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 1
, and identifies three other H-bonding interactions that, according to functional validation, are required for substrate recognition and efficient sequential catalysis.
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-Aβn 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.
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•Sequential γ-secretase cuts on APP generate progressively less stable E-S complexes•Unifying model for FAD: APP/PSEN mutations destabilize γ-secretase-Aβn complexes•FAD-like destabilization may increase the risk of sporadic Alzheimer’s disease•γ-Secretase-Aβn stabilizers, novel approach to target Aβ in Alzheimer’s disease
Sequential γ-secretase cuts on amyloid precursor protein (APP) generates progressively less stable enzyme-substrate (E-S) complexes. Alzheimer’s disease–causing mutations destabilize E-S complexes and thereby enhance amyloidogenic Aβ production.
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 β (Aβ)42 relative to Aβ40 by an unknown, possibly gain‐of‐toxic‐function, mechanism. However, many PSEN mutations paradoxically impair γ‐secretase and ‘loss‐of‐function’ mechanisms have also been postulated. Here, we use kinetic studies to demonstrate that FAD mutations affect Aβ generation via three different mechanisms, resulting in qualitative changes in the Aβ profiles, which are not limited to Aβ42. Loss of ε‐cleavage function is not generally observed among FAD mutants. On the other hand, γ‐secretase inhibitors used in the clinic appear to block the initial ε‐cleavage step, but unexpectedly affect more selectively Notch than APP processing, while modulators act as activators of the carboxypeptidase‐like (γ) 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 Aβ products, and suggest fundamental improvements for current drug development efforts.
Mutations in presenilin (PSEN) and amyloid precursor protein (APP) cause dominant early‐onset Alzheimer's disease (AD), but the mechanism involved is debated. Here, such mutations are shown to alter γ‐secretase activity, leading to changes in Aβ peptide cleavage patterns.
γ-Secretase complexes are involved in the generation of amyloid-β (Aβ) in the brain. Therefore, γ-secretase has been proposed as a potential therapeutic target in Alzheimer disease (AD). Targeting ...γ-secretase activity in AD requires the pharmacological dissociation of the processing of physiological relevant substrates and the generation of “toxic” Aβ. Previous reports suggest the differential targeting of γ-secretase complexes, based on their subunit composition, as a valid strategy. However, little is known about the biochemical properties of the different complexes, and key questions regarding their Aβ product profiles should be first addressed. Here, we expressed, purified, and analyzed, under the same conditions, the endopeptidase and carboxypeptidase-like activities of the four γ-secretase complexes present in humans. We find that the nature of the catalytic subunit in the complex affects both activities. Interestingly, PSEN2 complexes discriminate between the Aβ40 and Aβ38 production lines, indicating that Aβ generation in one or the other pathway can be dissociated. In contrast, the APH1 subunit mainly affects the carboxypeptidase-like activity, with APH1B complexes favoring the generation of longer Aβ peptides. In addition, we determined that expression of a single human γ-secretase complex in cell lines retains the intrinsic attributes of the protease while present in the membrane, providing validation for the in vitro studies. In conclusion, our data show that each γ-secretase complex produces a characteristic Aβ signature. The qualitative and quantitative differences between different γ-secretase complexes could be used to advance drug development in AD and other disorders.
γ-Secretase complexes generate amyloid-β (Aβ) in Alzheimer disease.
Aβ profiles of the four γ-secretase complexes expressed in humans show that PSEN regulates total peptide levels and the Aβ38 pathway, whereas APH1 affects mainly the efficiency of the carboxypeptidase-like activity.
γ-Secretase subunit composition regulates Aβ generation.
These intrinsic differences could be used to advance AD therapeutic development.
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/Aβn 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/Aβn during the sequential proteolysis. Our studies reveal that a direct interaction between NCT ectodomain and APPC99 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.
Synopsis
γ‐Secretase mediated cleavage of APP defines the length of Aβ peptides. Alzheimer's disease causing mutations destabilize γ‐secretase – APP interactions and thus promote the production of longer, amyloidogenic Aβs. Here, we investigated the molecular strategies securing γ‐secretase – APP interactions.
NCT ectodomain establishes a direct, short distance interaction with APP ectodomain.
NCT‐APP interface influences the stability of γ‐secretase – APP interactions and thereby modulates Aβ length.
NCT ectodomain influences the response towards compounds modulating Aβ length.
The data suggest a potential link between single nucleotide variants in NCSTN and AD risk.
A direct interaction between the gamma secretase subunit Nicastrin and APP regulates the stability and processivity of the γ‐secretase/substrate complex to affect Aβ length and Alzheimer disease pathogenicity.
Presenilin (PSEN) pathogenic mutations cause familial Alzheimer's disease (AD FAD) in an autosomal-dominant manner. The extent to which the healthy and diseased alleles influence each other to cause ...neurodegeneration remains unclear. In this study, we assessed γ-secretase activity in brain samples from 15 nondemented subjects, 22 FAD patients harboring nine different mutations in PSEN1, and 11 sporadic AD (SAD) patients. FAD and control brain samples had similar overall γ-secretase activity levels, and therefore, loss of overall (endopeptidase) γ-secretase function cannot be an essential part of the pathogenic mechanism. In contrast, impaired carboxypeptidase-like activity (γ-secretase dysfunction) is a constant feature in all FAD brains. Significantly, we demonstrate that pharmacological activation of the carboxypeptidase-like γ-secretase activity with γ-secretase modulators alleviates the mutant PSEN pathogenic effects. Most SAD cases display normal endo- and carboxypeptidase-like γ-secretase activities. However and interestingly, a few SAD patient samples display γ-secretase dysfunction, suggesting that γ-secretase may play a role in some SAD cases. In conclusion, our study highlights qualitative shifts in amyloid-β (Aβ) profiles as the common denominator in FAD and supports a model in which the healthy allele contributes with normal Aβ products and the diseased allele generates longer aggregation-prone peptides that act as seeds inducing toxic amyloid conformations.
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 APPC99 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 APPC99‐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 APPC99‐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.
Synopsis
Sequential proteolysis of amyloid precursor protein (APP) by γ‐secretase generates various amyloid‐β (Aβ) peptides, whose length correlates with pathogenicity of Alzheimer's disease (AD)‐associated mutations. Here, the ectodomain of the APP substrate is found to define Aβ length by promoting product release and destabilizing enzyme–substrate interactions.
Polar residues in the APPC99 ectodomain (APPC99‐ECD) drive product release by destabilizing enzyme–substrate interactions.
Increased hydrophobicity in the substrate ECD increases both efficiency and extent of sequential γ‐secretase‐mediated proteolysis of APP and Notch.
γ‐Secretase inhibitors (GSIs) DAPT and semagacestat act as high‐affinity competitors of substrates.
GSI‐mediated displacement of partially digested Aβ peptides, facilitated by the APPC99‐ECD, explains paradoxical increases in longer Aβ peptides.
Mitigation of APPC99‐ECD‐driven product release rescues the increased production of longer Aβ peptides linked to pathogenic variants in γ‐secretase and APP.
How γ‐secretase cleaves and processes the amyloid precursor protein depends on the hydrophobicity of its ectodomain, with implications for disease mechanism and drug discovery.
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.
Insights into drug‐target interactions and mode of action of an imidazole‐based γ‐secretase modulator may facilitate rational design of next‐generation compounds for treatment of Alzheimer's disease.
The structure and function of the gamma-secretase proteases are of great interest because of their crucial roles in cellular and disease processes. We established a novel purification protocol for ...the gamma-secretase complex that involves a conformation- and complex-specific nanobody, yielding highly pure and active enzyme. Using single particle electron microscopy, we analyzed the gamma-secretase structure and its conformational variability. Under steady-state conditions, the complex adopts three major conformations, which differ in overall compactness and relative position of the nicastrin ectodomain. Occupancy of the active or substrate-binding sites by inhibitors differentially stabilizes subpopulations of particles with compact conformations, whereas a mutation linked to familial Alzheimer disease results in enrichment of extended-conformation complexes with increased flexibility. Our study presents the csecretase complex as a dynamic population of interconverting conformations, involving rearrangements at the nanometer scale and a high level of structural interdependence between subunits. The fact that protease inhibition or clinical mutations, which affect amyloid beta (Abeta) generation, enrich for particular subpopulations of conformers indicates the functional relevance of the observed dynamic changes, which are likely to be instrumental for highly allosteric behavior of the enzyme.
Abstract
Background
Γ‐secretases are proteolytic switches at the membrane regulating multiple signaling cascades. Their dysfunction, resulting in enhanced generation of longer amyloid β (Aβ) peptides ...from the amyloid precursor protein (APP), leads to neurodegeneration in the context of Alzheimer’s disease (AD), while their inhibition causes neurodegenerative phenotypes in mice and cognitive worsening in AD patients treated with γ‐secretase inhibitors. The accumulation of Aβ in the brain is the earliest pathological hallmark of AD. Based on the proven affinity of Aβ peptides for γ‐secretases, we hypothesized that elevations in Aβ levels would promote an inhibitory‐feedback mechanism on γ‐secretases and impair downstream cell signaling events.
Method
We conducted rigorous kinetic analyses of γ‐secretase activity in the presence of a series of Aβ and p3 peptides, by quantifying the levels of intracellular domains generated from different γ‐secretase substrates in cell‐free assays. In addition, we determined the effects of these peptides on endogenous γ‐secretase activity in living neurons, using a ratiometric FRET‐based reporter and western blot analysis of the levels of immediate γ‐secretase substrates. Furthermore, we assessed the impact of Aβ and p3 peptides on γ‐secretase‐mediated, p75‐ and TrkA‐dependent downstream signaling via immunostaining for an apoptotic marker: cleaved caspase 3. Finally, we evaluated the impact of Aβ peptides on APP processing in synaptosome fractions derived from mouse brains.
Result
Our analyses showed that human Aβ42 inhibited γ‐secretase activity and accordingly caused accumulation of unprocessed γ‐secretase substrates in neuronal cells, i.e. CTFs of APP, p75 and pan‐cadherin. Remarkably, neither murine Aβ42 nor human p3 (17‐42) peptides exerted the inhibition. In TrkA signaling deficient PC12 cells and basal forebrain cholinergic neurons, Aβ1‐42‐mediated inhibition of γ‐secretase led to the accumulation of unprocessed p75‐CTFs and potentiated p75‐dependent cell death, mimicking the effects of γ‐secretase inhibitors.
Conclusion
We demonstrate that the pathologically relevant human Aβ1‐42 exerts product feedback inhibition on γ‐secretases, leading to dysregulation of downstream cellular signaling. These findings provide a novel conceptual framework for investigations of Aβ toxicity in the context of γ‐secretase‐dependent homeostatic signaling and raise the possibility that Aβ42‐mediated inactivation of these enzymes contributes to AD development.
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