NR AWQJ
AU Westergard,L.; Li,A.; Harris,D.A.
TI Using yeast to investigate the cytoprotective activity of PrP
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 CE-51
PT Konferenz-Poster
AB Numerous investigations implicate PrPc in neuroprotection from toxic or stressful insults. In cultured human neurons and mammalian cell lines, PrPc abrogates apoptosis induced by Bax, a pro-apoptotic member of the Bcl-2 family. Our results demonstrate that when PrPc is targeted to the secretory pathway of Saccharomyces cerevisiae by the addition of a modified signal peptide, it suppresses Bax-induced cell death. Deletion of the octapeptide repeat region of PrP did not affect Bax rescuing activity, indicating that copper binding to this region is not essential for rescue. However, deletion of a charged region (residues 23-31) encompassing an endocytic targeting motif partially eliminated activity, suggesting that PrP internalization may be required. Cytosolic PrP (23-231) failed to suppress Bax-induced death, indicating that protective activity requires PrP targeting to the secretory pathway. To track Bax localization we constructed an N-terminally GFP-tagged Bax protein. Preliminary results indicate that PrP co-expression enhances survival of GFP-Bax expressing yeast. In the majority of surviving cells, GFP-Bax translocates to mitochondria but the cells do not die. In control experiments, co-expression of GFP-Bax with Bcl-2 results in GFP-Bax localization to both the cytoplasm and mitochondria. These results imply that PrP inhibits Bax activity after translocation to mitochondria, perhaps by preventing oligomerization and formation of cytochrome c-releasing pores. Our research goal is to utilize yeast as a model system to investigate PrP mediated protection from Bax-induced cell death by dissecting the rescue mechanism, identifying other gene products involved, and determining their role in this pathway. The yeast system is advantageous because it offers the possibility of performing genetic screens to identify proteins of interest. Consistency between PrP function in the yeast and mammalian systems will allow us to extrapolate the information learned C about the protective function of PrP in yeast to the more physiologically relevant mammalian systems.
AD Department of Cell Biology and Physiology, Washington University School of Medicine St. Louis, Missouri, USA
SP englisch
PO Italien