Trans-dominant inhibition of prion propagation in vitro is not mediated by an accessory cofactor.
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 recomb...
| Main Authors: | , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Public Library of Science (PLoS)
2009-07-01
|
| Series: | PLoS Pathogens |
| Online Access: | http://europepmc.org/articles/PMC2713408?pdf=render |
| _version_ | 1818956326283247616 |
|---|---|
| author | James C Geoghegan Michael B Miller Aimee H Kwak Brent T Harris Surachai Supattapone |
| author_facet | James C Geoghegan Michael B Miller Aimee H Kwak Brent T Harris Surachai Supattapone |
| author_sort | James C Geoghegan |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-12-20T10:52:11Z |
| format | Article |
| id | doaj.art-d1cfab3da5f3407091079433b69ee043 |
| institution | Directory Open Access Journal |
| issn | 1553-7366 1553-7374 |
| language | English |
| last_indexed | 2024-12-20T10:52:11Z |
| publishDate | 2009-07-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS Pathogens |
| spelling | doaj.art-d1cfab3da5f3407091079433b69ee0432022-12-21T19:43:14ZengPublic Library of Science (PLoS)PLoS Pathogens1553-73661553-73742009-07-0157e100053510.1371/journal.ppat.1000535Trans-dominant inhibition of prion propagation in vitro is not mediated by an accessory cofactor.James C GeogheganMichael B MillerAimee H KwakBrent T HarrisSurachai SupattaponePrevious 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.http://europepmc.org/articles/PMC2713408?pdf=render |
| spellingShingle | James C Geoghegan Michael B Miller Aimee H Kwak Brent T Harris Surachai Supattapone Trans-dominant inhibition of prion propagation in vitro is not mediated by an accessory cofactor. PLoS Pathogens |
| title | Trans-dominant inhibition of prion propagation in vitro is not mediated by an accessory cofactor. |
| title_full | Trans-dominant inhibition of prion propagation in vitro is not mediated by an accessory cofactor. |
| title_fullStr | Trans-dominant inhibition of prion propagation in vitro is not mediated by an accessory cofactor. |
| title_full_unstemmed | Trans-dominant inhibition of prion propagation in vitro is not mediated by an accessory cofactor. |
| title_short | Trans-dominant inhibition of prion propagation in vitro is not mediated by an accessory cofactor. |
| title_sort | trans dominant inhibition of prion propagation in vitro is not mediated by an accessory cofactor |
| url | http://europepmc.org/articles/PMC2713408?pdf=render |
| work_keys_str_mv | AT jamescgeoghegan transdominantinhibitionofprionpropagationinvitroisnotmediatedbyanaccessorycofactor AT michaelbmiller transdominantinhibitionofprionpropagationinvitroisnotmediatedbyanaccessorycofactor AT aimeehkwak transdominantinhibitionofprionpropagationinvitroisnotmediatedbyanaccessorycofactor AT brenttharris transdominantinhibitionofprionpropagationinvitroisnotmediatedbyanaccessorycofactor AT surachaisupattapone transdominantinhibitionofprionpropagationinvitroisnotmediatedbyanaccessorycofactor |