NR AORZ
AU Drisaldi,B.; Coomaraswamy,J.; Mastrangelo,P.; Strome,R.; Yang,J.; Mount,H.T.J.; Westaway,D.
TI Genetic Mapping of Active Sites for Dpl/PrPc Interactions in a Cell Culture Assay
QU International Conference - Prion diseases: from basic research to intervention concepts - TSE-Forum, 08.10.-10.10.2003, Gasteig, München - Poster session - BR-118
PT Konferenz-Poster
AB
Doppel (Dpl) is a prion-like protein encoded by Prnd gene, displaying ~24% identity with PrPc. Dpl and PrPc are both GPI-anchored proteins, bear similar alpha-helical C-terminal domain structures and share an ability to bind copper ions in a selective manner in vitro.
Dpl expression in the CNS causes death of cerebellar neurons, determining a severe ataxic phenotype in all Dpl-expressing transgenic mice. Of note, Dpl's toxic effect upon cerebellar neurons is antagonized by wt PrPc. Based upon the established structural similarities between Dpl and PrPc, it has been suggested that analysis of Dpl induced neurodegeneration might mechanistic provide insights into both the cellular actions of prion proteins and the pathogenesis of genetic prion diseases.
After establishing a cell culture assay for Dpl/PrPc antagonism based upon transfection of cerebellar granule cells and measurement of apoptosis, we have mapped active sites in these two proteins.
Since PrP N-terminal deletion mutants are toxic when expressed in mice brain (PrPD32-121, PrPD32-136) and toxic Dpl resembles an N-terminal truncated version of PrP, we hypothesized that PrPs' neuroprotective region of PrP lay within the octarepeat sequences. PrP alleles lacking the octarepeat region (PrPD51-90) failed to rescue Dpl-induced toxicity unlike wtPrP alleles.
Moreover, Dpl deletion mutants were created to map Dpl toxic domain. The results of these assays indicated that toxicity mapped to the C-terminal region of the protein to sequences lying between codons 91 and 125, encompassing the kinked alphaB/B' helix region and partially overlapping a Cu binding region.
Remarkably, many Dpl alleles with mutations in residues required for posttranslational modification and mature folding retained full activity in this assay, arguing that Dpl-mediated neurotoxicity is not dependent on presenting GPI-anchored, N-glycosylated and natively folded Doppel on the cell-surface.
AD Bettina Drisaldi, Janaky Coomaraswamy, Peter Mastrangelo, Robert Strome, Jing Yang, Howard T.J. Mount, David Westaway, University of Toronto, Canada
SP englisch
PO Deutschland