Designed Helical Peptides as Functional Probes for γ-Secretase

gamma-Secretase is a membrane-embedded aspartyl protease complex with presenilin as the catalytic component that cleaves within the transmembrane domain (TMD) of >90 known substrates, including the amyloid precursor protein (APP) of Alzheimer's disease. Processing by gamma-secretase of the APP TMD produces the amyloid beta-peptide (A beta), including the 42-residue variant (A beta 42) that pathologically deposits in the Alzheimer brain. Complex proteolysis of APP substrate by gamma-secretase involves initial endoproteolysis and subsequent carboxypeptidase trimming, resulting in two pathways of A beta production: A beta 49 -> A beta 46 -> A beta 43 -> A beta 40 and A beta 48 -> A beta 45 -> A beta 42 -> A beta 38. Dominant mutations in APP and presenilin cause early onset familial Alzheimer's disease (FAD). Understanding how gamma-secretase processing of APP is altered in FAD is essential for elucidating pathogenic mechanisms in FAD and developing effective therapeutics. To improve our understanding, we designed synthetic APP-based TMD substrates as convenient functional probes for gamma-secretase. Installation of the helix-inducing residue alpha-aminoisobutyric acid provided full TMD helical substrates while also facilitating their synthesis and increasing the solubility of these highly hydrophobic peptides. Through mass spectrometric analysis of proteolytic products, synthetic substrates were identified that were processed in a manner that reproduced physiological processing of APP substrates. Validation of these substrates was accomplished through mutational variants, including the installation of two natural APP FAD mutations. These FAD mutations also resulted in increased levels of formation of A beta-like peptides corresponding to A beta 45 and longer, raising the question of whether the levels of such long A beta peptides are indeed increased and might contribute to FAD pathogenesis.