RESUMEN
The multidrug resistance protein MRP1 is an ATP-driven pump that confers resistance to chemotherapy. Previously, we have shown that intracellular substrates are recruited to a bipartite binding site when the transporter rests in an inward-facing conformation. A key question remains: how are high-affinity substrates transferred across the membrane and released outside the cell? Using electron cryomicroscopy, we show here that ATP binding opens the transport pathway to the extracellular space and reconfigures the substrate-binding site such that it relinquishes its affinity for substrate. Thus, substrate is released prior to ATP hydrolysis. With this result, we now have a complete description of the conformational cycle that enables substrate transfer in a eukaryotic ABC exporter.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/química , Adenosina Trifosfato/metabolismo , Leucotrieno C4/metabolismo , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Adenosina Trifosfato/química , Animales , Sitios de Unión , Células HEK293 , Humanos , Leucotrieno C4/química , Unión Proteica , Multimerización de Proteína , Células Sf9 , SpodopteraRESUMEN
Adenosine triphosphate-binding cassette (ABC) transporters, such as multidrug resistance protein 1 (MRP1), protect against cellular toxicity by exporting xenobiotic compounds across the plasma membrane. However, constitutive MRP1 function hinders drug delivery across the blood-brain barrier, and MRP1 overexpression in certain cancers leads to acquired multidrug resistance and chemotherapy failure. Small-molecule inhibitors have the potential to block substrate transport, but few show specificity for MRP1. Here we identify a macrocyclic peptide, named CPI1, which inhibits MRP1 with nanomolar potency but shows minimal inhibition of a related multidrug transporter P-glycoprotein. A cryoelectron microscopy (cryo-EM) structure at 3.27 Å resolution shows that CPI1 binds MRP1 at the same location as the physiological substrate leukotriene C4 (LTC4). Residues that interact with both ligands contain large, flexible sidechains that can form a variety of interactions, revealing how MRP1 recognizes multiple structurally unrelated molecules. CPI1 binding prevents the conformational changes necessary for adenosine triphosphate (ATP) hydrolysis and substrate transport, suggesting it may have potential as a therapeutic candidate.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP , Proteínas Asociadas a Resistencia a Múltiples Medicamentos , Adenosina Trifosfato/metabolismo , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/metabolismo , Transporte Biológico , Microscopía por Crioelectrón , Leucotrieno C4/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Péptidos/metabolismo , Péptidos Cíclicos/farmacologíaRESUMEN
BACKGROUND AND AIMS: The Glu504Lys polymorphism in the aldehyde dehydrogenase 2 (ALDH2) gene is closely associated with myocardial ischaemia/reperfusion injury (I/RI). The effects of ALDH2 on neutrophil extracellular trap (NET) formation (i.e. NETosis) during I/RI remain unknown. This study aimed to investigate the role of ALDH2 in NETosis in the pathogenesis of myocardial I/RI. METHODS: The mouse model of myocardial I/RI was constructed on wild-type, ALDH2 knockout, peptidylarginine deiminase 4 (Pad4) knockout, and ALDH2/PAD4 double knockout mice. Overall, 308 ST-elevation myocardial infarction patients after primary percutaneous coronary intervention were enrolled in the study. RESULTS: Enhanced NETosis was observed in human neutrophils carrying the ALDH2 genetic mutation and ischaemic myocardium of ALDH2 knockout mice compared with controls. PAD4 knockout or treatment with NETosis-targeting drugs (GSK484, DNase1) substantially attenuated the extent of myocardial damage, particularly in ALDH2 knockout. Mechanistically, ALDH2 deficiency increased damage-associated molecular pattern release and susceptibility to NET-induced damage during myocardial I/RI. ALDH2 deficiency induced NOX2-dependent NETosis via upregulating the endoplasmic reticulum stress/microsomal glutathione S-transferase 2/leukotriene C4 (LTC4) pathway. The Food and Drug Administration-approved LTC4 receptor antagonist pranlukast ameliorated I/RI by inhibiting NETosis in both wild-type and ALDH2 knockout mice. Serum myeloperoxidase-DNA complex and LTC4 levels exhibited the predictive effect on adverse left ventricular remodelling at 6 months after primary percutaneous coronary intervention in ST-elevation myocardial infarction patients. CONCLUSIONS: ALDH2 deficiency exacerbates myocardial I/RI by promoting NETosis via the endoplasmic reticulum stress/microsomal glutathione S-transferase 2/LTC4/NOX2 pathway. This study hints at the role of NETosis in the pathogenesis of myocardial I/RI, and pranlukast might be a potential therapeutic option for attenuating I/RI, particularly in individuals with the ALDH2 mutation.
Asunto(s)
Aldehído Deshidrogenasa Mitocondrial , Trampas Extracelulares , Leucotrieno C4 , Daño por Reperfusión Miocárdica , Animales , Femenino , Humanos , Masculino , Ratones , Persona de Mediana Edad , Aldehído Deshidrogenasa Mitocondrial/genética , Aldehído Deshidrogenasa Mitocondrial/metabolismo , Benzamidas , Benzodioxoles , Modelos Animales de Enfermedad , Trampas Extracelulares/metabolismo , Antagonistas de Leucotrieno/farmacología , Antagonistas de Leucotrieno/uso terapéutico , Leucotrieno C4/antagonistas & inhibidores , Leucotrieno C4/metabolismo , Ratones Noqueados , Daño por Reperfusión Miocárdica/prevención & control , Daño por Reperfusión Miocárdica/genética , Daño por Reperfusión Miocárdica/metabolismo , Neutrófilos/metabolismo , Arginina Deiminasa Proteína-Tipo 4/metabolismo , Infarto del Miocardio con Elevación del ST/metabolismoRESUMEN
Excitation-transcription coupling, linking stimulation at the cell surface to changes in nuclear gene expression, is conserved throughout eukaryotes. How closely related coexpressed transcription factors are differentially activated remains unclear. Here, we show that two Ca2+-dependent transcription factor isoforms, NFAT1 and NFAT4, require distinct sub-cellular InsP3 and Ca2+ signals for physiologically sustained activation. NFAT1 is stimulated by sub-plasmalemmal Ca2+ microdomains, whereas NFAT4 additionally requires Ca2+ mobilization from the inner nuclear envelope by nuclear InsP3 receptors. NFAT1 is rephosphorylated (deactivated) more slowly than NFAT4 in both cytoplasm and nucleus, enabling a more prolonged activation phase. Oscillations in cytoplasmic Ca2+, long considered the physiological form of Ca2+ signaling, play no role in activating either NFAT protein. Instead, effective sustained physiological activation of NFAT4 is tightly linked to oscillations in nuclear Ca2+. Our results show how gene expression can be controlled by coincident yet geographically distinct Ca2+ signals, generated by a freely diffusible InsP3 message.
Asunto(s)
Señalización del Calcio , Calcio/metabolismo , Fosfatos de Inositol/metabolismo , Factores de Transcripción NFATC/genética , Proteínas Recombinantes de Fusión/genética , Animales , Basófilos/citología , Basófilos/efectos de los fármacos , Basófilos/metabolismo , Bronquios/citología , Bronquios/efectos de los fármacos , Bronquios/metabolismo , Línea Celular , Línea Celular Tumoral , Inhibidores Enzimáticos/farmacología , Regulación de la Expresión Génica , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Receptores de Inositol 1,4,5-Trifosfato/genética , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Leucotrieno C4/farmacología , Factores de Transcripción NFATC/metabolismo , Transporte de Proteínas , Ratas , Proteínas Recombinantes de Fusión/metabolismo , Tapsigargina/farmacología , Transcripción GenéticaRESUMEN
Acute and chronic itch are burdensome manifestations of skin pathologies including allergic skin diseases and atopic dermatitis, but the underlying molecular mechanisms are not well understood. Cysteinyl leukotrienes (CysLTs), comprising LTC4, LTD4, and LTE4, are produced by immune cells during type 2 inflammation. Here, we uncover a role for LTC4 and its signaling through the CysLT receptor 2 (CysLT2R) in itch. Cysltr2 transcript is highly expressed in dorsal root ganglia (DRG) neurons linked to itch in mice. We also detected CYSLTR2 in a broad population of human DRG neurons. Injection of leukotriene C4 (LTC4) or its nonhydrolyzable form NMLTC4, but neither LTD4 nor LTE4, induced dose-dependent itch but not pain behaviors in mice. LTC4-mediated itch differed in bout duration and kinetics from pruritogens histamine, compound 48/80, and chloroquine. NMLTC4-induced itch was abrogated in mice deficient for Cysltr2 or when deficiency was restricted to radioresistant cells. Itch was unaffected in mice deficient for Cysltr1, Trpv1, or mast cells (WSh mice). CysLT2R played a role in itch in the MC903 mouse model of chronic itch and dermatitis, but not in models of dry skin or compound 48/80- or Alternaria-induced itch. In MC903-treated mice, CysLT levels increased in skin over time, and Cysltr2-/- mice showed decreased itch in the chronic phase of inflammation. Collectively, our study reveals that LTC4 acts through CysLT2R as its physiological receptor to induce itch, and CysLT2R contributes to itch in a model of dermatitis. Therefore, targeting CysLT signaling may be a promising approach to treat inflammatory itch.
Asunto(s)
Dermatitis Atópica/inmunología , Leucotrieno C4/metabolismo , Prurito/inmunología , Receptores de Leucotrienos/metabolismo , Piel/inervación , Animales , Enfermedad Crónica , Dermatitis Atópica/inducido químicamente , Dermatitis Atópica/complicaciones , Dermatitis Atópica/patología , Modelos Animales de Enfermedad , Ganglios Espinales/citología , Ganglios Espinales/metabolismo , Humanos , Ratones , Ratones Noqueados , Prurito/patología , Receptores de Leucotrienos/genética , Células Receptoras Sensoriales/metabolismo , Transducción de Señal/inmunología , Piel/patologíaRESUMEN
BACKGROUND: The 3 cysteinyl leukotrienes (cysLTs), leukotriene (LT) C4 (LTC4), LTD4, and LTE4, have different biologic half-lives, cellular targets, and receptor specificities. CysLT2R binds LTC4 and LTD4in vitro with similar affinities, but it displays a marked selectivity for LTC4in vivo. LTC4, but not LTD4, strongly potentiates allergen-induced pulmonary eosinophilia in mice through a CysLT2R-mediated, platelet- and IL-33-dependent pathway. OBJECTIVE: We sought to determine whether LTD4 functionally antagonizes LTC4 signaling at CysLT2R. METHODS: We used 2 different in vivo models of CysLT2R-dependent immunopathology, as well as ex vivo activation of mouse and human platelets. RESULTS: LTC4-induced CD62P expression; HMGB1 release; and secretions of thromboxane A2, CXCL7, and IL-33 by mouse platelets were all were blocked by a selective CysLT2R antagonist and inhibited by LTD4. These effects did not depend on CysLT1R. Inhaled LTD4 blocked LTC4-mediated potentiation of ovalbumin-induced eosinophilic inflammation; recruitment of platelet-adherent eosinophils; and increases in IL-33, IL-4, IL-5, and IL-13 levels in lung tissue. In contrast, the effect of administration of LTE4, the preferred ligand for CysLT3R, was additive with LTC4. The administration of LTD4 to Ptges-/- mice, which display enhanced LTC4 synthesis similar to that in aspirin-exacerbated respiratory disease, completely blocked the physiologic response to subsequent lysine-aspirin inhalation challenges, as well as increases in levels of IL-33, type 2 cytokines, and biochemical markers of mast cell and platelet activation. CONCLUSION: The conversion of LTC4 to LTD4 may limit the duration and extent of potentially deleterious signaling through CysLT2R, and it may contribute to the therapeutic properties of desensitization to aspirin in aspirin-exacerbated respiratory disease.
Asunto(s)
Plaquetas/inmunología , Leucotrieno C4/inmunología , Leucotrieno D4/inmunología , Pulmón/inmunología , Activación Plaquetaria/inmunología , Animales , Asma/inmunología , Cisteína/inmunología , Citocinas/inmunología , Leucotrieno E4/inmunología , Leucotrienos/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Eosinofilia Pulmonar/inmunología , Receptores de Leucotrienos/inmunologíaRESUMEN
Over the last several years it has become clear that higher order assemblies on membranes, exemplified by signalosomes, are a paradigm for the regulation of many membrane signaling processes. We have recently combined two-color direct stochastic optical reconstruction microscopy (dSTORM) with the (Clus-DoC) algorithm that combines cluster detection and colocalization analysis to observe the organization of 5-lipoxygenase (5-LO) and 5-lipoxygenase-activating protein (FLAP) into higher order assemblies on the nuclear envelope of mast cells; these assemblies were linked to leukotriene (LT) C4 production. In this study we investigated whether higher order assemblies of 5-LO and FLAP included cytosolic phospholipase A2 (cPLA2) and were linked to LTB4 production in murine neutrophils. Using two- and three-color dSTORM supported by fluorescence lifetime imaging microscopy we identified higher order assemblies containing 40 molecules (median) (IQR: 23, 87) of 5-LO, and 53 molecules (62, 156) of FLAP monomer. 98 (18, 154) molecules of cPLA2 were clustered with 5-LO, and 77 (33, 114) molecules of cPLA2 were associated with FLAP. These assemblies were tightly linked to LTB4 formation. The activation-dependent close associations of cPLA2, FLAP, and 5-LO in higher order assemblies on the nuclear envelope support a model in which arachidonic acid is generated by cPLA2 in apposition to FLAP, facilitating its transfer to 5-LO to initiate LT synthesis.
Asunto(s)
Proteínas Activadoras de la 5-Lipooxigenasa/metabolismo , Araquidonato 5-Lipooxigenasa/metabolismo , Leucotrieno C4/metabolismo , Neutrófilos/metabolismo , Proteínas Activadoras de la 5-Lipooxigenasa/análisis , Algoritmos , Animales , Araquidonato 5-Lipooxigenasa/análisis , Núcleo Celular/metabolismo , Células Cultivadas , Leucotrieno C4/análisis , Ratones , Ratones Endogámicos C57BL , Microscopía/métodos , Neutrófilos/citología , Imagen Óptica/métodosRESUMEN
Human multidrug resistance protein 1 (hMRP1) is an important member of the ATP-binding cassette (ABC) transporter superfamily. It can extrude a variety of anticancer drugs and physiological organic anions across the plasma membrane, which is activated by substrate binding, and is accompanied by large-scale cooperative movements between different domains. Currently, it remains unclear completely about how the specific interactions between hMRP1 and its substrate are and which critical residues are responsible for allosteric signal transduction. To the end, we first construct an inward-facing state of hMRP1 using homology modeling method, and then dock substrate proinflammatory agent leukotriene C4 (LTC4) to hMRP1 pocket. The result manifests LTC4 interacts with two parts of hMRP1 pocket, namely the positively charged pocket (P pocket) and hydrophobic pocket (H pocket), similar to its binding mode with bMRP1 (bovine MRP1). Additionally, we use the Gaussian network model (GNM)-based thermodynamic method proposed by us to identify the key residues whose perturbations markedly alter their binding free energy. Here the conventional GNM is improved with covalent/non-covalent interactions and secondary structure information considered (denoted as sscGNM). In the result, sscGNM improves the flexibility prediction, especially for the nucleotide binding domains with rich kinds of secondary structures. The 46 key residue clusters located in different subdomains are identified which are highly consistent with experimental observations. Furtherly, we explore the long-range cooperation within the transporter. This study is helpful for strengthening the understanding of the work mechanism in ABC exporters and can provide important information to scientists in drug design studies.
Asunto(s)
Adenosina Trifosfato/química , Leucotrieno C4/química , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/química , Adenosina Trifosfato/metabolismo , Sitio Alostérico , Animales , Bovinos , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Cinética , Leucotrieno C4/metabolismo , Simulación del Acoplamiento Molecular , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Transducción de Señal , Electricidad Estática , Homología Estructural de Proteína , Especificidad por Sustrato , TermodinámicaRESUMEN
Aspirin-exacerbated respiratory disease and some cases of chronic idiopathic urticaria are disorders in which increased baseline urinary excretion of leukotriene(LT)E4 further increases following aspirin administration. Increased urinary excretion of the metabolites of prostaglandin D2, 11ß-prostaglandin(PG)F2α and (2,3-dinor)-11ß-PGF2α, have been documented in systemic mastocytosis (SM) and in mast cell activation syndrome (MCAS). Symptoms due to increased baseline and/or episodic release of PGD2 can be prevented with aspirin, an inhibitor of cyclooxygenase (COX)1 and COX2. Here by retrospective chart review we discovered 8 of 10 patients with SM in whom normalization of an elevated urinary (2,3-dinor)-11ß-PGF2α occurred with aspirin therapy also had a parallel increased excretion of LTE4 by an average of nearly 13-fold. How widespread this phenomenon occurs in SM is unknown; however, this occurrence needs to be considered when interpreting changes in these urinary mast cell mediator metabolites during aspirin therapy.
Asunto(s)
Leucotrieno C4RESUMEN
Type I hypersensitivity is an immediate immune reaction that involves IgE-mediated activation of mast cells. Activated mast cells release chemical mediators, such as histamine and lipid mediators, which cause allergic reactions. Recent developments in detection devices have revealed that mast cells simultaneously release a wide variety of lipid mediators. Mounting evidence has revealed that mast cell-derived mediators exert both pro- and anti-inflammatory functions and positively and negatively regulate the development of allergic inflammation. This review presents the roles of major lipid mediators released from mast cells. Author believes this review will be helpful for a better understanding of the pathogenesis of allergic diseases and provide a new strategy for the diagnosis and treatment of allergic reactions.
Asunto(s)
Hipersensibilidad Inmediata/etiología , Metabolismo de los Lípidos/fisiología , Ácidos Grasos Insaturados , Liberación de Histamina , Humanos , Ácidos Hidroxieicosatetraenoicos , Hipersensibilidad Inmediata/diagnóstico , Hipersensibilidad Inmediata/terapia , Inmunoglobulina E , Inflamación , Leucotrieno B4 , Leucotrieno C4 , Mastocitos/metabolismo , Prostaglandina D2RESUMEN
Human mesenchymal stem cell (MSC) extracellular vesicles (EV) can reduce the severity of bacterial pneumonia, but little is known about the mechanisms underlying their antimicrobial activity. In the current study, we found that bacterial clearance induced by MSC EV in Escherichia coli pneumonia in C57BL/6 mice was associated with high levels of leukotriene (LT) B4 in the injured alveolus. More importantly, the antimicrobial effect of MSC EV was abrogated by cotreatment with a LTB4 BLT1 antagonist. To determine the role of MSC EV on LT metabolism, we measured the effect of MSC EV on a known ATP-binding cassette transporter, multidrug resistance-associated protein 1 (MRP1), and found that MSC EV suppressed MRP1 mRNA, protein, and pump function in LPS-stimulated Raw264.7 cells in vitro. The synthesis of LTB4 and LTC4 from LTA4 are competitive, and MRP1 is the efflux pump for LTC4 Inhibition of MRP1 will increase LTB4 production. In addition, administration of a nonspecific MRP1 inhibitor (MK-571) reduced LTC4 and subsequently increased LTB4 levels in C57BL/6 mice with acute lung injury, increasing overall antimicrobial activity. We previously found that the biological effects of MSC EV were through the transfer of its content, such as mRNA, microRNA, and proteins, to target cells. In the current study, miR-145 knockdown abolished the effect of MSC EV on the inhibition of MRP1 in vitro and the antimicrobial effect in vivo. In summary, MSC EV suppressed MRP1 activity through transfer of miR-145, thereby resulting in enhanced LTB4 production and antimicrobial activity through LTB4/BLT1 signaling.
Asunto(s)
Lesión Pulmonar Aguda , Infecciones por Escherichia coli , Escherichia coli/inmunología , Vesículas Extracelulares , Células Madre Mesenquimatosas/inmunología , Neumonía Bacteriana , Lesión Pulmonar Aguda/inmunología , Lesión Pulmonar Aguda/patología , Lesión Pulmonar Aguda/terapia , Animales , Infecciones por Escherichia coli/inmunología , Infecciones por Escherichia coli/terapia , Vesículas Extracelulares/inmunología , Vesículas Extracelulares/patología , Vesículas Extracelulares/trasplante , Humanos , Leucotrieno B4/inmunología , Leucotrieno C4/inmunología , Pulmón/inmunología , Pulmón/patología , Masculino , Células Madre Mesenquimatosas/patología , Ratones , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/antagonistas & inhibidores , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/inmunología , Neumonía Bacteriana/inmunología , Neumonía Bacteriana/patología , Neumonía Bacteriana/terapia , Propionatos/farmacología , Quinolinas/farmacología , Células RAW 264.7RESUMEN
BACKGROUND: The aim of this study was to evaluate the inhibitory effect of tamarixetin on the production of inflammatory mediators in IgE/antigen-induced mouse bone marrow-derived mast cells (BMMCs). MATERIALS AND METHODS: The effects of tamarixetin on mast cell activation were investigated with regard to degranulation, eicosanoid generation, Ca2+ influx, and immunoblotting of various signaling molecules. RESULTS: Tamarixetin effectively decreased degranulation and the eicosanoid generation such as leukotriene C4 and prostaglandin D2 in BMMCs. To elucidate the mechanism involved, we investigated the effect of tamarixetin on the phosphorylation of signal molecules. Tamarixetin inhibited the phosphorylation of Akt and its downstream signal molecules including IKK and nuclear factor κB. In addition, tamarixetin downregulated the phosphorylation of cytosolic phospholipase A2 (cPLA2) and p38 mitogen-activated protein kinase. CONCLUSIONS: Taken together, this study suggests that tamarixetin inhibits degranulation and eicosanoid generation through the PLCγ1 as well as Akt pathways in BMMCs, which would be potential for the prevention of allergic inflammatory diseases.
Asunto(s)
Degranulación de la Célula/efectos de los fármacos , Disacáridos/farmacología , Eicosanoides/biosíntesis , Mediadores de Inflamación/metabolismo , Inula/química , Mastocitos/efectos de los fármacos , Quercetina/análogos & derivados , Animales , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/metabolismo , Calcio/metabolismo , Leucotrieno C4/biosíntesis , Mastocitos/metabolismo , Mastocitos/fisiología , Ratones , Ratones Endogámicos BALB C , FN-kappa B/metabolismo , Fosfolipasa C gamma/metabolismo , Fosfolipasas A2/metabolismo , Fosforilación/efectos de los fármacos , Prostaglandina D2/biosíntesis , Proteínas Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Quercetina/farmacología , beta-N-Acetilhexosaminidasas/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
The human multidrug resistance protein 1 (hMRP1) transporter is implicated in cancer multidrug resistance as well as immune responses involving its physiologic substrate, glutathione (GSH)-conjugated leukotriene C4 (LTC4). LTC4 binds a bipartite site on hMRP1, which a recent cryoelectron microscopy structure of LTC4-bound bovine Mrp1 depicts as composed of a positively charged pocket and a hydrophobic (H) pocket that binds the GSH moiety and surrounds the fatty acid moiety, respectively, of LTC4. Here, we show that single Ala and Leu substitutions of H-pocket hMRP1-Met1093 have no effect on LTC4 binding or transport. Estrone 3-sulfate transport is also unaffected, but both hMRP1-Met1093 mutations eliminate estradiol glucuronide transport, demonstrating that these steroid conjugates have binding sites distinct from each other and from LTC4. To eliminate LTC4 transport by hMRP1, mutation of 3 H-pocket residues was required (W553/M1093/W1246A), indicating that H-pocket amino acids are key to the vastly different affinities of hMRP1 for LTC4vs. GSH alone. Unlike organic anion transport, hMRP1-mediated drug resistance was more diminished by Ala than Leu substitution of Met1093. Although our findings generally support a structure in which H-pocket residues bind the lipid tail of LTC4, their critical and differential role in the transport of conjugated estrogens and anticancer drugs remains unexplained.-Conseil, G., Arama-Chayoth, M., Tsfadia, Y., Cole, S. P. C. Structure-guided probing of the leukotriene C4 binding site in human multidrug resistance protein 1 (MRP1; ABCC1).
Asunto(s)
Leucotrieno C4/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/química , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Sitios de Unión/genética , Transporte Biológico Activo , Bovinos , Resistencia a Múltiples Medicamentos/genética , Estradiol/análogos & derivados , Estradiol/metabolismo , Estrona/análogos & derivados , Estrona/metabolismo , Células HEK293 , Humanos , Leucotrieno D4/metabolismo , Modelos Moleculares , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Mutagénesis Sitio-Dirigida , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología Estructural de ProteínaRESUMEN
BACKGROUND: Bronchiolitis is the main cause of hospitalization of children younger than 1 year; however, the immune mechanism of bronchiolitis is not completely understood. The aim of this study was to analyze the recovery of immune response after a bronchiolitis episode. METHODS: Forty-nine infants hospitalized with bronchiolitis diagnosis were enrolled. Nasopharyngeal aspirates (NPAs) were processed. Twenty-seven pro-inflammatory biomarkers linked to innate immunity, inflammation, and epithelial damage, as well as nitrites and lipid mediators, were evaluated in the NPA supernatant by ELISA (enzyme-linked immunosorbent assay) and Luminex. Also, 11 genes were analyzed in NPA cells by quantitative PCR. RESULTS: A widespread statistically significant decline of multiple pro-inflammatory parameters and cytokines were detected in the recovery period after respiratory infection: interferon-α2 (IFNα2), IFNγ, interleukin-10 (IL-10), IL-1ß, IL-8, IFN-γ-inducible protein-10, vascular endothelial growth factor, monocyte chemoattractant protein-1, macrophage inflammatory protein-1α (MIP-1α), and MIP-1ß. Supporting these results, a decreased nuclear factor-κB gene expression was observed (P = 0.0116). A significant diminution of cysteinyl leukotriene C4 (LTC4) soluble levels (P = 0.0319) and cyclooxygenase-2 (COX-2) gene expression were observed in the recovery sample. In children classified by post-bronchiolitis wheezing, LTC4 remains elevated in the NPA supernatant. CONCLUSIONS: After bronchiolitis, cytokines and biomarkers linked to innate immune response in NPA decrease significantly in the recovery period accompanied by a drop in LTC4 levels; however, this reduction was lower in infants with post-bronchiolitis wheezing.
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Inmunidad Adaptativa , Bronquiolitis/inmunología , Citocinas/metabolismo , Inmunidad Innata , Leucotrieno C4/metabolismo , Nasofaringe/inmunología , Biomarcadores/metabolismo , Bronquiolitis/diagnóstico , Bronquiolitis/metabolismo , Bronquiolitis/terapia , Citocinas/genética , Regulación hacia Abajo , Femenino , Humanos , Lactante , Masculino , Estudios Prospectivos , Factores de TiempoRESUMEN
Introduction: Nonsteroidal anti-inflammatory drugs (NSAIDs) intervene in the COX (cyclooxygenase) pathways which generate two important inflammation mediators, prostaglandins (PGs) and leukotriene (LTs). Contradictory claims regarding the effect of NSAIDs in asthmatic patients continues to be an issue. The present study investigated the effects of COX inhibitors on the responsiveness of the tracheal tract and on the levels of LTC4 and PGE2 in cells of the bronchoalveolar lavage fluid in an allergic guinea pig model.Materials and Methods: Adult male Dunkin-Hartley guinea pigs (250 - 300 g) were divided into seven groups of six animals each. Four COX inhibitors, aspirin (200 mg/kg and 20 mg/kg), indomethacin (10 mg/kg), ketoprofen (10 mg/kg), and celecoxib (25 mg/kg), were given orally on day 17 to allergy induced guinea pigs at 0, 12, and 24 h before ovalbumin challenge on day 18. PGF2 and LT4 were measured in the bronchoalveolar lavage fluid as well as inflammatory cell count and total protein. Tracheal responsiveness to acetylcholine (Ach) and histamine (His) also was evaluated.Results: An augment in the response of the trachea to Ach and His, as well as overt allergenic signs including short breath, wheezing and sneezing, was observed. The most significant increase in tracheal hyper-responsiveness was observed in the ketoprofen-treated group with similar but less pronounced changes observed in the indomethacin-treated group. Although some variables increased with the aspirin and celecoxib treatments, overall the tracheal sensitivity was reduced. Inflammatory cells including eosinophils and neutrophils corresponded to the changes observed for each treatment group.Conclusion: Ketoprofen and indomethacin increased the tracheal sensibility to Ach and His; therefore, their administration is not recommended in patients susceptible to allergy.
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Acetilcolina/farmacología , Inhibidores de la Ciclooxigenasa/efectos adversos , Dinoprostona/análisis , Histamina/farmacología , Hipersensibilidad/inmunología , Leucotrieno C4/análisis , Tráquea/efectos de los fármacos , Animales , Líquido del Lavado Bronquioalveolar/química , Líquido del Lavado Bronquioalveolar/inmunología , Inhibidores de la Ciclooxigenasa/uso terapéutico , Modelos Animales de Enfermedad , Cobayas , Hipersensibilidad/tratamiento farmacológico , Masculino , Ovalbúmina/inmunología , Tráquea/inmunologíaRESUMEN
Peroxisome proliferator-activated receptors (PPARs) are expressed in bovine uterus, and their agonists are arachidonic acid (AA) metabolites. We hypothesised that silencing of PPAR genes in bovine endometrial stromal cells (ESC) would change the intracellular signalling through PPAR and affect apoptosis after cell treatment with different AA metabolites. The study's aims are detection of apoptosis and examining the influence of prostaglandins and leukotrienes on apoptosis occurring in physiological ESC and cells with silenced PPAR (α, δ, and γ) genes. Silencing the PPARα and PPARδ genes in cells resulted in increased DNA fragmentation and mRNA and protein expression of caspase (CASP) -3 and -8 (P < 0.05). Neither DNA fragmentation nor the mRNA and protein expression of CASP3 and -8 in cells with silenced PPARγ gene were changed compared to physiological cells (P > 0.05). Among PPARs, PPARα and PPARδ appear to inhibit apoptosis, and AA metabolites, as PPAR agonists, modify this process in bovine ESC.
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Apoptosis/genética , Ácido Araquidónico/metabolismo , Endometrio/citología , Silenciador del Gen , Receptores Activados del Proliferador del Peroxisoma/deficiencia , Receptores Activados del Proliferador del Peroxisoma/genética , Animales , Apoptosis/efectos de los fármacos , Bovinos , Dinoprost/farmacología , Dinoprostona/farmacología , Femenino , Leucotrieno B4/farmacología , Leucotrieno C4/farmacologíaRESUMEN
Asthma is a complex disease that is promoted by dysregulated immunity and the presence of many cytokine and lipid mediators. Despite this, there is a paucity of data demonstrating the combined effects of multiple mediators in asthma pathogenesis. Group 2 innate lymphoid cells (ILC2s) have recently been shown to play important roles in the initiation of allergic inflammation; however, it is unclear whether lipid mediators, such as cysteinyl leukotrienes (CysLTs), which are present in asthma, could further amplify the effects of IL-33 on ILC2 activation and lung inflammation. In this article, we show that airway challenges with the parent CysLT, leukotriene C4 (LTC4), given in combination with low-dose IL-33 to naive wild-type mice, led to synergistic increases in airway Th2 cytokines, eosinophilia, and peribronchial inflammation compared with IL-33 alone. Further, the numbers of proliferating and cytokine-producing lung ILC2s were increased after challenge with both LTC4 and IL-33. Levels of CysLT1R, CysLT2R, and candidate leukotriene E4 receptor P2Y12 mRNAs were increased in ILC2s. The synergistic effect of LTC4 with IL-33 was completely dependent upon CysLT1R, because CysLT1R-/- mice, but not CysLT2R-/- mice, had abrogated responses. Further, CysLTs directly potentiated IL-5 and IL-13 production from purified ILC2s stimulated with IL-33 and resulted in NFAT1 nuclear translocation. Finally, CysLT1R-/- mice had reduced lung eosinophils and ILC2 responses after exposure to the fungal allergen Alternaria alternata Thus, CysLT1R promotes LTC4- and Alternaria-induced ILC2 activation and lung inflammation. These findings suggest that multiple pathways likely exist in asthma to activate ILC2s and propagate inflammatory responses.
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Inmunidad Innata , Interleucina-33/inmunología , Leucotrieno C4/metabolismo , Activación de Linfocitos , Linfocitos/inmunología , Neumonía/inmunología , Alérgenos/inmunología , Alternaria/inmunología , Animales , Asma/inmunología , Asma/fisiopatología , Citocinas/biosíntesis , Citocinas/inmunología , Citocinas/metabolismo , Eosinofilia/inmunología , Interleucina-33/administración & dosificación , Leucotrieno C4/inmunología , Pulmón/inmunología , Ratones , Neumonía/metabolismo , Receptores de Leucotrienos/administración & dosificación , Receptores de Leucotrienos/deficiencia , Receptores de Leucotrienos/genética , Receptores de Leucotrienos/inmunología , Receptores Purinérgicos P2Y12/genética , Receptores Purinérgicos P2Y12/inmunología , Células Th2/inmunologíaRESUMEN
PURPOSE OF REVIEW: This review will critically highlight the role of leukotrienes as mediators of renal diseases and drug nephrotoxicity. It will also discuss the recently identified mechanism of cysteinyl leukotrienes induction and action, and will propose clinical implementation of these findings. RECENT FINDINGS: Since last reviewed in 1994, leukotrienes were shown to mediate drug-associated nephrotoxicity, transplant rejection and morbidity in several models of renal diseases. Although leukotrienes may be released by various infiltrating leukocytes, a recent study demonstrated that cytotoxic agents trigger production of leukotriene C4 (LTC4) in mouse kidney cells by activating a biosynthetic pathway based on microsomal glutathione-S-transferase 2 (MGST2). LTC4 then elicits nuclear accumulation of hydrogen peroxide-generating NADPH oxidase 4, leading to oxidative DNA damage and cell death. LTC4 inhibitors, commonly used as systemic asthma drugs, alleviated drug-associated damage to proximal tubular cells and attenuated mouse morbidity. SUMMARY: Cysteinyl leukotrienes released by mast cells trigger the symptoms of asthma, including bronchoconstriction and vasoconstriction. Therefore, effective leukotriene inhibitors were approved as orally administered asthma drugs. The findings that leukotrienes mediate the cytotoxicity of nephrotoxic drugs, and are involved in numerous renal diseases, suggest that such asthma drugs may ameliorate drug-induced nephrotoxicity, as well as some renal diseases.
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Enfermedades Renales/inducido químicamente , Enfermedades Renales/metabolismo , Leucotrieno C4/metabolismo , Animales , Antiasmáticos/uso terapéutico , Muerte Celular , Cisteína/metabolismo , Daño del ADN , Glutatión Transferasa/metabolismo , Humanos , Antagonistas de Leucotrieno/uso terapéutico , Leucotrieno C4/antagonistas & inhibidores , Leucotrieno C4/biosíntesis , Leucotrienos/metabolismo , NADPH Oxidasa 4/metabolismo , Estrés OxidativoRESUMEN
Conventional allergic rhinitis (AR) treatments have limitations due to the lack of safety and complete cure strategy. We evaluated the effects of silent information regulator 1 (SIRT1), a multifunctional molecule involved in a variety of inflammatory pathways, on murine AR model. Ovalbumin (OVA)-induced murine model was constructed, and recombinant SIRT1 was administered into the nostril continuously. The expression of SIRT1 was measured at mRNA and protein levels, and the allergic symptoms were evaluated. Protein levels of OVA-specific IgE, leukotriene C4 (LTC4), eosinophil cation protein (ECP), prostaglandin D2 (PGD2), as well as different inflammatory cytokine mediators in the serum and nasal lavage fluid (NLF), were assessed by ELISA. The effects of SIRT1 on human primary nasal epithelial cells challenged with tumour necrosis factor (TNF)-α were also evaluated by investigating the HMGB1/TLR4 signalling pathway. Administration of SIRT1 significantly alleviated OVA-induced AR symptoms with lower numbers of sneezing and nasal rubbing events, decreased levels of OVA-specific IgE, LTC4, ECP, PGD2, less inflammatory cells and downregulated levels of Th2 type cytokines. SIRT1 also reduced the genes of HMGB1/TLR4 signalling pathway in the murine model and cultured human nasal epithelial cells. Expression of SIRT1 is impaired in OVA-induced AR model. The administration of SIRT1 alleviates the allergic symptoms of mice, regulates the production of pro-inflammatory mediators predominantly produced by Th2 cells in AR and attenuates expressions of proteins relevant to HMGB1/TLR4 signalling pathway. All the results showed that SIRT1 is promising as a therapeutic agent of AR.
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Proteína HMGB1/biosíntesis , Rinitis Alérgica/terapia , Sirtuina 1/uso terapéutico , Receptor Toll-Like 4/biosíntesis , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Regulación hacia Abajo , Proteína Catiónica del Eosinófilo/sangre , Femenino , Humanos , Inmunoglobulina E/sangre , Leucotrieno C4/sangre , Ratones , Ratones Endogámicos BALB C , Líquido del Lavado Nasal/química , Mucosa Nasal/citología , Ovalbúmina/efectos adversos , Prostaglandina D2/sangre , Proteínas Recombinantes/uso terapéutico , Rinitis Alérgica/inducido químicamente , Rinitis Alérgica/patología , Transducción de Señal/efectos de los fármacos , Células Th2/inmunología , Factor de Necrosis Tumoral alfa/efectos adversosRESUMEN
Receptor desensitization is a universal mechanism to turn off a biological response; in this process, the ability of a physiological trigger to activate a cell is lost despite the continued presence of the stimulus. Receptor desensitization of G-protein-coupled receptors involves uncoupling of the receptor from its G-protein or second-messenger pathway followed by receptor internalization. G-protein-coupled cysteinyl leukotriene type I (CysLT1) receptors regulate immune-cell function and CysLT1 receptors are an established therapeutic target for allergies, including asthma. Desensitization of CysLT1 receptors arises predominantly from protein-kinase-C-dependent phosphorylation of three serine residues in the receptor carboxy terminus. Physiological concentrations of the receptor agonist leukotriene C(4) (LTC(4)) evoke repetitive cytoplasmic Ca(2+) oscillations, reflecting regenerative Ca(2+) release from stores, which is sustained by Ca(2+) entry through store-operated calcium-release-activated calcium (CRAC) channels. CRAC channels are tightly linked to expression of the transcription factor c-fos, a regulator of numerous genes important to cell growth and development. Here we show that abolishing leukotriene receptor desensitization suppresses agonist-driven gene expression in a rat cell line. Mechanistically, stimulation of non-desensitizing receptors evoked prolonged inositol-trisphosphate-mediated Ca(2+) release, which led to accelerated Ca(2+)-dependent slow inactivation of CRAC channels and a subsequent loss of excitation-transcription coupling. Hence, rather than serving to turn off a biological response, reversible desensitization of a Ca(2+) mobilizing receptor acts as an 'on' switch, sustaining long-term signalling in the immune system.