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Sequential proteolysis of the amyloid precursor protein (APP) by γ-secretases generates amyloid-β (Aβ) peptides and defines the proportion of short-to-long Aβ peptides, which is tightly connected to Alzheimer's disease (AD) pathogenesis. Here, we study the mechanism that controls substrate processing by γ-secretases and Aβ peptide length. We found that polar interactions established by the APP C99 ectodomain (ECD), involving but not limited to its juxtamembrane region, restrain both the extent and degree of γ-secretases processive cleavage by destabilizing enzyme-substrate interactions. We show that increasing hydrophobicity, via mutation or ligand binding, at APP C99 -ECD attenuates substrate-driven product release and rescues the effects of Alzheimer's disease-associated pathogenic γ-secretase and APP variants on Aβ length. In addition, our study reveals that APP C99 -ECD facilitates the paradoxical production of longer Aβs caused by some γ-secretase inhibitors, which act as high-affinity competitors of the substrate. These findings assign a pivotal role to the substrate ECD in the sequential proteolysis by γ-secretases and suggest it as a sweet spot for the potential design of APP-targeting compounds selectively promoting its processing by these enzymes., (© 2023 The Authors. Published under the terms of the CC BY NC ND 4.0 license.)

γ-Secretase is an aspartyl intramembrane protease that cleaves the amyloid precursor protein (APP) involved in Alzheimer's disease pathology and other transmembrane proteins. Substrate-bound structures reveal a stable hybrid β-sheet immediately following the substrate scissile bond consisting of β1 and β2 from the enzyme and β3 from the substrate. Molecular dynamics simulations and enhanced sampling simulations demonstrate that the hybrid β-sheet stability is strongly correlated with the formation of a stable cleavage-compatible active geometry and it also controls water access to the active site. The hybrid β-sheet is only stable for substrates with 3 or more C-terminal residues beyond the scissile bond. The simulation model allowed us to predict the effect of Pro and Phe mutations that weaken the formation of the hybrid β-sheet which were confirmed by experimental testing. Our study provides a direct explanation why γ-secretase preferentially cleaves APP in steps of 3 residues and how the hybrid β-sheet facilitates γ-secretase proteolysis., (© 2023. The Author(s).)

γ-Secretase complexes (GSECs) are multimeric membrane proteases involved in a variety of physiological processes and linked to Alzheimer's disease (AD). Presenilin (PSEN, catalytic subunit), Nicastrin (NCT), Presenilin Enhancer 2 (PEN-2), and Anterior Pharynx Defective 1 (APH1) are the essential subunits of GSECs. Mutations in PSEN and the Amyloid Precursor Protein (APP) cause early-onset AD GSECs successively cut APP to generate amyloid-β (Aβ) peptides of various lengths. AD-causing mutations destabilize GSEC-APP/Aβ n interactions and thus enhance the production of longer Aβs, which elicit neurotoxic events underlying pathogenesis. Here, we investigated the molecular strategies that anchor GSEC and APP/Aβ n during the sequential proteolysis. Our studies reveal that a direct interaction between NCT ectodomain and APP C99 influences the stability of GSEC-Aβn assemblies and thereby modulates Aβ length. The data suggest a potential link between single-nucleotide variants in NCSTN and AD risk. Furthermore, our work indicates that an extracellular interface between the protease (NCT, PSEN) and the substrate (APP) represents the target for compounds (GSMs) modulating Aβ length. Our findings may guide future rationale-based drug discovery efforts., (© 2019 The Authors. Published under the terms of the CC BY NC ND 4.0 license.)

Alzheimer's disease (AD)-linked mutations in Presenilins (PSEN) and the amyloid precursor protein (APP) lead to production of longer amyloidogenic Aβ peptides. The shift in Aβ length is fundamental to the disease; however, the underlying mechanism remains elusive. Here, we show that substrate shortening progressively destabilizes the consecutive enzyme-substrate (E-S) complexes that characterize the sequential γ-secretase processing of APP. Remarkably, pathogenic PSEN or APP mutations further destabilize labile E-S complexes and thereby promote generation of longer Aβ peptides. Similarly, destabilization of wild-type E-S complexes by temperature, compounds, or detergent promotes release of amyloidogenic Aβ. In contrast, E-Aβ n stabilizers increase γ-secretase processivity. Our work presents a unifying model for how PSEN or APP mutations enhance amyloidogenic Aβ production, suggests that environmental factors may increase AD risk, and provides the theoretical basis for the development of γ-secretase/substrate stabilizing compounds for the prevention of AD., (Copyright © 2017. Published by Elsevier Inc.)

Deposition of amyloid-β (Aβ) peptides in the brain is a hallmark of Alzheimer's disease. Aβs are generated through sequential proteolysis of the amyloid precursor protein by the γ-secretase complexes (GSECs). Aβ peptide length, modulated by the Presenilin (PSEN) and APH-1 subunits of GSEC, is critical for Alzheimer's pathogenesis. Despite high relevance, mechanistic understanding of the proteolysis of Aβ, and its modulation by APH-1, remain incomplete. Here, we report cryo-EM structures of human GSEC (PSEN1/APH-1B) reconstituted into lipid nanodiscs in apo form and in complex with the intermediate Aβ46 substrate without cross-linking. We find that three non-conserved and structurally divergent APH-1 regions establish contacts with PSEN1, and that substrate-binding induces concerted rearrangements in one of the identified PSEN1/APH-1 interfaces, providing structural basis for APH-1 allosteric-like effects. In addition, the GSEC-Aβ46 structure reveals an interaction between Aβ46 and loop 1PSEN1, and identifies three other H-bonding interactions that, according to functional validation, are required for substrate recognition and efficient sequential catalysis. Incomplete proteolysis of amyloid-β (Aβ) peptides by γ-secretases is linked to Alzheimer's disease. Using cryo-EM and functional analyses, this study uncovers the γ-secretase – Aβ46 structure and unveils key interactions for efficient Aβ cleavage. [ABSTRACT FROM AUTHOR]

Alzheimer's disease (AD) pathogenesis has been linked to the accumulation of longer, aggregation‐prone amyloid β (Aβ) peptides in the brain. Γ‐secretases generate Aβ peptides from the amyloid precursor protein (APP). Γ‐secretase modulators (GSMs) promote the generation of shorter, less‐amyloidogenic Aβs and have therapeutic potential. However, poorly defined drug–target interactions and mechanisms of action have hampered their therapeutic development. Here, we investigate the interactions between the imidazole‐based GSM and its target γ‐secretase—APP using experimental and in silico approaches. We map the GSM binding site to the enzyme–substrate interface, define a drug‐binding mode that is consistent with functional and structural data, and provide molecular insights into the underlying mechanisms of action. In this respect, our analyses show that occupancy of a γ‐secretase (sub)pocket, mediating binding of the modulator's imidazole moiety, is sufficient to trigger allosteric rearrangements in γ‐secretase as well as stabilize enzyme–substrate interactions. Together, these findings may facilitate the rational design of new modulators of γ‐secretase with improved pharmacological properties. Synopsis: Modulators of γ‐secretase activity (GSMs) that shift amyloid‐β (Aβ) production towards the shorter non‐amyloidogenic peptides while sparing critical γ‐secretase‐mediated signalling cascades are promising agents in the fight against Alzheimer's disease. Here, insights into the underlying drug‐target interactions and GSM mode of action may help to overcome current limitations to their rational further therapeutic development. The binding pocket of a potent imidazole‐based GSM (GSM III) maps towards the γ‐secretase‐amyloid precursor protein (APP) (enzyme‐substrate) interface.The binding mode of GSM III at the γ‐secretase—APP interface reveals a dual mechanism of action, as activator and stabilizer of γ‐secretases.Occupancy of a sub‐pocket in γ‐secretase is sufficient to allosterically activate γ‐secretase and to stabilize enzyme—substrate interactions.This dual mode of action enhances the generation of short and non‐toxic Aβ peptides. [ABSTRACT FROM AUTHOR]

In vitro studies of autosomal dominant Alzheimer's disease implicate longer amyloid-β peptides in disease pathogenesis; however, less is known about the behaviour of these mutations in vivo. In this cross-sectional cohort study, we used liquid chromatography-tandem mass spectrometry to analyse 66 plasma samples from individuals who were at risk of inheriting a mutation or were symptomatic. We tested for differences in amyloid-β (Aβ)42:38, Aβ42:40 and Aβ38:40 ratios between presenilin 1 (PSEN1) and amyloid precursor protein (APP) carriers. We examined the relationship between plasma and in vitro models of amyloid-β processing and tested for associations with parental age at onset. Thirty-nine participants were mutation carriers (28 PSEN1 and 11 APP). Age- and sex-adjusted models showed marked differences in plasma amyloid-β between genotypes: higher Aβ42:38 in PSEN1 versus APP (P < 0.001) and non-carriers (P < 0.001); higher Aβ38:40 in APP versus PSEN1 (P < 0.001) and non-carriers (P < 0.001); while Aβ42:40 was higher in both mutation groups compared to non-carriers (both P < 0.001). Amyloid-β profiles were reasonably consistent in plasma and cell lines. Within the PSEN1 group, models demonstrated associations between Aβ42:38, Aβ42:40 and Aβ38:40 ratios and parental age at onset. In vivo differences in amyloid-β processing between PSEN1 and APP carriers provide insights into disease pathophysiology, which can inform therapy development. [ABSTRACT FROM AUTHOR]

The rising prevalence of Alzheimer's disease (AD), together with the lack of effective treatments, portray it as one of the major health challenges of our times. Untangling AD implies advancing the knowledge of the biology that gets disrupted during the disease while deciphering the molecular and cellular mechanisms leading to AD-related neurodegeneration. In fact, a solid mechanistic understanding of the disease processes stands as an essential prerequisite for the development of safe and effective treatments. Genetics has provided invaluable clues to the genesis of the disease by revealing deterministic genes - Presenilins (PSENs) and the Amyloid Precursor Protein (APP) - that, when affected, lead in an autosomal dominant manner to early-onset, familial AD (FAD). PSEN is the catalytic subunit of the membrane-embedded γ-secretase complexes, which act as proteolytic switches regulating key cell signalling cascades. Importantly, these intramembrane proteases are responsible for the production of Amyloid β (Aβ) peptides from APP. The convergence of pathogenic mutations on one functional pathway, the amyloidogenic cleavage of APP, strongly supports the significance of this process in AD pathogenesis. Here, we review and discuss the state-of-the-art knowledge of the molecular mechanisms underlying FAD, their implications for the sporadic form of the disease and for the development of safe AD therapeutics. [ABSTRACT FROM AUTHOR]

The mechanisms by which mutations in the presenilins (PSEN) or the amyloid precursor protein (APP) genes cause familial Alzheimer disease (FAD) are controversial. FAD mutations increase the release of amyloid ? (A?)42 relative to A?40 by an unknown, possibly gain-of-toxic-function, mechanism. However, many PSEN mutations paradoxically impair ?-secretase and 'loss-of-function' mechanisms have also been postulated. Here, we use kinetic studies to demonstrate that FAD mutations affect A? generation via three different mechanisms, resulting in qualitative changes in the A? profiles, which are not limited to A?42. Loss of ?-cleavage function is not generally observed among FAD mutants. On the other hand, ?-secretase inhibitors used in the clinic appear to block the initial ?-cleavage step, but unexpectedly affect more selectively Notch than APP processing, while modulators act as activators of the carboxypeptidase-like (?) activity. Overall, we provide a coherent explanation for the effect of different FAD mutations, demonstrating the importance of qualitative rather than quantitative changes in the A? products, and suggest fundamental improvements for current drug development efforts. [ABSTRACT FROM AUTHOR]

Summary Recently it was proposed that the familial Alzheimer’s disease (FAD) causing presenilin (PSEN) mutations PSEN1-L435F and PSEN1-C410Y do not support the generation of Aβ-peptides from the amyloid precursor protein (APP). This challenges the amyloid hypothesis and disagrees with previous work showing that PSEN1 FAD causing mutations generate invariably long Aβ and seed amyloid. We contrast here the proteolytic activities of these mutant PSEN alleles with the complete loss-of-function PSEN1-D257A allele. We find residual carboxy- and endo-peptidase γ-secretase activities, similar to the formerly characterized PSEN1-R278I. We conclude that the PSEN1-L435F and -C410Y mutations are extreme examples of the previously proposed “dysfunction” of γ-secretase that characterizes FAD-associated PSEN. This Matters Arising paper is in response to Xia et al. (2015) , published in Neuron . See also the response by Xia et al. (2016) , published in this issue. [ABSTRACT FROM AUTHOR]