Biochemical characterization of the gamma-secretase activity that produces beta-amyloid peptides.
Recent studies of gamma-secretase have pointed out that it may be comprised of a multisubunit complex with presenilin 1 and presenilin 2 as central components. Elucidation of the biochemical mechanism of this enzymatic activity will provide important information for developing gamma-secretase inhibitors in Alzheimer's disease therapy. Here we describe the biochemical characterization of gamma-secretase activities using a sensitive, membrane-based assay system. Membranes were isolated from 293 cells expressing C99, the substrate of gamma-secretase. Upon incubation at 37 degrees C, C99 is cleaved by the endogenous gamma-secretase, and Abeta peptides are liberated. Abeta40 and Abeta42 gamma-secretase activities are very similar in terms of their kinetic profiles and pH dependence, supporting the notion that a single enzyme is involved in both Abeta40 and Abeta42 production. Pepstatin A inhibited Abeta40 and Abeta42 gamma-secretase activities with similar potency. Peptide difluoroketone and peptide aldehyde inhibitors inhibited Abeta40 production in a dose-dependent fashion, enhanced Abeta42 production at low concentrations, and inhibited Abeta42 production at high concentrations. Although the selective increase of Abeta42 by low concentrations of peptide difluoroketone and peptide aldehyde inhibitors has been reported in intact cells, the finding that this phenomenon occurs in a membrane-based assay system suggests that these compounds increase Abeta42 by a direct effect on gamma-secretase. The ability of these compounds to increase Abeta42 production may reflect allosteric modulation of the gamma-secretase complex by a mechanism related to that responsible for the increase of Abeta42 production by mutations in presenilins.
Zhang
L
L
Department of Central Nervous System and Cardiovascular Research and Department of Structural Chemistry, Schering-Plough Research Institute, Kenilworth, New Jersey 07033, USA. lili.zhang@spcorp.com
Song
L
L
Terracina
G
G
Liu
Y
Y
Pramanik
B
B
Parker
E
E
D000544:Alzheimer Disease / Q000201:enzymology / Q000378:metabolism; D053829:Amyloid Precursor Protein Secretases; D016229:Amyloid beta-Peptides / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000378:metabolism; D016282:Aspartic Acid Endopeptidases; D020126:Brefeldin A / Q000494:pharmacology; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology / Q000378:metabolism; D002474:Cell-Free System; D010450:Endopeptidases / Q000737:chemistry* / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D006868:Hydrolysis; D007976:Leupeptins / Q000494:pharmacology; D008565:Membrane Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D009842:Oligopeptides / Q000494:pharmacology; D010446:Peptide Fragments / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000378:metabolism; D011480:Protease Inhibitors / Q000494:pharmacology; D021381:Protein Transport / Q000187:drug effects; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity
D016229:Amyloid beta-Peptides; D007976:Leupeptins; D008565:Membrane Proteins; D009842:Oligopeptides; D010446:Peptide Fragments; D011480:Protease Inhibitors; C078639:amyloid beta-protein (1-38); C078592:amyloid beta-protein (1-40); C075222:amyloid beta-protein (1-42); C080153:L 708286; D020126:Brefeldin A; D053829:Amyloid Precursor Protein Secretases; D010450:Endopeptidases; D016282:Aspartic Acid Endopeptidases; C509280:BACE1 protein, human; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde