Longer forms of amyloid β protein: Implications for the mechanism of intramembrane cleavage by γ-secretase

Abstract

γ-Cleavage of β-amyloid precursor protein (APP) in the middle of the cell membrane generates amyloid β protein (Aβ), and ε-cleavage, ∼ 10 residues downstream of the γ-cleavage site, releases the APP intracellular domain (AICD). A significant link between generation of Aβ and AICD and failure to detect AICD41-99 led us to hypothesize that ε-cleavage generates longer Aβs, which are then processed to Aβ40/42. Using newly developed gel systems and an N-end-specific monoclonal antibody, we have identified the longer Aβs (Aβ1- 43, Aβ1-45, Aβ1-46, and Aβ1-48) within the cells and in brain tissues. The production of these longer Aβs as well as Aβ40/42 is presenilin dependent and is suppressed by {1S-benzyl-4R-[1S- carbamoyl-2-phenylethylcarbamoyl-1S-3-methylbutylcarl bamoyl] -2R-hydroxy-5-phenylpentyl}carbamic acid tert-butyl ester, a transition state analog inhibitor for aspartyl protease. In contrast, N-[N-(3,5- difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester, a potent dipeptide γ-secretase inhibitor, builds up Aβ3-43 and Aβ1-46 intracellularly, which was also confirmed by mass spectrometry. Notably, suppression of Aβ40 appeared to lead to an increase in Aβ43, which in turn brings an increase in Aβ46, in a dose-dependent manner. We therefore propose an α-helical model in which longer Aβ species generated by ε-cleavage is cleaved at every three residues in its carboxyl portion.