BACE1- and BACE2-expressing Human Cells
We have set up stably transfected HEK293 cells overexpressing the β-secretases BACE1 and BACE2 either alone or in combination with wild-type β-amyloid precursor protein (βAPP). The characterization of the βAPP-derived catabolites indicates that cells expressing BACEs produce less genuine Aβ1– 40/42...
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Veröffentlicht in: | The Journal of biological chemistry 2003-07, Vol.278 (28), p.25859-25866 |
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Format: | Artikel |
Sprache: | eng |
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Zusammenfassung: | We have set up stably transfected HEK293 cells overexpressing the β-secretases BACE1 and BACE2 either alone or in combination with wild-type β-amyloid precursor protein (βAPP). The characterization of the βAPP-derived catabolites indicates that cells expressing BACEs produce less genuine Aβ1– 40/42 but higher amounts of secreted sAPPβ and N-terminal-truncated Aβ 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) βAPP 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 β-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 (Adisintegrin and metalloprotease 10), tumor necrosis α-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. |
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ISSN: | 0021-9258 1083-351X |
DOI: | 10.1074/jbc.M302622200 |