RESUMEN
Glial cell line-derived neurotrophic factor (GDNF) protects dopaminergic neurones against toxic and physical damage. In addition, GDNF promotes differentiation and structural integrity of dopaminergic neurones. Here we show that GDNF can support the function of primary dopaminergic neurones by triggering activation of GTP-cyclohydrolase I (GTPCH I), a key enzyme in catecholamine biosynthesis. GDNF stimulation of primary dopaminergic neurones expressing both tyrosine 3-monooxygenase and GTPCH I resulted in a dose-dependent doubling of GTPCH I activity, and a concomitant increase in tetrahydrobiopterin levels whereas tyrosine 3-monooxygenase activity was not altered. Actinomycin D, asan inhibitor of de novo biosynthesis, abolished any GDNF-mediated up-regulation of GTPCH I activity. However, GTPCH I mRNA levels in primary dopaminergic neurones were not altered by GDNF treatment, suggesting that the mode of action for that up-regulation is not directly connected to the regulation of GTPCH I transcription. We conclude that GDNF, in addition to its action in structural differentiation, also promotes differentiation regarding expression and enzymatic activity of a crucial component in the dopaminergic biosynthetic pathway.
Asunto(s)
Biopterinas/análogos & derivados , Biopterinas/metabolismo , Dopamina/metabolismo , GTP Ciclohidrolasa/metabolismo , Factores de Crecimiento Nervioso , Proteínas del Tejido Nervioso/farmacología , Neuronas/metabolismo , Animales , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Células Cultivadas , Dactinomicina/farmacología , Relación Dosis-Respuesta a Droga , Activación Enzimática/efectos de los fármacos , GTP Ciclohidrolasa/genética , Factor Neurotrófico Derivado de la Línea Celular Glial , Mesencéfalo/citología , Mesencéfalo/efectos de los fármacos , Mesencéfalo/embriología , Mesencéfalo/metabolismo , Neuronas/citología , Neuronas/efectos de los fármacos , Inhibidores de la Síntesis de la Proteína/farmacología , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Tirosina 3-Monooxigenasa/metabolismo , Regulación hacia Arriba/efectos de los fármacosRESUMEN
In the zebrafish, the peripheral neurons and the pigment cells are derived from the neural crest and share the pteridine pathway, which leads either to the cofactor tetrahydrobiopterin or to xanthophore pigments. The components of the pteridine pattern were identified as tetrahydrobiopterin, sepiapterin, 7-oxobiopterin, isoxanthopterin, and 2,4,7-trioxopteridine. The expression of GTP cyclohydrolase I activity during the first 24-h postfertilization, followed by 6-pyruvoyl-5,6,7,8-tetrahydropterin synthase and sepiapterin reductase, suggest an early supply of tetrahydrobiopterin for neurotransmitter synthesis in the neurons and for tyrosine supply in the melanophores. At 48-h postfertilization, sepiapterin formation branches off the de novo pathway of tetrahydrobiopterin synthesis. Sepiapterin, via 7,8-dihydrobiopterin and biopterin, serves as a precursor for the formation of 7-oxobiopterin, which may be further catabolized to isoxanthopterin and 2,4,7-trioxopteridine. Neither 7, 8-dihydrobiopterin nor biopterin is a substrate for xanthine oxidoreductase. In contrast, both of these compounds are oxidized at C-7 by a xanthine oxidase variant form, which is inactivated by KCN, but is insensitive to allopurinol. The oxidase and the dehydrogenase form of xanthine oxidoreductase as well as the xanthine oxidase variant have specific developmental patterns. It follows that GTP cyclohydrolase I, the formation of sepiapterin, and the xanthine oxidoreductase family control the pteridine pathway in the zebrafish.
Asunto(s)
Pteridinas/metabolismo , Oxidorreductasas de Alcohol/metabolismo , Animales , Cromatografía Líquida de Alta Presión , GTP Ciclohidrolasa/metabolismo , Liasas de Fósforo-Oxígeno/metabolismo , Xantina Deshidrogenasa/metabolismo , Pez CebraRESUMEN
GTP cyclohydrolase I controls the de novo pathway for the synthesis of tetrahydrobiopterin, which is the essential cofactor for tryptophan 5-monooxygenase and thus, for serotonin production. In mouse bone marrow-derived mast cells, the kit ligand selectively up-regulates GTP cyclohydrolase I activity (Ziegler, I., Hültner, L. , Egger, D., Kempkes, B., Mailhammer, R., Gillis, S., and Rödl, W. (1993) J. Biol. Chem. 268, 12544-12551). Immunoblot analysis now confirms that this long term enhancement is caused by increased expression of the enzyme. Furthermore we show that GTP cyclohydrolase I is subject to modification at the post-translational level. In vivo labeling with [32P]orthophosphate demonstrates that in primary mast cells and in transfected RBL-2H3 cells overexpressing GTP cyclohydrolase I, the enzyme exists in a phosphorylated form. Antigen binding to the high affinity receptor for IgE triggers an additional and transient phosphorylation of GTP cyclohydrolase I with a concomitant rise in its activity, and in consequence, cellular tetrahydrobiopterin levels increase. These events culminate 8 min after stimulation and can be mimicked by phorbol ester. The hyperphosphorylation is greatly reduced by the protein kinase C inhibitor Ro-31-8220. In vitro phosphorylation studies indicate that GTP cyclohydrolase I is a substrate for both casein kinase II and protein kinase C.
Asunto(s)
GTP Ciclohidrolasa/metabolismo , Mastocitos/enzimología , Receptores de IgE/metabolismo , Animales , Biopterinas/análogos & derivados , Biopterinas/biosíntesis , Quinasa de la Caseína II , Células Cultivadas , Inhibidores Enzimáticos/farmacología , Indoles/farmacología , Isoenzimas/antagonistas & inhibidores , Isoenzimas/metabolismo , Ratones , Fosforilación , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/metabolismo , Proteína Quinasa C-delta , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
Mutants of Escherichia coli unable to synthesize a functional pyruvate formate-lyase (PFL) are severely impaired in their capacity to grow by glucose fermentation. In a functional complementation assay designed to isolate the pfl gene from Clostridium butyricum, we fortuitously identified a gene that did not encode a PFL but nonetheless was able to complement the phenotypic defects caused by an E. coli pfl mutation. The clostridial gene encoded a basic 14. 5-kDa protein (TcbC) which, based on amino acid similarity and analysis of immediately adjacent DNA sequences, was part of a transposase exhibiting extensive similarity to the product of the site-specific transposon Tn554 from Staphylococcus aureus. Our studies revealed that the clostridial TcbC protein activated the transcription of the E. coli tdcABCDEFG operon, which encodes an anaerobic L-threonine-degradative pathway. Normally, anaerobic synthesis of the pathway is optimal when E. coli grows in the absence of catabolite-repressing sugars and in the presence of L-threonine. Although anaerobic control of pathway synthesis was maintained, TcbC alleviated glucose repression. One of the products encoded by the tdc operon, TdcE, has recently been shown to be a 2-keto acid formate-lyase (C. Hesslinger, S. A. Fairhurst, and G. Sawers, Mol. Microbiol. 27:477-492, 1998) that can accept pyruvate as an enzyme substrate. Here we show that TdcE is directly responsible for the restoration of fermentative growth to pfl mutants.
Asunto(s)
Acetiltransferasas/metabolismo , Aciltransferasas/metabolismo , Proteínas Bacterianas/genética , Proteínas de Escherichia coli , Escherichia coli/enzimología , Glucosa/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Fermentación , Eliminación de Gen , Oxígeno/metabolismo , Fenotipo , Plásmidos/genética , Regiones Promotoras Genéticas/genética , Transcripción GenéticaRESUMEN
An immunological analysis of an Escherichia coli strain unable to synthesize the main pyruvate formate-lyase enzyme Pfl revealed the existence of a weak, cross-reacting 85 kDa polypeptide that exhibited the characteristic oxygen-dependent fragmentation typical of a glycyl radical enzyme. Polypeptide fragmentation of this cross-reacting species was shown to be dependent on Pfl activase. Cloning and sequence analysis of the gene encoding this protein revealed that it coded for a new enzyme, termed TdcE, which has 82% identity with Pfl. On the basis of RNA analyses, the tdcE gene was shown to be part of a large operon that included the tdcABC genes, encoding an anaerobic threonine dehydratase, tdcD, coding for a propionate kinase, tdcF, the function of which is unknown, and the tdcG gene, which encodes a L-serine dehydratase. Expression of the tdcABCDEFG operon was strongly catabolite repressed. Enzyme studies showed that TdcE has both pyruvate formate-lyase and 2-ketobutyrate formate-lyase activity, whereas the TdcD protein is a new propionate/acetate kinase. By monitoring culture supernatants from various mutants using 1H nuclear magnetic resonance (NMR), we followed the anaerobic conversion of L-threonine to propionate. These studies confirmed that 2-ketobutyrate, the product of threonine deamination, is converted in vivo by TdcE to propionyl-CoA. These studies also revealed that Pfl and an as yet unidentified thiamine pyrophosphate-dependent enzyme(s) can perform this reaction. Double null mutants deficient in phosphotransacetylase (Pta) and acetate kinase (AckA) or AckA and TdcD were unable to metabolize threonine to propionate, indicating that propionyl-CoA and propionyl-phosphate are intermediates in the pathway and that ATP is generated during the conversion of propionyl-P to propionate by AckA or TdcD.
Asunto(s)
Aciltransferasas/metabolismo , Sistemas de Transporte de Aminoácidos Neutros , Proteínas de Escherichia coli , Escherichia coli/enzimología , Fosfotransferasas (aceptor de Grupo Carboxilo)/metabolismo , Propionatos/metabolismo , Treonina/metabolismo , Acetiltransferasas/genética , Acetiltransferasas/metabolismo , Anaerobiosis , Clonación Molecular , Proteínas de Unión al ADN/genética , Escherichia coli/genética , Fermentación , Mutación del Sistema de Lectura , Genes Bacterianos , Espectroscopía de Resonancia Magnética , Proteínas de Transporte de Membrana/genética , Transactivadores/genética , Transcripción GenéticaRESUMEN
Unlike other Escherichia coli K-12 strains, W3110 contains multiple copies of the insertion sequence IS5. Some of these IS5 elements have been involved in tandem duplication of a portion of the chromosome which includes, amonst others, the tdcABC-DEFG operon genes. The nucleotide sequence and insertion site of one of these elements, IS5P, was determined. It was shown that IS5P has inserted within the coding sequence of the tdcA gene and is flanked, not by the remaining portion of the tdcA gene, but by the extreme 3' end of the tdcD gene. In other E. coli K-12 strains the tdcD gene and three other genes, tdcE, tdcF and tdcG, all form part of the tdc operon. Our results demonstrate that during the duplication event the tdcABCgenes have been amplified and separated from the remaining genes tdcE, tdcF and tdcG of the operon, which are each present in single copy.