NR ARVW
AU Silverman,G.L.; Yao,H.; Strome,R.; Laplanche,J.L.; Foncin,J.F.; George-Hyslop,P.St; Fraser,P.E.; Westaway,D.
TI Proteins which interact with wild-type and mutant N-terminal domains of human PrPc
QU Journal of Neuropathology and Experimental Neurology 1997; 56(N5): 596 Nr. 99
PT Meeting Abstract
VT Additional octarepeat motifs in the conserved N-terminus of the prion protein (PrP) cause inherited diseases classified as CJD, GSS, or "variant prion disease". Mechanisms underlying such diseases are obscure but it has been suggested that the wild-type (wt) version of this domain binds to itself, to other proteins, and/or to Cu2+. The yeast two-hybrid system was used to test these hypotheses. A Gal4 DNA binding domain (BD):PrP23-98 fusion protein was created using a wt PrP cDNA, mutated to include a TGA stop codon at position 99. An analogous construct was created from a "GSS" PRNP allele with 8 additional octarepeats. wt and GSS Gal4BD:PrP fusion proteins of the predicted mass were expressed at similar levels in transfected yeast cells. Though Gal4BD:wtPrP23-98 transformants grew poorly, a total of 800,000 human brain cDNAs cloned in a Gal4 activation domain (AD) vector were screened as cotransformants with this "bait". Plasmids in about 80% of the recovered His+ clones encoded proteins which interacted with the Gal4 portion of the bait plasmid. 7 remaining clones interacted with the PrP region of the bait, and also with Gal4BD:mouse PrP23-98 bait. Three clones encoded novel cDNAs but interacted weakly with Gal4BD:PrP23-98 (as did a control Gal4AD:PrP23-98 plasmid). A plasmid encoding int-6, a nuclear protein, was retrieved twice. The two remaining clones interacted in a robust manner: clone 44 encoding a novel proline-rich protein, and clone 47 encoding a novel protein partially homologous to betaine-homocysteine methyltransferase (BHMT). The protein encoded by the Gal4BD:GSS PrP bait plasmid interacted more weakly with 6(7 clones retrieved by the wtPrP23-98 bait plasmid, but more strongly with clone 47 (lacZ assay comparable to a p53 + T-antigen control). These findings (i) do not strongly support a role for PrP's N-terminus in multimerization, (ii) establish that proteins can interact with PrP23-98 (which is predicted to have an extended random-coil conformation). and (iji) reveal that wt and GSS mutant PrP bait proteins behave differently. While it is unclear whether methyltransferases interact with PrPc in vivo, our data suggest that polypeptides which bind to PrPc's hydrophilic, multivalent N-terminus may be useful reagents for detecting prion proteins and altering their cellular trafficking.
ZR 0
SP englisch
AD University of Toronto, Ontario, and Hôpital St. Lazare, Hôpital de Ia Salpêtrière, Paris, France. (Sponsored by C. Bergeron*)
OR Prion-Krankheiten 7