Author response: The amyloid-beta forming tripeptide cleavage mechanism of γ-secretase

Abstract

g-secretase is responsible for the proteolysis of amyloid precursor protein (APP) into short, aggregation-prone amyloid-beta (Ab) peptides, which are centrally implicated in the pathogenesis of Alzheimer's disease (AD). Despite considerable interest in developing g-secretase targeting therapeutics for the treatment of AD, the precise mechanism by which g-secretase produces Ab has remained elusive. Herein, we demonstrate that g-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 g-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 Ab species.

Design of Substrate Transmembrane Mimetics as Structural Probes for γ-Secretase

“…Photoprobes TSA-Bpa-Bt (left) and HPI-Bpa-Bt (right) covalently label presenilin-1 (PS1) N-terminal fragment (NTF) at the active site and docking site, respectively. (a) TSA (17) but not HPI (2) decreased the labelling of PS1 NTF by the TSA photoprobe. (b) HPI (2) but not TSA (17) decreased the labelling PS1 NTF by the HPI photoprobe.…”

“…(a) TSA (17) but not HPI (2) decreased the labelling of PS1 NTF by the TSA photoprobe. (b) HPI (2) but not TSA (17) decreased the labelling PS1 NTF by the HPI photoprobe. (c, d).…”

“…[10][11][12][13] We have also reported helical peptide inhibitors (HPIs) that interact with a substrate docking exosite distinct from but proximal to the active site. [14][15] We recently demonstrated that substrate TMD is sufficient for highaffinity binding (K m < 100 nM) 16 and therefore sought peptide-based inhibitors that would mimic the entire TMD and interact with both the docking site and the active site. Specifically, we worked to couple an HPI to a TSA through a variable linker (Fig.…”

“…13 Residues P1', P2', and P3' are especially important for substrate recognition and processing. 17 In a purified enzyme assay, TSA 1 displayed an IC 50 of 41 nM (Table 1). HPI 2, containing helix-inducing α-aminoisobutyric acid (Aib) residues spaced apart to arrange the Aib residues along one face of the helix and presenting APP TMD residues to the enzyme along the rest of the helix, 14 showed comparable activity (IC 50 of 58 nM).…”

Design of substrate transmembrane mimetics as structural probes for γ-secretase

“…[10][11][12][13] We have also reported helical peptide inhibitors (HPIs) that interact with a substrate docking exosite distinct from but proximal to the active site. [14][15] We recently demonstrated that substrate TMD is sufficient for highaffinity binding (K m < 100 nM) 16 and therefore sought peptide-based inhibitors that would mimic the entire TMD and interact with both the docking site and the active site. Specifically, we worked to couple an HPI to a TSA through a variable linker (Fig.…”