RESUMEN
RATIONALE: 5-Hydroxytryptamine (5-HT) is involved in the pathogenesis of allergic airway inflammation (AAI). It is unclear, however, how 5-HT contributes to AAI and whether this depends on tryptophan hydroxylase (TPH) 1, the critical enzyme for peripheral 5-HT synthesis. OBJECTIVES: To elucidate the role of TPH1 and the peripheral source of 5-HT in asthma pathogenesis. METHODS: TPH1-deficient and TPH1-inhibitor-treated animals were challenged in ovalbumin and house dust mite models of AAI. Experiments with bone marrow chimera, mast cell-deficient animals, platelets transfusion, and bone marrow dendritic cells (BMDC) driven model of AAI were performed. 5-HT levels were measured in bronchoalveolar lavage fluid or serum of animals with AAI and in human asthma. MEASUREMENTS AND MAIN RESULTS: 5-HT levels are increased in bronchoalveolar lavage fluid of mice and people with asthma after allergen provocation. TPH1 deficiency and TPH1 inhibition reduced all cardinal features of AAI. Administration of exogenous 5-HT restored AAI in TPH1-deficient mice. The pivotal role of 5-HT production by structural cells was corroborated by bone marrow chimera experiments. Experiments in mast cell-deficient mice revealed that mast cells are not a source of 5-HT, whereas transfusion of platelets from wild-type and TPH1-deficient mice revealed that only platelets containing 5-HT enhanced AAI. Lack of endogenous 5-HT in vitro and in vivo was associated with an impaired Th2-priming capacity of BMDC. CONCLUSIONS: In summary, TPH1 deficiency or inhibition reduces AAI. Platelet- and not mast cell-derived 5-HT is pivotal in AAI, and lack of 5-HT leads to an impaired Th2-priming capacity of BMDC. Thus, targeting TPH1 could offer novel therapeutic options for asthma.
Asunto(s)
Asma/inmunología , Plaquetas/inmunología , Serotonina/metabolismo , Triptófano Hidroxilasa/inmunología , Animales , Asma/fisiopatología , Plaquetas/metabolismo , Líquido del Lavado Bronquioalveolar/inmunología , Células Dendríticas/inmunología , Humanos , Mastocitos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Ovalbúmina , Pyroglyphidae , Serotonina/biosíntesis , Serotonina/farmacología , Triptófano Hidroxilasa/antagonistas & inhibidores , Triptófano Hidroxilasa/deficienciaRESUMEN
BACKGROUND: Despite the potential of improving the delivery of epigenetic drugs, the subsequent assessment of changes in their epigenetic activity is largely dependent on the availability of a suitable and rapid screening bioassay. Here, we describe a cell-based assay system for screening gene reactivation. METHODS: A cell-based assay system (EPISSAY) was designed based on a silenced triple-mutated bacterial nitroreductase TMnfsB fused with Red-Fluorescent Protein (RFP) expressed in the non-malignant human breast cell line MCF10A. EPISSAY was validated using the target gene TXNIP, which has previously been shown to respond to epigenetic drugs. The potency of a epigenetic drug model, decitabine, formulated with PEGylated liposomes was also validated using this assay system. RESULTS: Following treatment with DNA methyltransferase (DNMT) and histone deacetylase (HDAC) inhibitors such as decitabine and vorinostat, increases in RFP expression were observed, indicating expression of RFP-TMnfsB. The EPISSAY system was then used to test the potency of decitabine, before and after PEGylated liposomal encapsulation. We observed a 50% higher potency of decitabine when encapsulated in PEGylated liposomes, which is likely to be due to its protection from rapid degradation. CONCLUSIONS: The EPISSAY bioassay system provides a novel and rapid system to compare the efficiencies of existing and newly formulated drugs that reactivate gene expression.
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Antimetabolitos Antineoplásicos/farmacología , Azacitidina/análogos & derivados , Mama/citología , Ensayos de Selección de Medicamentos Antitumorales/métodos , Proteínas Luminiscentes/metabolismo , Nitrorreductasas/metabolismo , Antineoplásicos/farmacología , Azacitidina/farmacología , Proteínas Portadoras/genética , Células Cultivadas , Citomegalovirus/genética , Metilación de ADN , Decitabina , Epigénesis Genética , Regulación de la Expresión Génica , Genes Reporteros , Vectores Genéticos , Humanos , Ácidos Hidroxámicos/farmacología , Proteínas Luminiscentes/genética , Nitrorreductasas/genética , Plásmidos , Regiones Promotoras Genéticas , ARN Mensajero/metabolismo , Proteínas Represoras/genética , Liposomas Unilamelares , Vorinostat , Proteína Fluorescente RojaRESUMEN
While serotonin (5-HT) co-localization with insulin in granules of pancreatic beta-cells was demonstrated more than three decades ago, its physiological role in the etiology of diabetes is still unclear. We combined biochemical and electrophysiological analyses of mice selectively deficient in peripheral tryptophan hydroxylase (Tph1-/-) and 5-HT to show that intracellular 5-HT regulates insulin secretion. We found that these mice are diabetic and have an impaired insulin secretion due to the lack of 5-HT in the pancreas. The pharmacological restoration of peripheral 5-HT levels rescued the impaired insulin secretion in vivo. These findings were further evidenced by patch clamp experiments with isolated Tph1-/- beta-cells, which clearly showed that the secretory defect is downstream of Ca(2+)-signaling and can be rescued by direct intracellular application of 5-HT via the clamp pipette. In elucidating the underlying mechanism further, we demonstrate the covalent coupling of 5-HT by transglutaminases during insulin exocytosis to two key players in insulin secretion, the small GTPases Rab3a and Rab27a. This renders them constitutively active in a receptor-independent signaling mechanism we have recently termed serotonylation. Concordantly, an inhibition of such activating serotonylation in beta-cells abates insulin secretion. We also observed inactivation of serotonylated Rab3a by enhanced proteasomal degradation, which is in line with the inactivation of other serotonylated GTPases. Our results demonstrate that 5-HT regulates insulin secretion by serotonylation of GTPases within pancreatic beta-cells and suggest that intracellular 5-HT functions in various microenvironments via this mechanism in concert with the known receptor-mediated signaling.
Asunto(s)
Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Procesamiento Proteico-Postraduccional , Serotonina/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Proteína de Unión al GTP rab3A/metabolismo , Animales , Línea Celular , Diabetes Mellitus/etiología , Diabetes Mellitus/metabolismo , Humanos , Secreción de Insulina , Espacio Intracelular/metabolismo , Ratones , Técnicas de Placa-Clamp , Ratas , Transglutaminasas/metabolismo , Triptófano Hidroxilasa/deficiencia , Proteínas rab27 de Unión a GTPRESUMEN
BACKGROUND: Bioinformatic analyses of expression control sequences in promoters of co-expressed or functionally related genes enable the discovery of common regulatory sequence motifs that might be involved in co-ordinated gene expression. By studying promoter sequences of the human ribosomal protein genes we recently identified a novel highly specific Localized Tandem Sequence Motif (LTSM). In this work we sought to identify additional genes and LTSM-binding proteins to elucidate potential regulatory mechanisms. RESULTS: Genome-wide analyses allowed finding a considerable number of additional LTSM-positive genes, the products of which are involved in translation, among them, translation initiation and elongation factors, and 5S rRNA. Electromobility shift assays then showed specific signals demonstrating the binding of protein complexes to LTSM in ribosomal protein gene promoters. Pull-down assays with LTSM-containing oligonucleotides and subsequent mass spectrometric analysis identified the related multifunctional nucleotide binding proteins NonO and SFPQ in the binding complex. Functional characterization then revealed that LTSM enhances the transcriptional activity of the promoters in dependency of the distance from the transcription start site. CONCLUSIONS: Our data demonstrate the power of bioinformatic analyses for the identification of biologically relevant sequence motifs. LTSM and the here found LTSM-binding proteins NonO and SFPQ were discovered through a synergistic combination of bioinformatic and biochemical methods and are regulators of the expression of a set of genes of the translational apparatus in a distance-dependent manner.
Asunto(s)
Elementos de Facilitación Genéticos , Proteínas Asociadas a Matriz Nuclear/metabolismo , Factores de Transcripción de Octámeros/metabolismo , Biosíntesis de Proteínas , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ADN , Humanos , Factor de Empalme Asociado a PTB , Regiones Promotoras Genéticas , Unión ProteicaRESUMEN
Mammary gland development is controlled by a dynamic interplay between endocrine hormones and locally produced factors. Biogenic monoamines (serotonin, dopamine, norepinephrine, and others) are an important class of bioregulatory molecules that have not been shown to participate in mammary development. Here we show that mammary glands stimulated by prolactin (PRL) express genes essential for serotonin biosynthesis (tryptophan hydroxylase [TPH] and aromatic amine decarboxylase). TPH mRNA was elevated during pregnancy and lactation, and serotonin was detected in the mammary epithelium and in milk. TPH was induced by PRL in mammosphere cultures and by milk stasis in nursing dams, suggesting that the gene is controlled by milk filling in the alveoli. Serotonin suppressed beta-casein gene expression and caused shrinkage of mammary alveoli. Conversely, TPH1 gene disruption or antiserotonergic drugs resulted in enhanced secretory features and alveolar dilation. Thus, autocrine-paracrine serotonin signaling is an important regulator of mammary homeostasis and early involution.
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Comunicación Autocrina/fisiología , Glándulas Mamarias Humanas/crecimiento & desarrollo , Comunicación Paracrina/fisiología , Prolactina/metabolismo , Serotonina/fisiología , Animales , Animales Recién Nacidos , Descarboxilasas de Aminoácido-L-Aromático/genética , Descarboxilasas de Aminoácido-L-Aromático/metabolismo , Comunicación Autocrina/efectos de los fármacos , Caseínas/genética , Caseínas/metabolismo , Células Cultivadas , Clonación Molecular , Diálisis , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Femenino , Fenclonina/farmacología , Regulación del Desarrollo de la Expresión Génica , Histología , Humanos , Ácido Hidroxiindolacético/metabolismo , Inmunohistoquímica , Hibridación in Situ , Lactalbúmina/genética , Lactalbúmina/metabolismo , Glándulas Mamarias Humanas/citología , Glándulas Mamarias Humanas/efectos de los fármacos , Metisergida/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Leche/metabolismo , Proteínas de la Leche/genética , Proteínas de la Leche/metabolismo , Mucinas/genética , Mucinas/metabolismo , Técnicas de Cultivo de Órganos , Comunicación Paracrina/efectos de los fármacos , Embarazo , Prolactina/deficiencia , Prolactina/genética , ARN Mensajero/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Antagonistas de la Serotonina/farmacología , Factores de Tiempo , Triptófano Hidroxilasa/genética , Triptófano Hidroxilasa/metabolismoRESUMEN
BACKGROUND: The nitroreductase/5-(azaridin-1-yl)-2,4-dinitrobenzamide (NTR/CB1954) enzyme/prodrug system is considered as a promising candidate for anti-cancer strategies by gene-directed enzyme prodrug therapy (GDEPT) and has recently entered clinical trials. It requires the genetic modification of tumor cells to express the E. coli enzyme nitroreductase that bioactivates the prodrug CB1954 to a powerful cytotoxin. This metabolite causes apoptotic cell death by DNA interstrand crosslinking. Enhancing the enzymatic NTR activity for CB1954 should improve the therapeutical potential of this enzyme-prodrug combination in cancer gene therapy. METHODS: We performed de novo synthesis of the bacterial nitroreductase gene adapting codon usage to mammalian preferences. The synthetic gene was investigated for its expression efficacy and ability to sensitize mammalian cells to CB1954 using western blotting analysis and cytotoxicity assays. RESULTS: In our study, we detected cytoplasmic protein aggregates by expressing GFP-tagged NTR in COS-7 cells, suggesting an impaired translation by divergent codon usage between prokaryotes and eukaryotes. Therefore, we generated a synthetic variant of the nitroreductase gene, called ntro, adapted for high-level expression in mammalian cells. A total of 144 silent base substitutions were made within the bacterial ntr gene to change its codon usage to mammalian preferences. The codon-optimized ntro either tagged to gfp or c-myc showed higher expression levels in mammalian cell lines. Furthermore, the ntro rendered several cell lines ten times more sensitive to the prodrug CB1954 and also resulted in an improved bystander effect. CONCLUSION: Our results show that codon optimization overcomes expression limitations of the bacterial ntr gene in mammalian cells, thereby improving the NTR/CB1954 system at translational level for cancer gene therapy in humans.
Asunto(s)
Expresión Génica , Genes Sintéticos , Nitrorreductasas/genética , Ingeniería de Proteínas , Secuencia de Aminoácidos , Animales , Antineoplásicos/farmacocinética , Aziridinas/farmacocinética , Secuencia de Bases , Biotransformación , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Codón , Humanos , Datos de Secuencia Molecular , Nitrorreductasas/metabolismo , Profármacos/farmacocinéticaRESUMEN
Mutations in the X chromosomal tRNA 2'Omethyltransferase FTSJ1 cause intellectual disability (ID). Although the gene is ubiquitously expressed affected individuals present no consistent clinical features beyond ID. In order to study the pathological mechanism involved in the aetiology of FTSJ1 deficiency-related cognitive impairment, we generated and characterized an Ftsj1 deficient mouse line based on the gene trapped stem cell line RRD143. Apart from an impaired learning capacity these mice presented with several statistically significantly altered features related to behaviour, pain sensing, bone and energy metabolism, the immune and the hormone system as well as gene expression. These findings show that Ftsj1 deficiency in mammals is not phenotypically restricted to the brain but affects various organ systems. Re-examination of ID patients with FTSJ1 mutations from two previously reported families showed that several features observed in the mouse model were recapitulated in some of the patients. Though the clinical spectrum related to Ftsj1 deficiency in mouse and man is variable, we suggest that an increased pain threshold may be more common in patients with FTSJ1 deficiency. Our findings demonstrate novel roles for Ftsj1 in maintaining proper cellular and tissue functions in a mammalian organism.
Asunto(s)
Modelos Animales de Enfermedad , Discapacidad Intelectual/etiología , Discapacidad Intelectual Ligada al Cromosoma X/genética , Metiltransferasas/fisiología , Mutación , Proteínas Nucleares/genética , ARNt Metiltransferasas/fisiología , Animales , Conducta Animal , Trastornos del Conocimiento/etiología , Trastornos del Conocimiento/patología , Familia , Femenino , Discapacidad Intelectual/patología , Masculino , Metiltransferasas/genética , Metiltransferasas/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Dolor Nociceptivo/etiología , Dolor Nociceptivo/patología , Proteínas Nucleares/metabolismo , ARNt Metiltransferasas/genética , ARNt Metiltransferasas/metabolismoRESUMEN
BACKGROUND: Tryptophan hydroxylase 2 (TPH2) is the rate-limiting enzyme in brain serotonin (5-HT) biosynthesis. Although dysfunction of 5-HT neurotransmission has been implicated in a variety of neuropsychiatric conditions, the human TPH2 promoter has not been characterized in vitro. METHODS: The functional relevance of TPH2 promoter polymorphisms was determined with luciferase assays in primary serotonergic neurons from rat raphe nuclei and in human small cell lung carcinoma cells (SHP-77 cells). We also investigated transcription factor binding to the variant promoter sequence with electrophoretic mobility shift assay (EMSA). RESULTS: The polymorphism rs11178997 of the human TPH2 promoter significantly reduced TPH2 transcriptional activity by 22% and 7% in primary serotonergic neurons and in SHP-77 cells, respectively. In contrast, no significant differences in promoter activity were observed for the G- and T-alleles of rs4570625. The EMSA revealed reduced binding of the transcription factor POU3F2 (also known as Brn-2, N-Oct-3) to the A-allele of the polymorphism rs11178997. Overexpression of POU3F2 resulted in a robust activation of the TPH2 promoter (2.7-fold). CONCLUSIONS: Our data suggest that the human TPH2 promoter polymorphism rs11178997 impacts on gene expression, which might have implications for the development and function of the serotonergic system in the brain.
Asunto(s)
Neuronas/enzimología , Regiones Promotoras Genéticas/genética , Núcleos del Rafe/enzimología , Serotonina/fisiología , Triptófano Hidroxilasa/genética , Animales , Northern Blotting , Western Blotting , Carcinoma de Células Pequeñas/enzimología , Carcinoma de Células Pequeñas/genética , Línea Celular Tumoral , ADN de Neoplasias/genética , Ensayo de Cambio de Movilidad Electroforética , Vectores Genéticos , Humanos , Inmunohistoquímica , Luciferasas/metabolismo , Polimorfismo Genético , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Núcleos del Rafe/citología , Ratas , TransfecciónRESUMEN
Classical neurotransmitters, including monoamines, acetylcholine, glutamate, GABA, and glycine, are loaded into synaptic vesicles by means of specific transporters. Vesicular monoamine transporters are under negative regulation by alpha subunits of trimeric G-proteins, including Galpha(o2) and Galpha(q). Furthermore, glutamate uptake, mediated by vesicular glutamate transporters (VGLUTs), is decreased by the nonhydrolysable GTP-analog guanylylimidodiphosphate. Using mutant mice lacking various Galpha subunits, including Galpha(o1), Galpha(o2), Galpha(q), and Galpha11, and a Galpha(o2)-specific monoclonal antibody, we now show that VGLUTs are exclusively regulated by Galpha(o2). G-protein activation does not affect the electrochemical proton gradient serving as driving force for neurotransmitter uptake; rather, Galpha(o2) exerts its action by specifically affecting the chloride dependence of VGLUTs. All VGLUTs show maximal activity at approximately 5 mm chloride. Activated Galpha(o2) shifts this maximum to lower chloride concentrations. In contrast, glutamate uptake by vesicles isolated from Galpha(o2-/-) mice have completely lost chloride activation. Thus, Galpha(o2) acts on a putative regulatory chloride binding domain that appears to modulate transport activity of vesicular glutamate transporters.
Asunto(s)
Cloruros/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/fisiología , Ácido Glutámico/metabolismo , Vesículas Sinápticas/metabolismo , Proteínas de Transporte Vesicular de Glutamato/metabolismo , Adenosina Trifosfato/farmacología , Animales , Anticuerpos/farmacología , Western Blotting/métodos , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/deficiencia , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/inmunología , Ácido Glutámico/farmacocinética , Ácido Glutámico/farmacología , Guanilil Imidodifosfato/farmacología , Ratones , Ratones Noqueados , Cloruro de Potasio/farmacología , Proteínas R-SNARE/metabolismo , Ratas , Vesículas Sinápticas/efectos de los fármacos , Proteína 25 Asociada a Sinaptosomas/inmunología , Tritio/farmacocinética , Proteínas de Transporte Vesicular de Glutamato/clasificaciónRESUMEN
Serotonin (5-hydroxytryptophan, 5-HT) is a neurotransmitter synthesized in the raphe nuclei of the brain stem and involved in the central control of food intake, sleep, and mood. Accordingly, dysfunction of the serotonin system has been implicated in the pathogenesis of psychiatric diseases. At the same time, serotonin is a peripheral hormone produced mainly by enterochromaffin cells in the intestine and stored in platelets, where it is involved in vasoconstriction, haemostasis, and the control of immune responses. Moreover, serotonin is a precursor for melatonin and is therefore synthesized in high amounts in the pineal gland. Tryptophan hydroxylase (TPH) catalyzes the rate limiting step in 5-HT synthesis. Until recently, only one gene encoding TPH was described for vertebrates. By gene targeting, we functionally ablated this gene in mice. To our surprise, the resulting animals, although being deficient for serotonin in the periphery and in the pineal gland, exhibited close to normal levels of 5-HT in the brain stem. This led us to the detection of a second TPH gene in the genome of humans, mice, and rats, called TPH2. This gene is predominantly expressed in the brain stem, while the classical TPH gene, now called TPH1, is expressed in the gut, pineal gland, spleen, and thymus. These findings clarify puzzling data, which have been collected over the last decades about partially purified TPH proteins with different characteristics and justify a new concept of the serotonin system. In fact, there are two serotonin systems in vertebrates, independently regulated and with distinct functions.
Asunto(s)
Isoenzimas/metabolismo , Serotonina/metabolismo , Triptófano Hidroxilasa/metabolismo , Secuencia de Aminoácidos , Animales , Humanos , Ratones , Datos de Secuencia Molecular , Ratas , Homología de Secuencia de AminoácidoRESUMEN
Posttranslational modifications (PTM) have been shown to be essential for protein function and signaling. Here we report the identification of a novel modification, protein transfer of histamine, and provide evidence for its function in G protein signaling. Histamine, known as neurotransmitter and mediator of the inflammatory response, was found incorporated into mastocytoma proteins. Histaminylation was dependent on transglutaminase II. Mass spectrometry confirmed histamine modification of the small and heterotrimeric G proteins Cdc42, Gαo1 and Gαq. The modification was specific for glutamine residues in the catalytic core, and triggered their constitutive activation. TGM2-mediated histaminylation is thus a novel PTM that functions in G protein signaling. Protein αmonoaminylations, thus including histaminylation, serotonylation, dopaminylation and norepinephrinylation, hence emerge as a novel class of regulatory PTMs.
Asunto(s)
Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Proteínas de Unión al GTP/metabolismo , Histamina/metabolismo , Procesamiento Proteico-Postraduccional , Transglutaminasas/metabolismo , Proteína de Unión al GTP cdc42/metabolismo , Secuencia de Aminoácidos , Animales , Dominio Catalítico , Línea Celular Tumoral , Escherichia coli/genética , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/genética , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/genética , Proteínas de Unión al GTP/genética , Expresión Génica , Glutamina/genética , Glutamina/metabolismo , Histamina/genética , Espectrometría de Masas , Mastocitoma/enzimología , Mastocitoma/genética , Ratones , Datos de Secuencia Molecular , Proteína Glutamina Gamma Glutamiltransferasa 2 , Proteoma/genética , Proteoma/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal/genética , Transglutaminasas/genética , Proteína de Unión al GTP cdc42/genéticaRESUMEN
Functional protein serotonylation is a newly recognized post-translational modification with the primary biogenic monoamine (PBMA) serotonin (5-HT). This covalent protein modification is catalyzed by transglutaminases (TGs) and, for example, acts in the constitutive activation of small GTPases. Multiple physiological roles have been identified since its description in 2003 and, importantly, deregulated serotonylation was shown in the etiology of bleeding disorders, primary pulmonary hypertension and diabetes. The PBMAs 5-HT, histamine, dopamine, and norepinephrine all act as neurotransmitters in the nervous system and as hormones in non-neuronal tissues, which points out their physiological importance. In analogy to serotonylation we have found that also the other PBMAs act through the TG-catalyzed mechanisms of 'histaminylation', 'dopaminylation' and 'norepinephrinylation'. Therefore, PBMAs deploy a considerable portion of their effects via protein monoaminylation in addition to their canonical receptor-mediated signaling. Here, the implications of these newly identified post-translational modifications are presented and discussed. Furthermore, the potential regulatory roles of protein monoaminylation in small GTPase, heterotrimeric G-protein and lipid signaling, as well as in modulating metabolic enzymes and nuclear processes, are critically assessed.
Asunto(s)
Monoaminas Biogénicas/metabolismo , Proteínas de Unión al GTP/metabolismo , Serotonina/metabolismo , Transglutaminasas/metabolismo , Animales , Dopamina/metabolismo , Evolución Molecular , Histamina/metabolismo , Humanos , Oxigenasas de Función Mixta/genética , Proteínas de Unión al GTP Monoméricas/metabolismo , Norepinefrina/metabolismo , Proteína Glutamina Gamma Glutamiltransferasa 2 , Procesamiento Proteico-Postraduccional , Receptores Acoplados a Proteínas G/fisiología , Especificidad por SustratoRESUMEN
Brain serotonin (5-HT) neurotransmission plays a key role in the regulation of mood and has been implicated in a variety of neuropsychiatric conditions. Tryptophan hydroxylase (TPH) is the rate-limiting enzyme in the biosynthesis of 5-HT. Recently, we discovered a second TPH isoform (TPH2) in vertebrates, including man, which is predominantly expressed in brain, while the previously known TPH isoform (TPH1) is primarly a non-neuronal enzyme. Overwhelming evidence now points to TPH2 as a candidate gene for 5-HT-related psychiatric disorders. To assess the role of TPH2 gene variability in the etiology of psychiatric diseases we performed cDNA sequence analysis of TPH2 transcripts from human post mortem amygdala samples obtained from individuals with psychiatric disorders (drug abuse, schizophrenia, suicide) and controls. Here we show that TPH2 exists in two alternatively spliced variants in the coding region, denoted TPH2a and TPH2b. Moreover, we found evidence that the pre-mRNAs of both splice variants are dynamically RNA-edited in a mutually exclusive manner. Kinetic studies with cell lines expressing recombinant TPH2 variants revealed a higher activity of the novel TPH2B protein compared with the previously known TPH2A, whereas RNA editing was shown to inhibit the enzymatic activity of both TPH2 splice variants. Therefore, our results strongly suggest a complex fine-tuning of central nervous system 5-HT biosynthesis by TPH2 alternative splicing and RNA editing. Finally, we present molecular and large-scale linkage data evidencing that deregulated alternative splicing and RNA editing is involved in the etiology of psychiatric diseases, such as suicidal behaviour.
Asunto(s)
Empalme Alternativo , Edición de ARN , Triptófano Hidroxilasa/genética , HumanosRESUMEN
Mutations in the human gene MCPH1 cause primary microcephaly associated with a unique cellular phenotype with premature chromosome condensation (PCC) in early G2 phase and delayed decondensation post-mitosis (PCC syndrome). The gene encodes the BRCT-domain containing protein microcephalin/BRIT1. Apart from its role in the regulation of chromosome condensation, the protein is involved in the cellular response to DNA damage. We report here on the first mouse model of impaired Mcph1-function. The model was established based on an embryonic stem cell line from BayGenomics (RR0608) containing a gene trap in intron 12 of the Mcph1 gene deleting the C-terminal BRCT-domain of the protein. Although residual wild type allele can be detected by quantitative real-time PCR cell cultures generated from mouse tissues bearing the homozygous gene trap mutation display the cellular phenotype of misregulated chromosome condensation that is characteristic for the human disorder, confirming defective Mcph1 function due to the gene trap mutation. While surprisingly the DNA damage response (formation of repair foci, chromosomal breakage, and G2/M checkpoint function after irradiation) appears to be largely normal in cell cultures derived from Mcph1(gt/gt) mice, the overall survival rates of the Mcph1(gt/gt) animals are significantly reduced compared to wild type and heterozygous mice. However, we could not detect clear signs of premature malignant disease development due to the perturbed Mcph1 function. Moreover, the animals show no obvious physical phenotype and no reduced fertility. Body and brain size are within the range of wild type controls. Gene expression on RNA and protein level did not reveal any specific pattern of differentially regulated genes. To the best of our knowledge this represents the first mammalian transgenic model displaying a defect in mitotic chromosome condensation and is also the first mouse model for impaired Mcph1-function.
Asunto(s)
Proteínas Cromosómicas no Histona/fisiología , Rotura Cromosómica , Cromosomas de los Mamíferos/genética , Daño del ADN , Animales , Encéfalo/metabolismo , Encéfalo/patología , Proteínas de Ciclo Celular , Proliferación Celular , Células Cultivadas , Proteínas Cromosómicas no Histona/deficiencia , Proteínas Cromosómicas no Histona/genética , Proteínas del Citoesqueleto , Electroforesis en Gel Bidimensional , Femenino , Fibroblastos/citología , Fibroblastos/metabolismo , Perfilación de la Expresión Génica , Humanos , Imagen por Resonancia Magnética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos , Ratones Noqueados , Modelos Animales , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteómica , Análisis de SupervivenciaRESUMEN
Serotonin is not only a neurotransmitter in the central nervous system, but also a ubiquitous hormone in the periphery involved in vasoconstriction and platelet function. Tryptophan hydroxylase is the rate-limiting enzyme in serotonin biosynthesis. By gene targeting, we have shown that serotonin is synthesized independently by two different tryptophan hydroxylase isoenzymes in peripheral tissues and neurons and identified a neuronal tryptophan hydroxylase isoform. Mice deficient in peripheral tryptophan hydroxylase (TPH1) and serotonin exhibit a reduced risk of thrombosis and thromboembolism. Therefore, we designed several antitph1 hammerhead miniribozymes and tested their cleavage activity against short synthetic Tph1 RNA substrates. In vitro cleavage studies demonstrated site-specific cleavage of Tph1 mRNA that was dependent on substrate/miniribozyme ratio and duration of exposure to miniribozyme. Interestingly, we detected different in vitro cleavage rates after we had cloned the miniribozymes into tRNA expression constructs, and found one with a high cleavage rate. We also demonstrated that this active tRNA-miniribozyme chimera is capable of selectively cleaving native Tph1 mRNA in vivo, with concomitant downregulation of the serotonin biosynthesis. Therefore, this Tph1-specific miniribozyme may provide a novel and effective form of gene therapy that may be applicable to a variety of thrombotic diseases.
Asunto(s)
Fibrinolíticos/farmacología , ARN Catalítico/farmacología , Triptófano Hidroxilasa/antagonistas & inhibidores , Animales , Secuencia de Bases , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Ratones , Datos de Secuencia Molecular , Conformación de Ácido Nucleico/efectos de los fármacos , ARN Mensajero/química , ARN de Transferencia/química , Termodinámica , Triptófano Hidroxilasa/genética , Triptófano Hidroxilasa/metabolismoRESUMEN
Tryptophan hydroxylase 1 catalyzes the rate-limiting step in the synthesis of serotonin in the periphery. Recently, it has been shown that expression of the tryptophan hydroxylase 1 gene is increased in lungs and pulmonary endothelial cells from patients with idiopathic pulmonary arterial hypertension. Here we investigated the effect of genetic deletion of tryptophan hydroxylase 1 on hypoxia-induced pulmonary arterial hypertension in mice by measuring pulmonary hemodynamics and pulmonary vascular remodeling before and after 2 weeks of hypoxia. In wild-type mice, hypoxia increased right ventricular pressure and pulmonary vascular remodeling. These effects of hypoxia were attenuated in the tryptophan hydroxylase 1-/-mice. Hypoxia increased right ventricular hypertrophy in both wild-type and tryptophan hydroxylase 1-/-mice suggesting that in vivo peripheral serotonin has a differential effect on the pulmonary vasculature and right ventricular hypertrophy. Contractile responses to serotonin were increased in pulmonary arteries from tryptophan hydroxylase 1-/-mice. Hypoxia increased serotonin-mediated contraction in vessels from the wild-type mice, but this was not further increased by hypoxia in the tryptophan hydroxylase 1-/-mice. In conclusion, these results indicate that tryptophan hydroxylase 1 and peripheral serotonin play an essential role in the development of hypoxia-induced elevations in pulmonary pressures and hypoxia-induced pulmonary vascular remodeling. In addition, the results suggest that, in mice, serotonin has differential effects on the pulmonary vasculature and right ventricular hypertrophy.
Asunto(s)
Hipertensión Pulmonar/etiología , Hipoxia/complicaciones , Triptófano Hidroxilasa/deficiencia , Animales , Hipertensión Pulmonar/fisiopatología , Hipertrofia Ventricular Derecha/etiología , Hipoxia/fisiopatología , Ratones , Ratones Noqueados , Presión , Arteria Pulmonar/efectos de los fármacos , Arteria Pulmonar/fisiopatología , Serotonina/farmacología , Serotoninérgicos/farmacología , Resistencia Vascular , Vasoconstricción , Función Ventricular DerechaRESUMEN
The liver can regenerate its volume after major tissue loss. In a mouse model of liver regeneration, thrombocytopenia, or impaired platelet activity resulted in the failure to initiate cellular proliferation in the liver. Platelets are major carriers of serotonin in the blood. In thrombocytopenic mice, a serotonin agonist reconstituted liver proliferation. The expression of 5-HT2A and 2B subtype serotonin receptors in the liver increased after hepatectomy. Antagonists of 5-HT2A and 2B receptors inhibited liver regeneration. Liver regeneration was also blunted in mice lacking tryptophan hydroxylase 1, which is the rate-limiting enzyme for the synthesis of peripheral serotonin. This failure of regeneration was rescued by reloading serotonin-free platelets with a serotonin precursor molecule. These results suggest that platelet-derived serotonin is involved in the initiation of liver regeneration.
Asunto(s)
Plaquetas/fisiología , Regeneración Hepática , Hígado/fisiología , Serotonina/fisiología , 5-Hidroxitriptófano/farmacología , Anfetaminas/farmacología , Animales , Plaquetas/metabolismo , Busulfano/farmacología , Proliferación Celular , Clopidogrel , Hepatectomía , Hepatocitos/citología , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Recuento de Plaquetas , Receptor de Serotonina 5-HT2A/metabolismo , Receptor de Serotonina 5-HT2B/metabolismo , Serotonina/sangre , Antagonistas del Receptor de Serotonina 5-HT2 , Trombocitopenia , Ticlopidina/análogos & derivados , Ticlopidina/farmacología , Triptófano Hidroxilasa/genética , Triptófano Hidroxilasa/metabolismoRESUMEN
ADP-ribosylation factor related protein 1 (ARFRP1) is a member of the ARF-family of GTPases which operate as molecular switches in the regulation of intracellular protein traffic. Deletion of the mouse Arfrp1 gene leads to embryonic lethality during early gastrulation, suggesting that ARFRP1 is required for cell adhesion-related processes. Here we show that ARFRP1 specifically controls targeting of ARL1 and its effector Golgin-245 to the trans-Golgi. GTP-bound ARFRP1 (ARFRP1-Q79L mutant) is associated with Golgi membranes and co-localized with the GTPase ARL1. In contrast, the guanine nucleotide exchange defective ARFRP1 mutant (ARFRP1-T31N) clusters within the cytosol. ARFRP1-T31N or depletion of endogenous ARFRP1 by RNA interference disrupts the Golgi association of ARL1 and of the GRIP-domain protein Golgin-245 and alters the distribution of a trans-Golgi network marker, syntaxin 6. In contrast, the targeting of two other Golgi-associated proteins, GM130 and giantin, was unaffected. Furthermore, in Arfrp1-/ - embryos ARL1 dislocated from Golgi membranes whereas it was associated with intracellular membranes in wild-type embryos. These data suggest that lethality of Arfrp1 knockout embryos is due to a specific disruption of protein targeting, e.g., of ARL1 and Golgin-245, to the Golgi.
Asunto(s)
Factor 1 de Ribosilacion-ADP/genética , Factores de Ribosilacion-ADP/metabolismo , Aparato de Golgi/metabolismo , Proteínas de la Membrana/metabolismo , Red trans-Golgi/metabolismo , Factor 1 de Ribosilacion-ADP/metabolismo , Animales , Autoantígenos/metabolismo , Embrión de Mamíferos/metabolismo , GTP Fosfohidrolasas/genética , Silenciador del Gen , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/química , Células HeLa , Humanos , Membranas Intracelulares/metabolismo , Ratones , Ratones Noqueados , Transporte de Proteínas , TransfecciónRESUMEN
Tryptophan hydroxylase (TPH) is the rate-limiting enzyme in serotonin biosynthesis. A novel gene, termed TPH2, has recently been described. This gene is preferentially expressed in the central nervous system, while the original TPH1 is the peripheral gene. We have expressed human tryptophan hydroxylase 2 (hTPH2) and two deletion mutants (NDelta150 and NDelta150/CDelta24) using isopropyl beta-D-thiogalactopyranoside-free autoinduction in Escherichia coli. This expression system produced active wild type TPH2 with relatively low solubility. The solubility was increased for mutants lacking the NH(2)-terminal regulatory domain. The solubility of hTPH2, NDelta150, and NDelta150/CDelta24 are 6.9, 62, and 97.5%, respectively. Removal of the regulatory domain also produced a more than 6-fold increase in enzyme stability (t((1/2)) at 37 degrees C). The wild type hTPH2, like other members of the aromatic amino acid hydroxylase superfamily, exists as a homotetramer (236 kDa on size exclusion chromatography). Similarly, NDelta150 also migrates as a tetramer (168 kDa). In contrast, removal of the NH(2)-terminal domain and the COOH-terminal, putative leucine zipper tetramerization domain produces monomeric enzyme (39 kDa). Interestingly, removal of the NH(2)-terminal regulatory domain did not affect the Michaelis constants for either substrate but did increase V(max) values. These data identify the NH(2)-terminal regulatory domain as the source of hTPH2 instability and reduced solubility.
Asunto(s)
Triptófano Hidroxilasa/química , Inducción Enzimática , Estabilidad de Enzimas , Humanos , Cinética , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Solubilidad , Triptófano Hidroxilasa/metabolismoRESUMEN
The activity of vesicular monoamine transporters (VMATs) is down-regulated by the G-protein alpha-subunits of G(o2) and G(q), but the signaling pathways are not known. We show here that no such regulation is observed when VMAT1 or VMAT2 are expressed in Chinese hamster ovary (CHO) cells. However, when the intracellular compartments of VMAT-expressing CHO cells are preloaded with different monoamines, transport becomes susceptible to G-protein-dependent regulation, with differences between the two transporter isoforms. Epinephrine induces G-protein-mediated inhibition of transmitter uptake in CHOVMAT1 cells but prevents inhibition induced by dopamine in CHOVMAT2 cells. Epinephrine also antagonizes G-protein-mediated inhibition of monoamine uptake by VMAT2 expressing platelets or synaptic vesicles. In CHOVMAT2 cells G-protein-mediated inhibition of monoamine uptake can be induced by 5-hydroxytryptamine (serotonin) 1B receptor agonists, whereas alpha1 receptor agonists modulate uptake into CHOVMAT1 cells. Accordingly, 5-hydroxytryptamine 1B receptor antagonists prevent G-protein-mediated inhibition of uptake in partially filled platelets and synaptic vesicles expressing VMAT2. CHO cells expressing VMAT mutants with a shortened first vesicular loop transport monoamines. However, no or a reduced G-protein regulation of uptake can be initiated. In conclusion, vesicular content is involved in the activation of vesicle associated G-proteins via a structure sensing the luminal monoamine content. The first luminal loop of VMATs may represent a G-protein-coupled receptor that adapts vesicular filling.