Limits...
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.

Show MeSH

Related in: MedlinePlus

Neuropathology of hamsters infected with prions derived from CHO-expressed PrP substrate.Representative histological fields of the brainstem region in control animals and animals inoculated with CHO-expressed in vitro-generated PrPSc molecules. Top row: normal 166 day old Syrian golden hamster, mock inoculated with day 15 sPMCA propagation reaction containing the cobalt-purified fraction from untransfected CHO cells. Bottom row: terminally ill hamster inoculated with Sc237-seeded day 15 sPMCA reaction containing cobalt-prepared PrPC expressed from CHO cells. Both reactions were seeded on day 0 with 0.01% Sc237-infected hamster brain homogenate. Hemotoxylin and eosin (H&E) and PrP immunohistochemical (3F4 antibody) staining are shown for each group. (Scale bar, 50 µm.)
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2713408&req=5

ppat-1000535-g002: Neuropathology of hamsters infected with prions derived from CHO-expressed PrP substrate.Representative histological fields of the brainstem region in control animals and animals inoculated with CHO-expressed in vitro-generated PrPSc molecules. Top row: normal 166 day old Syrian golden hamster, mock inoculated with day 15 sPMCA propagation reaction containing the cobalt-purified fraction from untransfected CHO cells. Bottom row: terminally ill hamster inoculated with Sc237-seeded day 15 sPMCA reaction containing cobalt-prepared PrPC expressed from CHO cells. Both reactions were seeded on day 0 with 0.01% Sc237-infected hamster brain homogenate. Hemotoxylin and eosin (H&E) and PrP immunohistochemical (3F4 antibody) staining are shown for each group. (Scale bar, 50 µm.)

Mentions: We also conducted three-rounds of serial propagation reactions using the CHO-expressed hamster PrPC substrate seeded with crude Sc237 hamster brain homogenate and supplemented with PrP0/0 brain homogenate (Figure 1C). The results show that HaPrPC was efficiently converted and propagated in this reaction, indicating that HaPrPC expressed in CHO cells is also a competent substrate for in vitro conversion into autocatalytic PrP (Figure 1C, top blot). In a separate reaction we carried out a seeded 15-round sPMCA propagation reaction with CHO-expressed wild type HaPrPC substrate in order to generate a sample containing autocatalytic HaPrPres, which was beyond limiting dilution of the original scrapie template. To determine whether this HaPrPres was infectious, the sample from round 15 of the propagation reaction was intracerebrally inoculated into wild type hamsters. All of the inoculated hamsters (6/6) developed clinical scrapie with a mean incubation time of 141±17 days, whereas hamsters inoculated with a buffer control remain healthy to date (0/5) >380 days post-inoculation. The brains of hamsters inoculated with CHO-derived PrPSc displayed spongiform degeneration and PrP deposition consistent with the diagnosis of scrapie (Figure 2). The vacuolation and PrP deposition profiles of the hamsters inoculated with Sc237-seeded, CHO-derived PrPSc were very similar to hamsters inoculated with Sc237-seeded brain-derived PrPSc (Figure S2A and B).


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)

Neuropathology of hamsters infected with prions derived from CHO-expressed PrP substrate.Representative histological fields of the brainstem region in control animals and animals inoculated with CHO-expressed in vitro-generated PrPSc molecules. Top row: normal 166 day old Syrian golden hamster, mock inoculated with day 15 sPMCA propagation reaction containing the cobalt-purified fraction from untransfected CHO cells. Bottom row: terminally ill hamster inoculated with Sc237-seeded day 15 sPMCA reaction containing cobalt-prepared PrPC expressed from CHO cells. Both reactions were seeded on day 0 with 0.01% Sc237-infected hamster brain homogenate. Hemotoxylin and eosin (H&E) and PrP immunohistochemical (3F4 antibody) staining are shown for each group. (Scale bar, 50 µm.)
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1000535-g002: Neuropathology of hamsters infected with prions derived from CHO-expressed PrP substrate.Representative histological fields of the brainstem region in control animals and animals inoculated with CHO-expressed in vitro-generated PrPSc molecules. Top row: normal 166 day old Syrian golden hamster, mock inoculated with day 15 sPMCA propagation reaction containing the cobalt-purified fraction from untransfected CHO cells. Bottom row: terminally ill hamster inoculated with Sc237-seeded day 15 sPMCA reaction containing cobalt-prepared PrPC expressed from CHO cells. Both reactions were seeded on day 0 with 0.01% Sc237-infected hamster brain homogenate. Hemotoxylin and eosin (H&E) and PrP immunohistochemical (3F4 antibody) staining are shown for each group. (Scale bar, 50 µm.)
Mentions: We also conducted three-rounds of serial propagation reactions using the CHO-expressed hamster PrPC substrate seeded with crude Sc237 hamster brain homogenate and supplemented with PrP0/0 brain homogenate (Figure 1C). The results show that HaPrPC was efficiently converted and propagated in this reaction, indicating that HaPrPC expressed in CHO cells is also a competent substrate for in vitro conversion into autocatalytic PrP (Figure 1C, top blot). In a separate reaction we carried out a seeded 15-round sPMCA propagation reaction with CHO-expressed wild type HaPrPC substrate in order to generate a sample containing autocatalytic HaPrPres, which was beyond limiting dilution of the original scrapie template. To determine whether this HaPrPres was infectious, the sample from round 15 of the propagation reaction was intracerebrally inoculated into wild type hamsters. All of the inoculated hamsters (6/6) developed clinical scrapie with a mean incubation time of 141±17 days, whereas hamsters inoculated with a buffer control remain healthy to date (0/5) >380 days post-inoculation. The brains of hamsters inoculated with CHO-derived PrPSc displayed spongiform degeneration and PrP deposition consistent with the diagnosis of scrapie (Figure 2). The vacuolation and PrP deposition profiles of the hamsters inoculated with Sc237-seeded, CHO-derived PrPSc were very similar to hamsters inoculated with Sc237-seeded brain-derived PrPSc (Figure S2A and B).

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