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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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Related in: MedlinePlus

Model of trans-dominant inhibition of prion formation.This diagram illustrates the proposed model of competition between various PrP substrate molecules for binding to a nascent seeding site on newly formed PrPSc molecules. Legend: Sc = PrPSc; WT = wild type PrPC; mutant PrP molecules labeled according to residue numbering for mouse PrP. The relative affinities of various PrP molecules for the nascent seeding site on the growing PrPSc polymer are indicated schematically by the length and curvature of the cartoon protrusion, which is meant to represent the NSS binding domain, and which likely includes the residue 170–174 loop domain.
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ppat-1000535-g007: Model of trans-dominant inhibition of prion formation.This diagram illustrates the proposed model of competition between various PrP substrate molecules for binding to a nascent seeding site on newly formed PrPSc molecules. Legend: Sc = PrPSc; WT = wild type PrPC; mutant PrP molecules labeled according to residue numbering for mouse PrP. The relative affinities of various PrP molecules for the nascent seeding site on the growing PrPSc polymer are indicated schematically by the length and curvature of the cartoon protrusion, which is meant to represent the NSS binding domain, and which likely includes the residue 170–174 loop domain.

Mentions: Collectively, the data presented in this manuscript refute the hypothesis that protein X or any other accessory cofactor is required for dominant negative inhibition of prion formation. Moreover, the ability of the Q172R mutant to inhibit hamster prion formation in a purified system even when WT HaPrP substrate was pre-incubated with both poly(A) RNA and scrapie template suggests that mutant PrP inhibits the conversion of WT PrP by blocking a nascent seeding site (NSS) on newly formed, PMCA-generated PrPSc molecules (Figure 7). Our data also show complete dissociation between conversion ability and trans-dominant inhibitory activity. Most notably, we showed that Q219K HaPrP is able both to convert into PrPres itself and to inhibit the conversion of WT PrP in trans. Thus, we can conclude that the mechanism of trans-dominant inhibition does not require a “negative” mutation, i.e. a PrP molecule that is unable to convert into PrPres itself.


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)

Model of trans-dominant inhibition of prion formation.This diagram illustrates the proposed model of competition between various PrP substrate molecules for binding to a nascent seeding site on newly formed PrPSc molecules. Legend: Sc = PrPSc; WT = wild type PrPC; mutant PrP molecules labeled according to residue numbering for mouse PrP. The relative affinities of various PrP molecules for the nascent seeding site on the growing PrPSc polymer are indicated schematically by the length and curvature of the cartoon protrusion, which is meant to represent the NSS binding domain, and which likely includes the residue 170–174 loop domain.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1000535-g007: Model of trans-dominant inhibition of prion formation.This diagram illustrates the proposed model of competition between various PrP substrate molecules for binding to a nascent seeding site on newly formed PrPSc molecules. Legend: Sc = PrPSc; WT = wild type PrPC; mutant PrP molecules labeled according to residue numbering for mouse PrP. The relative affinities of various PrP molecules for the nascent seeding site on the growing PrPSc polymer are indicated schematically by the length and curvature of the cartoon protrusion, which is meant to represent the NSS binding domain, and which likely includes the residue 170–174 loop domain.
Mentions: Collectively, the data presented in this manuscript refute the hypothesis that protein X or any other accessory cofactor is required for dominant negative inhibition of prion formation. Moreover, the ability of the Q172R mutant to inhibit hamster prion formation in a purified system even when WT HaPrP substrate was pre-incubated with both poly(A) RNA and scrapie template suggests that mutant PrP inhibits the conversion of WT PrP by blocking a nascent seeding site (NSS) on newly formed, PMCA-generated PrPSc molecules (Figure 7). Our data also show complete dissociation between conversion ability and trans-dominant inhibitory activity. Most notably, we showed that Q219K HaPrP is able both to convert into PrPres itself and to inhibit the conversion of WT PrP in trans. Thus, we can conclude that the mechanism of trans-dominant inhibition does not require a “negative” mutation, i.e. a PrP molecule that is unable to convert into PrPres itself.

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