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

• Published in: Journal of Alzheimer's disease Vol. 7; no. 2; p. 139
• Main Authors: Shi, Xiao-Ping, Tugusheva, Katherine, Bruce, James E, Lucka, Adam, Chen-Dodson, Elizabeth, Hu, Binghua, Wu, Guo-Xin, Price, Eric, Register, Robert B, Lineberger, Janet, Miller, Ron, Tang, Mei-Jy, Espeseth, Amy, Kahana, Jason, Wolfe, Abigail, Crouthamel, Ming-Chih, Sankaranarayanan, Sethu, Simon, Adam, Chen, Lin, Lai, Ming-Tain, Pietrak, Beth, DiMuzio, Jillian, Li, Yueming, Xu, Min, Huang, Qian, Garsky, Victor, Sardana, Mohinder K, Hazuda, Daria J
• Format: Journal Article
• Language: English
• Published: Netherlands 01-04-2005
• Subjects: Alzheimer Disease - enzymology; Alzheimer Disease - genetics; Alzheimer Disease - immunology; Amyloid beta-Peptides - antagonists & inhibitors; Amyloid beta-Peptides - biosynthesis; Amyloid beta-Peptides - genetics; Amyloid beta-Protein Precursor - genetics; Amyloid Precursor Protein Secretases; Animals; Antibodies, Monoclonal - immunology; Aspartic Acid Endopeptidases - genetics; Aspartic Acid Endopeptidases - metabolism; Disease Models, Animal; Endopeptidases; Enzyme Activation - physiology; Fibroblasts - metabolism; Gene Expression Regulation, Enzymologic; In Vitro Techniques; Mice; Molecular Sequence Data; Peptide Fragments - antagonists & inhibitors; Peptide Fragments - biosynthesis; Peptide Fragments - genetics; Point Mutation - genetics; Substrate Specificity; Transfection
• ISSN: 1387-2877
• DOI: 10.3233/JAD-2005-7207

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.