Positive-negative epitope-tagging of beta amyloid precursor protein to identify inhibitors of A beta processing.
Brain Res Mol Brain Res
; 84(1-2): 115-26, 2000 Dec 08.
Article
em En
| MEDLINE
| ID: mdl-11113538
ABSTRACT
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.
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Endopeptidases
/
Inibidores de Proteases
/
Proteínas Recombinantes de Fusão
/
Processamento de Proteína Pós-Traducional
/
Precursor de Proteína beta-Amiloide
/
Epitopos
Tipo de estudo:
Diagnostic_studies
Limite:
Humans
Idioma:
En
Revista:
Brain Res Mol Brain Res
Assunto da revista:
BIOLOGIA MOLECULAR
/
CEREBRO
Ano de publicação:
2000
Tipo de documento:
Article
País de afiliação:
Estados Unidos