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.
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.
Shi
Xiao-Ping
XP
Department of Biological Chemistry, Merck Research Laboratories, West Point, PA 19486, USA. xiao-ping_shi@merck.com
Tugusheva
Katherine
K
Bruce
James E
JE
Lucka
Adam
A
Chen-Dodson
Elizabeth
E
Hu
Binghua
B
Wu
Guo-Xin
GX
Price
Eric
E
Register
Robert B
RB
Lineberger
Janet
J
Miller
Ron
R
Tang
Mei-Jy
MJ
Espeseth
Amy
A
Kahana
Jason
J
Wolfe
Abigail
A
Crouthamel
Ming-Chih
MC
Sankaranarayanan
Sethu
S
Simon
Adam
A
Chen
Lin
L
Lai
Ming-Tain
MT
Pietrak
Beth
B
DiMuzio
Jillian
J
Li
Yueming
Y
Xu
Min
M
Huang
Qian
Q
Garsky
Victor
V
Sardana
Mohinder K
MK
Hazuda
Daria J
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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
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