Familial Alzheimer's disease mutations in amyloid protein precursor alter proteolysis by γ-secretase to increase amyloid β-peptides of ≥45 residues

Abstract

Production of amyloid β-protein (Aβ) is carried out by the membrane-embedded γ-secretase complex. Mutations in the transmembrane domain of amyloid β-protein precursor (APP) associated with early-onset familial Alzheimer's disease (FAD) can alter the ratio of aggregation-prone 42-residue Aβ (Aβ42) to 40-residue Aβ (Aβ40). However, APP substrate is proteolyzed processively by γ-secretase along two pathways: Aβ49→Aβ46→Aβ43→Aβ40 and Aβ48→Aβ45→Aβ42→Aβ38. Effects of FAD mutations on each proteolytic step are unknown, largely due to difficulties in detecting and quantifying longer Aβ peptides. To address this, we carried out systematic and quantitative analyses of all tri- and tetrapeptide coproducts from proteolysis of wild-type and 14 FAD-mutant APP substrates by purified γ-secretase. These small peptides, including FAD-mutant forms, were detected by tandem mass spectrometry and quantified by establishing concentration curves for each of 32 standards. APP intracellular domain (AICD) coproducts were quantified by immunoblot, and the ratio of AICD products corresponding to Aβ48 and Aβ49 was determined by mass spectrometry. Levels of individual Aβ peptides were determined by subtracting levels of peptide coproducts associated with degradation from those associated with production. This method was validated for Aβ40 and Aβ42 by specific ELISAs and production of equimolar levels of Aβ and AICD. Not all mutant substrates led to increased Aβ42/40. However, all 14 disease-causing mutations led to inefficient processing of longer forms of Aβ ≥ 45 residues. In addition, the effects of certain mutations provided insight into the mechanism of processive proteolysis: intermediate Aβ peptides apparently remain bound for subsequent trimming and are not released and reassociated.

Keywords: amyloid β-protein (Aβ); amyloid β-protein precursor (APP); intramembrane proteolysis; mass spectrometry; pathogenesis.