γ-Secretase: Successive tripeptide and tetrapeptide release from the transmembrane domain of β-carboxyl terminal fragment

Abstract

Amyloid β protein (Aβ), a pathogenic molecule associated with Alzheimer's disease, is produced by γ-secretase, which cleaves the β-carboxyl terminal fragment (βCTF) of β-amyloid precursor protein in the middle of its transmembrane domain. How the cleavage proceeds within the membrane has long been enigmatic. We hypothesized previously that βCTF is cleaved first at the membrane-cytoplasm boundary, producing two long Aβs, Aβ48 and Aβ49, which are processed further by releasing three residues at each step to produce Aβ42 and Aβ40, respectively. To test this hypothesis, we used liquid chromatography tandem mass spectrometry (LC-MS/MS) to quantify the specific tripeptides that are postulated to be released. Using CHAPSO (3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxyl-1-propanesulfonate)- reconstituted γ-secretase system, we confirmed that Aβ49 is converted to Aβ43/40 by successively releasing two or three tripeptides and that A β48 is converted to Aβ 42/38 by successively releasing two tripeptides or these plus an additional tetrapeptide. Most unexpectedly, LC-MS/MS quantification revealed an induction period, 3-4 min, in the generation of peptides. When extrapolated, each time line for each tripeptide appears to intercept the same point on the x-axis. According to numerical simulation based on the successive reaction kinetics, the induction period exists. These results strongly suggest that Aβ is generated through the stepwise processing of βCTF by γ-secretase. Copyright © 2009 Society for Neuroscience.