NR AWFD
AU Galkin,A.P.; Rubel,A.A.; Saifitdinova,A.F.; Lada,A.G.; Petrova,I.T.; Inge-Vechtomov,S.G.; Chernoff,Y.O.
TI Phenotypic detection of the prion-like alterations of mammalian PrP protein in the yeast model
QU International Conference - Prion 2006: Strategies, advances and trends towards protection of society - 3.10.-6.10.2006, Torino, Italy, Lingotto Conference Centre - Poster sessions GEN-10
PT Konferenz-Poster
AB Mammalian prion diseases associated with formation of the abnormal aggregated isoform of Prion Protein (PrPsc) are incurable and fatal. Factors regulating prion propagation remain unknown to date. We have generated a yeast-based system for screening the agents involved in regulation of PrP conformational switches. Our system is based on the properties of yeast prion protein Sup35, which is a translation termination factor. Yeast cells containing a prion form of Sup35 exhibit nonsense suppression, detectable in the strains with reporter nonsense mutations as a growth on selective media. Prion properties of Sup35 are exclusively determined by the N-terminal prion domain. We have substituted the prion domain of Sup35 with a mouse sequence coding for the 90-231aa fragment of PrP. Yeast strain bearing a plasmid with PrnP-SUP35MC gene under the control of copper-inducible promoter, and containing a deletion of the chromosomal copy of SUP35 has been generated. Chimeric PrP-Sup35MC protein compensates for the termination function of Sup35 in our system. However, derivatives exhibiting nonsense suppression have been selected. We have shown that nonsense suppression is a result of partial inactivation of the PrP-Sup35MC protein. The S+ heritable, functionally defective state of PrP-Sup35MC designated as [PrPs+] possesses all characteristics of a yeast prion, such as reversible curability by GuHCl, non-Mendelian inheritance and cytoplasmic infectivity. PrP-Sup35MCp in prion isoform is more resistant to proteinase K digestion, compared to the same protein in a non-prion form. This provides the first example of the PrP-based prion formation in the organism other than mammals. Our phenotypic assay could be employed to screen for the proteins and agents affecting PrP prionization. Supported by grants: Fogarty TW006965-01A1, NIH R01GM58763, RFBR 49002, CRDF ST-012.
AD A.P. Galkin, A.A. Rubel, A.F. Saifitdinova, A.G. Lada, I.T. Petrova, S.G. Inge-Vechtomov: Dept. of Genetics, St.-Petersburg State University, St.-Petersburg, Russia; Y.O. Chernoff: School of Biology and Institute of Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, USA. E-mail: apgalkin@yahoo.com
SP englisch
PO Italien