David M. Bolduc

γ-secretase is responsible for the proteolysis of amyloid precursor protein (APP) into short, aggregation-prone amyloid-beta (Aβ) peptides, which are centrally implicated in the pathogenesis of Alzheimer's disease (AD). Despite considerable interest in developing γ-secretase targeting therapeutics for the treatment of AD, the precise mechanism by which γ-secretase produces Aβ has remained elusive. Herein, we demonstrate that γ-secretase catalysis is driven by the stabilization of an enzyme-substrate scission complex via three distinct amino-acid-binding pockets in the enzyme's active site, providing the mechanism by which γ-secretase preferentially cleaves APP in three amino acid increments. Substrate occupancy of these three pockets occurs after initial substrate binding but precedes catalysis, suggesting a conformational change in substrate may be required for cleavage. We uncover and exploit substrate cleavage preferences dictated by these three pockets to investigate the mechanism by which familial Alzheimer's disease mutations within APP increase the production of pathogenic Aβ species.

γ-Secretase is an intramembrane-cleaving protease that processes many type-I integral membrane proteins within the lipid bilayer, an event preceded by shedding of most of the substrate's ectodomain by α- or β-secretases. The mechanism by which γ-secretase selectively recognizes and recruits ectodomain-shed substrates for catalysis remains unclear. In contrast to previous reports that substrate is actively recruited for catalysis when its remaining short ectodomain interacts with the nicastrin component of γ-secretase, we find that substrate ectodomain is entirely dispensable for cleavage. Instead, γ-secretase-substrate binding is driven by an apparent tight-binding interaction derived from substrate transmembrane domain, a mechanism in stark contrast to rhomboid--another family of intramembrane-cleaving proteases. Disruption of the nicastrin fold allows for more efficient cleavage of substrates retaining longer ectodomains, indicating that nicastrin actively excludes larger substrates through steric hindrance, thus serving as a molecular gatekeeper for substrate binding and catalysis.

Proceedings of the National Academy of Sciences (PNAS), a peer reviewed journal of the National Academy of Sciences (NAS) - an authoritative source of high-impact, original research that broadly spans the biological, physical, and social sciences.

Individuals with Alzheimer’s disease generally have deposits known as “amyloid plaques” in the brain. These plaques are made up of a mixture of molecules called amyloid beta peptides that clump together and are thought to be a key cause of the disease. The amyloid beta peptides vary in size; the larger peptides tend to be more prone to forming clumps than the smaller ones and are thus more toxic to the brain. An enzyme called gamma-secretase makes amyloid beta peptides by cutting up a protein called APP. Proteins are made of chains of building blocks called amino acids and studies using a technique called mass spectrometry show that gamma-secretase cuts APP in segments of three amino acids at a time. The size of the amyloid beta peptides produced is determined by the positions in APP that gamma-secretase selects to cut. Therefore, understanding how the enzyme works could provide new opportunities for developing drugs to treat Alzheimer’s disease. Here, Bolduc et al. found that the human gamma-secretase enzyme has sites that amino acids in APP can bind to that help to guide the enzyme to cut APP by three amino acids at a time. These binding sites control where the enzyme cuts APP and therefore determines which amyloid peptides are produced. Previous studies have linked several naturally occurring mutations in the gene encoding APP to inherited forms of Alzheimer’s disease. Bolduc et al. now reveal that several of these mutations affect the places that gamma-secretase cuts APP to produce amyloid peptides. These findings may be helpful for developing drugs that could manipulate gamma-secretase to produce smaller, less harmful amyloid peptides. Gamma-secretase can cut many other proteins, and so a future challenge will be to find out if the enzyme cuts these other proteins in the same way that it cuts APP.