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.

Abnormal production and accumulation of amyloid-beta peptide (A beta) plays a major role in the pathogenesis of Alzheimer's disease (AD). beta-secretase (BACE1) is responsible for the cleavage at the beta-site in amyloid beta protein precursor (A beta PP/APP) to generate the N-terminus of A beta. Here we report the stepwise identification and characterization of a novel APP-beta-site mutant, "NFEW (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 A beta peptides in BACEI-KO mouse fibroblast cells. The production of A beta 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 BACE I 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 "NFEW mutant is not only an enhanced substrate for BACE1 in vitro, but also a specific substrate for BACE1 in cells.