gamma-Secretase, evidence for multiple proteolytic activities and influence of membrane positioning of substrate on generation of amyloid beta peptides of varying length.
gamma-Secretase activity is the final cleavage event that releases the amyloid beta peptide (Abeta) from the beta-secretase cleaved carboxyl-terminal fragment of the amyloid beta protein precursor (APP). No protease responsible for this highly unusual, purportedly intramembranous, cleavage has been definitively identified. We examined the substrate specificity of gamma-secretase by mutating various residues within or adjacent to the transmembrane domain of the APP and then analyzing Abeta production from cells transfected with these mutant APPs by enzyme-linked immunosorbent assay and mass spectrometry. Abeta production was also analyzed from a subset of transmembrane domain APP mutants that showed dramatic shifts in gamma-secretase cleavage in the presence or absence of pepstatin, an inhibitor of gamma-secretase activity. These studies demonstrate that gamma-secretase's cleavage specificity is primarily determined by location of the gamma-secretase cleavage site of APP with respect to the membrane, and that gamma-secretase activity is due to the action of multiple proteases exhibiting both a pepstatin- sensitive activity and a pepstatin-insensitive activity. Given that gamma-secretase is a major therapeutic target in Alzheimer's disease these studies provide important information with respect to the mechanism of Abeta production that will direct efforts to isolate the gamma-secretases and potentially to develop effective therapeutic inhibitors of pathologically relevant gamma-secretase activities.
Murphy
M P
MP
Department of Pharmacology, Mayo Clinic Jacksonville, Jacksonville, Florida 32224, USA.
Hickman
L J
LJ
Eckman
C B
CB
Uljon
S N
SN
Wang
R
R
Golde
T E
TE
D000595:Amino Acid Sequence; D053829:Amyloid Precursor Protein Secretases; D016229:Amyloid beta-Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D017931:DNA Primers; D010450:Endopeptidases / Q000378:metabolism*; D006868:Hydrolysis; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D016229:Amyloid beta-Peptides; D017931:DNA Primers; D010446:Peptide Fragments; D053829:Amyloid Precursor Protein Secretases; D010450:Endopeptidases