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1.
Nat Commun ; 11(1): 5205, 2020 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-33060564

RESUMO

Growth hormone-releasing hormone (GHRH) regulates the secretion of growth hormone that virtually controls metabolism and growth of every tissue through its binding to the cognate receptor (GHRHR). Malfunction in GHRHR signaling is associated with abnormal growth, making GHRHR an attractive therapeutic target against dwarfism (e.g., isolated growth hormone deficiency, IGHD), gigantism, lipodystrophy and certain cancers. Here, we report the cryo-electron microscopy (cryo-EM) structure of the human GHRHR bound to its endogenous ligand and the stimulatory G protein at 2.6 Å. This high-resolution structure reveals a characteristic hormone recognition pattern of GHRH by GHRHR, where the α-helical GHRH forms an extensive and continuous network of interactions involving all the extracellular loops (ECLs), all the transmembrane (TM) helices except TM4, and the extracellular domain (ECD) of GHRHR, especially the N-terminus of GHRH that engages a broad set of specific interactions with the receptor. Mutagenesis and molecular dynamics (MD) simulations uncover detailed mechanisms by which IGHD-causing mutations lead to the impairment of GHRHR function. Our findings provide insights into the molecular basis of peptide recognition and receptor activation, thereby facilitating the development of structure-based drug discovery and precision medicine.


Assuntos
Hormônio Liberador de Hormônio do Crescimento/química , Hormônio Liberador de Hormônio do Crescimento/metabolismo , Receptores de Neuropeptídeos/química , Receptores de Neuropeptídeos/metabolismo , Receptores de Hormônios Reguladores de Hormônio Hipofisário/química , Receptores de Hormônios Reguladores de Hormônio Hipofisário/metabolismo , Sítios de Ligação , Microscopia Crioeletrônica , Nanismo Hipofisário/genética , Proteínas de Ligação ao GTP , Hormônio Liberador de Hormônio do Crescimento/deficiência , Humanos , Simulação de Dinâmica Molecular , Mutagênese , Mutação , Conformação Proteica , Conformação Proteica em alfa-Hélice , Receptores de Neuropeptídeos/genética , Receptores de Hormônios Reguladores de Hormônio Hipofisário/genética , Transdução de Sinais
2.
Chem Biol Interact ; 330: 109248, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-32871113

RESUMO

Dextromethorphan (DM) is a cough suppressant available in many prescribed and over-the-counter medications. Adverse reactions induced by DM have been regularly reported, including allergic skin reactions in some cases. However, the underlying mechanisms of local anaphylaxis induced by DM have not been elucidated. In this study, we found that DM could activate mast cells to increase calcium mobilization and release ß-hexosaminidase, histamine, tumor necrosis factor-α, MCP-1, and IL-8 in a dose-dependent manner. The allergic reactions were confirmed by hind paw swelling and extravasation assay in vivo. Furthermore, DM was revealed to induce local anaphylaxis via MRGPRX2 by the mast cell-deficient kitW-sh/W-sh mice and MRGPRX2 knockdown mast cells. And the MRGPRX2-HEK293/CMC analysis and frontal analysis also showed that DM has a considerable affinity with MRGPRX2. Together, our findings suggest that close monitoring should be drawn on patients with DM for its potential anaphylaxis via MRGPRX2.


Assuntos
Anafilaxia/induzido quimicamente , Dextrometorfano/efeitos adversos , Mastócitos/efeitos dos fármacos , Receptores Acoplados a Proteínas-G/metabolismo , Animais , Antitussígenos/efeitos adversos , Células Cultivadas , Dextrometorfano/toxicidade , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Mastócitos/imunologia , Mastócitos/metabolismo , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/metabolismo , Receptores de Neuropeptídeos/metabolismo
3.
J Headache Pain ; 21(1): 87, 2020 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-32640973

RESUMO

BACKGROUND: Stimulation of trigeminovascular pathway is widely used to establish the headache animal model. Headache is a common neurological disorder, in which symptomatic attacks are mediated by calcitonin-gene-related peptide (CGRP). CGRP is synthesized and released from the trigeminal ganglion to transmit pain signals under stimulation. On the other hand, Neuropeptide FF (NPFF) is a candidate transmitter/modulator for migraine, and stimulation of its receptor, NPFFR2, increases the expression and release of CGRP in mice sensory neurons. Here, we investigate the impact of NPFFR2 on trigeminal CGRP level in a capsaicin-induced headache mouse model. METHODS: Mice were intracisternally injected with capsaicin into the cisterna magna to activate the trigeminovascular pathway and induce headache symptoms. Mice pretreated with Npffr2-shRNA or NPFFR2 knockouts were adopted to test the impact of NPFFR2 on capsaicin-induced CGRP upregulation in trigeminal ganglion. The gene silencing effect of Npffr2-shRNA in trigeminal ganglion was confirmed by real-time PCR. Trigeminal CGRP level was determined by immunofluorescence staining, and the percentage of CGRP-positive cell was calculated after setting the signal intensity threshold by Image J software. Amount of trigeminal CGRP in NPFFR2 overexpressed mice was also measured by CGRP ELISA. FINDINGS: Infusion of capsaicin into the cisterna magna upregulated the CGRP in trigeminal ganglion and induced spontaneous pain behaviors including the reduction of locomotor activity and the increase of freezing behavior. Intracisternal injection of Npffr2-shRNA reduced the mRNA of Npffr2 in trigeminal ganglion. Mice pretreatment with Npffr2-shRNA prevented capsaicin-induced CGRP upregulation in trigeminal ganglion. Similarly, CGRP upregulation was also reduced in NPFFR2 knockout mice. On the contrary, trigeminal CGRP was increased in NPFFR2 overexpressed mice. CONCLUSIONS: Reducing the level of NPFFR2 leads to the downregulation of capsaicin-induced CGRP in trigeminal ganglion, which would consequently attenuate the activation of trigeminovascular pathway. Thus, NPFFR2 could serve as a potential target for neuromodulation of cephalic pain.


Assuntos
Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Capsaicina/farmacologia , Receptores de Neuropeptídeos/metabolismo , Gânglio Trigeminal/metabolismo , Animais , Calcitonina/genética , Modelos Animais de Doenças , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Transtornos de Enxaqueca/metabolismo , Neurônios Aferentes/metabolismo , Ratos , Regulação para Cima/efeitos dos fármacos
4.
Rev Med Suisse ; 16(689): 679-682, 2020 Apr 08.
Artigo em Francês | MEDLINE | ID: mdl-32270934

RESUMO

Since the description in 2015 of the MRGPRX2 receptor on mast cells, responsible for pseudo-allergies, our knowledge of this type of allergy-like drug reaction is growing, as has the list of drugs -supposed to be able to induce this type of reaction. Unlike IgE--mediated reactions, these pseudoallergic reactions do not require a prior sensitization, are dose-dependent and predictable, and could be prevented, if the offending drug has to be re-administered, -simply with a reduced rate of perfusion or dose. Genetic factors seem to play a role in the predisposition to this type of reactions, but we do not yet have clinically available tools to diagnose them. This literature review summarizes the discoveries of the last 4 years in this field that seem to challenge many dogmas in allergology.


Assuntos
Degranulação Celular , Hipersensibilidade a Drogas , Mastócitos/metabolismo , Degranulação Celular/efeitos dos fármacos , Relação Dose-Resposta Imunológica , Hipersensibilidade a Drogas/genética , Hipersensibilidade a Drogas/metabolismo , Humanos , Proteínas do Tecido Nervoso/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Receptores de Neuropeptídeos/metabolismo
5.
PLoS Biol ; 18(3): e3000614, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32126082

RESUMO

The reproductive hormones that trigger oocyte meiotic maturation and release from the ovary vary greatly between animal species. Identification of receptors for these maturation-inducing hormones (MIHs) and understanding how they initiate the largely conserved maturation process remain important challenges. In hydrozoan cnidarians including the jellyfish Clytia hemisphaerica, MIH comprises neuropeptides released from somatic cells of the gonad. We identified the receptor (MIHR) for these MIH neuropeptides in Clytia using cell culture-based "deorphanization" of candidate oocyte-expressed G protein-coupled receptors (GPCRs). MIHR mutant jellyfish generated using CRISPR-Cas9 editing had severe defects in gamete development or in spawning both in males and females. Female gonads, or oocytes isolated from MIHR mutants, failed to respond to synthetic MIH. Treatment with the cAMP analogue Br-cAMP to mimic cAMP rise at maturation onset rescued meiotic maturation and spawning. Injection of inhibitory antibodies to the alpha subunit of the Gs heterodimeric protein (GαS) into wild-type oocytes phenocopied the MIHR mutants. These results provide the molecular links between MIH stimulation and meiotic maturation initiation in hydrozoan oocytes. Molecular phylogeny grouped Clytia MIHR with a subset of bilaterian neuropeptide receptors, including neuropeptide Y, gonadotropin inhibitory hormone (GnIH), pyroglutamylated RFamide, and luqin, all upstream regulators of sexual reproduction. This identification and functional characterization of a cnidarian peptide GPCR advances our understanding of oocyte maturation initiation and sheds light on the evolution of neuropeptide-hormone systems.


Assuntos
Hidrozoários/fisiologia , Neuropeptídeos/metabolismo , Oócitos/fisiologia , Receptores Acoplados a Proteínas-G/metabolismo , Animais , Animais Geneticamente Modificados , Sistemas CRISPR-Cas , AMP Cíclico/metabolismo , Feminino , Expressão Gênica , Hidrozoários/genética , Masculino , Mutação , Filogenia , Receptores Acoplados a Proteínas-G/genética , Receptores de Neuropeptídeos/genética , Receptores de Neuropeptídeos/metabolismo
6.
Nat Commun ; 11(1): 1450, 2020 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-32193370

RESUMO

Olfactory and metabolic dysfunctions are intertwined phenomena associated with obesity and neurodegenerative diseases; yet how mechanistically olfaction regulates metabolic homeostasis remains unclear. Specificity of olfactory perception integrates diverse environmental odors and olfactory neurons expressing different receptors. Here, we report that specific but not all olfactory neurons actively regulate fat metabolism without affecting eating behaviors in Caenorhabditis elegans, and identified specific odors that reduce fat mobilization via inhibiting these neurons. Optogenetic activation or inhibition of the responsible olfactory neural circuit promotes the loss or gain of fat storage, respectively. Furthermore, we discovered that FLP-1 neuropeptide released from this olfactory neural circuit signals through peripheral NPR-4/neuropeptide receptor, SGK-1/serum- and glucocorticoid-inducible kinase, and specific isoforms of DAF-16/FOXO transcription factor to regulate fat storage. Our work reveals molecular mechanisms underlying olfactory regulation of fat metabolism, and suggests the association between olfactory perception specificity of each individual and his/her susceptibility to the development of obesity.


Assuntos
Comportamento Alimentar/fisiologia , Metabolismo dos Lipídeos/fisiologia , Sistemas Neurossecretores/metabolismo , Obesidade/metabolismo , Percepção Olfatória/fisiologia , Animais , Animais Geneticamente Modificados , Butanonas/química , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Modelos Animais de Doenças , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Humanos , Neurônios/metabolismo , Neuropeptídeos/metabolismo , Odorantes , Optogenética , Proteínas Serina-Treonina Quinases/metabolismo , Receptores de Neuropeptídeos/metabolismo
7.
Proc Natl Acad Sci U S A ; 117(11): 6067-6074, 2020 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-32123064

RESUMO

Ocular inflammation is a major cause of visual impairment attributed to dysregulation of the immune system. Previously, we have shown that the receptor for growth-hormone-releasing hormone (GHRH-R) affects multiple inflammatory processes. To clarify the pathological roles of GHRH-R in acute ocular inflammation, we investigated the inflammatory cascades mediated by this receptor. In human ciliary epithelial cells, the NF-κB subunit p65 was phosphorylated in response to stimulation with lipopolysaccharide (LPS), resulting in transcriptional up-regulation of GHRH-R. Bioinformatics analysis and coimmunoprecipitation showed that GHRH-R had a direct interaction with JAK2. JAK2, but not JAK1, JAK3, and TYK2, was elevated in ciliary body and iris after treatment with LPS in a rat model of endotoxin-induced uveitis. This elevation augmented the phosphorylation of STAT3 and production of proinflammatory factors, including IL-6, IL-17A, COX2, and iNOS. In explants of iris and ciliary body, the GHRH-R antagonist, MIA-602, suppressed phosphorylation of STAT3 and attenuated expression of downstream proinflammatory factors after LPS treatment. A similar suppression of STAT3 phosphorylation was observed in human ciliary epithelial cells. In vivo studies showed that blocking of the GHRH-R/JAK2/STAT3 axis with the JAK inhibitor Ruxolitinib alleviated partially the LPS-induced acute ocular inflammation by reducing inflammatory cells and protein leakage in the aqueous humor and by repressing expression of STAT3 target genes in rat ciliary body and iris and in human ciliary epithelial cells. Our findings indicate a functional role of the GHRH-R/JAK2/STAT3-signaling axis in acute anterior uveitis and suggest a therapeutic strategy based on treatment with antagonists targeting this signaling pathway.


Assuntos
Células Epiteliais/patologia , Receptores de Neuropeptídeos/metabolismo , Receptores de Hormônios Reguladores de Hormônio Hipofisário/metabolismo , Transdução de Sinais/imunologia , Uveíte/patologia , Animais , Linhagem Celular , Corpo Ciliar/citologia , Modelos Animais de Doenças , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/imunologia , Humanos , Janus Quinase 2/metabolismo , Lipopolissacarídeos/imunologia , Masculino , Pirazóis/farmacologia , Pirazóis/uso terapêutico , Ratos , Receptores de Neuropeptídeos/antagonistas & inibidores , Receptores de Neuropeptídeos/imunologia , Receptores de Hormônios Reguladores de Hormônio Hipofisário/antagonistas & inibidores , Receptores de Hormônios Reguladores de Hormônio Hipofisário/imunologia , Fator de Transcrição STAT3/metabolismo , Sermorelina/análogos & derivados , Sermorelina/farmacologia , Sermorelina/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Uveíte/tratamento farmacológico , Uveíte/imunologia
8.
Phytomedicine ; 68: 153149, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32032836

RESUMO

BACKGROUND: Mast cells (MCs) are crucial effectors in allergic disorders by secreting inflammatory mediators. The Mas-related G-protein-coupled receptor X2 (Mrgprx2) was shown to have a key role in IgE-independent allergic reactions. Therefore, potential drug candidates that directly target Mrgprx2 could be used to treat pseudo-allergic diseases. Shikonin, an active ingredient derived from Lithospermum erythrorhizon Sieb. et Zucc has been used for its anti-inflammatory properties since ancient China. PURPOSE: To investigate the inhibitory effects of Shikonin on IgE-independent allergy both in vitro and in vivo, as well as the mechanism underlying its effects. METHODS/STUDY DESIGNS: The anti-anaphylactoid activity of Shikonin was evaluated in PCA and systemic anaphylaxis models, Calcium imaging was used to assess intracellular Ca2+ mobilization. The release of cytokines and chemokines was measured using enzyme immunoassay kits. Western blot analysis was conducted to investigate the molecules of PLCγ-PKC-IP3 signaling pathway. The analytical method of surface plasmon resonance was employed to study the interaction between Shikonin and potential target protein Mrgprx2. RESULTS: Shikonin can suppress compound 48/80 (C48/80)-induced PCA, active systemic anaphylaxis, and MCs degranulation in mice in a dose-dependent manner. In addition, Shikonin reduced C48/80-induced calcium flux and suppressed LAD2 cell degranulation via PLCγ-PKC-IP3 signaling pathway. Moreover, Shikonin was found to inhibit C48/80-induced Mrgprx2 expression in HEK cells, displaying specific interactions with the Mrgprx2 protein. CONCLUSION: Shikonin could be a potential antagonist of Mrgprx2, thereby inhibiting pseudo-allergic reactions through Ca2+ mobilization.


Assuntos
Anafilaxia/tratamento farmacológico , Hipersensibilidade/tratamento farmacológico , Naftoquinonas/farmacologia , Proteínas do Tecido Nervoso/imunologia , Receptores Acoplados a Proteínas-G/imunologia , Receptores de Neuropeptídeos/imunologia , Anafilaxia/induzido quimicamente , Animais , Cálcio/metabolismo , Degranulação Celular/efeitos dos fármacos , Linhagem Celular , Quimiocinas/metabolismo , Citocinas/metabolismo , Humanos , Hipersensibilidade/imunologia , Masculino , Mastócitos/efeitos dos fármacos , Mastócitos/metabolismo , Camundongos Endogâmicos C57BL , Naftoquinonas/química , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Fosfolipase C gama/metabolismo , Receptores Acoplados a Proteínas-G/antagonistas & inibidores , Receptores Acoplados a Proteínas-G/química , Receptores Acoplados a Proteínas-G/metabolismo , Receptores de Neuropeptídeos/antagonistas & inibidores , Receptores de Neuropeptídeos/química , Receptores de Neuropeptídeos/metabolismo , Secretagogos/toxicidade , p-Metoxi-N-metilfenetilamina/toxicidade
9.
Immunity ; 52(2): 404-416.e5, 2020 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-32049054

RESUMO

Mast cells are rare tissue-resident cells of importance to human allergies. To understand the structural basis of principle mast cell functions, we analyzed the proteome of primary human and mouse mast cells by quantitative mass spectrometry. We identified a mast-cell-specific proteome signature, indicative of a unique lineage, only distantly related to other immune cell types, including innate immune cells. Proteome comparison between human and mouse suggested evolutionary conservation of core mast cell functions. In addition to specific proteases and proteins associated with degranulation and proteoglycan biosynthesis, mast cells expressed proteins potentially involved in interactions with neurons and neurotransmitter metabolism, including cell adhesion molecules, ion channels, and G protein coupled receptors. Toward targeted cell ablation in severe allergic diseases, we used MRGPRX2 for mast cell depletion in human skin biopsies. These proteome analyses suggest a unique role of mast cells in the immune system, probably intertwined with the nervous system.


Assuntos
Mastócitos/citologia , Mastócitos/imunologia , Animais , Biomarcadores/metabolismo , Degranulação Celular , Linhagem da Célula , Células Cultivadas , Tecido Conjuntivo/imunologia , Humanos , Imunoterapia , Mastócitos/metabolismo , Proteínas de Membrana/imunologia , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/imunologia , Proteínas do Tecido Nervoso/metabolismo , Neuroimunomodulação , Proteoglicanas/biossíntese , Proteoma , Receptores Acoplados a Proteínas-G/imunologia , Receptores Acoplados a Proteínas-G/metabolismo , Receptores de Neuropeptídeos/imunologia , Receptores de Neuropeptídeos/metabolismo , Pele/imunologia
10.
Biochem Biophys Res Commun ; 524(4): 890-894, 2020 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-32057362

RESUMO

Osteoporosis is a disease of low bone mass that places individuals at enhanced risk for fracture, disability, and death. Osteoporosis rates are expected to rise significantly in the coming decades yet there are limited pharmacological treatment options, particularly for long-term management of this chronic condition. The drug development pipeline is relatively bereft of new strategies, causing an urgent and unmet need for developing new strategies and targets for treating osteoporosis. Here, we examine a lesser-studied bone remodeling pathway, Neuromedin U (NMU), which is expressed in the bone microenvironment along with its cognate receptors NMU receptor 1 (NMUR1) and 2 (NMUR2). We independently corroborate a prior report that global loss of NMU expression leads to high bone mass and test the hypothesis that NMU negatively regulates osteoblast differentiation. Consistent with this, in vitro studies reveal NMU represses osteoblastic differentiation of osteogenic precursors but, in contrast, promotes osteoblastic marker expression, proliferation and activity of osteoblast-like cells. Phospho-profiling arrays were used to detail differential signaling outcomes that may underlie the opposite responses of these cell types. Collectively, our findings indicate that NMU exerts cell-type-specific responses to regulate osteoblast differentiation and activity.


Assuntos
Neuropeptídeos/genética , Osteoblastos/metabolismo , Osteoporose/genética , Fosfoproteínas/genética , Receptores Acoplados a Proteínas-G/genética , Receptores de Neuropeptídeos/genética , Receptores de Neurotransmissores/genética , Animais , Densidade Óssea , Osso e Ossos/metabolismo , Osso e Ossos/patologia , Diferenciação Celular , Linhagem Celular , Modelos Animais de Doenças , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Camundongos , Camundongos Knockout , Neuropeptídeos/metabolismo , Osteoblastos/patologia , Osteogênese/genética , Osteoporose/metabolismo , Osteoporose/patologia , Fosfoproteínas/classificação , Fosfoproteínas/metabolismo , Fosforilação , Receptores Acoplados a Proteínas-G/metabolismo , Receptores de Neuropeptídeos/metabolismo , Receptores de Neurotransmissores/metabolismo , Transdução de Sinais
11.
PLoS One ; 15(1): e0227666, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31945099

RESUMO

Species-specific sex pheromones play key roles in moth sexual communication. Although the general pathway of Type-I sex pheromone biosynthesis is well established, only a handful of genes encoding enzymes involved in this pathway have been characterized. Streltzoviella insularis is a destructive wood-boring pest of many street trees in China, and the female sex pheromone of this species comprises a blend of (Z)-3-tetradecenyl acetate, (E)-3-tetradecenyl acetate, and (Z)-5-dodecenyl acetate. This organism therefore provides an excellent model for research on the diversity of genes and molecular mechanisms involved in pheromone production. Herein, we assembled the pheromone gland transcriptome of S. insularis by next-generation sequencing and identified 74 genes encoding candidate key enzymes involved in the fatty acid biosynthesis, ß-oxidation, and functional group modification. In addition, tissue expression patterns further showed that an acetyl-CoA carboxylase and two desaturases were highly expressed in the pheromone glands compared with the other tissues, indicating possible roles in S. insularis sex pheromone biosynthesis. Finally, we proposed putative S. insularis biosynthetic pathways for sex pheromone components and highlighted candidate genes. Our findings lay a solid foundation for understanding the molecular mechanisms underpinning S. insularis sex pheromone biosynthesis, and provide potential targets for disrupting chemical communication that could assist the development of novel pest control methods.


Assuntos
Genes de Insetos , Mariposas/genética , Mariposas/metabolismo , Atrativos Sexuais/biossíntese , Atrativos Sexuais/genética , Acetil-CoA Carboxilase/genética , Acetil-CoA Carboxilase/metabolismo , Acetiltransferases/genética , Acetiltransferases/metabolismo , Álcool Desidrogenase/genética , Álcool Desidrogenase/metabolismo , Aldeído Oxirredutases/genética , Aldeído Oxirredutases/metabolismo , Aldeído Redutase/genética , Aldeído Redutase/metabolismo , Animais , Vias Biossintéticas/genética , China , Ácidos Graxos Dessaturases/genética , Ácidos Graxos Dessaturases/metabolismo , Ácido Graxo Sintases/genética , Ácido Graxo Sintases/metabolismo , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Filogenia , Receptores de Neuropeptídeos/genética , Receptores de Neuropeptídeos/metabolismo , Glândulas Odoríferas/metabolismo , Análise de Sequência de RNA , Transcriptoma
12.
FASEB J ; 34(1): 133-147, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31914613

RESUMO

Neuromedin U (NMU), a highly conserved peptide in mammals, is involved in a wide variety of physiological processes. NMU, which is synthesized in ß-cells and co-localizes with insulin, directly acts on ß-cells via NMU receptor 1 (NMUR1) to suppress glucose-stimulated insulin secretion (GSIS). The mechanism underlying this insulinostatic effect has yet to be elucidated. We observed that NMU caused mitochondrial dysfunction by impairing mitochondrial biogenesis, respiration, and mitochondrial Ca2+ uptake in ß-cell-derived MIN6-K8 cells. NMU administration induced the endoplasmic reticulum (ER) stress, as reflected by the activation of ER stress signaling pathways involving ATF6, XBP-1s, and PERK-ATF4-CHOP. Nmu knockdown in MIN6-K8 cells increased the number of insulin granules and improved mitochondrial biogenesis and function. NMU was upregulated in both the islets of db/db mice and palmitate-treated MIN6-K8 cells. Our results highlight the crucial role of NMU in the maintenance of ß-cell function and glucose metabolism through regulation of mitochondria dysfunction and ER stress. In pathological stages that develop into diabetes, upregulation of NMU could suppress the insulin secretion by inducing mitochondrial dysfunction and ER stress, which may contribute to subsequent ß-cell dysfunction.


Assuntos
Estresse do Retículo Endoplasmático/efeitos dos fármacos , Células Secretoras de Insulina/efeitos dos fármacos , Insulina/metabolismo , Mitocôndrias/efeitos dos fármacos , Neuropeptídeos/farmacologia , Animais , Apoptose , Cálcio , Linhagem Celular Tumoral , Regulação da Expressão Gênica/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Insulinoma , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Interferência de RNA , Espécies Reativas de Oxigênio , Receptores Acoplados a Proteínas-G/genética , Receptores Acoplados a Proteínas-G/metabolismo , Receptores de Neuropeptídeos/genética , Receptores de Neuropeptídeos/metabolismo , Receptores de Neurotransmissores/genética , Receptores de Neurotransmissores/metabolismo
13.
Phytother Res ; 34(2): 401-408, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31667930

RESUMO

Mas-related G protein-coupled receptor-X2 (MRGPRX2) expressed on mast cells (MCs) has been shown to be a pivotal target for pseudo-allergic diseases. Therefore, MRGPRX2 might be a therapeutic target for allergic contact dermatitis, atopic dermatitis, and red man syndrome. Paeoniflorin (PF) was reported to have an antiinflammatory effect in neuroinflammation, enteritis, and so forth. In this study, we investigated the anti-pseudo-allergic effect of PF and the underlying molecular mechanisms. Our results showed that PF can suppress compound 48/80 (C48/80)-induced PCA and MCs degranulation in vivo, in a dose-dependent manner. Moreover, PF can reduce C48/80-induced calcium influx and suppress MC degranulation and chemokines release in vitro. PF can downregulate the phosphorylation levels of key kinases in PLCγ-regulated calcium influx and subsequent cytokine synthesis pathways. Our study revealed that PF could inhibit C48/80-induced allergic responses both in vivo and in vitro. As such, it may be regarded as a novel inhibitor for preventing MRGPRX2-mediated allergic diseases.


Assuntos
Antialérgicos/uso terapêutico , Sinalização do Cálcio/efeitos dos fármacos , Glucosídeos/uso terapêutico , Hipersensibilidade/tratamento farmacológico , Mastócitos/efeitos dos fármacos , Monoterpenos/uso terapêutico , Proteínas do Tecido Nervoso/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Receptores de Neuropeptídeos/metabolismo , Animais , Degranulação Celular/efeitos dos fármacos , Quimiocinas/metabolismo , Citocinas/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL
14.
Arch Insect Biochem Physiol ; 103(2): e21625, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31565815

RESUMO

In insects, neuropeptides constitute a group of signaling molecules that act in regulation of multiple physiological and behavioral processes by binding to their corresponding receptors. On the basis of the bioinformatic approaches, we screened the genomic and transcriptomic data of the parasitoid wasp, Pteromalus puparum, and annotated 36 neuropeptide precursor genes and 33 neuropeptide receptor genes. Compared to the number of precursor genes in Bombyx mori (Lepidoptera), Chilo suppressalis (Lepidoptera), Drosophila melanogaster (Diptera), Nilaparvata lugens (Hemiptera), Apis mellifera (Hymenoptera), and Tribolium castaneum (Coleoptera), P. puparum (Hymenoptera) has the lowest number of neuropeptide precursor genes. This lower number may relate to its parasitic life cycle. Transcriptomic data of embryos, larvae, pupae, adults, venom glands, salivary glands, ovaries, and the remaining carcass revealed stage-, sex-, and tissue-specific expression patterns of the neuropeptides, and their receptors. These data provided basic information about the identity and expression profiles of neuropeptides and their receptors that are required to functionally address their biological significance in an endoparasitoid wasp.


Assuntos
Proteínas de Insetos/genética , Neuropeptídeos/genética , Receptores de Neuropeptídeos/genética , Vespas/genética , Sequência de Aminoácidos , Animais , Embrião não Mamífero/química , Embrião não Mamífero/metabolismo , Feminino , Perfilação da Expressão Gênica , Proteínas de Insetos/química , Proteínas de Insetos/metabolismo , Larva/genética , Larva/metabolismo , Masculino , Neuropeptídeos/química , Neuropeptídeos/metabolismo , Filogenia , Pupa/genética , Pupa/metabolismo , Receptores de Neuropeptídeos/química , Receptores de Neuropeptídeos/metabolismo , Alinhamento de Sequência , Vespas/crescimento & desenvolvimento , Vespas/metabolismo
15.
Gen Comp Endocrinol ; 285: 113294, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31585115

RESUMO

Motilin and ghrelin were identified in the pheasant by molecular cloning, and the actions of both peptides on the contractility of gastrointestinal (GI) strips were examined in vitro. Molecular cloning indicated that the deduced amino acid sequences of the pheasant motilin and ghrelin were a 22-amino acid peptide, FVPFFTQSDIQKMQEKERIKGQ, and a 26-amino acid peptide, GSSFLSPAYKNIQQQKDTRKPTGRLH, respectively. In in vitro studies using pheasant GI strips, chicken motilin caused contraction of the proventriculus and small intestine, whereas the crop and colon were insensitive. Human motilin, but not erythromycin, caused contraction of small intestine. Chicken motilin-induced contractions in the proventriculus and ileum were not inhibited by a mammalian motilin receptor antagonist, GM109. Neither atropine (a cholinergic receptor antagonist) nor tetrodotoxin (a neuron blocker) inhibited the responses of chicken motilin in the ileum but both drugs decreased the responses to motilin in the proventriculus, suggesting that the contractile mechanisms of motilin in the proventriculus was neurogenic, different from that of the small intestine (myogenic). On the other hand, chicken and quail ghrelin did not cause contraction in any regions of pheasant GI tract. Since interaction of ghrelin and motilin has been reported in the house musk shrew, interaction of two peptides was examined. The chicken motilin-induced contractions were not modified by ghrelin, and ghrelin also did not cause any contraction under the presence of motilin, suggesting the absence of interaction in both peptides. In conclusion, both the motilin system and ghrelin system are present in the pheasant. Regulation of GI motility by motilin might be common in avian species. However, absence of ghrelin actions in any GI regions suggests the avian species-related difference in regulation of GI contractility by ghrelin.


Assuntos
Aves/metabolismo , Trato Gastrointestinal/fisiologia , Grelina/farmacologia , Motilina/farmacologia , Contração Muscular/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Atropina/farmacologia , Sequência de Bases , Galinhas , Clonagem Molecular , Feminino , Motilidade Gastrointestinal/efeitos dos fármacos , Motilidade Gastrointestinal/fisiologia , Trato Gastrointestinal/efeitos dos fármacos , Grelina/química , Grelina/genética , Humanos , Masculino , Motilina/química , Motilina/genética , Proventrículo/efeitos dos fármacos , Codorniz , Ratos , Receptores dos Hormônios Gastrointestinais/metabolismo , Receptores de Neuropeptídeos/metabolismo , Tetrodotoxina/farmacologia
16.
Neuron ; 105(5): 909-920.e5, 2020 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-31879163

RESUMO

Dopamine neurons of the ventral tegmental area (VTA) regulate reward association and motivation. It remains unclear whether there are distinct dopamine populations to mediate these functions. Using mouse genetics, we isolated two populations of dopamine-producing VTA neurons with divergent projections to the nucleus accumbens (NAc) core and shell. Inhibition of VTA-core-projecting neurons disrupted Pavlovian reward learning, and activation of these cells promoted the acquisition of an instrumental response. VTA-shell-projecting neurons did not regulate Pavlovian reward learning and could not facilitate acquisition of an instrumental response, but their activation could drive robust responding in a previously learned instrumental task. Both populations are activated simultaneously by cues, actions, and rewards, and this co-activation is required for robust reinforcement of behavior. Thus, there are functionally distinct dopamine populations in the VTA for promoting motivation and reward association, which operate on the same timescale to optimize behavioral reinforcement.


Assuntos
Associação , Condicionamento Clássico/fisiologia , Neurônios Dopaminérgicos/fisiologia , Motivação , Núcleo Accumbens/fisiologia , Recompensa , Área Tegmentar Ventral/fisiologia , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Dependovirus , Neurônios Dopaminérgicos/citologia , Neurônios Dopaminérgicos/metabolismo , Vetores Genéticos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Camundongos , Vias Neurais/fisiologia , Núcleo Accumbens/citologia , Receptores de Neuropeptídeos/genética , Receptores de Neuropeptídeos/metabolismo , Reforço Psicológico , Área Tegmentar Ventral/citologia
17.
Int J Mol Sci ; 20(21)2019 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-31652731

RESUMO

The neuropeptide substance P (SP) contributes to neurogenic inflammation through the activation of human mast cells via Mas-related G protein-coupled receptor-X2 (MRGPRX2). Using pertussis toxins and YM-254890, we demonstrated that SP induces Ca2+ mobilization and degranulation via both the Gαi and Gαq family of G proteins in rat basophilic leukemia (RBL-2H3) cells stably expressing MRGPRX2. To determine the roles of MRGPRX2's transmembrane (TM) and intracellular domains on SP-induced responses, we utilized information obtained from both structural modeling and naturally occurring MRGPRX2 missense variants. We found that highly conserved residues in TM6 (I225) and TM7 (Y279) of MRGPRX2 are essential for SP-induced Ca2+ mobilization and degranulation in transiently transfected RBL-2H3 cells. Cells expressing missense variants in the receptor's conserved residues (V123F and V282M) as well as intracellular loops (R138C and R141C) failed to respond to SP. By contrast, replacement of all five Ser/Thr residues with Ala and missense variants (S325L and L329Q) in MRGPRX2's carboxyl-terminus resulted in enhanced mast cell activation by SP when compared to the wild-type receptor. These findings suggest that MRGPRX2 utilizes conserved residues in its TM domains and intracellular loops for coupling to G proteins and likely undergoes desensitization via phosphorylation at Ser/Thr residues in its carboxyl-terminus. Furthermore, identification of gain and loss of function MRGPRX2 variants has important clinical implications for SP-mediated neurogenic inflammation and other chronic inflammatory diseases.


Assuntos
Mutação com Ganho de Função , Mutação com Perda de Função , Mastócitos/metabolismo , Proteínas do Tecido Nervoso/química , Receptores Acoplados a Proteínas-G/química , Receptores de Neuropeptídeos/química , Substância P/metabolismo , Animais , Cálcio/metabolismo , Linhagem Celular Tumoral , Sequência Conservada , Humanos , Mutação de Sentido Incorreto , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Domínios Proteicos , Ratos , Receptores Acoplados a Proteínas-G/genética , Receptores Acoplados a Proteínas-G/metabolismo , Receptores de Neuropeptídeos/genética , Receptores de Neuropeptídeos/metabolismo
18.
J Dermatol Sci ; 95(3): 99-106, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31558225

RESUMO

BACKGROUND: Thimerosal has been used as a preservative in many products which may cause contact dermatitis. It is the second most common allergen in positive patch test reactions, though being a clinical irrelevant allergen. Thimerosal-induced contact dermatitis is generally considered to be a delayed-type hypersensitivity reaction, but it is difficult to explain the fact that most patients develop an allergic reaction upon first encounter with thimerosal. Recent studies have demonstrated the association between Mas-related G protein coupled receptor X2 (MRGPRX2) and pseudo-allergic reactions which occur at the first contact with stimulation. This suggests the possibility that thimerosal may cause contact dermatitis via MRGPRX2 mediated mechanism. OBJECTIVES: To investigate the role of Mas-related G-protein coupled receptor B2 (MrgprB2)/MRGPRX2 in contact dermatitis induced by thimerosal. METHODS: Thimerosal induced pseudo-allergic reactions via MrgprB2/ MRGPRX2 were investigated using a novel skin pseudo-allergic reaction mouse model, footpad swelling and extravasation assays in vivo and mast cell degranulation assay in vitro. RESULTS: Thimerosal induced contact dermatitis in dorsal skin and footpad swelling in wild-type mice, but had no significant effect in MrgprB2-knockout mice. Thimerosal-induced dermatitis is characterized by infiltration of inflammatory cells and elevation of serum histamine and inflammatory cytokines, rather than elevation of serum IgE level. Thimerosal increased the intracellular Ca2+ concentration in HEK293 cells overexpressing MrgprB2/MRGPRX2. Downregulation of MRGPRX2 resulted in the reduced degranulation of LAD2 human mast cells. CONCLUSIONS: MrgprB2 mediates thimerosal-induced mast cell degranulation and pseudo-allergic reaction in mice. MRGPRX2 may be a key contributor to human contact dermatitis.


Assuntos
Dermatite de Contato/etiologia , Hipersensibilidade Tardia/etiologia , Mastócitos/efeitos dos fármacos , Proteínas do Tecido Nervoso/metabolismo , Conservantes Farmacêuticos/toxicidade , Receptores Acoplados a Proteínas-G/metabolismo , Receptores de Neuropeptídeos/metabolismo , Timerosal/efeitos adversos , Administração Cutânea , Animais , Degranulação Celular/efeitos dos fármacos , Dermatite de Contato/patologia , Modelos Animais de Doenças , Células HEK293 , Humanos , Hipersensibilidade Tardia/patologia , Masculino , Mastócitos/patologia , Camundongos , Camundongos Knockout , Conservantes Farmacêuticos/administração & dosagem , Receptores Acoplados a Proteínas-G/genética , Timerosal/administração & dosagem
19.
Acta Derm Venereol ; 99(12): 1131-1135, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31449313

RESUMO

Most canine visits to veterinarians are related to skin diseases with itch being the chief complaint. Historically, several itch-inducing molecules and pathways have been identified in mice, but whether or not these are similar in dogs is not yet known. Herein, we set out to study the expression of pruritogenic neuropeptides, their cognate receptors with a limited functional validation thereof using a multidisciplinary approach. We demonstrated the expression of somatostatin and other major neuropeptides and receptors in canine dorsal root ganglia neurons. Next, we showed that interleukin-31, serotonin, and histamine activate such neurons. Furthermore, we demonstrated the physiological release of somatostatin from dog dorsal root ganglia neurons in response to several endogenous itch mediators. In summary, our results provide the first evidence that dogs use similar pruritogenic pathways to those characterized in mice and we thus identify multiple targets for the future treatment of itch in dogs.


Assuntos
Gânglios Espinais/metabolismo , Neuropeptídeos/metabolismo , Prurido/metabolismo , Receptores de Neuropeptídeos/metabolismo , Medula Espinal/metabolismo , Animais , Sinalização do Cálcio , Células Cultivadas , Cães , Feminino , Gânglios Espinais/fisiopatologia , Regulação da Expressão Gênica , Masculino , Neuropeptídeos/genética , Prurido/genética , Prurido/fisiopatologia , Receptores de Neuropeptídeos/genética , Medula Espinal/fisiopatologia
20.
Endocrinology ; 160(11): 2673-2691, 2019 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-31436800

RESUMO

Isolated growth hormone deficiency type II (IGHD2) is mainly caused by heterozygous splice-site mutations in intron 3 of the GH1 gene. A dominant-negative effect of the mutant GH lacking exon 3 on wild-type GH secretion has been proposed; however, the molecular mechanisms involved are elusive. To uncover the molecular systems underlying GH deficiency in IGHD2, we established IGHD2 model mice, which carry both wild-type and mutant copies of the human GH1 gene, replacing each of the endogenous mouse Gh loci. Our IGHD2 model mice exhibited growth retardation along with intact cellular architecture and mildly activated endoplasmic reticulum stress in the pituitary gland, caused by decreased GH-releasing hormone receptor (Ghrhr) and Gh gene promoter activities. Decreased Ghrhr and Gh promoter activities were likely caused by reduced levels of nuclear CREB3L2, which was demonstrated to stimulate Ghrhr and Gh promoter activity. To our knowledge, this is the first in vivo study to reveal a novel molecular mechanism of GH deficiency in IGHD2, representing a new paradigm that differs from widely accepted models.


Assuntos
Nanismo Hipofisário/etiologia , Hormônio do Crescimento/metabolismo , Receptores de Neuropeptídeos/metabolismo , Receptores de Hormônios Reguladores de Hormônio Hipofisário/metabolismo , Animais , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Nanismo Hipofisário/patologia , Feminino , Hormônio do Crescimento/genética , Humanos , Masculino , Camundongos , Hipófise/metabolismo , Hipófise/ultraestrutura , Regiões Promotoras Genéticas , Receptores de Neuropeptídeos/genética , Receptores de Hormônios Reguladores de Hormônio Hipofisário/genética
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