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Phenotypic Detection of Mouse PRP Aggregation in Yeast


Prions are protein isoforms that propagate themselves by converting the normal protein, encoded by the same maintenance gene, into a prion form. The aggregation-prone prion isoform of PrP protein is implicated in fatal and incurabe infectious neurodegenerative diseases in mammals and humans. Some of these diseases are of huge importance, for example 'mad cow' disease (transmissible to humans) which caused significant damage in Europe and was recently detected in US. <P> Animal model systems for prion diseases are expensive and laborious, making large-scale in vivo screening of antiprion agents essentially impossible. In yeast, mammalian PrP can form aggregates that acquire some biochemical characteristics of a prion. This suggests that yeast could be used as a model to study PrP aggregation.<P> Yeast translation termination factor Sup35 was used to develop a phenotypic assay for PrP aggregation in yeast. The N-proximal domain of Sup35 possesses prion properties. This domain was substituted with a mouse sequence, coding for the portion of PrP. In yeast, transient overproduction of such a chimeric protein, named PrP-Sup35MC, induced partial loss of function of the Sup35, leading to the translation termination defect (easily detectable in the specially designed strains) and accompanied by aggregation. Partially non-functional state of PrP-Sup35MC is maintained in mitotic divisions, resembling some patterns of the endogenous prion isoform of full-size Sup35. <P> It is hypothesized that PrP-Sup35MC aggregates are generated due to conversion of the PrP region into a prion state. This hypothesis will be tested. In addition, as aggregated PrP-Sup35MC certainly resembles at least some patterns of a prion, it will be used as a powerful tool for large-scale screening of the proteins, peptides and chemicals counteracting prion aggregation. <P> This research will complement one of the aims of the parental NIH grant, dealing with identification of the mammalian chaperones that influence prions, and may also lead to identification of the new potential anti-prion treatments.

Chernoff, Yury
Georgia Institute of Technology
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