γ-Secretase associated with lipid rafts: multiple interactive pathways in the stepwise processing of ß-carboxyl-terminal fragment.

ABSTRACT

γ-Secretase generates amyloid ß-protein (Aß), a pathogenic molecule in Alzheimer disease, through the intramembrane cleavage of the ß-carboxyl-terminal fragment (ßCTF) of ß-amyloid precursor protein. We previously showed the framework of the γ-secretase cleavage, i.e. the stepwise successive processing of ßCTF at every three (or four) amino acids. However, the membrane integrity of γ-secretase was not taken into consideration because of the use of the 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonic acid-solubilized reconstituted γ-secretase system. Here, we sought to address how the membrane-integrated γ-secretase cleaves ßCTF by using γ-secretase associated with lipid rafts. Quantitative analyses using liquid chromatography-tandem mass spectrometry of the ßCTF transmembrane domain-derived peptides released along with Aß generation revealed that the raft-associated γ-secretase cleaves ßCTF in a stepwise sequential manner, but novel penta- and hexapeptides as well as tri- and tetrapeptides are released. The cropping of these peptides links the two major tripeptide-cleaving pathways generating Aß40 and Aß42 at several points, implying that there are multiple interactive pathways for the stepwise cleavages of ßCTF. It should be noted that Aß38 and Aß43 are generated through three routes, and γ-secretase modulator 1 enhances all the three routes generating Aß38, which results in decreases in Aß42 and Aß43 and an increase in Aß38. These observations indicate that multiple interactive pathways for stepwise successive processing by γ-secretase define the species and quantity of Aß produced.