Novel mutations introduced at the β-site of amyloid β protein precursor enhance the production of amyloid β peptide by BACE1 in vitro and in cells

Article type: Research Article

Authors: Shi, Xiao-Ping^{a; *} | Tugusheva, Katherine^a | Bruce, James E.^b | Lucka, Adam^a | Chen-Dodson, Elizabeth^a | Hu, Binghua^a | Wu, Guo-Xin^a | Price, Eric^a | Register, Robert B.^a | Lineberger, Janet^a | Miller, Ron^a | Tang, Mei-Jy^a | Espeseth, Amy^a | Kahana, Jason^a | Wolfe, Abigail^a | Crouthamel, Ming-Chih^a | Sankaranarayanan, Sethu^a | Simon, Adam^a | Chen, Lin^a | Lai, Ming-Tain^a | Pietrak, Beth^a | DiMuzio, Jillian^a | Li, Yueming^c | Xu, Min^a | Huang, Qian^a | Garsky, Victor^d | Sardana, Mohinder K.^a | Hazuda, Daria J.^a

Affiliations: [a] Department of Biological Chemistry, Merck Research Laboratories, West Point, PA 19486, USA | [b] Department of Chemistry, Washington State University, Pullman, WA 99164-4630, USA | [c] RRL-617A, Box 459, Memorial Sloan-Kettering Cancer Center, 1275, York Ave., New York, NY 10021, USA | [d] Department of Medicinal Chemistry, Merck Research Laboratories, West Point, PA 19486, USA

Correspondence: [*] Corresponding author: Xiao-Ping Shi, WP16-205, Department of Biological Chemistry, Merck Research Laboratories, West Point, PA 19486, USA. Tel.: +1 215 652 3622; Fax: +1 215 652 0264; E-mail: xiao-ping_shi@merck.com.

Note: [] Communicated by Yuan Luo.

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.

Keywords: Alzheimer's disease, BACE, β-secretases, APP

DOI: 10.3233/JAD-2005-7207

Journal: Journal of Alzheimer's Disease, vol. 7, no. 2, pp. 139-148, 2005