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.

We showed previously that cells expressing wild-type (WT) ␤-amyloid precursor protein (APP) or coexpressing WTAPP and WT presenilin (PS) 1/2 produced APP intracellular domains (AICD) 49 -99 and 50 -99, with the latter predominating. On the other hand, the cells expressing mutant (MT) APP or coexpressing WTAPP and MTPS1/2 produced a greater proportion of AICD-(49 -99) than AICD-(50 -99). In addition, the expression of amyloid ␤-protein (A␤) 49 in cells resulted in predominant production of A␤40 and that of A␤48 leads to preferential production of A␤42. These observations suggest that -cleavage and ␥-cleavage are interrelated. To determine the stoichiometry between A␤ and AICD, we have established a 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonic acid-solubilized ␥-secretase assay system that exhibits high specific activity. By using this assay system, we have shown that equal amounts of A␤ and AICD are produced from ␤-carboxyl-terminal fragment (C99) by ␥-secretase, irrespective of WT or MTAPP and PS1/2. Although various A␤ species, including A␤40, A␤42, A␤43, A␤45, A␤48, and A␤49, are generated, only two species of AICD, AICD-(49 -99) and AICD-(50 -99), are detected. We also have found that M233T MTPS1 produced only one species of AICD, AICD-(49 -99), and only one for its counterpart, A␤48, in contrast to WT and other MTPS1s. These strongly suggest that -cleavage is the primary event, and the produced A␤48 and A␤49 rapidly undergo ␥-cleavage, resulting in generation of various A␤ species.Amyloid ␤-protein (A␤) 4 is the major component of senile plaques, one of the neuropathological hallmarks of Alzheimer disease (AD). Current data indicate that A␤ forms large fibrous aggregates that may not be toxic to the cells, but its intermediates, diffusible oligomers, exhibit neuronal toxicity, supporting the view that A␤ is a real culprit for AD. This 38 -43-amino acid residue, a small protein, is derived from ␤-amyloid precursor protein (APP) through successive cleavages by ␤-and ␥-secretases (1). ␤-Secretase is a membrane-bound aspartyl protease and produces a carboxyl-terminal fragment (␤CTF or C99) of APP by cleaving its luminal portion (2). Cumulative evidence indicates that ␥-secretase is also an aspartyl protease, a high molecular mass protein complex composed of four different membrane proteins, Aph-1, nicastrin, Pen-2, and presenilin (PS) 1/2 (3-5). ␥-Secretase cleaves C99 in the middle of its transmembrane domain (␥-cleavage), leading to release of A␤. The mechanism of A␤ production is controversial, mainly because the hydrolytic event is postulated to occur in the very hydrophobic environment of the lipid bilayer.Besides ␥-cleavage sites, we and other groups identified novel cleavage sites close to the membrane/cytoplasmic boundary of APP (-cleavage) (6 -8). -Cleavage sites of C99 are analogous to the ␥-secretasemediated Notch S3 cleavage site (9). As in Notch signaling, -cleavage results in release of the APP intracellular domains (AICD) 49 -99 and 50 -99, which translocate to the nucleus an...

Deciphering the mechanism by which the relative Aβ42(43) to total Aβ ratio is regulated is central to understanding Alzheimer disease (AD) etiology; however, the mechanisms underlying changes in the Aβ42(43) ratio caused by familial mutations and γ-secretase modulators (GSMs) are unclear. Here, we show in vitro and in living cells that presenilin (PS)/γ-secretase cleaves Aβ42 into Aβ38, and Aβ43 into Aβ40 or Aβ38. Approximately 40% of Aβ38 is derived from Aβ43. Aβ42(43) cleavage is involved in the regulation of the Aβ42(43) ratio in living cells. GSMs increase the cleavage of PS/γ-secretase-bound Aβ42 (increase k(cat)) and slow its dissociation from the enzyme (decrease k(b)), whereas PS1 mutants and inverse GSMs show the opposite effects. Therefore, we suggest a concept to describe the Aβ42(43) production process and propose how GSMs act, and we suggest that a loss of PS/γ-secretase function to cleave Aβ42(43) may initiate AD and might represent a therapeutic target.

Background: Intramembranous cleavages of ␤-carboxyl-terminal fragment (␤CTF) by ␥-secretase generate amyloid ␤-protein (A␤). Results: Three-to six-residue peptides are released successively along with A␤ generation by lipid raft-associated ␥-secretase. Conclusion: ␥-Secretase cleaves ␤CTF through multiple interactive pathways for stepwise successive processing to generate A␤.Significance: This cleavage model provides insights into the precise molecular mechanism of A␤ generation.