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1.
Nature ; 626(7998): 427-434, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38081299

RESUMO

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.


Assuntos
Microscopia Crioeletrônica , Proteínas Vesiculares de Transporte de Monoamina , Humanos , Sítios de Ligação , Citoplasma/efeitos dos fármacos , Citoplasma/metabolismo , Ketanserina/química , Ketanserina/metabolismo , Ketanserina/farmacologia , Reserpina/química , Reserpina/metabolismo , Reserpina/farmacologia , Serotonina/química , Serotonina/metabolismo , Especificidade por Substrato , Tetrabenazina/química , Tetrabenazina/metabolismo , Tetrabenazina/farmacologia , Proteínas Vesiculares de Transporte de Monoamina/antagonistas & inibidores , Proteínas Vesiculares de Transporte de Monoamina/química , Proteínas Vesiculares de Transporte de Monoamina/metabolismo , Proteínas Vesiculares de Transporte de Monoamina/ultraestrutura
2.
Nature ; 629(8010): 235-243, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38499039

RESUMO

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.


Assuntos
Monoaminas Biogênicas , Interações Medicamentosas , Proteínas Vesiculares de Transporte de Monoamina , Humanos , 1-Metil-4-fenilpiridínio/química , 1-Metil-4-fenilpiridínio/metabolismo , 1-Metil-4-fenilpiridínio/farmacologia , Anfetamina/química , Anfetamina/farmacologia , Anfetamina/metabolismo , Sítios de Ligação , Monoaminas Biogênicas/química , Monoaminas Biogênicas/metabolismo , Microscopia Crioeletrônica , Dopamina/química , Dopamina/metabolismo , Modelos Moleculares , Norepinefrina/química , Norepinefrina/metabolismo , Ligação Proteica , Prótons , Reserpina/farmacologia , Reserpina/química , Reserpina/metabolismo , Serotonina/química , Serotonina/metabolismo , Especificidade por Substrato , Proteínas Vesiculares de Transporte de Monoamina/química , Proteínas Vesiculares de Transporte de Monoamina/metabolismo , Proteínas Vesiculares de Transporte de Monoamina/ultraestrutura
3.
EMBO J ; 43(20): 4451-4471, 2024 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-39232129

RESUMO

Multimeric membrane proteins are produced in the endoplasmic reticulum and transported to their target membranes which, for ion channels, is typically the plasma membrane. Despite the availability of many fully assembled channel structures, our understanding of assembly intermediates, multimer assembly mechanisms, and potential functions of non-standard assemblies is limited. We demonstrate that the pentameric ligand-gated serotonin 5-HT3A receptor (5-HT3AR) can assemble to tetrameric forms and report the structures of the tetramers in plasma membranes of cell-derived microvesicles and in membrane memetics using cryo-electron microscopy and tomography. The tetrameric structures have near-symmetric transmembrane domains, and asymmetric extracellular domains, and can bind serotonin molecules. Computer simulations, based on our cryo-EM structures, were used to decipher the assembly pathway of pentameric 5-HT3R and suggest a potential functional role for the tetrameric receptors.


Assuntos
Microscopia Crioeletrônica , Multimerização Proteica , Receptores 5-HT3 de Serotonina , Receptores 5-HT3 de Serotonina/metabolismo , Receptores 5-HT3 de Serotonina/química , Receptores 5-HT3 de Serotonina/genética , Humanos , Membrana Celular/metabolismo , Serotonina/metabolismo , Serotonina/química , Animais , Células HEK293 , Modelos Moleculares
4.
J Proteome Res ; 23(10): 4457-4466, 2024 Oct 04.
Artigo em Inglês | MEDLINE | ID: mdl-39208062

RESUMO

Serotonylation has been identified as a novel protein posttranslational modification for decades, where an isopeptide bond is formed between the glutamine residue and serotonin through transamination. Transglutaminase 2 (also known as TGM2 or TGase2) was proven to act as the main "writer" enzyme for this PTM, and a number of key regulatory proteins (including small GTPases, fibronectin, fibrinogen, serotonin transporter, and histone H3) have been characterized as the substrates of serotonylation. However, due to the lack of pan-specific antibodies for serotonylated glutamine, the precise enrichment and proteomic profiling of serotonylation still remain challenging. In our previous research, we developed an aryldiazonium probe to specifically label protein serotonylation in a bioorthogonal manner, which depended on a pH-controlled chemoselective rapid azo-coupling reaction. Here, we report the application of a photoactive aryldiazonium-biotin probe for the global profiling of serotonylation proteome in cancer cells. Thus, over 1,000 serotonylated proteins were identified from HCT 116 cells, many of which are highly related to carcinogenesis. Moreover, a number of modification sites of these serotonylated proteins were determined, attributed to the successful application of our chemical proteomic approach. Overall, these findings provided new insights into the significant association between cellular protein serotonylation and cancer development, further suggesting that to target TGM2-mediated monoaminylation may serve as a promising strategy for cancer therapeutics.


Assuntos
Proteína 2 Glutamina gama-Glutamiltransferase , Processamento de Proteína Pós-Traducional , Proteoma , Proteômica , Transglutaminases , Humanos , Proteoma/análise , Concentração de Íons de Hidrogênio , Transglutaminases/metabolismo , Proteína 2 Glutamina gama-Glutamiltransferase/metabolismo , Proteômica/métodos , Células HCT116 , Proteínas de Ligação ao GTP/metabolismo , Biotina/química , Biotina/análogos & derivados , Biotina/metabolismo , Serotonina/análogos & derivados , Serotonina/metabolismo , Serotonina/química , Serotonina/análise , Compostos Azo/química , Glutamina/metabolismo , Glutamina/química , Neoplasias/metabolismo
5.
Small ; 20(39): e2307410, 2024 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-38778499

RESUMO

The detection of monoamine neurotransmitters is of paramount importance as the neurotransmitters are the chemical messengers regulating the gut-brain axis (GBA). It requires real-time, ultrasensitive, and selective sensing of the neurotransmitters in the gastric/intestinal fluid. However, multi-components present in the gastric/intestinal fluid make sensing challenging to achieve in terms of ultra-high sensitivity and selectivity. Herein, an approach is introduced to utilize vanadium single atom catalytic (SAC) centers in van der Waals MoS2 (V-MoS2) to selectively detect real-time serotonin (5-HT) in artificial gastric/intestinal fluid. The synergetic effect of V-SACs and the surface S-bonds on the MoS2 surface, enables an extremely wide range of 5-HT detection (from 1 pM to 100 µM), with optimum selectivity and interference resistance. By combining density functional theory calculations and scanning transmission electron microscopy, it is concluded that the V-SACs embedded in the MoS2 network create active sites that greatly facilitate the charge exchange between the material and the 5-HT molecules. This result allows the 5-HT detection in GBA studies to be more reliable, and the material tunability provides a general platform to achieve real-time and multi-component detection of other monoamine neurotransmitters in GBA such as dopamine and norepinephrine.


Assuntos
Encéfalo , Dissulfetos , Molibdênio , Neurotransmissores , Serotonina , Vanádio , Neurotransmissores/metabolismo , Dissulfetos/química , Molibdênio/química , Encéfalo/metabolismo , Serotonina/metabolismo , Serotonina/química , Vanádio/química
6.
Chembiochem ; 25(9): e202400069, 2024 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-38504591

RESUMO

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.


Assuntos
Arilalquilamina N-Acetiltransferase , Serotonina , Arilalquilamina N-Acetiltransferase/metabolismo , Arilalquilamina N-Acetiltransferase/genética , Arilalquilamina N-Acetiltransferase/química , Serotonina/metabolismo , Serotonina/química , Serotonina/biossíntese , Animais , Acetilação , Engenharia de Proteínas , Suínos
7.
Phys Chem Chem Phys ; 26(18): 13751-13761, 2024 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-38683175

RESUMO

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.


Assuntos
Simulação de Dinâmica Molecular , Neurotransmissores , Serotonina , Radiação Terahertz , Ácido gama-Aminobutírico , Neurotransmissores/química , Ácido gama-Aminobutírico/química , Serotonina/química , Serotonina/metabolismo , Nicotina/química , Epinefrina/química
8.
Nature ; 563(7730): 270-274, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30401837

RESUMO

The 5-HT3A serotonin receptor1, a cationic pentameric ligand-gated ion channel (pLGIC), is the clinical target for management of nausea and vomiting associated with radiation and chemotherapies2. Upon binding, serotonin induces a global conformational change that encompasses the ligand-binding extracellular domain (ECD), the transmembrane domain (TMD) and the intracellular domain (ICD), the molecular details of which are unclear. Here we present two serotonin-bound structures of the full-length 5-HT3A receptor in distinct conformations at 3.32 Å and 3.89 Å resolution that reveal the mechanism underlying channel activation. In comparison to the apo 5-HT3A receptor, serotonin-bound states underwent a large twisting motion in the ECD and TMD, leading to the opening of a 165 Å permeation pathway. Notably, this motion results in the creation of lateral portals for ion permeation at the interface of the TMD and ICD. Combined with molecular dynamics simulations, these structures provide novel insights into conformational coupling across domains and functional modulation.


Assuntos
Microscopia Crioeletrônica , Receptores 5-HT3 de Serotonina/química , Receptores 5-HT3 de Serotonina/ultraestrutura , Serotonina/química , Serotonina/metabolismo , Animais , Apoproteínas/química , Apoproteínas/metabolismo , Apoproteínas/ultraestrutura , Sítios de Ligação , Condutividade Elétrica , Feminino , Ativação do Canal Iônico , Transporte de Íons , Camundongos , Simulação de Dinâmica Molecular , Movimento , Conformação Proteica , Receptores 5-HT3 de Serotonina/genética , Receptores 5-HT3 de Serotonina/metabolismo , Xenopus laevis
9.
Nature ; 563(7730): 275-279, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30401839

RESUMO

The serotonin 5-HT3 receptor is a pentameric ligand-gated ion channel (pLGIC). It belongs to a large family of receptors that function as allosteric signal transducers across the plasma membrane1,2; upon binding of neurotransmitter molecules to extracellular sites, the receptors undergo complex conformational transitions that result in transient opening of a pore permeable to ions. 5-HT3 receptors are therapeutic targets for emesis and nausea, irritable bowel syndrome and depression3. In spite of several reported pLGIC structures4-8, no clear unifying view has emerged on the conformational transitions involved in channel gating. Here we report four cryo-electron microscopy structures of the full-length mouse 5-HT3 receptor in complex with the anti-emetic drug tropisetron, with serotonin, and with serotonin and a positive allosteric modulator, at resolutions ranging from 3.2 Å to 4.5 Å. The tropisetron-bound structure resembles those obtained with an inhibitory nanobody5 or without ligand9. The other structures include an 'open' state and two ligand-bound states. We present computational insights into the dynamics of the structures, their pore hydration and free-energy profiles, and characterize movements at the gate level and cation accessibility in the pore. Together, these data deepen our understanding of the gating mechanism of pLGICs and capture ligand binding in unprecedented detail.


Assuntos
Microscopia Crioeletrônica , Receptores 5-HT3 de Serotonina/química , Receptores 5-HT3 de Serotonina/ultraestrutura , Regulação Alostérica/efeitos dos fármacos , Animais , Sítios de Ligação , Ativação do Canal Iônico , Ligantes , Camundongos , Simulação de Dinâmica Molecular , Movimento/efeitos dos fármacos , Conformação Proteica/efeitos dos fármacos , Receptores 5-HT3 de Serotonina/metabolismo , Serotonina/química , Serotonina/metabolismo , Antagonistas do Receptor 5-HT3 de Serotonina/farmacologia , Anticorpos de Domínio Único/farmacologia , Termodinâmica , Tropizetrona/química , Tropizetrona/metabolismo , Tropizetrona/farmacologia
10.
Angew Chem Int Ed Engl ; 63(41): e202407308, 2024 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-38995157

RESUMO

The intrinsic correlation between depression and serotonin (5-HT) is a highly debated topic, with significant implications for the diagnosis, treatment, and advancement of drugs targeting neurological disorders. To address this important question, it is of utmost priority to understand the action mechanism of serotonin in depression through fluorescence imaging studies. However, the development of efficient molecular probes for serotonin is hindered by the lack of responsive sites with high selectivity for serotonin at the present time. Herein, we developed the first highly selective serotonin responsive site, 3-mercaptopropionate, utilizing thiol-ene click cascade nucleophilic reactions. The novel responsive site was then employed to construct the powerful molecular probe SJ-5-HT for imaging the serotonin level changes in the depression cells and brain tissues. Importantly, the imaging studies reveal that the level of serotonin in patients with depression may not be the primary factor, while the ability of neurons in patients with depression to release serotonin appears to be more critical. Additionally, this serotonin release capability correlates strongly with the levels of mTOR (intracellular mammalian target of rapamycin). These discoveries could offer valuable insights into the molecular mechanisms underpinning depression and furnish mTOR as a novel direction for the advancement of antidepressant therapies.


Assuntos
Depressão , Corantes Fluorescentes , Serotonina , Compostos de Sulfidrila , Serotonina/metabolismo , Serotonina/química , Corantes Fluorescentes/química , Humanos , Compostos de Sulfidrila/química , Depressão/tratamento farmacológico , Depressão/metabolismo , Química Click , Estrutura Molecular , Animais , Imagem Óptica
11.
J Biol Chem ; 298(3): 101613, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-35065961

RESUMO

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.


Assuntos
Proteínas da Membrana Plasmática de Transporte de Serotonina , Serotonina , Citalopram/química , Citalopram/farmacologia , Humanos , Simulação de Dinâmica Molecular , Domínios Proteicos , Estrutura Secundária de Proteína , Serotonina/química , Serotonina/metabolismo , Proteínas da Membrana Plasmática de Transporte de Serotonina/química , Proteínas da Membrana Plasmática de Transporte de Serotonina/metabolismo , Inibidores Seletivos de Recaptação de Serotonina/química , Inibidores Seletivos de Recaptação de Serotonina/farmacologia , Relação Estrutura-Atividade
12.
J Chem Inf Model ; 63(4): 1196-1207, 2023 02 27.
Artigo em Inglês | MEDLINE | ID: mdl-36757760

RESUMO

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.


Assuntos
Simulação de Dinâmica Molecular , Serotonina , Serotonina/química , Serotonina/metabolismo , Microscopia Crioeletrônica , Estrutura Secundária de Proteína , Transporte de Íons , Receptores 5-HT3 de Serotonina/química , Receptores 5-HT3 de Serotonina/metabolismo
13.
Proc Natl Acad Sci U S A ; 117(7): 3848-3857, 2020 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-32024760

RESUMO

l-tryptophan (Trp), an essential amino acid for mammals, is the precursor of a wide array of immunomodulatory metabolites produced by the kynurenine and serotonin pathways. The kynurenine pathway is a paramount source of several immunoregulatory metabolites, including l-kynurenine (Kyn), the main product of indoleamine 2,3-dioxygenase 1 (IDO1) that catalyzes the rate-limiting step of the pathway. In the serotonin pathway, the metabolite N-acetylserotonin (NAS) has been shown to possess antioxidant, antiinflammatory, and neuroprotective properties in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). However, little is known about the exact mode of action of the serotonin metabolite and the possible interplay between the 2 Trp metabolic pathways. Prompted by the discovery that NAS neuroprotective effects in EAE are abrogated in mice lacking IDO1 expression, we investigated the NAS mode of action in neuroinflammation. We found that NAS directly binds IDO1 and acts as a positive allosteric modulator (PAM) of the IDO1 enzyme in vitro and in vivo. As a result, increased Kyn will activate the ligand-activated transcription factor aryl hydrocarbon receptor and, consequently, antiinflammatory and immunoregulatory effects. Because NAS also increased IDO1 activity in peripheral blood mononuclear cells of a significant proportion of MS patients, our data may set the basis for the development of IDO1 PAMs as first-in-class drugs in autoimmune/neuroinflammatory diseases.


Assuntos
Encefalomielite Autoimune Experimental/enzimologia , Encefalomielite Autoimune Experimental/metabolismo , Indolamina-Pirrol 2,3,-Dioxigenase/química , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Regulação Alostérica , Sítio Alostérico , Animais , Biocatálise , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/genética , Feminino , Humanos , Indolamina-Pirrol 2,3,-Dioxigenase/genética , Cinurenina/metabolismo , Leucócitos Mononucleares/metabolismo , Masculino , Camundongos Knockout , Esclerose Múltipla/enzimologia , Esclerose Múltipla/genética , Esclerose Múltipla/metabolismo , Serotonina/análogos & derivados , Serotonina/química , Serotonina/metabolismo , Triptofano/metabolismo
14.
J Biol Chem ; 297(1): 100863, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34118233

RESUMO

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.


Assuntos
Proteínas da Membrana Plasmática de Transporte de Dopamina/genética , Proteínas de Membrana Transportadoras/genética , Conformação Proteica/efeitos dos fármacos , Inibidores Seletivos de Recaptação de Serotonina/farmacologia , Proteínas da Membrana Plasmática de Transporte de Serotonina/genética , Sequência de Aminoácidos/genética , Animais , Sítios de Ligação/efeitos dos fármacos , Células COS , Chlorocebus aethiops , Proteínas da Membrana Plasmática de Transporte de Dopamina/antagonistas & inibidores , Proteínas da Membrana Plasmática de Transporte de Dopamina/ultraestrutura , Células HEK293 , Humanos , Ligantes , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/ultraestrutura , Técnicas de Patch-Clamp , Domínios Proteicos/genética , Serotonina/química , Serotonina/genética , Proteínas da Membrana Plasmática de Transporte de Serotonina/efeitos dos fármacos , Proteínas da Membrana Plasmática de Transporte de Serotonina/ultraestrutura , Inibidores Seletivos de Recaptação de Serotonina/química
15.
Nat Chem Biol ; 16(1): 77-86, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31792443

RESUMO

Membrane receptors sense and transduce extracellular stimuli into intracellular signaling responses but the molecular underpinnings remain poorly understood. We report a computational approach for designing protein allosteric signaling functions. By combining molecular dynamics simulations and design calculations, the method engineers amino-acid 'microswitches' at allosteric sites that modulate receptor stability or long-range coupling, to reprogram specific signaling properties. We designed 36 dopamine D2 receptor variants, whose constitutive and ligand-induced signaling agreed well with our predictions, repurposed the D2 receptor into a serotonin biosensor and predicted the signaling effects of more than 100 known G-protein-coupled receptor (GPCR) mutations. Our results reveal the existence of distinct classes of allosteric microswitches and pathways that define an unforeseen molecular mechanism of regulation and evolution of GPCR signaling. Our approach enables the rational design of allosteric receptors with enhanced stability and function to facilitate structural characterization, and reprogram cellular signaling in synthetic biology and cell engineering applications.


Assuntos
Engenharia de Proteínas , Receptores de Dopamina D2/química , Receptores de Dopamina D2/genética , Transdução de Sinais , Regulação Alostérica/efeitos dos fármacos , Sítio Alostérico , Motivos de Aminoácidos , Técnicas Biossensoriais , Simulação por Computador , Análise Mutacional de DNA , Células HEK293 , Humanos , Concentração Inibidora 50 , Cinética , Ligantes , Simulação de Dinâmica Molecular , Mutagênese , Serotonina/química , Software
16.
Histochem Cell Biol ; 155(6): 623-636, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33608804

RESUMO

5-HT containing enteroendocrine cells (EEC), the most abundant type of EEC in the gut, regulate many functions including motility, secretion and inflammatory responses. We examined the morphologies of 5-HT cells from stomach to rectum, patterns of hormone co-expression in the stomach and colon, and the relationship of 5-HT cells with nerve fibres. We also reviewed some of the relevant literature. The morphologies of 5-HT cells were distinct, depending on their location in the gut. A noticeable feature of some 5-HT cells in the antrum and colon was their long basal processes, which resembled processes of neurons, whereas 5-HT cells in the small intestinal mucosa lacked basal processes. In the stomach, numerous 5-HT cells, including cells with basal processes, were identified as enterochromaffin-like cells by their expression of histidine decarboxylase. In the colon, we observed a small number of 5-HT cells that were in close contact with, but distinct from, oxyntomodulin (OXM) and PYY immunoreactive EEC. We did not find specific relationships between nerve fibres and the processes of colonic 5-HT cells. We conclude that five major features, i.e., gut region, morphology, hormone content, receptor repertoire and cell lineage, can be used to define 5-HT cells.


Assuntos
Células Enteroendócrinas/metabolismo , Trato Gastrointestinal/metabolismo , Fibras Nervosas/metabolismo , Hormônios Peptídicos/metabolismo , Serotonina/metabolismo , Animais , Células Enteroendócrinas/citologia , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Hormônios Peptídicos/análise , Serotonina/química
17.
Bioorg Med Chem Lett ; 40: 127909, 2021 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-33705900

RESUMO

A series of benzoisoxazoleylpiperidine derivatives were synthesized by using the multi-target strategies and their potent affinities for dopamine (DA), serotonin (5-HT) and human histamine H3 receptors have been evaluated. Of these compounds, the promising candidate 4w displayed high affinities for D2, D3, 5-HT1A, 5-HT2A and H3, a moderate affinity for 5-HT6, negligible effects on the human ether-a-go-go-related gene (hERG) channel, low affinities for off-target receptors (5-HT2C, adrenergic α1 and H1). In addition, the animal behavioral study revealed that, compared to risperidone, compound 4w significantly inhibited apomorphine-induced climbing and MK-801-induced movement behaviors with a high threshold for catalepsy and low liabilities for weight gain and hyperprolactinemia. Results from the conditioned avoidance response test and novel object recognition task demonstrated that 4w had pro-cognitive effects. Thus, the antipsychotic drug-like activities of 4w indicate that it may be a potential polypharmacological antipsychotic candidate drug.


Assuntos
Antipsicóticos/química , Cognição/efeitos dos fármacos , Piperidinas/química , Animais , Antipsicóticos/farmacologia , Comportamento Animal , Dopamina/química , Desenho de Fármacos , Humanos , Hiperprolactinemia/metabolismo , Camundongos , Modelos Animais , Movimento/efeitos dos fármacos , Piperidinas/farmacologia , Ligação Proteica , Receptores Histamínicos H3/química , Risperidona/farmacologia , Serotonina/química , Relação Estrutura-Atividade , Aumento de Peso
18.
Mediators Inflamm ; 2021: 6652791, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34557056

RESUMO

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.


Assuntos
Anti-Inflamatórios/farmacologia , Quimiocina CCL17/antagonistas & inibidores , Quimiocina CCL22/antagonistas & inibidores , Inibidores de Ciclo-Oxigenase 2/farmacologia , Inibidores de Lipoxigenase/farmacologia , Extratos Vegetais/farmacologia , Serotonina/análogos & derivados , Pele/efeitos dos fármacos , Zea mays/química , Células Cultivadas , Quimiocina CCL17/química , Quimiocina CCL22/química , Humanos , Simulação de Acoplamento Molecular , Extratos Vegetais/análise , Serotonina/química , Serotonina/farmacologia
19.
Mikrochim Acta ; 188(4): 146, 2021 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-33792757

RESUMO

A sensitive and selective voltammetric biosensor composed of layer-by-layer (LbL) self-assembly of positively charged poly(diallyldimethylammonium)-wrapped oxidized single-walled carbon nanotubes (PDDA-oSWCNTs), negatively charged serotonin (5-hydroxytryptamine, 5-HT)-specific aptamer, and tyrosinase on Au nanoparticles deposited screen printed carbon electrode was developed for measurement of 5-HT. Surface characteristics of 5-HT biosensor were explored using scanning electron microscopy, X-ray photoelectron spectroscopy, and electrochemical impedance spectroscopy. The respective effects of 5-HT-specific aptamer and oSWCNTs on the detection of 5-HT were investigated by differential pulse voltammetry (DPV). The peak current at the potential of 0.29 V (vs. Ag/AgCl) increased with respect to 5-HT concentration resulting in two dynamic ranges from 0.05 to 0.5 and 1 to 20 µM with a limit of detection of 2 nM from the LbL biosensor in buffer solution, which were better than those without the LbL of aptamer and oSWCNTs. The developed biosensor was applied to the direct determination of 5-HT concentrations in undiluted healthy control and Internet gaming disorder serum samples. The results were verified by comparison with those from liquid chromatography-mass spectrometric analyses.


Assuntos
Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais/métodos , DNA/química , Técnicas Eletroquímicas/métodos , Nanocompostos/química , Serotonina/sangue , Agaricales/enzimologia , Enzimas Imobilizadas/química , Ouro/química , Humanos , Transtorno de Adição à Internet/sangue , Transtorno de Adição à Internet/diagnóstico , Limite de Detecção , Nanopartículas Metálicas/química , Monofenol Mono-Oxigenase/química , Nanotubos de Carbono/química , Polietilenos/química , Compostos de Amônio Quaternário/química , Serotonina/química
20.
Int J Mol Sci ; 22(11)2021 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-34072751

RESUMO

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.


Assuntos
Peptídeos beta-Amiloides/metabolismo , Serotonina/metabolismo , Albumina Sérica Humana/metabolismo , Doença de Alzheimer , Peptídeos beta-Amiloides/química , Sítios de Ligação , Humanos , Ligantes , Conformação Molecular , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Ligação Proteica , Multimerização Proteica , Serotonina/química , Albumina Sérica Humana/química , Relação Estrutura-Atividade , Temperatura
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