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1.
J Phys Chem B ; 128(20): 4975-4985, 2024 May 23.
Article in English | MEDLINE | ID: mdl-38743687

ABSTRACT

The primary event in chemical neurotransmission involves the fusion of a membrane-limited vesicle at the plasma membrane and the subsequent release of its chemical neurotransmitter cargo. The cargo itself is not known to have any effect on the fusion event. However, amphiphilic monoamine neurotransmitters (e.g., serotonin and dopamine) are known to strongly interact with lipid bilayers and to affect their mechanical properties, which can in principle impact membrane-mediated processes. Here, we probe whether serotonin can enhance the association and fusion of artificial lipid vesicles in vitro. We employ fluorescence correlation spectroscopy and total internal reflection fluorescence microscopy to measure the attachment and fusion of vesicles whose lipid compositions mimic the major lipid components of synaptic vesicles. We find that the association between vesicles and supported lipid bilayers is strongly enhanced in a serotonin dose-dependent manner, and this drives an increase in the rate of spontaneous fusion. Molecular dynamics simulations and fluorescence spectroscopy data show that serotonin insertion increases the water content of the hydrophobic part of the bilayer. This suggests that the enhanced membrane association is likely driven by an energetically favorable drying transition. Other monoamines, such as dopamine and norepinephrine, but not other related species, such as tryptophan, show similar effects on membrane association. Our results reveal a lipid bilayer-mediated mechanism by which monoamines can themselves modulate vesicle fusion, potentially adding to the control toolbox for the tightly regulated process of neurotransmission in vivo.


Subject(s)
Lipid Bilayers , Molecular Dynamics Simulation , Serotonin , Lipid Bilayers/chemistry , Lipid Bilayers/metabolism , Serotonin/chemistry , Serotonin/metabolism , Membrane Fusion , Synaptic Vesicles/metabolism , Synaptic Vesicles/chemistry , Spectrometry, Fluorescence , Hydrophobic and Hydrophilic Interactions
2.
Chem Commun (Camb) ; 60(37): 4926-4929, 2024 May 02.
Article in English | MEDLINE | ID: mdl-38629227

ABSTRACT

A turn-on fluorescence aptasensing approach for the highly sensitive and selective determination of 5-HT was proposed via target-induced knot displacement. 5-HT can be determined in a range from 0.5 nM to 100 nM with a limit of detection as low as 0.1 nM and a low dissociation constant of 2.3 nM.


Subject(s)
Aptamers, Nucleotide , Biosensing Techniques , Fluorescent Dyes , Serotonin , Spectrometry, Fluorescence , Aptamers, Nucleotide/chemistry , Serotonin/analysis , Serotonin/chemistry , Biosensing Techniques/methods , Fluorescent Dyes/chemistry , Limit of Detection , Humans , Fluorescence
3.
Phys Chem Chem Phys ; 26(18): 13751-13761, 2024 May 08.
Article in English | MEDLINE | ID: mdl-38683175

ABSTRACT

Understanding the dynamics of neurotransmitters is crucial for unraveling synaptic transmission mechanisms in neuroscience. In this study, we investigated the impact of terahertz (THz) waves on the aggregation of four common neurotransmitters through all-atom molecular dynamics (MD) simulations. The simulations revealed enhanced nicotine (NCT) aggregation under 11.05 and 21.44 THz, with a minimal effect at 42.55 THz. Structural analysis further indicated strengthened intermolecular interactions and weakened hydration effects under specific THz stimulation. In addition, enhanced aggregation was observed at stronger field strengths, particularly at 21.44 THz. Furthermore, similar investigations on epinephrine (EPI), 5-hydroxytryptamine (5-HT), and γ-aminobutyric acid (GABA) corroborated these findings. Notably, EPI showed increased aggregation at 19.05 THz, emphasizing the influence of vibrational modes on aggregation. However, 5-HT and GABA, with charged or hydrophilic functional groups, exhibited minimal aggregation under THz stimulation. The present study sheds some light on neurotransmitter responses to THz waves, offering implications for neuroscience and interdisciplinary applications.


Subject(s)
Molecular Dynamics Simulation , Neurotransmitter Agents , Serotonin , Terahertz Radiation , gamma-Aminobutyric Acid , Neurotransmitter Agents/chemistry , gamma-Aminobutyric Acid/chemistry , Serotonin/chemistry , Serotonin/metabolism , Nicotine/chemistry , Epinephrine/chemistry
4.
Nature ; 629(8010): 235-243, 2024 May.
Article in English | MEDLINE | ID: mdl-38499039

ABSTRACT

Biogenic monoamines-vital transmitters orchestrating neurological, endocrinal and immunological functions1-5-are stored in secretory vesicles by vesicular monoamine transporters (VMATs) for controlled quantal release6,7. Harnessing proton antiport, VMATs enrich monoamines around 10,000-fold and sequester neurotoxicants to protect neurons8-10. VMATs are targeted by an arsenal of therapeutic drugs and imaging agents to treat and monitor neurodegenerative disorders, hypertension and drug addiction1,8,11-16. However, the structural mechanisms underlying these actions remain unclear. Here we report eight cryo-electron microscopy structures of human VMAT1 in unbound form and in complex with four monoamines (dopamine, noradrenaline, serotonin and histamine), the Parkinsonism-inducing MPP+, the psychostimulant amphetamine and the antihypertensive drug reserpine. Reserpine binding captures a cytoplasmic-open conformation, whereas the other structures show a lumenal-open conformation stabilized by extensive gating interactions. The favoured transition to this lumenal-open state contributes to monoamine accumulation, while protonation facilitates the cytoplasmic-open transition and concurrently prevents monoamine binding to avoid unintended depletion. Monoamines and neurotoxicants share a binding pocket that possesses polar sites for specificity and a wrist-and-fist shape for versatility. Variations in this pocket explain substrate preferences across the SLC18 family. Overall, these structural insights and supporting functional studies elucidate the mechanism of vesicular monoamine transport and provide the basis to develop therapeutics for neurodegenerative diseases and substance abuse.


Subject(s)
Biogenic Monoamines , Drug Interactions , Vesicular Monoamine Transport Proteins , Humans , 1-Methyl-4-phenylpyridinium/chemistry , 1-Methyl-4-phenylpyridinium/metabolism , 1-Methyl-4-phenylpyridinium/pharmacology , Amphetamine/chemistry , Amphetamine/pharmacology , Amphetamine/metabolism , Binding Sites , Biogenic Monoamines/chemistry , Biogenic Monoamines/metabolism , Cryoelectron Microscopy , Dopamine/chemistry , Dopamine/metabolism , Models, Molecular , Norepinephrine/chemistry , Norepinephrine/metabolism , Protein Binding , Protons , Reserpine/pharmacology , Reserpine/chemistry , Reserpine/metabolism , Serotonin/chemistry , Serotonin/metabolism , Substrate Specificity , Vesicular Monoamine Transport Proteins/chemistry , Vesicular Monoamine Transport Proteins/metabolism , Vesicular Monoamine Transport Proteins/ultrastructure
5.
Chembiochem ; 25(9): e202400069, 2024 May 02.
Article in English | MEDLINE | ID: mdl-38504591

ABSTRACT

Arylalkylamine N-acetyltransferase (AANAT) serves as a key enzyme in the biosynthesis of melatonin by transforming 5-hydroxytryptamine (5-HT) to N-acetyl-5-hydroxytryptamine (NAS), while its low activity may hinder melatonin yield. In this study, a novel AANAT derived from Sus scrofa (SsAANAT) was identified through data mining using 5-HT as a model substrate, and a rational design of SsAANAT was conducted in the quest to improving its activity. After four rounds of mutagenesis procedures, a triple combinatorial dominant mutant M3 was successfully obtained. Compared to the parent enzyme, the conversion of the whole-cell reaction bearing the best variant M3 exhibted an increase from 50 % to 99 % in the transformation of 5-HT into NAS. Additionally, its catalytic efficiency (kcat/Km) was enhanced by 2-fold while retaining the thermostability (Tm>45 °C). In the up-scaled reaction with a substrate loading of 50 mM, the whole-cell system incorporating variant M3 achieved a 99 % conversion of 5-HT in 30 h with an 80 % yield. Molecular dynamics simulations were ultilized to shed light on the origin of improved activity. This study broadens the repertoire of AANAT for the efficient biosynthesis of melatonin.


Subject(s)
Arylalkylamine N-Acetyltransferase , Serotonin , Arylalkylamine N-Acetyltransferase/metabolism , Arylalkylamine N-Acetyltransferase/genetics , Arylalkylamine N-Acetyltransferase/chemistry , Serotonin/metabolism , Serotonin/chemistry , Serotonin/biosynthesis , Animals , Acetylation , Protein Engineering , Swine
6.
Nat Nanotechnol ; 19(5): 660-667, 2024 May.
Article in English | MEDLINE | ID: mdl-38233588

ABSTRACT

Small molecules such as neurotransmitters are critical for biochemical functions in living systems. While conventional ultraviolet-visible spectroscopy and mass spectrometry lack portability and are unsuitable for time-resolved measurements in situ, techniques such as amperometry and traditional field-effect detection require a large ensemble of molecules to reach detectable signal levels. Here we demonstrate the potential of carbon-nanotube-based single-molecule field-effect transistors (smFETs), which can detect the charge on a single molecule, as a new platform for recognizing and assaying small molecules. smFETs are formed by the covalent attachment of a probe molecule, in our case a DNA aptamer, to a carbon nanotube. Conformation changes on binding are manifest as discrete changes in the nanotube electrical conductance. By monitoring the kinetics of conformational changes in a binding aptamer, we show that smFETs can detect and quantify serotonin at the single-molecule level, providing unique insights into the dynamics of the aptamer-ligand system. In particular, we show the involvement of G-quadruplex formation and the disruption of the native hairpin structure in the conformational changes of the serotonin-aptamer complex. The smFET is a label-free approach to analysing molecular interactions at the single-molecule level with high temporal resolution, providing additional insights into complex biological processes.


Subject(s)
Aptamers, Nucleotide , Nanotubes, Carbon , Serotonin , Transistors, Electronic , Aptamers, Nucleotide/chemistry , Nanotubes, Carbon/chemistry , Kinetics , Ligands , Serotonin/chemistry , Serotonin/metabolism , Biosensing Techniques/methods , Biosensing Techniques/instrumentation
7.
J Biomol Struct Dyn ; 42(2): 993-1014, 2024.
Article in English | MEDLINE | ID: mdl-37021485

ABSTRACT

The human serotonin transporters (hSERTs) are neurotransmitter sodium symporters of the aminergic G protein-coupled receptors, regulating the synaptic serotonin and neuropharmacological processes related to neuropsychiatric disorders, notably, depression. Selective serotonin reuptake inhibitors (SSRIs) such as fluoxetine and (S)-citalopram are competitive inhibitors of hSERTs and are commonly the first-line medications for major depressive disorder (MDD). However, treatment-resistance and unpleasant aftereffects constitute their clinical drawbacks. Interestingly, vilazodone emerged with polypharmacological (competitive and allosteric) inhibitions on hSERTs, amenable to improved efficacy. However, its application usually warrants adjuvant/combination therapy, another subject of critical adverse events. Thus, the discovery of alternatives with polypharmacological potentials (one-drug-multiple-target) and improved safety remains essential. In this study, carbazole analogues from chemical libraries were explored using docking and molecular dynamics (MD) simulation. Selectively, two IBScreen ligands, STOCK3S-30866 and STOCK1N-37454 predictively bound to the active pockets and expanded boundaries (extracellular vestibules) of the hSERTs more potently than vilazodone and (S)-citalopram. For instance, the two ligands showed docking scores of -9.52 and -9.59 kcal/mol and MM-GBSA scores of -92.96 and -65.66 kcal/mol respectively compared to vilazodone's respective scores of -7.828 and -59.27 against the central active site of the hSERT (PDB 7LWD). Similarly, the two ligands also docked to the allosteric pocket (PDB 5I73) with scores of -8.15 and -8.40 kcal/mol and MM-GBSA of -96.14 and -68.46 kcal/mol whereas (S)-citalopram has -6.90 and -69.39 kcal/mol respectively. The ligands also conferred conformational stability on the receptors during 100 ns MD simulations and displayed interesting ADMET profiles, representing promising hSERT modulators for MDD upon experimental validation.Communicated by Ramaswamy H. Sarma.


Subject(s)
Depressive Disorder, Major , Serotonin Plasma Membrane Transport Proteins , Humans , Serotonin Plasma Membrane Transport Proteins/chemistry , Serotonin Plasma Membrane Transport Proteins/metabolism , Vilazodone Hydrochloride , Citalopram/pharmacology , Citalopram/metabolism , Serotonin/chemistry , Serotonin/metabolism , Molecular Dynamics Simulation , Carbazoles/pharmacology , Molecular Docking Simulation
8.
Nature ; 626(7998): 427-434, 2024 Feb.
Article in English | MEDLINE | ID: mdl-38081299

ABSTRACT

Vesicular monoamine transporter 2 (VMAT2) accumulates monoamines in presynaptic vesicles for storage and exocytotic release, and has a vital role in monoaminergic neurotransmission1-3. Dysfunction of monoaminergic systems causes many neurological and psychiatric disorders, including Parkinson's disease, hyperkinetic movement disorders and depression4-6. Suppressing VMAT2 with reserpine and tetrabenazine alleviates symptoms of hypertension and Huntington's disease7,8, respectively. Here we describe cryo-electron microscopy structures of human VMAT2 complexed with serotonin and three clinical drugs at 3.5-2.8 Å, demonstrating the structural basis for transport and inhibition. Reserpine and ketanserin occupy the substrate-binding pocket and lock VMAT2 in cytoplasm-facing and lumen-facing states, respectively, whereas tetrabenazine binds in a VMAT2-specific pocket and traps VMAT2 in an occluded state. The structures in three distinct states also reveal the structural basis of the VMAT2 transport cycle. Our study establishes a structural foundation for the mechanistic understanding of substrate recognition, transport, drug inhibition and pharmacology of VMAT2 while shedding light on the rational design of potential therapeutic agents.


Subject(s)
Cryoelectron Microscopy , Vesicular Monoamine Transport Proteins , Humans , Binding Sites , Cytoplasm/drug effects , Cytoplasm/metabolism , Ketanserin/chemistry , Ketanserin/metabolism , Ketanserin/pharmacology , Reserpine/chemistry , Reserpine/metabolism , Reserpine/pharmacology , Serotonin/chemistry , Serotonin/metabolism , Substrate Specificity , Tetrabenazine/chemistry , Tetrabenazine/metabolism , Tetrabenazine/pharmacology , Vesicular Monoamine Transport Proteins/antagonists & inhibitors , Vesicular Monoamine Transport Proteins/chemistry , Vesicular Monoamine Transport Proteins/metabolism , Vesicular Monoamine Transport Proteins/ultrastructure
9.
Sci Rep ; 13(1): 10239, 2023 06 23.
Article in English | MEDLINE | ID: mdl-37353529

ABSTRACT

In this work, CoNiWO4 nanocomposite was used as an electrochemical sensor for the simultaneous electrochemical detection of tramadol and serotonin. The nanocomposite was synthesized using a hydrothermal method and characterized via XRD, SEM, TGA, Zeta, UV, and FTIR. The sensor was developed by depositing CoNiWO4-NPs onto the glassy carbon electrode surface. Tramadol and serotonin were detected by employing cyclic voltammetry (CV), differential pulse voltammetry (DPV), electrochemical impedance spectroscopy (EIS), and chronoamperometry. Analytes were detected at different pH, concentrations, and scan rates. The prepared sensor showed a 0-60 µM linear range, with a LOD of 0.71 µM and 4.29 µM and LOQ of 14.3 µM and 2.3 µM for serotonin and tramadol, respectively. Finally, the modified electrode (CoNiWO4-GCE) was applied to determine tramadol and serotonin in biological samples.


Subject(s)
Tramadol , Serotonin/chemistry , Nickel/chemistry , Electrochemical Techniques/methods , Limit of Detection , Analgesics , Electrodes , Neurotransmitter Agents
10.
J Chem Inf Model ; 63(4): 1196-1207, 2023 02 27.
Article in English | MEDLINE | ID: mdl-36757760

ABSTRACT

Pentameric ligand-gated ion channels play an important role in mediating fast neurotransmissions. As a member of this receptor family, cation-selective 5-HT3 receptors are a clinical target for treating nausea and vomiting associated with chemotherapy and radiation therapy (Thompson and Lummis, 2006). Multiple cryo-electron microscopy (cryo-EM) structures of 5-HT3 receptors have been determined in distinct functional states (e.g., open, closed, etc.) (Basak et al., 2018; Basak et al., 2018; Polovinkin et al., 2018; Zhang et al., 2015). However, recent work has shown that the transmembrane pores of the open 5-HT3 receptor structures rapidly collapse and become artificially asymmetric in molecular dynamics (MD) simulations. To avoid this hydrophobic collapse, Dämgen and Biggin developed an equilibration protocol that led to a stable open state structure of the glycine receptor in MD simulations (Dämgen and Biggin, 2020). However, the protocol failed to yield open-like structures of the 5-HT3 receptor in our simulations. Here, we present a refined equilibration protocol that involves the rearrangement of the transmembrane helices to achieve stable open state structures of the 5-HT3 receptor that allow both water and ion permeation through the channel. Notably, channel gating is mediated through collective movement of the transmembrane helices, involving not only pore lining M2 helices but also their cross-talk with the adjacent M1 and M3 helices. Thus, the successful application of our refined equilibration protocol underscores the importance of the conformational coupling between the transmembrane helices in stabilizing open-like structures of the 5-HT3 receptor.


Subject(s)
Molecular Dynamics Simulation , Serotonin , Serotonin/chemistry , Serotonin/metabolism , Cryoelectron Microscopy , Protein Structure, Secondary , Ion Transport , Receptors, Serotonin, 5-HT3/chemistry , Receptors, Serotonin, 5-HT3/metabolism
11.
Article in English | MEDLINE | ID: mdl-36078233

ABSTRACT

The aim of this study was to evaluate the influence of ß-endorphins and serotonin on the course of treatment, disease-free time, and overall survival of patients with ovarian cancer. This study may contribute to the identification of modifiable factors that may influence the treatment of ovarian cancer. The research was carried out in a group of 162 patients of which 139 respondents were included in the research; ovarian cancer was diagnosed in 78 of these patients. The study consisted of three stages. In the first stage of diagnostics, a survey among the patients was carried out. In the second stage-5 mL of blood was collected from each patient (n = 139) in the preoperative period to determine the concentration of ß-endorphin and serotonin. In the third stage-blood samples were collected from those patients who had completed chemotherapy treatment or had surgery. Concentrations of ß-endorphin and serotonin were measured by the Luminex method, using the commercial Luminex Human Discovery Assay kit. The average age of the patients was 62.99 years. The level of ß-endorphin significantly differs among patients diagnosed with ovarian cancer and among patients in the control group (202.86; SD-15.78 vs. 302.00; SD-24.49). A lower level of ß-endorphins was found in the patients with a recurrence of the neoplastic process compared to those without recurrence (178.84; SD-12.98 vs. 205.66; SD-13.37). On the other hand, the level of serotonin before chemotherapy was higher in the group of people with disease recurrence compared to those without recurrence (141.53; SD-15.33 vs. 134.99; SD-10.08). Statistically significantly positive correlations were found between the level of ß-endorphin and both disease-free time (ß-endorphin levels before chemotherapy: rho Spearman 0.379, p < 0.027; ß-endorphin levels after chemotherapy: rho Spearman 0.734 p < 0.001) and survival time (ß-endorphin levels before chemotherapy: rho Spearman 0.267, p < 0.018; ß-endorphin levels after chemotherapy: rho Spearman 0.654 p < 0.001). 1. The levels of serotonin and ß-endorphin levels are significantly related to ovarian cancer and change during treatment. 2. High mean preoperative concentrations of ß-endorphins were significantly related to overall survival and disease-free time.


Subject(s)
Endorphins , Ovarian Neoplasms , Serotonin , beta-Endorphin , Biological Factors , Endorphins/chemistry , Endorphins/metabolism , Female , Humans , Middle Aged , Ovarian Neoplasms/drug therapy , Serotonin/chemistry , Serotonin/metabolism , beta-Endorphin/metabolism
12.
J Biol Chem ; 298(3): 101613, 2022 03.
Article in English | MEDLINE | ID: mdl-35065961

ABSTRACT

The human serotonin transporter (hSERT) terminates neurotransmission by removing serotonin (5HT) from the synaptic cleft, an essential process for proper functioning of serotonergic neurons. Structures of the hSERT have revealed its molecular architecture in four conformations, including the outward-open and occluded states, and show the transporter's engagement with co-transported ions and the binding mode of inhibitors. In this study, we investigated the molecular mechanism by which the hSERT occludes and sequesters the substrate 5HT. This first step of substrate uptake into cells is a structural change consisting of the transition from the outward-open to the occluded state. Inhibitors such as the antidepressants citalopram, fluoxetine, and sertraline inhibit this step of the transport cycle. Using molecular dynamics simulations, we reached a fully occluded state, in which the transporter-bound 5HT becomes fully shielded from both sides of the membrane by two closed hydrophobic gates. Analysis of 5HT-triggered occlusion showed that bound 5HT serves as an essential trigger for transporter occlusion. Moreover, simulations revealed a complex sequence of steps and showed that movements of bundle domain helices are only partially correlated. 5HT-triggered occlusion is initially dominated by movements of transmembrane helix 1b, while in the final step, only transmembrane helix 6a moves and relaxes an intermediate change in its secondary structure.


Subject(s)
Serotonin Plasma Membrane Transport Proteins , Serotonin , Citalopram/chemistry , Citalopram/pharmacology , Humans , Molecular Dynamics Simulation , Protein Domains , Protein Structure, Secondary , Serotonin/chemistry , Serotonin/metabolism , Serotonin Plasma Membrane Transport Proteins/chemistry , Serotonin Plasma Membrane Transport Proteins/metabolism , Selective Serotonin Reuptake Inhibitors/chemistry , Selective Serotonin Reuptake Inhibitors/pharmacology , Structure-Activity Relationship
13.
Science ; 375(6579): 403-411, 2022 01 28.
Article in English | MEDLINE | ID: mdl-35084960

ABSTRACT

Drugs that target the human serotonin 2A receptor (5-HT2AR) are used to treat neuropsychiatric diseases; however, many have hallucinogenic effects, hampering their use. Here, we present structures of 5-HT2AR complexed with the psychedelic drugs psilocin (the active metabolite of psilocybin) and d-lysergic acid diethylamide (LSD), as well as the endogenous neurotransmitter serotonin and the nonhallucinogenic psychedelic analog lisuride. Serotonin and psilocin display a second binding mode in addition to the canonical mode, which enabled the design of the psychedelic IHCH-7113 (a substructure of antipsychotic lumateperone) and several 5-HT2AR ß-arrestin-biased agonists that displayed antidepressant-like activity in mice but without hallucinogenic effects. The 5-HT2AR complex structures presented herein and the resulting insights provide a solid foundation for the structure-based design of safe and effective nonhallucinogenic psychedelic analogs with therapeutic effects.


Subject(s)
Antidepressive Agents/pharmacology , Drug Design , Hallucinogens/chemistry , Hallucinogens/pharmacology , Receptor, Serotonin, 5-HT2A/chemistry , Animals , Antidepressive Agents/chemistry , Antidepressive Agents/metabolism , Arrestin/metabolism , Binding Sites , Crystallography, X-Ray , Hallucinations/chemically induced , Hallucinogens/metabolism , Heterocyclic Compounds, 4 or More Rings/chemistry , Humans , Ligands , Lisuride/chemistry , Lisuride/metabolism , Lysergic Acid Diethylamide/chemistry , Lysergic Acid Diethylamide/metabolism , Mice , Protein Conformation , Psilocybin/analogs & derivatives , Psilocybin/chemistry , Psilocybin/metabolism , Receptor, Serotonin, 5-HT2A/metabolism , Serotonin/chemistry , Serotonin/metabolism , Signal Transduction , Structure-Activity Relationship
14.
Mediators Inflamm ; 2021: 6652791, 2021.
Article in English | MEDLINE | ID: mdl-34557056

ABSTRACT

Thymus and Activation-Regulated Chemokine (TARC/CCL17) and Macrophage-Derived Chemokine (MDC/CCL22) are two key chemokines exerting their biological effect via binding and activating a common receptor CCR4, expressed at the surface of type 2 helper T (Th2) cells. By recruiting Th2 cells in the dermis, CCL17 and CCL22 promote the development of inflammation in atopic skin. The aim of this research was to develop a plant extract whose biological properties, when applied topically, could be beneficial for people with atopic-prone skin. The strategy which was followed consisted in identifying ligands able to neutralize the biological activity of CCL17 and CCL22. Thus, an in silico molecular modeling and a generic screening assay were developed to screen natural molecules binding and blocking these two chemokines. N-Feruloylserotonin was identified as a neutraligand of CCL22 in these experiments. A cornflower extract containing N-feruloylserotonin was selected for further in vitro tests: the gene expression modulation of inflammation biomarkers induced by CCL17 or CCL22 in the presence or absence of this extract was assessed in the HaCaT keratinocyte cell line. Additionally, the same cornflower extract in another vehicle was evaluated in parallel with N-feruloylserotonin for cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX) enzymatic cellular inhibition. The cornflower extract was shown to neutralize the two chemokines in vitro, inhibited COX-2 and 5-LOX, and demonstrated anti-inflammatory activities due mainly to the presence of N-feruloylserotonin. Although these findings would need to be confirmed in an in vivo study, the in vitro studies lay the foundation to explain the benefits of the cornflower extract when applied topically to individuals with atopic-prone skin.


Subject(s)
Anti-Inflammatory Agents/pharmacology , Chemokine CCL17/antagonists & inhibitors , Chemokine CCL22/antagonists & inhibitors , Cyclooxygenase 2 Inhibitors/pharmacology , Lipoxygenase Inhibitors/pharmacology , Plant Extracts/pharmacology , Serotonin/analogs & derivatives , Skin/drug effects , Zea mays/chemistry , Cells, Cultured , Chemokine CCL17/chemistry , Chemokine CCL22/chemistry , Humans , Molecular Docking Simulation , Plant Extracts/analysis , Serotonin/chemistry , Serotonin/pharmacology
15.
Angew Chem Int Ed Engl ; 60(44): 23552-23556, 2021 10 25.
Article in English | MEDLINE | ID: mdl-34363735

ABSTRACT

Electrochemical methods were used to explore the exocytotic nature of serotonin (5-HT) release in human carcinoid BON cells, an in vitro human enterochromaffin cell model, to understand the mechanisms operating the release of gut-derived 5-HT in the intestinal mucosal epithelium. We show that the fractional vesicular 5-HT release in BON cells is 80 % compared to previous work in pancreatic beta cells (34 %). The fractional release increased from 80 % in control BON cells to 87 % with 5-HT preincubation and nearly 100 % with the combination of 5-HT and the 5-HT4 autoreceptor agonist, cisapride. Thus, partial release is the primary mechanism of exocytosis in BON cells, resulting in a variable amount of the vesicular content being released. Factors that control secretion of 5-HT from enterochromaffin cells or BON cells are important as partial release provides a mechanism for development of effective therapeutic strategies to treat gastrointestinal diseases.


Subject(s)
Electrochemical Techniques , Enterochromaffin Cells/drug effects , Nanotechnology , Serotonin/pharmacology , Drug Liberation , Exocytosis/drug effects , Gastrointestinal Diseases/drug therapy , Humans , Serotonin/chemistry
16.
Biochim Biophys Acta Mol Cell Biol Lipids ; 1866(12): 159044, 2021 12.
Article in English | MEDLINE | ID: mdl-34450265

ABSTRACT

During analysis of components of baobab (Adansonia digitata) seed oil, several new fluorescent compounds were detected in HPLC chromatograms that were not found previously in any seed oils investigated so far. After preparative isolation of these compounds, structural analysis by NMR spectroscopy, UHPLC-HR-MS, GC-FID and spectroscopic methods were applied and allowed identification of these substances as series of N-acylserotonins containing saturated C22 to C26 fatty acids with minor contribution of C27 to C30 homologues. The main component was N-lignocerylserotonin and the content of odd carbon-atom-number fatty acids was unusually high among the homologues. The suggested structure of the investigated compounds was additionally confirmed by their chemical synthesis. Synthetic N-acylserotonins showed pronounced inhibition of membrane lipid peroxidation of liposomes prepared from chloroplast lipids, especially when the peroxidation was initiated by a water-soluble azo-initiator, AIPH. Comparative studies of the reaction rate constants of the N-acylserotonins and tocopherols with a stable radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) in solvents of different polarity revealed that N-acylserotonins showed similar activity to δ-tocopherol in this respect. The described compounds have been not reported before either in plants or in animals. This indicates that we have identified a new class of plant lipids with antioxidant properties that could have promising pharmacological activities.


Subject(s)
Adansonia/chemistry , Antioxidants/chemistry , Lipids/chemistry , Serotonin/chemistry , Antioxidants/isolation & purification , Antioxidants/pharmacology , Chromatography, High Pressure Liquid , Lipid Peroxidation/drug effects , Lipids/isolation & purification , Lipids/pharmacology , Lipolysis , Magnetic Resonance Spectroscopy , Plant Oils/chemistry , Seeds/chemistry , Serotonin/analogs & derivatives , Serotonin/isolation & purification , Serotonin/pharmacology , Water/chemistry
17.
Int J Mol Sci ; 22(11)2021 May 31.
Article in English | MEDLINE | ID: mdl-34072751

ABSTRACT

Prevention of amyloid ß peptide (Aß) deposition via facilitation of Aß binding to its natural depot, human serum albumin (HSA), is a promising approach to preclude Alzheimer's disease (AD) onset and progression. Previously, we demonstrated the ability of natural HSA ligands, fatty acids, to improve the affinity of this protein to monomeric Aß by a factor of 3 (BBRC, 510(2), 248-253). Using plasmon resonance spectroscopy, we show here that another HSA ligand related to AD pathogenesis, serotonin (SRO), increases the affinity of the Aß monomer to HSA by a factor of 7/17 for Aß40/Aß42, respectively. Meanwhile, the structurally homologous SRO precursor, tryptophan (TRP), does not affect HSA's affinity to monomeric Aß, despite slowdown of the association and dissociation processes. Crosslinking with glutaraldehyde and dynamic light scattering experiments reveal that, compared with the TRP-induced effects, SRO binding causes more marked changes in the quaternary structure of HSA. Furthermore, molecular docking reveals distinct structural differences between SRO/TRP complexes with HSA. The disintegration of the serotonergic system during AD pathogenesis may contribute to Aß release from HSA in the central nervous system due to impairment of the SRO-mediated Aß trapping by HSA.


Subject(s)
Amyloid beta-Peptides/metabolism , Serotonin/metabolism , Serum Albumin, Human/metabolism , Alzheimer Disease , Amyloid beta-Peptides/chemistry , Binding Sites , Humans , Ligands , Molecular Conformation , Molecular Docking Simulation , Molecular Dynamics Simulation , Protein Binding , Protein Multimerization , Serotonin/chemistry , Serum Albumin, Human/chemistry , Structure-Activity Relationship , Temperature
19.
J Biol Chem ; 297(1): 100863, 2021 07.
Article in English | MEDLINE | ID: mdl-34118233

ABSTRACT

The serotonin transporter (SERT) shapes serotonergic neurotransmission by retrieving its eponymous substrate from the synaptic cleft. Ligands that discriminate between SERT and its close relative, the dopamine transporter DAT, differ in their association rate constant rather than their dissociation rate. The structural basis for this phenomenon is not known. Here we examined the hypothesis that the extracellular loops 2 (EL2) and 4 (EL4) limit access to the ligand-binding site of SERT. We employed an antibody directed against EL4 (residues 388-400) and the antibody fragments 8B6 scFv (directed against EL2 and EL4) and 15B8 Fab (directed against EL2) and analyzed their effects on the transport cycle of and inhibitor binding to SERT. Electrophysiological recordings showed that the EL4 antibody and 8B6 scFv impeded the initial substrate-induced transition from the outward to the inward-facing conformation but not the forward cycling mode of SERT. In contrast, binding of radiolabeled inhibitors to SERT was enhanced by either EL4- or EL2-directed antibodies. We confirmed this observation by determining the association and dissociation rate of the DAT-selective inhibitor methylphenidate via electrophysiological recordings; occupancy of EL2 with 15B8 Fab enhanced the affinity of SERT for methylphenidate by accelerating its binding. Based on these observations, we conclude that (i) EL4 undergoes a major movement during the transition from the outward to the inward-facing state, and (ii) EL2 and EL4 limit access of inhibitors to the binding of SERT, thus acting as a selectivity filter. This insight has repercussions for drug development.


Subject(s)
Dopamine Plasma Membrane Transport Proteins/genetics , Membrane Transport Proteins/genetics , Protein Conformation/drug effects , Selective Serotonin Reuptake Inhibitors/pharmacology , Serotonin Plasma Membrane Transport Proteins/genetics , Amino Acid Sequence/genetics , Animals , Binding Sites/drug effects , COS Cells , Chlorocebus aethiops , Dopamine Plasma Membrane Transport Proteins/antagonists & inhibitors , Dopamine Plasma Membrane Transport Proteins/ultrastructure , HEK293 Cells , Humans , Ligands , Membrane Transport Proteins/chemistry , Membrane Transport Proteins/ultrastructure , Patch-Clamp Techniques , Protein Domains/genetics , Serotonin/chemistry , Serotonin/genetics , Serotonin Plasma Membrane Transport Proteins/drug effects , Serotonin Plasma Membrane Transport Proteins/ultrastructure , Selective Serotonin Reuptake Inhibitors/chemistry
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