BACE1- and BACE2-expressing Human Cells CHARACTERIZATION OF β-AMYLOID PRECURSOR PROTEIN-DERIVED CATABOLITES, DESIGN OF A NOVEL FLUORIMETRIC ASSAY, AND IDENTIFICATION OF NEW IN VITRO INHIBITORS

摘要: We have set up stably transfected HEK293 cells overexpressing the beta-secretases BACE1 and BACE2 either alone or in combination with wild-type beta-amyloid precursor protein (betaAPP). The characterization of betaAPP-derived catabolites indicates that expressing BACEs produce less genuine Abeta1- 40/42 but higher amounts secreted sAPPbeta N-terminal-truncated Abeta species. This was accompanied by a concomitant modulation C-terminal counterpart products C89 C79 for BACE2, respectively. These were used to novel BACE assay based on two quenched fluorimetric substrates mimicking (JMV2235) Swedish-mutated (JMV2236) betaAPP sequences targeted activities. show activities are enhanced Swedish mutation maximal at pH 4.5. specificity this double beta-secretase activity demonstrated means cathepsin D, "false positive" candidate. Thus, D unable cleave preferentially JMV2236-mutated substrate. selectivity also emphasized lack JMV cleavage triggered other "secretases" candidates such as ADAM10 (A disintegrin metalloprotease 10), tumor necrosis alpha-converting enzyme, presenilins 1 2. Finally, screen putative vitro inhibitors. identified series statine-derived dose-dependently inhibited IC50 micromolar range, some which displaying BACE2.