title: Novel mutations introduced at the beta-site of amyloid beta protein precursor enhance the production of amyloid beta peptide by BACE1 in vitro and in cells.
abstract: Abnormal production and accumulation of amyloid-beta peptide (Abeta) plays a major role in the pathogenesis of Alzheimer's disease (AD). beta-secretase (BACE1) is responsible for the cleavage at thebeta-site in amyloid beta protein precursor (AbetaPP/APP) to generate the N-terminus of Abeta. Here we report the stepwise identification and characterization of a novel APP-beta-site mutant, "NFEV" (APP_NFEV) in vitro and in cells. In vitro, the APP_NFEV exhibits 100-fold enhanced cleavage rate relative to the "wild-type" substrate (APPwt) and 10-fold increase relative to the Swedish-type mutation variant (APPsw). In cells, it was preferably cleaved among 24 APP beta-site mutations tested. More importantly, the APP_NFEV mutant failed to generate any detectable Abeta peptides in BACE1-KO mouse fibroblast cells. The production of Abeta peptides was restored by co-transfecting human BACE1, demonstrating that BACE1 is the only enzyme responsible for the processing of APP_NFEV in these cells. Analysis of APP_NFEV cleavage products secreted in the media revealed that in cells BACE1 cleaves APP_NFEV at the position between NF and EV, identical to that observed in vitro. A BACE inhibitor blocked the processing of the APP_NFEV beta-site in vitro and in cells. Our data indicates that the "NFEV" mutant is not only an enhanced substrate for BACE1 in vitro, but also a specific substrate for BACE1 in cells.
authors.affiliation: Department of Biological Chemistry, Merck Research Laboratories, West Point, PA 19486, USA. xiao-ping_shi@merck.com
authors.forename: Xiao-Ping
authors.initials: XP
authors.lastname: Shi
authors.forename: Katherine
authors.initials: K
authors.lastname: Tugusheva
authors.forename: James E
authors.initials: JE
authors.lastname: Bruce
authors.forename: Adam
authors.initials: A
authors.lastname: Lucka
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Chen-Dodson
authors.forename: Binghua
authors.initials: B
authors.lastname: Hu
authors.forename: Guo-Xin
authors.initials: GX
authors.lastname: Wu
authors.forename: Eric
authors.initials: E
authors.lastname: Price
authors.forename: Robert B
authors.initials: RB
authors.lastname: Register
authors.forename: Janet
authors.initials: J
authors.lastname: Lineberger
authors.forename: Ron
authors.initials: R
authors.lastname: Miller
authors.forename: Mei-Jy
authors.initials: MJ
authors.lastname: Tang
authors.forename: Amy
authors.initials: A
authors.lastname: Espeseth
authors.forename: Jason
authors.initials: J
authors.lastname: Kahana
authors.forename: Abigail
authors.initials: A
authors.lastname: Wolfe
authors.forename: Ming-Chih
authors.initials: MC
authors.lastname: Crouthamel
authors.forename: Sethu
authors.initials: S
authors.lastname: Sankaranarayanan
authors.forename: Adam
authors.initials: A
authors.lastname: Simon
authors.forename: Lin
authors.initials: L
authors.lastname: Chen
authors.forename: Ming-Tain
authors.initials: MT
authors.lastname: Lai
authors.forename: Beth
authors.initials: B
authors.lastname: Pietrak
authors.forename: Jillian
authors.initials: J
authors.lastname: DiMuzio
authors.forename: Yueming
authors.initials: Y
authors.lastname: Li
authors.forename: Min
authors.initials: M
authors.lastname: Xu
authors.forename: Qian
authors.initials: Q
authors.lastname: Huang
authors.forename: Victor
authors.initials: V
authors.lastname: Garsky
authors.forename: Mohinder K
authors.initials: MK
authors.lastname: Sardana
authors.forename: Daria J
authors.initials: DJ
authors.lastname: Hazuda
mesh_terms: D000544:Alzheimer Disease / Q000201:enzymology / Q000235:genetics / Q000276:immunology; D053829:Amyloid Precursor Protein Secretases; D016229:Amyloid beta-Peptides / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics; D016564:Amyloid beta-Protein Precursor / Q000235:genetics*; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D016282:Aspartic Acid Endopeptidases / Q000235:genetics* / Q000378:metabolism; D004195:Disease Models, Animal; D010450:Endopeptidases; D004789:Enzyme Activation / Q000502:physiology; D005347:Fibroblasts / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D066298:In Vitro Techniques; D051379:Mice; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics; D017354:Point Mutation / Q000235:genetics*; D013379:Substrate Specificity; D014162:Transfection
chemical_list: D016229:Amyloid beta-Peptides; D016564:Amyloid beta-Protein Precursor; D000911:Antibodies, Monoclonal; D010446:Peptide Fragments; C500436:amyloid beta-peptide (5-40); D053829:Amyloid Precursor Protein Secretases; D010450:Endopeptidases; D016282:Aspartic Acid Endopeptidases; C509284:BACE2 protein, human; C509280:BACE1 protein, human; C509282:Bace1 protein, mouse