BACE1- and BACE2-expressing human cells: characterization of beta-amyloid precursor protein-derived catabolites, design of a novel fluorimetric assay, and identification of new in vitro inhibitors.
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 the betaAPP-derived catabolites indicates that cells expressing BACEs produce less genuine Abeta1- 40/42 but higher amounts of secreted sAPPbeta and N-terminal-truncated Abeta species. This was accompanied by a concomitant modulation of the C-terminal counterpart products C89 and C79 for BACE1 and BACE2, respectively. These cells were used to set up a novel BACE assay based on two quenched fluorimetric substrates mimicking the wild-type (JMV2235) and Swedish-mutated (JMV2236) betaAPP sequences targeted by BACE activities. We show that BACEs activities are enhanced by the Swedish mutation and maximal at pH 4.5. The specificity of this double assay for genuine beta-secretase activity was demonstrated by means of cathepsin D, a "false positive" BACE candidate. Thus, cathepsin D was unable to cleave preferentially the JMV2236-mutated substrate. The selectivity of the assay was also emphasized by the lack of JMV cleavage triggered by other "secretases" candidates such as ADAM10 (A disintegrin and metalloprotease 10), tumor necrosis alpha-converting enzyme, and presenilins 1 and 2. Finally, the assay was used to screen for putative in vitro BACE inhibitors. We identified a series of statine-derived sequences that dose-dependently inhibited BACE1 and BACE2 activities with IC50 in the micromolar range, some of which displaying selectivity for either BACE1 or BACE2.
Institut de Pharmacologie Moléculaire et Cellulaire of Centre National de la Recherche Scientifique, UMR6097, 06560 Valbonne, France.
David
D
Andrau
Cecile
C
Dumanchin-Njock
Erwan
E
Ayral
Jean
J
Vizzavona
Mike
M
Farzan
Michel
M
Boisbrun
Pierre
P
Fulcrand
Jean-Francois
JF
Hernandez
Jean
J
Martinez
Solveig
S
Lefranc-Jullien
Frederic
F
Checler
D053829:Amyloid Precursor Protein Secretases; D016564:Amyloid beta-Protein Precursor / Q000235:genetics / Q000378:metabolism*; D016282:Aspartic Acid Endopeptidases / Q000096:biosynthesis*; D002402:Cathepsin D / Q000378:metabolism; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D010450:Endopeptidases; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D020128:Inhibitory Concentration 50; D007700:Kinetics; D008956:Models, Chemical; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013997:Time Factors; D014162:Transfection
D016564:Amyloid beta-Protein Precursor; D004791:Enzyme Inhibitors; D010455:Peptides; D053829:Amyloid Precursor Protein Secretases; D010450:Endopeptidases; D016282:Aspartic Acid Endopeptidases; C509284:BACE2 protein, human; C509280:BACE1 protein, human; D002402:Cathepsin D