Trans-Dominant Inhibition of Prion Propagation In Vitro Is Not Mediated by an Accessory Cofactor

PLoS Pathogens

Michael B. Miller¹  Aimee H. Kwak¹  James C. Geoghegan¹  Surachai Supattapone¹  Brent T. Harris² 

[1] Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire, United States of America;Department of Pathology, Dartmouth Medical School, Hanover, New Hampshire, United States of America

关键词: CHO cells;    Hamsters;    Scrapie;    Animal prion diseases;    Cofactors (biochemistry);    Prion diseases;    Proteases;    Mouse models;   

DOI  :  10.1371/journal.ppat.1000535

学科分类：生物科学（综合）

来源: Public Library of Science

【 摘 要 】

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 PrPC 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) PrPC is pre-incubated with poly(A) RNA and PrPSc template, and (3) Q172R is the only hamster PrP mutant tested that fails to convert into PrPSc 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 PrPSc molecules, most likely through an epitope containing residue 172.

【 授权许可】

CC BY