimolar concentrations (+Mutant, lanes 8–11), as indicated, were subjected to three rounds of serial propagation. An arrowhead demarks the ∼25 kDa PK-resistant T215K HaPrP species. All reactions were reconstituted with Prnp0/0 brain homogenate. In all blots, a sample containing wild type or mutant PrP substrate not subjected to proteinase K digestion is shown in the lanes preceding the corresponding PK-digested samples as a reference for comparison of electrophoretic mobility (−PK PrP, WT, or Mut). All other samples were subjected to limited proteolysis with proteinase K (25 µg/ml for 30 min. for mouse, 50 µg/ml for 1 hr. for hamster) at 37°C (+PK).

Mentions: To study the properties of dominant negative mutant PrP molecules in vitro we began by expressing wild type mouse and hamster PrPC in CHO cells, which do not express detectable levels of endogenous HaPrPC protein [41]. Comparison of CHO-expressed PrPC molecules to brain-derived PrPC by SDS-PAGE revealed that the PrPC expressed in CHO cells migrated between 26 and 43 kDa, a broader electrophoretic mobility pattern than brain-derived PrPC, which migrated between 26 kDa and ∼37 kDa (Figure S1, lane 1 vs. 2–5 and lane 6 vs. 7–10). Deglycosylation of CHO-expressed MoPrPC with peptide-N-glycosidase F (PNGase F) revealed that the increase in apparent molecular weight was due to more extensive glycosylation, as the core polypeptide of CHO-expressed and brain-derived MoPrPC migrated equivalently on an SDS-PAGE gel (data not shown). We partially purified the mouse and hamster PrPC from CHO cells by cobalt-affinity column chromatography for use as sPMCA substrates. We first conducted three-rounds of serial propagation reactions using CHO-expressed mouse PrPC originally seeded with crude RML murine scrapie brain homogenate and reconstituted with PrP0/0 brain homogenate, which contains cofactors that facilitate the conversion of brain-derived HaPrPC in sPMCA reactions [42] (Figure 1A). The results show that MoPrPC was efficiently converted and propagated in this reaction, indicating that MoPrPC expressed in CHO cells is a competent substrate for in vitro conversion into a protease-resistant and autocatalytic form of PrP (Figure 1A, 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)

sPMCA propagation with CHO-expressed PrP substrates and Prnp0/0 brain homogenate.(A) Reactions containing either wild type or mutant mouse (Mo) PrP substrate alone were originally seeded with RML scrapie brain homogenate and propagated for three rounds of sPMCA. (B) Reactions containing either wild type MoPrPC substrate alone (−Mutant, lanes 2–5) or in combination with either Q171R, V214I, V214K, or Q218K MoPrP mutant substrates at approximately equimolar concentrations (+Mutant, lanes 8–11), as indicated, were subjected to three rounds of serial propagation. (C) Reactions containing either wild type or mutant hamster (Ha) PrP substrate alone were originally seeded with Sc237 scrapie brain homogenate and propagated for three rounds of sPMCA reconstituted with Prnp0/0 brain homogenate. (D) Reactions containing either wild type HaPrP substrates alone (−Mutant, lanes 2–5) or in combination with either Q172R, T215K, or Q219K HaPrP mutant substrates at approximately equimolar concentrations (+Mutant, lanes 8–11), as indicated, were subjected to three rounds of serial propagation. An arrowhead demarks the ∼25 kDa PK-resistant T215K HaPrP species. All reactions were reconstituted with Prnp0/0 brain homogenate. In all blots, a sample containing wild type or mutant PrP substrate not subjected to proteinase K digestion is shown in the lanes preceding the corresponding PK-digested samples as a reference for comparison of electrophoretic mobility (−PK PrP, WT, or Mut). All other samples were subjected to limited proteolysis with proteinase K (25 µg/ml for 30 min. for mouse, 50 µg/ml for 1 hr. for hamster) at 37°C (+PK).
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1000535-g001: sPMCA propagation with CHO-expressed PrP substrates and Prnp0/0 brain homogenate.(A) Reactions containing either wild type or mutant mouse (Mo) PrP substrate alone were originally seeded with RML scrapie brain homogenate and propagated for three rounds of sPMCA. (B) Reactions containing either wild type MoPrPC substrate alone (−Mutant, lanes 2–5) or in combination with either Q171R, V214I, V214K, or Q218K MoPrP mutant substrates at approximately equimolar concentrations (+Mutant, lanes 8–11), as indicated, were subjected to three rounds of serial propagation. (C) Reactions containing either wild type or mutant hamster (Ha) PrP substrate alone were originally seeded with Sc237 scrapie brain homogenate and propagated for three rounds of sPMCA reconstituted with Prnp0/0 brain homogenate. (D) Reactions containing either wild type HaPrP substrates alone (−Mutant, lanes 2–5) or in combination with either Q172R, T215K, or Q219K HaPrP mutant substrates at approximately equimolar concentrations (+Mutant, lanes 8–11), as indicated, were subjected to three rounds of serial propagation. An arrowhead demarks the ∼25 kDa PK-resistant T215K HaPrP species. All reactions were reconstituted with Prnp0/0 brain homogenate. In all blots, a sample containing wild type or mutant PrP substrate not subjected to proteinase K digestion is shown in the lanes preceding the corresponding PK-digested samples as a reference for comparison of electrophoretic mobility (−PK PrP, WT, or Mut). All other samples were subjected to limited proteolysis with proteinase K (25 µg/ml for 30 min. for mouse, 50 µg/ml for 1 hr. for hamster) at 37°C (+PK).
Mentions: To study the properties of dominant negative mutant PrP molecules in vitro we began by expressing wild type mouse and hamster PrPC in CHO cells, which do not express detectable levels of endogenous HaPrPC protein [41]. Comparison of CHO-expressed PrPC molecules to brain-derived PrPC by SDS-PAGE revealed that the PrPC expressed in CHO cells migrated between 26 and 43 kDa, a broader electrophoretic mobility pattern than brain-derived PrPC, which migrated between 26 kDa and ∼37 kDa (Figure S1, lane 1 vs. 2–5 and lane 6 vs. 7–10). Deglycosylation of CHO-expressed MoPrPC with peptide-N-glycosidase F (PNGase F) revealed that the increase in apparent molecular weight was due to more extensive glycosylation, as the core polypeptide of CHO-expressed and brain-derived MoPrPC migrated equivalently on an SDS-PAGE gel (data not shown). We partially purified the mouse and hamster PrPC from CHO cells by cobalt-affinity column chromatography for use as sPMCA substrates. We first conducted three-rounds of serial propagation reactions using CHO-expressed mouse PrPC originally seeded with crude RML murine scrapie brain homogenate and reconstituted with PrP0/0 brain homogenate, which contains cofactors that facilitate the conversion of brain-derived HaPrPC in sPMCA reactions [42] (Figure 1A). The results show that MoPrPC was efficiently converted and propagated in this reaction, indicating that MoPrPC expressed in CHO cells is a competent substrate for in vitro conversion into a protease-resistant and autocatalytic form of PrP (Figure 1A, 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