ABSTRACT
Ginkgo biloba (GB) extracts have been used in clinical studies as an alternative therapy for Alzheimer's disease (AD), but the exact bioaction mechanism has not yet been elucidated. In this work, an in silico study on GB metabolites was carried out using SwissTargetPrediction to determine the proteins associated with AD. The resulting proteins, AChE, MAO-A, MAO-B, ß-secretase and γ-secretase, were studied by molecular docking, resulting in the finding that kaempferol, quercetin, and luteolin have multitarget potential against AD. These compounds also exhibit antioxidant activity towards reactive oxygen species (ROS), so antioxidant tests were performed on the extracts using the DPPH and ABTS techniques. The ethanol and ethyl acetate GB extracts showed an important inhibition percentage, higher than 80%, at a dose of 0.01 mg/mL. The effect of GB extracts on AD resulted in multitarget action through two pathways: firstly, inhibiting enzymes responsible for degrading neurotransmitters and forming amyloid plaques; secondly, decreasing ROS in the central nervous system (CNS), reducing its deterioration, and promoting the formation of amyloid plaques. The results of this work demonstrate the great potential of GB as a medicinal plant.
ABSTRACT
Background: Infiltration is a life-threatening growth pattern in malignant astrocytomas and a significant cause of therapy resistance. It results in the tumor cell spreading deeply into the surrounding brain tissue, fostering tumor recurrence and making complete surgical resection impossible. We need to thoroughly understand the mechanisms underlying diffuse infiltration to develop effective therapies. Methods: We integrated in vitro and in vivo functional assays, RNA sequencing, clinical, and expression information from public data sets to investigate the role of ADAM23 expression coupling astrocytoma's growth and motility. Results: ADAM23 downregulation resulted in increased infiltration, reduced tumor growth, and improved overall survival in astrocytomas. Additionally, we show that ADAM23 deficiency induces γ-secretase (GS) complex activity, contributing to the production and deposition of the Amyloid-ß and release of NICD. Finally, GS ablation in ADAM23-low astrocytomas induced a significant inhibitory effect on the invasive programs. Conclusions: Our findings reveal a role for ADAM23 in regulating the balance between cell proliferation and invasiveness in astrocytoma cells, proposing GS inhibition as a therapeutic option in ADAM23 low-expressing astrocytomas.
ABSTRACT
γ-Secretase (GS) is an intramembrane aspartyl protease that participates in the sequential cleavage of C99 to generate different isoforms of the amyloid-ß (Aß) peptides that are associated with the development of Alzheimer's disease. Due to its importance in the proteolytic processing of C99 by GS, we performed pH replica exchange molecular dynamics (pH-REMD) simulations of GS in its apo and substrate-bound forms to sample the protonation states of the catalytic dyad. We found that the catalytic dyad is deprotonated at physiological pH in our apo form, but the presence of the substrate at the active site displaces its monoprotonated state toward physiological pH. Our results show that Asp257 acts as the general base and Asp385 as the general acid during the cleavage mechanism. We identified different amino acids such as Lys265, Arg269, and the PAL motif interacting with the catalytic dyad and promoting changes in its acid-base behavior. Finally, we also found a significant pKa shift of Glu280 related to the internalization of TM6-CT in the GS-apo form. Our study provides critical mechanistic insight into the GS mechanism and the basis for future research on the genesis of Aß peptides and the development of Alzheimer's disease.
Subject(s)
Alzheimer Disease , Amyloid Precursor Protein Secretases , Humans , Amyloid Precursor Protein Secretases/metabolism , Alzheimer Disease/metabolism , Amyloid beta-Peptides/chemistry , Catalysis , Molecular Dynamics Simulation , Amyloid beta-Protein Precursor/metabolismABSTRACT
Nicastrin (NICT) is a transmembrane protein physically associated with the polytypical aspartyl protease presenilin that plays a vital role in the correct localization and stabilization of presenilin to the membrane-bound γ-secretase complex. This complex is involved in the regulation of a wide range of cellular events, including cell signaling and the regulation of endocytosed membrane proteins for their trafficking and protein processing. Methods: In Trypanosoma cruzi, the causal agent of the Chagas disease, a NICT-like protein (Tc/NICT) was identified with a short C-terminus orthologous to the human protein, a large ectodomain (ECD) with numerous glycosylation sites and a single-core transmembrane domain containing a putative TM-domain (457GSVGA461) important for the γ-secretase complex activity. Results: Using the Spot-synthesis strategy with Chagasic patient sera, five extracellular epitopes were identified and synthetic forms were used to generate rabbit anti-Tc/NICT polyclonal serum that recognized a ~72-kDa molecule in immunoblots of T. cruzi epimastigote extracts. Confocal microscopy suggests that Tc/NICT is localized in the flagellar pocket, which is consistent with data from our previous studies with a T. cruzi presenilin-like protein. Phylogenetically, Tc/NICT was localized within a subgroup with the T. rangeli protein that is clearly detached from the other Trypanosomatidae, such as T. brucei. These results, together with a comparative analysis of the selected peptide sequence regions between the T. cruzi and mammalian proteins, suggest a divergence from the human NICT that might be relevant to Chagas disease pathology. As a whole, our data show that a NICT-like protein is expressed in the infective and replicative stages of T. cruzi and may be considered further evidence for a γ-secretase complex in trypanosomatids.
ABSTRACT
γ-Secretase (GS) is one of the most attractive molecular targets for the treatment of Alzheimer's disease (AD). Its key role in the final step of amyloid-ß peptides generation and its relationship in the cascade of events for disease development have caught the attention of many pharmaceutical groups. Over the past years, different inhibitors and modulators have been evaluated as promising therapeutics against AD. However, despite the great chemical diversity of the reported compounds, a global classification and visual representation of the chemical space for GS inhibitors and modulators remain unavailable. In the present work, we carried out a two-dimensional (2D) chemical space analysis from different classes and subclasses of GS inhibitors and modulators based on their structural similarity. Along with the novel structural information available for GS complexes, our analysis opens the possibility to identify compounds with high molecular similarity, critical to finding new chemical structures through the optimization of existing compounds and relating them with a potential binding site.
Subject(s)
Alzheimer Disease , Amyloid Precursor Protein Secretases , Alzheimer Disease/drug therapy , Amyloid Precursor Protein Secretases/metabolism , Amyloid beta-Peptides , Binding Sites , Enzyme Inhibitors/pharmacology , HumansABSTRACT
Alzheimer's disease (AD) is associated with marked atrophy of the cerebral cortex and accumulation of amyloid plaques and neurofibrillary tangles. Amyloid plaques are formed by oligomers of amyloid-ß (Aß) in the brain, with a length of 42 and 40 amino acids. α-secretase cleaves amyloid-ß protein precursor (AßPP) producing the membrane-bound fragment CTFα and the soluble fragment sAßPPα with neuroprotective activity; ß-secretase produces membrane-bound fragment CTFß and a soluble fragment sAßPPß. After α-secretase cleavage of AßPP, γ-secretase cleaves CTFα to produce the cytoplasmic fragment AICD and P3 in the non-amyloidogenic pathway. CTFß is cleaved by γ-secretase producing AICD as well as Aß in amyloidogenic pathways. In the last years, the study of natural products and synthetic compounds, such as α-secretase activity enhancers, ß-secretase inhibitors (BACE-1), and γ-secretase activity modulators, have been the focus of pharmaceuticals and researchers. Drugs were improved regarding solubility, blood-brain barrier penetration, selectivity, and potency decreasing Aß42. In this regard, BACE-1 inhibitors, such as Atabecestat, NB-360, Umibecestat, PF-06751979 Verubecestat, LY2886721, Lanabecestat, LY2811376 and Elenbecestat, were submitted to phase I-III clinical trials. However, inhibition of Aß production did not recover cognitive functions or reverse disease progress. Novel strategies are being developed, aiming at a partial reduction of Aß production, such as the development of γ-secretase modulators or α-secretase activity enhancers. Such therapeutic tools shall focus on slowing down or minimizing the progression of neuronal damage. Here, we summarize structures and activities of the latest compounds designed for AD treatment, with remarkable in vitro, in vivo, and clinical phase activities.
Subject(s)
Alzheimer Disease/drug therapy , Amyloid Precursor Protein Secretases/antagonists & inhibitors , Amyloid Precursor Protein Secretases/metabolism , Neuroprotective Agents/therapeutic use , Animals , HumansABSTRACT
Anterior pharynx-defective 1A (APH-1A) is a seven transmembrane component of γ-secretase (GS), an aspartyl protease enzyme involved in the production of toxic amyloid-ß peptides in Alzheimer's disease patients. Cryo-electron microscopy structures of the enzyme complex revealed a central cavity in its APH-1A component, similar to water-containing cavities in G-protein coupled receptors (GPCRs). In this work, we performed molecular dynamics and umbrella sampling simulations to understand the role of the APH-1A cavity in the GS complex. Our results suggest that APH-1A is able to store water molecules in its inner cavity and transport some of them between cell spaces. Additionally, APH-1A allows the influx of extracellular cations into a central hydrophilic cavity but cannot transport them into the intracellular space. Overall, this study seeks to describe an alternative APH-1A function in GS besides its complex stabilization role and provide novel approaches to understand the functioning of the GS enzyme.
Subject(s)
Amyloid Precursor Protein Secretases/chemistry , Endopeptidases/chemistry , Membrane Proteins/chemistry , Humans , Molecular Dynamics Simulation , WaterABSTRACT
DAPT is a potent γ-secretase (GS) inhibitor that blocks the production of short amyloid-ß (Aß) peptides. Aggregation and oligomerization of Aß peptides have been associated with the development and progression of Alzheimer's disease. A recent cryo-electron microscopy density map disclosed DAPT binding at the GS active site. In this study, we employed the density map data to assign a possible binding pose of DAPT to characterize its dynamic behavior through different molecular dynamics simulation approaches. Our simulations showed a high preference of DAPT for the intramembrane region of the protein and that its entry site is located between TM2 and TM3 of PS1. DAPT interaction with the active site led to a decreased flexibility of key PS1 regions related to the recognition and internalization of GS substrates. Moreover, our study showed that the proximity of DAPT to the catalytic aspartic acids should be able to modify its protonation states, preventing the enzyme from reaching its active form. These results provide valuable information toward understanding the molecular mechanism of a GS inhibitor for the development of novel Alzheimer's disease treatments.
Subject(s)
Amyloid Precursor Protein Secretases/metabolism , Diamines/chemistry , Enzyme Inhibitors/chemistry , Thiazoles/chemistry , Amino Acid Sequence , Amyloid beta-Peptides/chemistry , Aspartic Acid/chemistry , Catalytic Domain , Molecular Dynamics Simulation , Protein Binding , Protein Conformation , ThermodynamicsABSTRACT
γ-secretase is an intra-membrane aspartyl protease involved in the production of amyloid-ß peptides. Aberrant cleavage of the 99-residue C-terminal fragment of the amyloid precursor protein leads to the formation of a 42-amino-acid isoform (Aß42). Further oligomerization and aggregation of this isoform is implicated in the onset and progression of Alzheimer's disease. Recent elucidation of γ-secretase by cryo-electron microscopy techniques have opened a new horizon in the structural and dynamic characterization of the enzyme. Currently, only a few molecular dynamics studies have been carried out to explore the mechanism of substrate recognition and entry, or the transition between active and inactive states of the catalytic subunit. Herein, we briefly review the computational approaches and their most relevant findings. The general picture of the current GS simulation studies will open new questions to understand the behavior of the enzyme dynamics and explain the modulation mechanisms for the treatment of Alzheimer's disease.
Subject(s)
Amyloid Precursor Protein Secretases/metabolism , Models, Molecular , Amyloid beta-Protein Precursor/chemistry , Amyloid beta-Protein Precursor/metabolism , Humans , Peptide Fragments/metabolism , Protein TransportABSTRACT
A wide variety of cellular processes and signaling events are regulated by the proteolytic enzyme γ-secretase. Notch-1 is one of the substrates of γ-secretase and its role in the regulation of muscle differentiation has been well described. Importantly, besides Notch-1, a number of proteins have been identified to undergo proteolysis by γ-secretase. To date, the specific role of γ-secretase during embryonic skeletal muscle differentiation has not been studied. Therefore, we address this question through the analysis of in vitro grown chick myogenic cells during the formation of multinucleated myotubes. The γ-secretase inhibitor DAPT (N-N[-(3,5-Difluorophenacetyl-l-alanyl)]-S-328 phenylglycine-t-butyl-ester) induces muscle hypertrophy. Knockdown of Notch-1 using siRNA specific to chick shows no significant effect in myotube size, suggesting that γ-secretase-dependent effects on muscle hypertrophy in chick myogenic cells are Notch-1-independent. We also investigate the effects of γ-secretase inhibition in the whole proteomic profile of chick myogenic cells. We identified 276 differentially expressed proteins from Label-free proteomic approach. Data overview of interaction network obtained from STRING show that after γ-secretase inhibition cells exhibited imbalance in protein metabolism, cytoskeleton/adhesion, and Sonic Hedgehog signaling. The collection of these results provides new insights into the role of γ-secretase in skeletal muscle hypertrophy.
Subject(s)
Amyloid Precursor Protein Secretases/antagonists & inhibitors , Avian Proteins/metabolism , Diamines/toxicity , Hypertrophy/veterinary , Muscle Proteins/metabolism , Muscular Diseases/veterinary , Receptors, Notch/metabolism , Thiazoles/toxicity , Animals , Cells, Cultured , Chick Embryo , Hypertrophy/chemically induced , Hypertrophy/physiopathology , Muscular Diseases/chemically induced , Muscular Diseases/physiopathology , Protein Interaction Maps , Proteomics , Signal TransductionABSTRACT
Presenilin 1 (PSEN1) and presenilin 2 (PSEN2) genes encode the major component of y-secretase, which is responsible for sequential proteolytic cleavages of amyloid precursor proteins and the subsequent formation of amyloid-ß peptides. 150 RNA samples from the entorhinal cortex, auditory cortex and hippocampal regions of individuals with Alzheimer's disease (AD) and controls elderly subjects were analyzed with using real-time rtPCR. There were no differences between groups for PSEN1 expression. PSEN2 was significantly downregulated in the auditory cortex of AD patients when compared to controls and when compared to other brain regions of the patients. Alteration in PSEN2 expression may be a risk factor for AD.