title: Random mutagenesis of presenilin-1 identifies novel mutants exclusively generating long amyloid beta-peptides.
abstract: Familial Alzheimer disease-causing mutations in the presenilins increase production of longer pathogenic amyloid beta-peptides (A beta(42/43)) by altering gamma-secretase activity. The mechanism underlying this effect remains unknown, although it has been proposed that heteromeric macromolecular complexes containing presenilins mediate gamma-secretase cleavage of the amyloid beta-precursor protein. Using a random mutagenesis screen of presenilin-1 (PS1) for PS1 endoproteolysis-impairing mutations, we identified five unique mutants, including R278I-PS1 and L435H-PS1, that exclusively generated a high level of A beta43, but did not support physiological PS1 endoproteolysis or A beta40 generation. These mutants did not measurably alter the molecular size or subcellular localization of PS1 complexes. Pharmacological studies indicated that the up-regulation of activity for A beta43 generation by these mutations was not further enhanced by the difluoroketone inhibitor DFK167 and was refractory to inhibition by sulindac sulfide. These results suggest that PS1 mutations can lead to a wide spectrum of changes in the activity and specificity of gamma-secretase and that the effects of PS1 mutations and gamma-secretase inhibitors on the specificity are mediated through a common mechanism.
authors.affiliation: Molecular Neuroscience Research Center, Shiga University of Medical Science, Otsu, Shiga 520-2192, Japan.
authors.forename: Yoshifumi
authors.initials: Y
authors.lastname: Nakaya
authors.forename: Takuya
authors.initials: T
authors.lastname: Yamane
authors.forename: Hirohisa
authors.initials: H
authors.lastname: Shiraishi
authors.forename: Hua-Qin
authors.initials: HQ
authors.lastname: Wang
authors.forename: Etsuro
authors.initials: E
authors.lastname: Matsubara
authors.forename: Toru
authors.initials: T
authors.lastname: Sato
authors.forename: Georgia
authors.initials: G
authors.lastname: Dolios
authors.forename: Rong
authors.initials: R
authors.lastname: Wang
authors.forename: Bart
authors.initials: B
authors.lastname: De Strooper
authors.forename: Mikio
authors.initials: M
authors.lastname: Shoji
authors.forename: Hiroto
authors.initials: H
authors.lastname: Komano
authors.forename: Katsuhiko
authors.initials: K
authors.lastname: Yanagisawa
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Ihara
authors.forename: Paul
authors.initials: P
authors.lastname: Fraser
authors.forename: Peter
authors.initials: P
authors.lastname: St George-Hyslop
authors.forename: Masaki
authors.initials: M
authors.lastname: Nishimura
mesh_terms: D000495:Allosteric Site; D016229:Amyloid beta-Peptides / Q000737:chemistry*; D016564:Amyloid beta-Protein Precursor / Q000737:chemistry; D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002499:Centrifugation, Density Gradient; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay; D005347:Fibroblasts / Q000378:metabolism; D005990:Glycerol / Q000494:pharmacology; D006801:Humans; D015151:Immunoblotting; D007150:Immunohistochemistry; D047468:Immunoprecipitation; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism / Q000502:physiology*; D051379:Mice; D016296:Mutagenesis; D009154:Mutation; D020125:Mutation, Missense; D053764:Presenilin-1; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry; D051880:Receptors, Notch; D012190:Retroviridae / Q000235:genetics; D013347:Subcellular Fractions / Q000378:metabolism; D013467:Sulindac / Q000031:analogs & derivatives* / Q000494:pharmacology; D014162:Transfection; D015854:Up-Regulation
chemical_list: D016229:Amyloid beta-Peptides; D016564:Amyloid beta-Protein Precursor; D018076:DNA, Complementary; D008565:Membrane Proteins; C508853:PSEN1 protein, human; D053764:Presenilin-1; D020033:Protein Isoforms; D011506:Proteins; D051880:Receptors, Notch; D013467:Sulindac; C025462:sulindac sulfide; D005990:Glycerol