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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.
J Alzheimers Dis. 2005 Apr; 7(2):139-48; discussion 173-80.JA

Abstract

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.

Authors+Show Affiliations

Department of Biological Chemistry, Merck Research Laboratories, West Point, PA 19486, USA. xiao-ping_shi@merck.comNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article

Language

eng

PubMed ID

15851852

Citation

Shi, Xiao-Ping, et al. "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." Journal of Alzheimer's Disease : JAD, vol. 7, no. 2, 2005, pp. 139-48; discussion 173-80.
Shi XP, Tugusheva K, Bruce JE, et al. 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. J Alzheimers Dis. 2005;7(2):139-48; discussion 173-80.
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., ... Hazuda, D. J. (2005). 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. Journal of Alzheimer's Disease : JAD, 7(2), 139-48; discussion 173-80.
Shi XP, et al. 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. J Alzheimers Dis. 2005;7(2):139-48; discussion 173-80. PubMed PMID: 15851852.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - 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. AU - Shi,Xiao-Ping, AU - Tugusheva,Katherine, AU - Bruce,James E, AU - Lucka,Adam, AU - Chen-Dodson,Elizabeth, AU - Hu,Binghua, AU - Wu,Guo-Xin, AU - Price,Eric, AU - Register,Robert B, AU - Lineberger,Janet, AU - Miller,Ron, AU - Tang,Mei-Jy, AU - Espeseth,Amy, AU - Kahana,Jason, AU - Wolfe,Abigail, AU - Crouthamel,Ming-Chih, AU - Sankaranarayanan,Sethu, AU - Simon,Adam, AU - Chen,Lin, AU - Lai,Ming-Tain, AU - Pietrak,Beth, AU - DiMuzio,Jillian, AU - Li,Yueming, AU - Xu,Min, AU - Huang,Qian, AU - Garsky,Victor, AU - Sardana,Mohinder K, AU - Hazuda,Daria J, PY - 2005/4/27/pubmed PY - 2005/8/16/medline PY - 2005/4/27/entrez SP - 139-48; discussion 173-80 JF - Journal of Alzheimer's disease : JAD JO - J Alzheimers Dis VL - 7 IS - 2 N2 - 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. SN - 1387-2877 UR - https://www.unboundmedicine.com/medline/citation/15851852/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_ L2 - https://content.iospress.com/openurl?genre=article&issn=1387-2877&volume=7&issue=2&spage=139 DB - PRIME DP - Unbound Medicine ER -