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.
Article Type:
Research Article
Abstract:
Abnormal production and accumulation of amyloid-β peptide (Aβ) plays a major role in the pathogenesis of Alzheimer's disease (AD). β-secretase (BACE1) is responsible for the cleavage at the β-site in amyloid β protein precursor (AβPP/APP) to generate the N-terminus of Aβ. Here we report the stepwise identification and characterization of a novel APP-β-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 β-site mutations tested. More importantly,
…the APP_NFEV mutant failed to generate any detectable Aβ peptides in BACE1-KO mouse fibroblast cells. The production of Aβ 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 β-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.
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Keywords: Alzheimer's disease, BACE, β-secretases, APP
DOI: 10.3233/JAD-2005-7207
Citation: Journal of Alzheimer's Disease,
vol. 7, no. 2, pp. 139-148, 2005
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