γ-Secretase: Successive Tripeptide and Tetrapeptide Release from the Transmembrane Domain of β-Carboxyl Terminal Fragment

Abstract

Amyloid ␤ protein (A␤), a pathogenic molecule associated with Alzheimer's disease, is produced by ␥-secretase, which cleaves the ␤-carboxyl terminal fragment (␤CTF) of ␤-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 ␤CTF is cleaved first at the membranecytoplasm boundary, producing two long A␤s, A␤ 48 and A␤ 49 , which are processed further by releasing three residues at each step to produce A␤ 42 and A␤ 40 , 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 ␥-secretase system, we confirmed that A␤ 49 is converted to A␤ 43/40 by successively releasing two or three tripeptides and that A␤ 48 is converted to A␤ 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 A␤ is generated through the stepwise processing of ␤CTF by ␥-secretase.

Potent amyloidogenicity and pathogenicity of Aβ43

“…Taken together, the R278I mutation inhibits Aβ43 to Aβ40 conversion leading to increased Aβ43 levels and concomitant decrease of Aβ40, without altering Aβ42 levels. A similar Aβ-processing pathway has been previously described by Takami et al 9 ( Fig. 2k and Supplementary Fig.…”

“…In contrast, the carboxyl-terminal amino acid of Aβ43 is threonine, which carries a hydrophilic alcohol group (together with a hydrophobic methyl group) and thus could reverse the hydrophobicity of Aβ42. Therefore, the amyloidogenicity of Aβ43, a natural product of γ-secretase activity 8,9 , has remained elusive.…”

“…A number of studies 8,9 demonstrated that γ/ε-cleavage by γ-secretase activity controls the fate of the C-terminal end. Aβ43, generated from Aβ49 via Aβ46, is subsequently converted to Aβ40 by γ-secretase whereas Aβ42 is independently generated from Aβ48 via Aβ45.…”

Evidence that Enzyme Processivity Mediates Differential Aβ Production by PS1 and PS2

“…1B ) and it is well documented that PS1 generates more Aβ than PS2 [8-18]. Previous work indicates that γ-secretase cleaves APP in a processive manner such that ε-site cleavage (resulting in AICD release) precedes γ-site cleavage (resulting in Aβ release) [25, 37]. In addition, several distinct PS1 and PS2 familial mutations influence the relative production of AICD and Aβ [10, 38, 39], supporting the notion that the production of AICD and Aβ are dissociable.…”

“…Recent work indicates that γ-secretase cleaves the transmembrane domain of APP in a processive fashion [25, 26], whereby the AICD is released first by γ-secretase cleavage at the APP ε-site and the predominant secreted product, Aβ 40 , is subsequently released as a result of processive cleavages. Furthermore, the final cleavage site in the transmembrane domain is limited by a charged basic residue at the luminal-transmembrane boundary of substrates [27], culminating in the cleavage of APP at the intramembrane γ-site corresponding to the carboxy-terminus of Aβ 40 .…”