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.
Colorectal cancer (CRC) cells undergo the remodeling of intracellular Ca
homeostasis, which contributes to cancer hallmarks such as enhanced proliferation, invasion and survival. Ca
remodeling ...includes critical changes in store-operated Ca
entry (SOCE) and Ca
store content. Some changes have been investigated at the molecular level. However, since nearly 100 genes are involved in intracellular Ca
transport, a comprehensive view of Ca
remodeling in CRC is lacking. We have used Next Generation Sequencing (NGS) to investigate differences in expression of 77 selected gene transcripts involved in intracellular Ca
transport in CRC. To this end, mRNA from normal human colonic NCM460 cells and human colon cancer HT29 cells was isolated and used as a template for transcriptomic sequencing and expression analysis using Ion Torrent technology. After data transformation and filtering, exploratory analysis revealed that both cell types were well segregated. In addition, differential gene expression using R and bioconductor packages show significant differences in expression of selected voltage-operated Ca
channels and store-operated Ca
entry players, transient receptor potential (TRP) channels, Ca
release channels, Ca
pumps, Na⁺/Ca
exchanger isoforms and genes involved in mitochondrial Ca
transport. These data provide the first comprehensive transcriptomic analysis of Ca
remodeling in CRC.
The protein corona formed on the surface of a nanoparticle in a biological medium determines its behavior in vivo. Herein, iron oxide nanoparticles containing the same core and shell, but bearing two ...different surface coatings, either glucose or poly(ethylene glycol), were evaluated. The nanoparticles’ protein adsorption, in vitro degradation, and in vivo biodistribution and biotransformation over four months were investigated. Although both types of nanoparticles bound similar amounts of proteins in vitro, the differences in the protein corona composition correlated to the nanoparticles biodistribution in vivo. Interestingly, in vitro degradation studies demonstrated faster degradation for nanoparticles functionalized with glucose, whereas the in vivo results were opposite with accelerated biodegradation and clearance of the nanoparticles functionalized with poly(ethylene glycol). Therefore, the variation in the degradation rate observed in vivo could be related not only to the molecules attached to the surface, but also with the associated protein corona, as the key role of the adsorbed proteins on the magnetic core degradation has been demonstrated in vitro.
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.
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.
Cerebral accumulation of amyloid-β (Aβ) initiates molecular and cellular cascades that lead to Alzheimer’s disease (AD). However, amyloid deposition does not invariably lead to dementia. ...Amyloid-positive but cognitively unaffected (AP-CU) individuals present widespread amyloid pathology, suggesting that molecular signatures more complex than the total amyloid burden are required to better differentiate AD from AP-CU cases. Motivated by the essential role of Aβ and the key lipid involvement in AD pathogenesis, we applied multimodal mass spectrometry imaging (MSI) and machine learning (ML) to investigate amyloid plaque heterogeneity, regarding Aβ and lipid composition, in AP-CU versus AD brain samples at the single-plaque level. Instead of focusing on a population mean, our analytical approach allowed the investigation of large populations of plaques at the single-plaque level. We found that different (sub)populations of amyloid plaques, differing in Aβ and lipid composition, coexist in the brain samples studied. The integration of MSI data with ML-based feature extraction further revealed that plaque-associated gangliosides GM2 and GM1, as well as Aβ1–38, but not Aβ1–42, are relevant differentiators between the investigated pathologies. The pinpointed differences may guide further fundamental research investigating the role of amyloid plaque heterogeneity in AD pathogenesis/progression and may provide molecular clues for further development of emerging immunotherapies to effectively target toxic amyloid assemblies in AD therapy. Our study exemplifies how an integrative analytical strategy facilitates the unraveling of complex biochemical phenomena, advancing our understanding of AD from an analytical perspective and offering potential avenues for the refinement of diagnostic tools.
γ-Secretases are a group of widely expressed, intramembrane-cleaving proteases involved in many physiological processes. Their clinical relevance comes from their involvement in Alzheimer's disease, ...cancer, and other disorders. A clinical trial with the wide-spectrum γ-secretase inhibitor semagacestat has, however, demonstrated that global inhibition of all γ-secretases causes serious toxicity. Evolving insights suggest that selective inhibition of one of these proteases, or more subtle modulation of γ-secretases by stimulating their carboxypeptidase-like activity but sparing their endopeptidase activity, are potentially highly interesting approaches. The rapidly growing knowledge of regulation, assembly, and specificity of these intriguing protein complexes and the potential advent of high-resolution structural information could dramatically change the perspective on safe and efficacious γ-secretase inhibition in various disorders.
Inhibition of γ-secretase, an intramembrane protease, to reduce secretion of Amyloid-β (Aβ) peptides has been considered for treating Alzheimer’s disease. However, γ-secretase inhibitors suffer from ...severe side effects. As an alternative, γ-secretase modulators (GSM) reduce the generation of toxic peptides by enhancing the cleavage processivity without diminishing the enzyme activity. Starting from a known γ-secretase structure without substrate but in complex with an E2012 GSM, we generated a structural model that included a bound Aβ43 peptide and studied interactions among enzyme, substrate, GSM, and lipids. Our result suggests that E2012 binding at the enzyme–substrate–membrane interface attenuates the membrane distortion by shielding the substrate–membrane interaction. The model predicts that the E2012 modulation is charge-dependent and explains the preserved hydrogen acceptor and the aromatic ring observed in many imidazole-based GSM. Predicted effects of γ-secretase mutations on E2012 modulation were confirmed experimentally. We anticipate that the study will facilitate the future development of effective GSMs.
Magnetic hyperthermia (MH) was used to treat a murine model of pancreatic cancer. This type of cancer is generally characterized by the presence of dense stroma that acts as a barrier for ...chemotherapeutic treatments. Several alternating magnetic field (AMF) conditions were evaluated using three-dimensional (3D) cell culture models loaded with magnetic nanoparticles (MNPs) to determine which conditions were producing a strong effect on the cell viability. Once the optimal AMF conditions were selected, in vivo experiments were carried out using similar frequency and field amplitude parameters. A marker of the immune response activation, calreticulin (CALR), was evaluated in cells from a xenograft tumor model after the MH treatment. Moreover, the distribution of nanoparticles within the tumor tissue was assessed by histological analysis of tumor sections, observing that the exposure to the alternating magnetic field resulted in the migration of particles toward the inner parts of the tumor. Finally, a relationship between an inadequate body biodistribution of the particles after their intratumoral injection and a significant decrease in the effectiveness of the MH treatment was found. Animals in which most of the particles remained in the tumor area after injection showed higher reductions in the tumor volume growth in comparison with those animals in which part of the particles were found also in the liver and spleen. Therefore, our results point out several factors that should be considered to improve the treatment effectiveness of pancreatic cancer by magnetic hyperthermia.
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