Abstract
The amyloid precursor protein (APP) undergoes two consecutive cleavages by different proteases, beta-secretase and gamma-secretase, leading to the release of an amyloidogenic 4 kDa fragment called amyloid beta (Abeta). Combining immunoprecipitation and mass spectrometry, we characterized soluble Abeta in cultured cell media of mouse neuroblastoma N2a cells and double hAPP/hBACE-1 transfected HEK293. The major Abeta isoforms detected were Abeta11-34, Abeta1-34, Abeta11-40 and Abeta1-40. In this study, we demonstrate that overexpression of human beta-secretase (BACE-1) in HEK293 cells resulted in predominant Abeta cleavage at position Glu(11) rather than Asp(1), as well as increased production of Abeta(x)-34, but not Abeta(x)-40. Incubation of cells with a specific gamma-secretase inhibitor suggests that cleavage of APP at Leu(34) could be mediated by gamma-secretase itself or by a gamma-secretase dependent process.
MeSH terms
-
Amyloid Precursor Protein Secretases
-
Amyloid beta-Peptides / analysis
-
Amyloid beta-Peptides / antagonists & inhibitors
-
Amyloid beta-Peptides / biosynthesis*
-
Amyloid beta-Peptides / chemistry
-
Amyloid beta-Protein Precursor / chemistry
-
Amyloid beta-Protein Precursor / genetics
-
Amyloid beta-Protein Precursor / metabolism
-
Animals
-
Aspartic Acid Endopeptidases / genetics
-
Aspartic Acid Endopeptidases / pharmacology
-
Cell Line
-
Culture Media / chemistry
-
Endopeptidases / drug effects
-
Endopeptidases / physiology*
-
Enzyme Inhibitors / pharmacology
-
Humans
-
Mice
-
Peptide Fragments / analysis
-
Peptide Fragments / antagonists & inhibitors
-
Peptide Fragments / biosynthesis*
-
Protein Isoforms / analysis
-
Solubility
-
Transfection
Substances
-
Amyloid beta-Peptides
-
Amyloid beta-Protein Precursor
-
Culture Media
-
Enzyme Inhibitors
-
Peptide Fragments
-
Protein Isoforms
-
amyloid beta-protein (1-34)
-
Amyloid Precursor Protein Secretases
-
Endopeptidases
-
Aspartic Acid Endopeptidases
-
BACE1 protein, human
-
Bace1 protein, mouse