RESUMEN
The notion that neutrophils exist as a homogeneous population is being replaced with the knowledge that neutrophils adopt different functional states. Neutrophils can have a pro-inflammatory phenotype or an anti-inflammatory state, but how these states are regulated remains unclear. Here, we demonstrated that the neutrophil-expressed G-protein-coupled receptor (GPCR) Mrgpra1 is a negative regulator of neutrophil bactericidal functions. Mrgpra1-mediated signaling was driven by its ligand, neuropeptide FF (NPFF), which dictated the balance between pro- and anti-inflammatory programming. Specifically, the Mrgpra1-NPFF axis counter-regulated interferon (IFN) γ-mediated neutrophil polarization during acute lung infection by favoring an alternative-like polarization, suggesting that it may act to balance overzealous neutrophilic responses. Distinct, cross-regulated populations of neutrophils were the primary source of NPFF and IFNγ during infection. As a subset of neutrophils at steady state expressed NPFF, these findings could have broad implications in various infectious and inflammatory diseases. Therefore, a neutrophil-intrinsic pathway determines their cellular fate, function, and magnitude of infection.
Asunto(s)
Infecciones Bacterianas , Neuropéptidos , Humanos , Receptores de Neuropéptido/metabolismo , Neutrófilos/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , AntiinflamatoriosRESUMEN
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.
Asunto(s)
Mastocitos/citología , Mastocitos/inmunología , Animales , Biomarcadores/metabolismo , Degranulación de la Célula , Linaje de la Célula , Células Cultivadas , Tejido Conectivo/inmunología , Humanos , Inmunoterapia , Mastocitos/metabolismo , Proteínas de la Membrana/inmunología , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Proteínas del Tejido Nervioso/inmunología , Proteínas del Tejido Nervioso/metabolismo , Neuroinmunomodulación , Proteoglicanos/biosíntesis , Proteoma , Receptores Acoplados a Proteínas G/inmunología , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Neuropéptido/inmunología , Receptores de Neuropéptido/metabolismo , Piel/inmunologíaRESUMEN
Classical itch studies have focused on immunoglobulin E (IgE)-mediated mast cell activation and histamine release. Recently, members of the Mas-related G-protein-coupled receptor (Mrgpr) family have been identified as mast cell receptors, but their role in itch is unclear. Here, we report that mast cell activation via Mrgprb2 evoked non-histaminergic itch in mice independently of the IgE-Fc epsilon RI (FcεRI)-histamine axis. Compared with IgE-FcεRI stimulation, Mrgprb2 activation of mast cells was distinct in both released substances (histamine, serotonin, and tryptase) and the pattern of activated itch-sensory neurons. Mrgprb2 deficiency decreased itch in multiple preclinical models of allergic contact dermatitis (ACD), a pruritic inflammatory skin disorder, and both mast cell number and PAMP1-20 concentrations (agonist of the human Mrgprb2 homolog, MRGPRX2) were increased in human ACD skin. These findings suggest that this pathway may represent a therapeutic target for treating ACD and mast-cell-associated itch disorders in which antihistamines are ineffective.
Asunto(s)
Mastocitos/inmunología , Proteínas del Tejido Nervioso/metabolismo , Prurito/patología , Receptores Acoplados a Proteínas G/metabolismo , Receptores de IgE/metabolismo , Receptores de Neuropéptido/metabolismo , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Animales , Femenino , Histamina/metabolismo , Antagonistas de los Receptores Histamínicos/uso terapéutico , Humanos , Inmunoglobulina E/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana Edad , Fragmentos de Péptidos/metabolismo , Receptores Acoplados a Proteínas G/genética , Serotonina/metabolismo , Piel/metabolismo , Triptasas/metabolismo , Adulto JovenRESUMEN
Peripheral sensory neurons recognize diverse noxious stimuli, including microbial products and allergens traditionally thought to be targets of the mammalian immune system. Activation of sensory neurons by these stimuli leads to pain and itch responses as well as the release of neuropeptides that interact with their cognate receptors expressed on immune cells, such as dendritic cells (DCs). Neuronal control of immune cell function through neuropeptide release not only affects local inflammatory responses but can impact adaptive immune responses through downstream effects on T cell priming. Numerous neuropeptide receptors are expressed by DCs but only a few have been characterized, presenting opportunities for further investigation of the pathways by which cutaneous neuroimmune interactions modulate host immunity.
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Células Receptoras Sensoriales , Piel , Humanos , Animales , Células Receptoras Sensoriales/inmunología , Células Receptoras Sensoriales/metabolismo , Células Receptoras Sensoriales/fisiología , Piel/inmunología , Neuropéptidos/metabolismo , Neuropéptidos/inmunología , Células Dendríticas/inmunología , Neuroinmunomodulación , Receptores de Neuropéptido/metabolismo , Receptores de Neuropéptido/inmunologíaRESUMEN
In the clades of animals that diverged from the bony fish, a group of Mas-related G-protein-coupled receptors (MRGPRs) evolved that have an active role in itch and allergic signals1,2. As an MRGPR, MRGPRX2 is known to sense basic secretagogues (agents that promote secretion) and is involved in itch signals and eliciting pseudoallergic reactions3-6. MRGPRX2 has been targeted by drug development efforts to prevent the side effects induced by certain drugs or to treat allergic diseases. Here we report a set of cryo-electron microscopy structures of the MRGPRX2-Gi1 trimer in complex with polycationic compound 48/80 or with inflammatory peptides. The structures of the MRGPRX2-Gi1 complex exhibited shallow, solvent-exposed ligand-binding pockets. We identified key common structural features of MRGPRX2 and describe a consensus motif for peptidic allergens. Beneath the ligand-binding pocket, the unusual kink formation at transmembrane domain 6 (TM6) and the replacement of the general toggle switch from Trp6.48 to Gly6.48 (superscript annotations as per Ballesteros-Weinstein nomenclature) suggest a distinct activation process. We characterized the interfaces of MRGPRX2 and the Gi trimer, and mapped the residues associated with key single-nucleotide polymorphisms on both the ligand and G-protein interfaces of MRGPRX2. Collectively, our results provide a structural basis for the sensing of cationic allergens by MRGPRX2, potentially facilitating the rational design of therapies to prevent unwanted pseudoallergic reactions.
Asunto(s)
Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/metabolismo , Prurito/metabolismo , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Neuropéptido/química , Receptores de Neuropéptido/metabolismo , Alérgenos/inmunología , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Sitios de Unión , Secuencia de Consenso , Microscopía por Crioelectrón , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Humanos , Modelos Moleculares , Proteínas del Tejido Nervioso/inmunología , Proteínas del Tejido Nervioso/ultraestructura , Receptores Acoplados a Proteínas G/inmunología , Receptores Acoplados a Proteínas G/ultraestructura , Receptores de Neuropéptido/inmunología , Receptores de Neuropéptido/ultraestructuraRESUMEN
The MRGPRX family of receptors (MRGPRX1-4) is a family of mas-related G-protein-coupled receptors that have evolved relatively recently1. Of these, MRGPRX2 and MRGPRX4 are key physiological and pathological mediators of itch and related mast cell-mediated hypersensitivity reactions2-5. MRGPRX2 couples to both Gi and Gq in mast cells6. Here we describe agonist-stabilized structures of MRGPRX2 coupled to Gi1 and Gq in ternary complexes with the endogenous peptide cortistatin-14 and with a synthetic agonist probe, respectively, and the development of potent antagonist probes for MRGPRX2. We also describe a specific MRGPRX4 agonist and the structure of this agonist in a complex with MRGPRX4 and Gq. Together, these findings should accelerate the structure-guided discovery of therapeutic agents for pain, itch and mast cell-mediated hypersensitivity.
Asunto(s)
Microscopía por Crioelectrón , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Proteínas del Tejido Nervioso/química , Prurito/metabolismo , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Receptores Acoplados a Proteínas G/química , Receptores de Neuropéptido/antagonistas & inhibidores , Receptores de Neuropéptido/química , Agonismo Inverso de Drogas , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/química , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/ultraestructura , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/química , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/ultraestructura , Humanos , Modelos Moleculares , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Tejido Nervioso/ultraestructura , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/ultraestructura , Receptores de Neuropéptido/metabolismo , Receptores de Neuropéptido/ultraestructuraRESUMEN
Internal physiological states influence behavioral decisions. We have investigated the underlying cellular and molecular mechanisms at the first olfactory synapse for starvation modulation of food-search behavior in Drosophila. We found that a local signal by short neuropeptide F (sNPF) and a global metabolic cue by insulin are integrated at specific odorant receptor neurons (ORNs) to modulate olfactory sensitivity. Results from two-photon calcium imaging show that starvation increases presynaptic activity via intraglomerular sNPF signaling. Expression of sNPF and its receptor (sNPFR1) in Or42b neurons is necessary for starvation-induced food-search behavior. Presynaptic facilitation in Or42b neurons is sufficient to mimic starvation-like behavior in fed flies. Furthermore, starvation elevates the transcription level of sNPFR1 but not that of sNPF, and insulin signaling suppresses sNPFR1 expression. Thus, starvation increases expression of sNPFR1 to change the odor map, resulting in more robust food-search behavior.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila/fisiología , Neuropéptidos/metabolismo , Receptores de Neuropéptido/metabolismo , Receptores Odorantes/metabolismo , Transducción de Señal , Animales , Antenas de Artrópodos/metabolismo , Femenino , Odorantes , Células Receptoras Sensoriales/metabolismo , Inanición/metabolismo , Sinapsis/metabolismoRESUMEN
Human prefrontal cortex (hPFC) is a complex brain region involved in cognitive and emotional processes and several psychiatric disorders. Here, we present an overview of the distribution of the peptidergic systems in 17 subregions of hPFC and three reference cortices obtained by microdissection and based on RNA sequencing and RNAscope methods integrated with published single-cell transcriptomics data. We detected expression of 60 neuropeptides and 60 neuropeptide receptors in at least one of the hPFC subregions. The results reveal that the peptidergic landscape in PFC consists of closely located and functionally different subregions with unique peptide/transmitter-related profiles. Neuropeptide-rich PFC subregions were identified, encompassing regions from anterior cingulate cortex/orbitofrontal gyrus. Furthermore, marked differences in gene expression exist between different PFC regions (>5-fold; cocaine and amphetamine-regulated transcript peptide) as well as between PFC regions and reference regions, for example, for somatostatin and several receptors. We suggest that the present approach allows definition of, still hypothetical, microcircuits exemplified by glutamatergic neurons expressing a peptide cotransmitter either as an agonist (hypocretin/orexin) or antagonist (galanin). Specific neuropeptide receptors have been identified as possible targets for neuronal afferents and, interestingly, peripheral blood-borne peptide hormones (leptin, adiponectin, gastric inhibitory peptide, glucagon-like peptides, and peptide YY). Together with other recent publications, our results support the view that neuropeptide systems may play an important role in hPFC and underpin the concept that neuropeptide signaling helps stabilize circuit connectivity and fine-tune/modulate PFC functions executed during health and disease.
Asunto(s)
Neuropéptidos , Corteza Prefrontal , Receptores de Neuropéptido , Femenino , Perfilación de la Expresión Génica , Humanos , Masculino , Neuropéptidos/genética , Neuropéptidos/metabolismo , Corteza Prefrontal/metabolismo , Receptores de Neuropéptido/genética , Receptores de Neuropéptido/metabolismoRESUMEN
Mas-related G protein-coupled receptor X2 (MrgprX2) plays a crucial role in anaphylactoid reactions and allergic diseases. Some antagonists with reasonable potency and selectivity have been reported. Cell membrane chromatography (CMC) is effective for discovering ligands. Protein-tag-based CMC models (e.g., SNAP tags and HALO tags) have enhanced performance but also increased nonspecific adsorption of small molecules. The Avi tag, a short peptide sequence, binds biotin specifically via BirA catalysis. Our study showed that 2-iminobiotin (IB) can be a BirA substrate, enabling the development of a new cell membrane stationary phase (CMSP) based on the chemical properties (modifying carboxyl silica gel and specifically labeling the Avi tag) of IB. First, we constructed the MrgprX2-Avi-tag HEK293T cell line. Next, we synthesized IB-modified silica gel (SiO2-IB) stepwise. Finally, we immobilized Avi-tagged MrgprX2 cell membranes on SiO2-IB under BirA catalysis. We characterized the developed CMSP and used it to establish a MrgprX2-Avi-tag/CMC-HPLC/MS two-dimensional screening platform, successfully screening vitexicarpin fromViticis Fructus extract via a 2D/CMC platform. In vitro and in vivo experiments confirmed that vitexicarpin targets the MrgprX2 receptor, demonstrating antiallergic effects. Our IB-Avi tag-based CMC approach effectively decreased nonspecific adsorption of the screening materials. The Avi-tag-based 2D/CMC platform is suitable for screening potential drug candidates.
Asunto(s)
Membrana Celular , Receptores Acoplados a Proteínas G , Humanos , Receptores Acoplados a Proteínas G/metabolismo , Células HEK293 , Membrana Celular/metabolismo , Animales , Receptores de Neuropéptido/metabolismo , Receptores de Neuropéptido/antagonistas & inhibidores , Ratones , Cromatografía Líquida de Alta Presión , Proteínas del Tejido NerviosoRESUMEN
Insects have about 50 neuropeptide genes and about 70 genes, coding for neuropeptide G protein-coupled receptors (GPCRs). An important, but small family of evolutionarily related insect neuropeptides consists of adipokinetic hormone (AKH), corazonin, and AKH/corazonin-related peptide (ACP). Normally, insects have one specific GPCR for each of these neuropeptides. The tick Ixodes scapularis is not an insect, but belongs to the subphylum Chelicerata, which comprises ticks, scorpions, mites, spiders, and horseshoe crabs. Many of the neuropeptides and neuropeptide GPCRs occurring in insects, also occur in chelicerates, illustrating that insects and chelicerates are evolutionarily closely related. The tick I. scapularis is an ectoparasite and health risk for humans, because it infects its human host with dangerous pathogens during a blood meal. Understanding the biology of ticks will help researchers to prevent tick-borne diseases. By annotating the I. scapularis genome sequence, we previously found that ticks contain as many as five genes, coding for presumed ACP receptors. In the current paper, we cloned these receptors and expressed each of them in Chinese Hamster Ovary (CHO) cells. Each expressed receptor was activated by nanomolar concentrations of ACP, demonstrating that all five receptors were functional ACP receptors. Phylogenetic tree analyses showed that the cloned tick ACP receptors were mostly related to insect ACP receptors and, next, to insect AKH receptors, suggesting that ACP receptor genes and AKH receptor genes originated by gene duplications from a common ancestor. Similar duplications have probably occurred for the ligand genes, during a process of ligand/receptor co-evolution. Interestingly, chelicerates, in contrast to all other arthropods, do not have AKH or AKH receptor genes. Therefore, the ancestor of chelicerates might have lost AKH and AKH receptor genes and functionally replaced them by ACP and ACP receptor genes. For the small family of AKH, ACP, and corazonin receptors and their ligands, gene losses and gene gains occur frequently between the various ecdysozoan clades. Tardigrades, for example, which are well known for their survival in extreme environments, have as many as ten corazonin receptor genes and six corazonin peptide genes, while insects only have one of each, or none.
Asunto(s)
Hormonas de Insectos , Ixodes , Neuropéptidos , Oligopéptidos , Ácido Pirrolidona Carboxílico , Receptores Acoplados a Proteínas G , Animales , Neuropéptidos/metabolismo , Neuropéptidos/genética , Hormonas de Insectos/metabolismo , Hormonas de Insectos/genética , Ixodes/metabolismo , Ixodes/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/genética , Oligopéptidos/metabolismo , Oligopéptidos/genética , Oligopéptidos/química , Ácido Pirrolidona Carboxílico/análogos & derivados , Ácido Pirrolidona Carboxílico/metabolismo , Filogenia , Secuencia de Aminoácidos , Cricetulus , Células CHO , Proteínas de Insectos/genética , Proteínas de Insectos/metabolismo , Receptores de Neuropéptido/metabolismo , Receptores de Neuropéptido/genéticaRESUMEN
Motilin (MLN) is a peptide hormone originally isolated from the mucosa of the porcine intestine. Its orthologs have been identified in various vertebrates. Although MLN regulates gastrointestinal motility in tetrapods from amphibians to mammals, recent studies indicate that MLN is not involved in the regulation of isolated intestinal motility in zebrafish, at least in vitro. To determine the unknown function of MLN in teleosts, we examined the expression of MLN and the MLN receptor (MLNR) at the cellular level in Japanese medaka (Oryzias latipes). Quantitative PCR revealed that mln mRNA was limitedly expressed in the gut, whereas mlnr mRNA was not detected in the gut but was expressed in the brain and kidney. By in situ hybridization and immunohistochemistry, mlnr mRNA was detected in the dopaminergic neurons of the area postrema in the brain and the noradrenaline-producing cells in the interrenal gland of the kidney. Furthermore, we observed efferent projections of mlnr-expressing dopaminergic neurons in the lobus vagi (XL) and nucleus motorius nervi vagi (NXm) of the medulla oblongata by establishing a transgenic medaka expressing the enhanced green fluorescence protein driven by the mlnr promoter. The expression of dopamine receptor mRNAs in the XL and cholinergic neurons in NXm was confirmed by in situ hybridization. These results indicate novel sites of MLN activity other than the gastrointestinal tract. MLN may exert central and peripheral actions through the regulation of catecholamine release in medaka.
Asunto(s)
Motilina , Oryzias , Receptores de la Hormona Gastrointestinal , Animales , Oryzias/metabolismo , Oryzias/genética , Receptores de la Hormona Gastrointestinal/metabolismo , Receptores de la Hormona Gastrointestinal/genética , Motilina/metabolismo , Receptores de Neuropéptido/metabolismo , Receptores de Neuropéptido/genética , ARN Mensajero/metabolismo , ARN Mensajero/genética , Animales Modificados Genéticamente , Neuronas Dopaminérgicas/metabolismo , Encéfalo/metabolismoRESUMEN
Growth hormone-releasing hormone (GHRH) has been widely shown to stimulate growth hormone (GH) production via binding to GHRH receptor GHRHR in various species of vertebrates, but information regarding the functional roles of GHRH and GHRHR in the protochordate amphioxus remains rather scarce. We showed here that two mature peptides, BjGHRH-1 and BjGHRH-2, encoded by BjGHRH precursor, and a single BjGHRHR protein were identified in the amphioxus Branchiostoma. japonicum. Like the distribution profiles of vertebrate GHRHs and GHRHRs, both the genes Bjghrh and Bjghrhr were widely expressed in the different tissues of amphioxus, including in the cerebral vesicle, Hatschek's pit, neural tube, gill, hepatic caecum, notochord, testis and ovary. Moreover, both BjGHRH-1 and BjGHRH-2 interacted with BjGHRHR, and triggered the cAMP/PKA signal pathway in a dose-dependent manner. Importantly, BjGHRH-1 and BjGHRH-2 were both able to activate the expression of GH-like gene in the cells of Hatschek's pit. These indicate that a functional vertebrate-like GHRH-GHRHR axis had already emerged in amphioxus, which is a seminal innovation making physiological divergence including reproduction, growth, metabolism, stress and osmoregulation possible during the early evolution of vertebrates.
Asunto(s)
Hormona Liberadora de Hormona del Crecimiento , Anfioxos , Receptores de Neuropéptido , Receptores de Hormona Reguladora de Hormona Hipofisaria , Animales , Anfioxos/metabolismo , Anfioxos/genética , Receptores de Neuropéptido/metabolismo , Receptores de Neuropéptido/genética , Hormona Liberadora de Hormona del Crecimiento/metabolismo , Hormona Liberadora de Hormona del Crecimiento/genética , Receptores de Hormona Reguladora de Hormona Hipofisaria/metabolismo , Receptores de Hormona Reguladora de Hormona Hipofisaria/genética , Sistema Hipotálamo-Hipofisario/metabolismoRESUMEN
Mas-related G protein-coupled receptor X2 (MrgprX2) is acknowledged as a mast cell-specific receptor, playing a crucial role in orchestrating anaphylactoid responses through mast cell degranulation. It holds promise as a target for regulating allergic and inflammatory diseases mediated by mast cells. Polygonum cuspidatum (PC) has shown notable anti-anaphylactoid effects, while its pharmacologically active components remain unclear. In this study, we successfully utilized MrgprX2 high-expressing cell membrane chromatography (CMC), in conjunction with liquid chromatography-mass spectrometry (LC-MS), to identify active anti-anaphylactoid components in PC. Our study pinpointed polydatin, resveratrol, and emodin-8-O-ß-d-glucoside as potential anti-anaphylactoid compounds in PC. Their anti-anaphylactoid activities were evaluated through ß-aminohexosidase and histamine release assays, demonstrating a concentration-dependent inhibition for both ß-aminohexosidase and histamine release. This approach, integrating MrgprX2 high-expression CMC with LC-MS, proves effective in screening potential anti-anaphylactoid ingredients in natural herbal medicines. The findings from this study illuminated the anti-anaphylactoid properties of specific components in PC and provided an efficient method for the drug development of natural products.
Asunto(s)
Fallopia japonica , Receptores Acoplados a Proteínas G , Receptores de Neuropéptido , Receptores Acoplados a Proteínas G/metabolismo , Fallopia japonica/química , Receptores de Neuropéptido/metabolismo , Receptores de Neuropéptido/antagonistas & inhibidores , Humanos , Espectrometría de Masas , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Membrana Celular/química , Cromatografía Liquida , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Mastocitos/efectos de los fármacos , Mastocitos/metabolismo , Extractos Vegetales/farmacología , Extractos Vegetales/química , Glucósidos/farmacología , Glucósidos/química , Glucósidos/análisis , Estructura Molecular , Cromatografía Líquida con Espectrometría de MasasRESUMEN
BACKGROUND: Prurigo nodularis (PN) is a chronic inflammatory skin disorder that is characterized by extremely itchy nodules. Proadrenomedullin N-terminal 20 (PAMP) activates mast cell degranulation via Mas-related G protein-coupled receptor X2 (MRGPRX2), which is associated with pruritus in allergic contact dermatitis. However, the mechanisms underlying the action of PAMP and MRGPRX2 in PN remain unclear. OBJECTIVE: To determine the role of PAMP-induced mast cell activation via MRGPRX2 (mouse homologous Mrgprb2) in PN. METHODS: The expression of PAMP and the number of MRGPRX2-expressing mast cells in the skin biopsies of patients with PN, atopic dermatitis (AD), and healthy participants were analyzed using immunohistochemistry and immunofluorescence, respectively. The biphasic response of PAMP9-20 mediated by Mrgprb2 in mouse peritoneal mast cells (PMC) was validated in vitro using qRT-PCR, ELISA, flow cytometry, and siRNA techniques. RESULTS: PAMP expression and the number of MRGPRX2+ mast cells in lesional PN skin, but not in AD, were elevated compared to healthy skin. PAMP9-20 mediates the immediate and delayed phase responses of PMC, such as degranulation, histamine and ß-hexosaminidase release, and secretion of inflammatory factors such as CCL2, TNF-α, and GM-CSF. These effects were inhibited when Mrgprb2 expression was silenced. Silencing Mrgprb2 did not affect the biphasic response of PMC that was induced by IgE-FcεRI activation. CONCLUSIONS: The results show that PAMP mediates mouse mast cell activation via Mrgprb2, which may be involved in the pathogenesis of PN. The PAMP/ Mrgprb2 pathway, independent of classical IgE signaling, could be developed as a candidate drug target for treating PN.
Asunto(s)
Dermatitis Atópica , Prurigo , Receptores Acoplados a Proteínas G , Animales , Humanos , Ratones , Adrenomedulina/metabolismo , Dermatitis Atópica/patología , Inmunoglobulina E/metabolismo , Mastocitos/metabolismo , Mastocitos/patología , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Prurigo/metabolismo , Prurigo/patología , Prurito , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Neuropéptido/genética , Receptores de Neuropéptido/metabolismo , Piel/metabolismoRESUMEN
Optic neuropathies are leading causes of irreversible visual impairment and blindness, currently affecting more than 100 million people worldwide. Glaucoma is a group of optic neuropathies attributed to progressive degeneration of retinal ganglion cells (RGCs). We have previously demonstrated an increase in survival of RGCs by the activation of macrophages, whereas the inhibition of macrophages was involved in the alleviation on endotoxin-induced inflammation by antagonist of growth hormone-releasing hormone (GHRH). Herein, we hypothesized that GHRH receptor (GHRH-R) signaling could be involved in the survival of RGCs mediated by inflammation. We found the expression of GHRH-R in RGCs of adult rat retina. After optic nerve crush, subcutaneous application of GHRH agonist MR-409 or antagonist MIA-602 promoted the survival of RGCs. Both the GHRH agonist and antagonist increased the phosphorylation of Akt in the retina, but only agonist MR-409 promoted microglia activation in the retina. The antagonist MIA-602 reduced significantly the expression of inflammation-related genes Il1b, Il6, and Tnf Moreover, agonist MR-409 further enhanced the promotion of RGC survival by lens injury or zymosan-induced macrophage activation, whereas antagonist MIA-602 attenuated the enhancement in RGC survival. Our findings reveal the protective effect of agonistic analogs of GHRH on RGCs in rats after optic nerve injury and its additive effect to macrophage activation, indicating a therapeutic potential of GHRH agonists for the protection of RGCs against optic neuropathies especially in glaucoma.
Asunto(s)
Hormona Liberadora de Hormona del Crecimiento/agonistas , Macrófagos/patología , Neuroprotección , Traumatismos del Nervio Óptico/patología , Células Ganglionares de la Retina/patología , Animales , Supervivencia Celular/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Hormona del Crecimiento/metabolismo , Hormona Liberadora de Hormona del Crecimiento/antagonistas & inhibidores , Inflamación/genética , Inflamación/patología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Masculino , Microglía/efectos de los fármacos , Microglía/metabolismo , Microglía/patología , Neuroprotección/efectos de los fármacos , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas Endogámicas F344 , Receptores de Neuropéptido/metabolismo , Receptores de Hormona Reguladora de Hormona Hipofisaria/metabolismo , Células Ganglionares de la Retina/efectos de los fármacos , Células Ganglionares de la Retina/metabolismo , Factor de Transcripción STAT3/metabolismo , Sermorelina/análogos & derivados , Sermorelina/farmacología , Transducción de Señal/efectos de los fármacos , Cuerpo Vítreo/efectos de los fármacos , Cuerpo Vítreo/metabolismo , Zimosan/farmacologíaRESUMEN
BACKGROUND: Activation of Mas-related G protein-coupled receptor X2 (MRGPRX2) is a crucial non-IgE pathway for mast cell activation associated with allergic reactions and inflammation. Only a few peptides and small compounds targeting MRGPRX2 have been reported, with limited information on their pharmacologic activity. OBJECTIVE: We sought to develop novel small molecule MRGPRX2 antagonists to treat MRGPRX2-mediated allergies and inflammation. METHODS: A computational approach was used to design novel small molecules as MRGPRX2 antagonists. The short-listed molecules were synthesized and characterized by liquid chromatography and mass spectrometry as well as nuclear magnetic resonance. Inhibitory activity on MRGPRX2 signaling was assessed in vitro by using functional bioassays (ß-hexosaminidase, calcium flux, and chemokine synthesis) and receptor activation assays (ß-arrestin recruitment and Western blot analysis) in human LAD-2 mast cells and HTLA cells. In vivo effects of the novel MRGPRX2 antagonists were assessed using a mouse model of acute allergy and systemic anaphylaxis. RESULTS: The novel small molecules demonstrated higher binding affinity with MRGPRX2 in the docking study. The half-maximal inhibitory concentration is in the low micromolar range (5-21 µM). The small molecules inhibited not only the early phase of mast cell activation but also the late phase, associated with chemokine and prostaglandin release. Further, Western blot analysis revealed inhibition of downstream phospholipase C-γ, extracellular signal-regulated protein kinase 1/2, and Akt signaling pathway. Moreover, in the mouse models of allergies, small molecule administration effectively blocks acute, systemic allergic reactions and inflammation and prevents systemic anaphylaxis. CONCLUSION: The small molecules might hold a significant therapeutic promise to treat MRGPRX2-mediated allergies and inflammation.
Asunto(s)
Anafilaxia , Animales , Ratones , Humanos , Anafilaxia/patología , Modelos Animales de Enfermedad , Receptores Acoplados a Proteínas G/metabolismo , Quimiocinas/metabolismo , Mastocitos/patología , Inflamación/patología , Receptores de Neuropéptido/metabolismo , Degranulación de la Célula , Proteínas del Tejido Nervioso/metabolismoRESUMEN
BACKGROUND: Drug-induced anaphylaxis is triggered by the direct stimulation of mast cells (MCs) via Mas-related G protein-coupled receptor X2 (MRGPRX2; mouse ortholog MRGPRB2). However, the precise mechanism that links MRGPRX2/B2 to MC degranulation is poorly understood. Dedicator of cytokinesis 2 (DOCK2) is a Rac activator predominantly expressed in hematopoietic cells. Although DOCK2 regulates migration and activation of leukocytes, its role in MCs remains unknown. OBJECTIVE: We aimed to elucidate whether-and if so, how-DOCK2 is involved in MRGPRX2/B2-mediated MC degranulation and anaphylaxis. METHODS: Induction of drug-induced systemic and cutaneous anaphylaxis was compared between wild-type and DOCK2-deficient mice. In addition, genetic or pharmacologic inactivation of DOCK2 in human and murine MCs was used to reveal its role in MRGPRX2/B2-mediated signal transduction and degranulation. RESULTS: Induction of MC degranulation and anaphylaxis by compound 48/80 and ciprofloxacin was severely attenuated in the absence of DOCK2. Although calcium influx and phosphorylation of several signaling molecules were unaffected, MRGPRB2-mediated Rac activation and phosphorylation of p21-activated kinase 1 (PAK1) were impaired in DOCK2-deficient MCs. Similar results were obtained when mice or MCs were treated with small-molecule inhibitors that bind to the catalytic domain of DOCK2 and inhibit Rac activation. CONCLUSION: DOCK2 regulates MRGPRX2/B2-mediated MC degranulation through Rac activation and PAK1 phosphorylation, thereby indicating that the DOCK2-Rac-PAK1 axis could be a target for preventing drug-induced anaphylaxis.
Asunto(s)
Anafilaxia , Hipersensibilidad a las Drogas , Humanos , Ratones , Animales , Anafilaxia/inducido químicamente , Degranulación de la Célula , Mastocitos/metabolismo , Receptores de Neuropéptido/genética , Receptores de Neuropéptido/metabolismo , Hipersensibilidad a las Drogas/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismoRESUMEN
Hematological and oncological diseases are still among the leading causes of childhood mortality. Expression of growth hormone-releasing hormone (GHRH) and its receptors (GHRH-R) has been previously demonstrated in various human tumors, but very limited findings are available about the presence and potential function of GHRH-Rs in oncological and hematological disorders of children. In this study, we aimed to investigate the expression of mRNA for GHRH and splice variant 1 (SV) of GHRH-R in 15 pediatric hematological/oncological specimens by RT-PCR. The presence and binding characteristics of GHRH-R protein were also studied by Western blot and ligand competition assays. Of the fifteen specimens studied, eleven pediatric samples (73%) showed the expression of mRNA for GHRH. These eleven samples also expressed mRNA for GHRH receptor SV1. GHRH-R protein was found to be expressed in two benign tumor samples and five malignant tumors examined by Western blot. The presence of specific, high affinity binding sites on GHRH-R was demonstrated in all of the seven human pediatric solid tumor samples investigated. Our results show that the expression of GHRH and SV1 of GHRH-R in hemato-oncological diseases in children can pave the way for further investigation of GHRH-Rs as potential molecular targets for diagnosis and therapy.
Asunto(s)
Hormona Liberadora de Hormona del Crecimiento , Neoplasias , Receptores de Neuropéptido , Receptores de Hormona Reguladora de Hormona Hipofisaria , Humanos , Receptores de Hormona Reguladora de Hormona Hipofisaria/genética , Receptores de Hormona Reguladora de Hormona Hipofisaria/metabolismo , Niño , Masculino , Femenino , Receptores de Neuropéptido/genética , Receptores de Neuropéptido/metabolismo , Proyectos Piloto , Hormona Liberadora de Hormona del Crecimiento/genética , Hormona Liberadora de Hormona del Crecimiento/metabolismo , Preescolar , Adolescente , Neoplasias/genética , Neoplasias/metabolismo , Hungría , Lactante , ARN Mensajero/genética , ARN Mensajero/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Enfermedades Hematológicas/genética , Enfermedades Hematológicas/metabolismo , Estudios de CohortesRESUMEN
Orexin-A and orexin-B, also named hypocretin-1 and hypocretin-2, are two hypothalamic neuropeptides highly conserved across mammalian species. Their effects are mediated by two distinct G protein-coupled receptors, namely orexin receptor type 1 (OX1-R) and type 2 (OX2-R), which share 64% amino acid identity. Given the wide expression of OX-Rs in different central nervous system and peripheral areas and the several pathophysiological functions in which they are involved, including sleep-wake cycle regulation (mainly mediated by OX2-R), emotion, panic-like behaviors, anxiety/stress, food intake, and energy homeostasis (mainly mediated by OX1-R), both subtypes represent targets of interest for many structure-activity relationship (SAR) campaigns carried out by pharmaceutical companies and academies. However, before 2017 the research was predominantly directed towards dual-orexin ligands, and limited chemotypes were investigated. Analytical characterizations, including resolved structures for both OX1-R and OX2-R in complex with agonists and antagonists, have improved the understanding of the molecular basis of receptor recognition and are assets for medicinal chemists in the design of subtype-selective ligands. This review is focused on the medicinal chemistry aspects of small molecules acting as dual or subtype selective OX1-R/OX2-R agonists and antagonists belonging to different chemotypes and developed in the last years, including radiolabeled OX-R ligands for molecular imaging. Moreover, the pharmacological effects of the most studied ligands in different neuropsychiatric diseases, such as sleep, mood, substance use, and eating disorders, as well as pain, have been discussed. Poly-pharmacology applications and multitarget ligands have also been considered.
Asunto(s)
Neuropéptidos , Humanos , Animales , Receptores de Orexina/metabolismo , Ligandos , Orexinas , Neuropéptidos/metabolismo , Neuropéptidos/farmacología , Receptores Acoplados a Proteínas G , Sistema Nervioso Central , Receptores de Neuropéptido/metabolismo , Mamíferos/metabolismoRESUMEN
G protein-coupled receptors (GPCRs) are a major class of membrane receptors that modulate a wide range of physiological functions. These receptors transmit extracellular signals, including secreted bioactive peptides, to intracellular signaling pathways. The nematode Caenorhabditis elegans has FMRFamide-like peptides, which are one of the most diverse neuropeptide families, some of which modulate larval development through GPCRs. In this study, we identified the GPCR neuropeptide receptor (NPR)-15, which modulates C. elegans larval development. Our molecular genetic analyses indicated the following: 1) NPR-15 mainly functions in ASI neurons, which predominantly regulate larval development, 2) NPR-15 interacts with GPA-4, a C. elegans Gα subunit, and 3) NPR-15, along with GPA-4, modulates larval development by regulating the production and secretion of the transforming growth factor-ß (TGF-ß)-like protein DAF-7. The present study is the first report to demonstrate the importance of a GPCR to the direct regulation of a TGF-ß-like protein.