NR AROZ
AU Drisaldi,B.; Coomaraswamy,J.; Mastrangelo,P.; Strome,B.; Yang,J.; Watts,J.C.; Chishti,M.A.; Marvi,M.; Windl,O.; Ahrens,R.; Major,F.; Sy,M.S.; Kretzschmar,H.A.; Fraser,P.E.; Mount,H.T.J.; Westaway,D.
TI Genetic mapping of activity determinants within cellular prion proteins: N-terminal modules in PrPc offset pro-apoptotic activity of the Doppel helix B/B' region.
QU The Journal of Biological Chemistry 2004 Dec 31; 279(53): 55443-54
PT journal article
AB The PrP-like Doppel (Dpl) protein causes apoptotic death of cerebellar neurons in transgenic mice, a process prevented by expression of the wild type (wt) cellular prion protein, PrPc. Internally deleted forms of PrPc resembling Dpl such as PrPDelta32-121 produce a similar PrPc-sensitive pro-apoptotic phenotype in transgenic mice. Here we demonstrate that these phenotypic attributes of wt Dpl, wt PrPc, and PrPDelta132-121 can be accurately recapitulated by transfected mouse cerebellar granule cell cultures. This system was then explored by mutagenesis of the co-expressed prion proteins to reveal functional determinants. By this means, neuroprotective activity of wt PrPc was shown to be nullified by a deletion of the N-terminal charged region implicated in endocytosis and retrograde axonal transport (PrPDelta23-28), by deletion of all five octarepeats (PrPDelta51-90), or by glycine replacement of four octarepeat histidine residues required for selective binding of copper ions (Prnp"H/G"). In the case of Dpl, overlapping deletions defined a requirement for the gene interval encoding helices B and B' (DplDelta101-125). These data suggest contributions of copper binding and neuronal trafficking to wt PrPc function in vivo and place constraints upon current hypotheses to explain Dpl/PrPc antagonism by competitive ligand binding. Further implementation of this assay should provide a fuller understanding of the attributes and subcellular localizations required for activity of these enigmatic proteins.
MH Alleles; Animals; *Apoptosis; Cell Death; Cell Line, Tumor; Cerebellum/metabolism; Chromosome Mapping; Copper; DNA Mutational Analysis; Endocytosis; Gene Deletion; Glycine/chemistry; Green Fluorescent Proteins/metabolism; Ions; Mice; Mice, Transgenic; Microscopy, Fluorescence; Models, Genetic; Mutation; Neurons/metabolism/pathology; Phenotype; Plasmids/metabolism; Point Mutation; PrPc Proteins/chemistry/*genetics/*physiology; Prions/chemistry/*genetics/*physiology; Protein Binding; Protein Structure, Tertiary; Research Support, Non-U.S. Gov't; Transfection; Transgenes
AD Centre for Research in Neurodegenerative Diseases, Tanz Neuroscience Building, 6 Queen's Park Crescent West, University of Toronto, Toronto, Ontario M5S 3H2, Canada.
SP englisch
PO USA