Amyloid β-protein production and metabolism of intramembrane regin of APPthrough stepwise processing.

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To learn about the molecular mechanism of intramemebrane cleavage of APP by γ-secretase, we have tested the tripeptide hypothesis that Aβ40/42 is produced from longer Aβ that is generated by ε-cleavage through stepwise processing at every three residues along the α-helical structure of the intramembrane region (J Neurosci 25, 436-445, 2005).1. We examined whether longer Aβ (Aβ46) is processed to Aβ43 and Aβ40 in a γ-secretase-dependent manner. CHO cells treated with DAPT caused an accumulation of Aβ46, which was fractionated into lipid raft by sucrose density gradient centrifugation. Incubating the Aβ46-accumulating raft in the presence of L685,458 prevented AICD generation and resulted in a large decrease in the level of Aβ46 and the concomitant appearance of Aβ40 and Aβ43, but not Aβ42. The amount of newly produced Aβ40 and Aβ43 was roughly equivalent to the decrease in the amount of Aβ46. Further addition of DAPT suppressed the production of Aβ40/43 and abolished the decrease in the amount of Aβ46. These data indicate that preaccumulated Aβ46 is processed by γ-secretase to Aβ40/43, but not to Aβ42 in the raft.2. We sought to identify by LC-MS/MS particular tripeptides that should be released from the transmembrane domain of APP according to the tripeptide hypothesis. Using cell -free Aβ generation system with the membrane prepared from CHO cells, we found that the five tripeptides, IAT, VIV, ITL, TVI, and VIT, were generated during incubation. The production of these tripeptides was significantly suppressed by either L685, 458 or DAPT. These results strongly suggest that those five tripeptides are generated by γ-secretase.