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APP substrate ectodomain defines amyloid-β peptide length by restraining γ-secretase processivity and facilitating product release

ABSTRACT: Sequential proteolysis of the amyloid precursor protein (APP) by γ-secretases (GSECs) generates amyloid-β (Aβ) 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 controlling substrate processing by GSECs and defining product 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 GSEC processive cleavage by destabilizing enzyme-substrate (E-S) interactions. We show that increasing hydrophobicity at APPC99 ECD - due to mutation or ligand binding - attenuates this substrate-driven product release mechanism, and rescues the effects that AD pathogenic variants exert on Aβ profiles. In addition, our study reveals that APPC99-ECD facilitates the paradoxical production of longer Aβs caused by some GSEC inhibitors that act as high-affinity competitors to the substrate.These findings assign a pivotal role to the substrate ECD in the sequential proteolysis by GSEC and suggest it as a sweet spot for the potential design of APP targeting compounds selectively promoting its processing by GSEC.

SUBMITTER: Prof. Lucía Chávez-Gutiérrez

PROVIDER: S-SCDT-10_15252-EMBJ_2023114372 | biostudies-other |

REPOSITORIES: biostudies-other

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APP substrate ectodomain defines amyloid-β peptide length by restraining γ-secretase processivity and facilitating product release.

Project description: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.

| S-EPMC10690472 | biostudies-literature

Independent relationship between amyloid precursor protein (APP) dimerization and ?-secretase processivity.

Project description:Altered production of ?-amyloid (A?) from the amyloid precursor protein (APP) is closely associated with Alzheimer's disease (AD). APP has a number of homo- and hetero-dimerizing domains, and studies have suggested that dimerization of ?-secretase derived APP carboxyl terminal fragment (CTF?, C99) impairs processive cleavage by ?-secretase increasing production of long A?s (e.g., A?1-42, 43). Other studies report that APP CTF? dimers are not ?-secretase substrates. We revisited this issue due to observations made with an artificial APP mutant referred to as 3xK-APP, which contains three lysine residues at the border of the APP ectodomain and transmembrane domain (TMD). This mutant, which dramatically increases production of long A?, was found to form SDS-stable APP dimers, once again suggesting a mechanistic link between dimerization and increased production of long A?. To further evaluate how multimerization of substrate affects both initial ?-secretase cleavage and subsequent processivity, we generated recombinant wild type- (WT) and 3xK-C100 substrates, isolated monomeric, dimeric and trimeric forms of these proteins, and evaluated both ?-cleavage site utilization and A? production. These show that multimerization significantly impedes ?-secretase cleavage, irrespective of substrate sequence. Further, the monomeric form of the 3xK-C100 mutant increased long A? production without altering the initial ?-cleavage utilization. These data confirm and extend previous studies showing that dimeric substrates are not efficient ?-secretase substrates, and demonstrate that primary sequence determinants within APP substrate alter ?-secretase processivity.

| S-EPMC4211736 | biostudies-literature

?-Synuclein increases ?-amyloid secretion by promoting ?-/?-secretase processing of APP.

Project description:?-Synuclein misfolding and aggregation is often accompanied by ?-amyloid deposition in some neurodegenerative diseases. We hypothesised that ?-synuclein promotes ?-amyloid production from APP. ?-Amyloid levels and APP amyloidogenic processing were investigated in neuronal cell lines stably overexpressing wildtype and mutant ?-synuclein. ?-Secretase activity and ?-secretase expression were also measured. We show that ?-synuclein expression induces ?-amyloid secretion and amyloidogenic processing of APP in neuronal cell lines. Certain mutations of ?-synuclein potentiate APP amyloidogenic processing. ?-Secretase activity was not enhanced by wildtype ?-synuclein expression, however ?-secretase protein levels were induced. Furthermore, a correlation between ?-synuclein and ?-secretase protein was seen in rat brain striata. Iron chelation abolishes the effect of ?-synuclein on neuronal cell ?-amyloid secretion, whereas overexpression of the ferrireductase enzyme Steap3 is robustly pro-amyloidogenic. We propose that ?-synuclein promotes ?-amyloid formation by modulating ?-cleavage of APP, and that this is potentially mediated by the levels of reduced iron and oxidative stress.

| S-EPMC5302447 | biostudies-literature

Lysine 624 of the amyloid precursor protein (APP) is a critical determinant of amyloid ? peptide length: support for a sequential model of ?-secretase intramembrane proteolysis and regulation by the amyloid ? precursor protein (APP) juxtamembrane region.

Project description:?-Secretase is a multiprotein intramembrane cleaving aspartyl protease (I-CLiP) that catalyzes the final cleavage of the amyloid ? precursor protein (APP) to release the amyloid ? peptide (A?). A? is the primary component of senile plaques in Alzheimer's disease (AD), and its mechanism of production has been studied intensely. ?-Secretase executes multiple cleavages within the transmembrane domain of APP, with cleavages producing A? and the APP intracellular domain (AICD), referred to as ? and ?, respectively. The heterogeneous nature of the ? cleavage that produces various A? peptides is highly relevant to AD, as increased production of A? 1-42 is genetically and biochemically linked to the development of AD. We have identified an amino acid in the juxtamembrane region of APP, lysine 624, on the basis of APP695 numbering (position 28 relative to A?) that plays a critical role in determining the final length of A? peptides released by ?-secretase. Mutation of this lysine to alanine (K28A) shifts the primary site of ?-secretase cleavage from 1-40 to 1-33 without significant changes to ? cleavage. These results further support a model where ? cleavage occurs first, followed by sequential proteolysis of the remaining transmembrane fragment, but extend these observations by demonstrating that charged residues at the luminal boundary of the APP transmembrane domain limit processivity of ?-secretase.

| S-EPMC3220543 | biostudies-literature

Membrane lipid remodeling modulates γ-secretase processivity.

Project description:Imbalances in the amounts of amyloid-β peptides (Aβ) generated by the membrane proteases β- and γ-secretase are considered as a trigger of Alzheimer's disease (AD). Cell-free studies of γ-secretase have shown that increasing membrane thickness modulates Aβ generation but it has remained unclear if these effects are translatable to cells. Here we show that the very long-chain fatty acid erucic acid (EA) triggers acyl chain remodeling in AD cell models, resulting in substantial lipidome alterations which included increased esterification of EA in membrane lipids. Membrane remodeling enhanced γ-secretase processivity, resulting in the increased production of the potentially beneficial Aβ37 and/or Aβ38 species in multiple cell lines. Unexpectedly, we found that the membrane remodeling stimulated total Aβ secretion by cells expressing WT γ-secretase but lowered it for cells expressing an aggressive familial AD mutant γ-secretase. We conclude that EA-mediated modulation of membrane composition is accompanied by complex lipid homeostatic changes that can impact amyloidogenic processing in different ways and elicit distinct γ-secretase responses, providing critical implications for lipid-based AD treatment strategies.

| S-EPMC10070668 | biostudies-literature

Acute gamma-secretase inhibition of nonhuman primate CNS shifts amyloid precursor protein (APP) metabolism from amyloid-beta production to alternative APP fragments without amyloid-beta rebound.

Project description:The accumulation of amyloid beta (Abeta) in Alzheimer's disease is caused by an imbalance of production and clearance, which leads to increased soluble Abeta species and extracellular plaque formation in the brain. Multiple Abeta-lowering therapies are currently in development: an important goal is to characterize the molecular mechanisms of action and effects on physiological processing of Abeta, as well as other amyloid precursor protein (APP) metabolites, in models which approximate human Abeta physiology. To this end, we report the translation of the human in vivo stable-isotope-labeling kinetics (SILK) method to a rhesus monkey cisterna magna ported (CMP) nonhuman primate model, and use the model to test the mechanisms of action of a gamma-secretase inhibitor (GSI). A major concern of inhibiting the enzymes which produce Abeta (beta- and gamma-secretase) is that precursors of Abeta may accumulate and cause a rapid increase in Abeta production when enzyme inhibition discontinues. In this study, the GSI MK-0752 was administered to conscious CMP rhesus monkeys in conjunction with in vivo stable-isotope-labeling, and dose-dependently reduced newly generated CNS Abeta. In contrast to systemic Abeta metabolism, CNS Abeta production was not increased after the GSI was cleared. These results indicate that most of the CNS APP was metabolized to products other than Abeta, including C-terminal truncated forms of Abeta: 1-14, 1-15 and 1-16; this demonstrates an alternative degradation pathway for CNS amyloid precursor protein during gamma-secretase inhibition.

| S-EPMC2913973 | biostudies-literature

Amyloid precursor protein (APP) processing genes and cerebrospinal fluid APP cleavage product levels in Alzheimer's disease.

Project description:The aim of this exploratory investigation was to determine if genetic variation within amyloid precursor protein (APP) or its processing enzymes correlates with APP cleavage product levels: APP?, APP? or A?42, in cerebrospinal fluid (CSF) of cognitively normal subjects or Alzheimer's disease (AD) patients. Cognitively normal control subjects (n = 170) and AD patients (n = 92) were genotyped for 19 putative regulatory tagging SNPs within 9 genes (APP, ADAM10, BACE1, BACE2, PSEN1, PSEN2, PEN2, NCSTN and APH1B) involved in the APP processing pathway. SNP genotypes were tested for their association with CSF APP?, APP?, and A?42, AD risk and age-at-onset while taking into account age, gender, race and APOE ?4. After adjusting for multiple comparisons, a significant association was found between ADAM10 SNP rs514049 and APP? levels. In controls, the rs514049 CC genotype had higher APP? levels than the CA, AA collapsed genotype, whereas the opposite effect was seen in AD patients. These results suggest that genetic variation within ADAM10, an APP processing gene, influences CSF APP? levels in an AD specific manner.

| S-EPMC3065534 | biostudies-literature

Extracellular interface between APP and Nicastrin regulates Aβ length and response to γ-secretase modulators.

Project description: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.

| S-EPMC6576158 | biostudies-literature

Eta-secretase-like processing of the amyloid precursor protein (APP) by the rhomboid protease RHBDL4.

Project description:The amyloid precursor protein (APP) is a key protein in Alzheimer's disease synthesized in the endoplasmic reticulum (ER) and translocated to the plasma membrane where it undergoes proteolytic cleavages by several proteases. Conversely, to other known proteases, we previously elucidated rhomboid protease RHBDL4 as a novel APP processing enzyme where several cleavages likely occur already in the ER. Interestingly, the pattern of RHBDL4-derived large APP C-terminal fragments resembles those generated by the η-secretase or MT5-MMP, which was described to generate so-called Aη fragments. The similarity in large APP C-terminal fragments between both proteases raised the question of whether RHBDL4 may contribute to η-secretase activity and Aη-like fragments. Here, we identified two cleavage sites of RHBDL4 in APP by mass spectrometry, which, intriguingly, lie in close proximity to the MT5-MMP cleavage sites. Indeed, we observed that RHBDL4 generates Aη-like fragments in vitro without contributions of α-, β-, or γ-secretases. Such Aη-like fragments are likely generated in the ER since RHBDL4-derived APP-C-terminal fragments do not reach the cell surface. Inherited, familial APP mutations appear to not affect this processing pathway. In RHBDL4 knockout mice, we observed increased cerebral full-length APP in comparison to wild type (WT) in support of RHBDL4 being a physiologically relevant protease for APP. Furthermore, we found secreted Aη fragments in dissociated mixed cortical cultures from WT mice, however significantly fewer Aη fragments in RHBDL4 knockout cultures. Our data underscores that RHBDL4 contributes to the η-secretease-like processing of APP and that RHBDL4 is a physiologically relevant protease for APP.

| S-EPMC11345391 | biostudies-literature

Interaction of the cytosolic domains of sorLA/LR11 with the amyloid precursor protein (APP) and beta-secretase beta-site APP-cleaving enzyme.

Project description:sorLA is a recently identified neuronal receptor for amyloid precursor protein (APP) that is known to interact with APP and affect its intracellular transport and processing. Decreased levels of sorLA in the brain of Alzheimer's disease (AD) patients and elevated levels of amyloid-beta peptide (Abeta) in sorLA-deficient mice point to the importance of the receptor in this neurodegenerative disorder. We analyzed APP cleavage in an APP-shedding assay and found that both sorLA and, surprisingly, a sorLA tail construct inhibited APP cleavage in a beta-site APP-cleaving enzyme (BACE)-dependent manner. In line with this finding, sorLA and the sorLA tail significantly reduced secreted Abeta levels when BACE was overexpressed, suggesting that sorLA influences beta-cleavage. To understand the effect of sorLA on APP cleavage by BACE, we analyzed whether sorLA interacts with APP and/or BACE. Because both full-length sorLA and sorLA C-terminal tail constructs were functionally relevant for APP processing, we analyzed sorLA-APP for a potential cytoplasmatic interaction domain. sorLA and C99 coimmunoprecipitated, pointing toward the existence of a new cytoplasmatic interaction site between sorLA and APP. Moreover, sorLA and BACE also coimmunoprecipitate. Thus, sorLA interacts both with BACE and APP and might therefore directly affect BACE-APP complex formation. To test whether sorLA impacts BACE-APP interactions, we used a fluorescence resonance energy transfer assay to evaluate BACE-APP interactions in cells. We discovered that sorLA significantly reduced BACE-APP interactions in Golgi. We postulate that sorLA acts as a trafficking receptor that prevents BACE-APP interactions and hence BACE cleavage of APP.

| S-EPMC6674411 | biostudies-literature

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