Intramembrane proteolysis of ß-amyloid precursor protein by γ-secretase is an unusually slow process.

Intramembrane proteolysis has emerged as a key mechanism required for membrane proteostasis and cellular signaling. One of the intramembrane-cleaving proteases (I-CLiPs), γ-secretase, is also intimately implicated in Alzheimer's disease, a major neurodegenerative disease and leading cause of dementia. High-resolution crystal structural analyses have revealed that I-CLiPs harbor their active sites buried deeply in the membrane bilayer. Surprisingly, however, the key kinetic constants of these proteases, turnover number kcat and catalytic efficiency kcat/KM, are largely unknown. By investigating the kinetics of intramembrane cleavage of the Alzheimer's disease-associated ß-amyloid precursor protein in vitro and in human embryonic kidney cells, we show that γ-secretase is a very slow protease with a kcat value similar to those determined recently for rhomboid-type I-CLiPs. Our results indicate that low turnover numbers may be a general feature of I-CLiPs.

Generation of Alzheimer disease-associated amyloid ß42/43 peptide by γ-secretase can be inhibited directly by modulation of membrane thickness.

Pathogenic generation of amyloid ß-peptide (Aß) by sequential cleavage of ß-amyloid precursor protein (APP) by ß- and γ-secretases is widely believed to causally underlie Alzheimer disease (AD). ß-Secretase initially cleaves APP thereby generating a membrane-bound APP C-terminal fragment, from which γ-secretase subsequently liberates 37-43-amino acid long Aß species. Although the latter cleavages are intramembranous and although lipid alterations have been implicated in AD, little is known of how the γ-secretase-mediated release of the various Aß species, in particular that of the pathogenic longer variants Aß(42) and Aß(43), is affected by the lipid environment. Using a cell-free system, we have directly and systematically investigated the activity of γ-secretase reconstituted in defined model membranes of different thicknesses. We found that bilayer thickness is a critical parameter affecting both total activity as well as cleavage specificity of γ-secretase. Whereas the generation of the pathogenic Aß(42/43) species was markedly attenuated in thick membranes, that of the major and rather benign Aß(40) species was enhanced. Moreover, the increased production of Aß(42/43) by familial AD mutants of presenilin 1, the catalytic subunit of γ-secretase, could be substantially lowered in thick membranes. Our data demonstrate an effective modulation of γ-secretase activity by membrane thickness, which may provide an approach to lower the generation of the pathogenic Aß(42/43) species.

Novel γ-secretase enzyme modulators directly target presenilin protein.

γ-Secretase is essential for the generation of the neurotoxic 42-amino acid amyloid ß-peptide (Aß(42)). The aggregation-prone hydrophobic peptide, which is deposited in Alzheimer disease (AD) patient brain, is generated from a C-terminal fragment of the ß-amyloid precursor protein by an intramembrane cleavage of γ-secretase. Because Aß(42) is widely believed to trigger AD pathogenesis, γ-secretase is a key AD drug target. Unlike inhibitors of the enzyme, γ-secretase modulators (GSMs) selectively lower Aß(42) without interfering with the physiological function of γ-secretase. The molecular target(s) of GSMs and hence the mechanism of GSM action are not established. Here we demonstrate by using a biotinylated photocross-linkable derivative of highly potent novel second generation GSMs that γ-secretase is a direct target of GSMs. The GSM photoprobe specifically bound to the N-terminal fragment of presenilin, the catalytic subunit of γ-secretase, but not to other γ-secretase subunits. Binding was differentially competed by GSMs of diverse structural classes, indicating the existence of overlapping/multiple GSM binding sites or allosteric alteration of the photoprobe binding site. The ß-amyloid precursor protein C-terminal fragment previously implicated as the GSM binding site was not targeted by the compound. The identification of presenilin as the molecular target of GSMs directly establishes allosteric modulation of enzyme activity as a mechanism of GSM action and may contribute to the development of therapeutically active GSMs for the treatment of AD.