Positive-negative epitope-tagging of beta amyloid precursor protein to identify inhibitors of A beta processing.
In this report, a novel positive-negative epitope tagging approach was developed to study the cellular processing of beta amyloid precursor protein (beta APP). Amino acids centered around the alpha-secretase cleavage site within the A beta sequence were replaced with residues comprising an epitope for which high-affinity monoclonal antibodies are commercially available. The resulting mutant beta APP cDNAs were expressed in human embryonic kidney cells (HEK 293). Cleavage of labeled beta APP by beta- and gamma-secretase(s) results in the release of an epitope-tagged A beta peptide, whereas cleavage by alpha-secretase results in destruction of the epitope. Highly sensitive and specific immunoassays were developed to study processing of this labeled beta APP via the amyloidogenic pathway. Secretion of epitope-tagged A beta was prevented by MDL 28170, a previously described gamma-secretase inhibitor. Confocal microscopic studies revealed that processing and cellular trafficking of epitope-tagged beta APP was not different from wild-type beta APP. These results suggest that positive-negative epitope-tagged beta APP is normally processed within the cell and may be used to identify secretase inhibitors as therapeutics for Alzheimer's disease.
Seiffert
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D
E400/3253, Department of Chemical Enzymology, DuPont Pharmaceuticals Company, 198880-0400, Wilmington, DE 19880-0400, USA. dietmar.a.sieffert@dupontpharma.com
Mitchell
T
T
Stern
A M
AM
Roach
A
A
Zhan
Y
Y
Grzanna
R
R
D000544:Alzheimer Disease / Q000188:drug therapy / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D053829:Amyloid Precursor Protein Secretases; D016564:Amyloid beta-Protein Precursor / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D000911:Antibodies, Monoclonal / Q000276:immunology; D016282:Aspartic Acid Endopeptidases; D015153:Blotting, Western; D002460:Cell Line; D017077:Culture Media, Conditioned / Q000737:chemistry / Q000378:metabolism; D004151:Dipeptides / Q000494:pharmacology; D010450:Endopeptidases / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000378:metabolism; D011480:Protease Inhibitors / Q000032:analysis / Q000494:pharmacology* / Q000627:therapeutic use; D011499:Protein Processing, Post-Translational / Q000187:drug effects*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D012680:Sensitivity and Specificity; D014162:Transfection
D016564:Amyloid beta-Protein Precursor; D000911:Antibodies, Monoclonal; D017077:Culture Media, Conditioned; D004151:Dipeptides; D000939:Epitopes; D007527:Isoenzymes; D011480:Protease Inhibitors; D011993:Recombinant Fusion Proteins; D053829:Amyloid Precursor Protein Secretases; D010450:Endopeptidases; D016282:Aspartic Acid Endopeptidases; C509280:BACE1 protein, human; C058076:calpain inhibitor III