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 Journal Article
| Authors: | Shi, X. P.; Tugusheva, K.; Bruce, J. E.; Lucka, A.; Chen-Dodson, E.; Hu, B.; Wu, G. X.; Price, E.; Register, R. B.; Lineberger, J.; Miller, R.; Tang, M. J.; Espeseth, A.; Kahana, J.; Wolfe, A.; Crouthamel, M. C.; Sankaranarayanan, S.; Simon, A.; Chen, L.; Lai, M. T.; Pietrak, B.; DiMuzio, J.; Li, Y.; Xu, M.; Huang, Q.; Garsky, V.; Sardana, M. K.; Hazuda, D. J. |
| Article Title: | 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 |
| 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 theβ-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. © 2005 - IOS Press and the authors. All rights reserved. |
| Keywords: | controlled study; human cell; nonhuman; protein function; protein analysis; animal cell; mouse; animals; mice; protein degradation; enzyme activation; enzyme substrate; transfection; wild type; antibodies, monoclonal; molecular sequence data; cell culture; enzyme analysis; peptide fragments; substrate specificity; fibroblast; fibroblasts; point mutation; disease models, animal; alzheimer disease; protein modification; amyloid precursor protein secretases; amyloid beta-protein; endopeptidases; gene expression regulation, enzymologic; alzheimer's disease; amyloid beta protein; amyloid beta-protein precursor; beta secretase; aspartic endopeptidases; app; β-secretases; bace; beta secretase inhibitor |
| Journal Title: | Journal of Alzheimer's Disease |
| Volume: | 7 |
| Issue: | 2 |
| ISSN: | 1387-2877 |
| Publisher: | IOS Press |
| Date Published: | 2005-04-01 |
| Start Page: | 139 |
| End Page: | 148 |
| Language: | English |
| PUBMED: | 15851852 |
| PROVIDER: | scopus |
| DOI/URL: | |
| Notes: | --- - "Cited By (since 1996): 23" - "Export Date: 24 October 2012" - "CODEN: JADIF" - "Source: Scopus" |
