RESUMEN
Thymidylate synthase (TS) may undergo phosphorylation endogenously in mammalian cells, and as a recombinant protein expressed in bacterial cells, as indicated by the reaction of purified enzyme protein with Pro-Q® Diamond Phosphoprotein Gel Stain (PGS). With recombinant human, mouse, rat, Trichinella spiralis and Caenorhabditis elegans TSs, expressed in Escherichia coli, the phosphorylated, compared to non-phosphorylated recombinant enzyme forms, showed a decrease in Vmax(app), bound their cognate mRNA (only rat enzyme studied), and repressed translation of their own and several heterologous mRNAs (human, rat and mouse enzymes studied). However, attempts to determine the modification site(s), whether endogenously expressed in mammalian cells, or recombinant proteins, did not lead to unequivocal results. Comparative ESI-MS/analysis of IEF fractions of TS preparations from parental and FdUrd-resistant mouse leukemia L1210 cells, differing in sensitivity to inactivation by FdUMP, demonstrated phosphorylation of Ser(10) and Ser(16) in the resistant enzyme only, although PGS staining pointed to the modification of both L1210 TS proteins. The TS proteins phosphorylated in bacterial cells were shown by (31)P NMR to be modified only on histidine residues, like potassium phosphoramidate (KPA)-phosphorylated TS proteins. NanoLC-MS/MS, enabling the use of CID and ETD peptide fragmentation methods, identified several phosphohistidine residues, but certain phosphoserine and phosphothreonine residues were also implicated. Molecular dynamics studies, based on the mouse TS crystal structure, allowed one to assess potential of several phosphorylated histidine residues to affect catalytic activity, the effect being phosphorylation site dependent.
Asunto(s)
Timidilato Sintasa/metabolismo , Animales , Línea Celular Tumoral , Ratones , Fosforilación , ConejosRESUMEN
Localization of thymidylate synthase protein in Trichinella spiralis and Caenorhabditis elegans development was followed with the use of confocal microscopy, revealing similar expression patterns in both nematode species. In T. spiralis premature muscle larvae and C. elegans dauer, L3 and L4 larvae, thymidylate synthase was detected in the nerve ring and gonad primordia, as well as T. spiralis stichosome and C. elegans pharyngeal glandular cells. In developmentally arrested T. spiralis muscle larvae, the enzyme was found localized to the gonad primordia and stichosome. High enzyme level was also observed in the embryos developing in uteri of T. spiralis female adult and C. elegans hermaphrodite forms. In the case of T. spiralis adult forms, thymidylate synthase was detected in stichosome, along esophagus wall, as well as in egg and sperm cells. While the enzyme protein present in the embryos remains in accord with its known association with proliferating systems, thymidylate synthase presence in the nerve ring, and reproductive and secretory (T. spiralis stichosomal and C. elegans pharyngeal glandular cells) systems, points to a state of cell cycle-arrest, also known to preserve the enzyme protein.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/enzimología , Larva/enzimología , Timidilato Sintasa/metabolismo , Trichinella spiralis/enzimología , Animales , Caenorhabditis elegans/citología , Caenorhabditis elegans/genética , Caenorhabditis elegans/crecimiento & desarrollo , Proteínas de Caenorhabditis elegans/genética , Esófago/citología , Esófago/enzimología , Femenino , Regulación del Desarrollo de la Expresión Génica , Larva/citología , Larva/genética , Larva/crecimiento & desarrollo , Masculino , Microscopía Confocal , Sistema Nervioso/citología , Sistema Nervioso/enzimología , Sistema Nervioso/crecimiento & desarrollo , Especificidad de Órganos , Óvulo/citología , Óvulo/enzimología , Óvulo/crecimiento & desarrollo , Transporte de Proteínas , Espermatocitos/citología , Espermatocitos/enzimología , Espermatocitos/crecimiento & desarrollo , Timidilato Sintasa/genética , Trichinella spiralis/citología , Trichinella spiralis/genética , Trichinella spiralis/crecimiento & desarrolloRESUMEN
Highly purified preparations of thymidylate synthase, isolated from calf thymus, and L1210 parental and FdUrd-resistant cells, were found to be nitrated, as indicated by a specific reaction with anti-nitro-tyrosine antibodies, suggesting this modification to appear endogenously in normal and tumor tissues. Each human, mouse and Ceanorhabditis elegans recombinant TS preparation, incubated in vitro in the presence of NaHCO(3), NaNO(2) and H(2)O(2) at pH 7.5, underwent tyrosine nitration, leading to a V(max)(app) 2-fold lower following nitration of 1 (with human or C. elegans TS) or 2 (with mouse TS) tyrosine residues per monomer. Enzyme interactions with dUMP, meTHF or 5-fluoro-dUMP were not distinctly influenced. Nitration under the same conditions of model tripeptides of a general formula H(2)N-Gly-X-Gly-COOH (X = Phe, Tyr, Trp, Lys, Arg, His, Ser, Thr, Cys, Gly), monitored by NMR spectroscopy, showed formation of nitro-species only for H-Gly-Tyr-Gly-OH and H-Gly-Phe-Gly-OH peptides, the chemical shifts for nitrated H-Gly-Tyr-Gly-OH peptide being in a very good agreement with the strongest peak found in (15)N-(1)H HMBC spectrum of nitrated protein. MS analysis of nitrated human and C. elegans proteins revealed several thymidylate synthase-derived peptides containing nitro-tyrosine (at positions 33, 65, 135, 213, 230, 258 and 301 in the human enzyme) and oxidized cysteine (human protein Cys(210), with catalytically critical Cys(195) remaining apparently unmodified) residues.
Asunto(s)
Timidilato Sintasa/metabolismo , Tirosina/metabolismo , Animales , Caenorhabditis elegans/enzimología , Bovinos , Línea Celular Tumoral , Humanos , Ratones , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Timidilato Sintasa/química , Timidilato Sintasa/aislamiento & purificación , Timo/enzimología , Tirosina/químicaRESUMEN
To establish whether NADPH oxidase activation, responsible for previously demonstrated Trichinella spiralis-induced respiratory burst, results from assembling of membrane and cytosolic NADPH oxidase components and/or increased expression of the oxidase complex proteins, the superoxide anion production and expression of the regulatory p47(phox) subunit were measured in cultured alveolar macrophages obtained during T. spiralis infection of guinea pigs. The results demonstrate for the first time helminth parasite-infection-induced stimulation of NADPH oxidase p47(phox) subunit protein expression, with the effect being decreased by in vivo treatment with cyclosporin A, previously shown to inhibit T. spiralis infection-induced respiratory burst in guinea-pig alveolar macrophages. However, although the expression of the p47(phox) subunit protein remained induced during secondary infection, it was accompanied by superoxide anion production that was significantly suppressed in comparison with that observed during primary infection, suggesting suppressive action of T. spiralis on host's alveolar macrophage immune response, presumably connected with NADPH oxidase complex activity attenuation.
Asunto(s)
Macrófagos Alveolares/parasitología , Fosfoproteínas/biosíntesis , Trichinella spiralis/fisiología , Animales , Células Cultivadas , Ciclosporina/farmacología , Cobayas , Inmunosupresores/farmacología , Macrófagos Alveolares/efectos de los fármacos , Macrófagos Alveolares/metabolismo , NADPH Oxidasas/metabolismo , Estallido Respiratorio , Superóxidos/metabolismoRESUMEN
Mouse thymidylate synthase R209K (a mutation corresponding to R218K in Lactobacillus casei), overexpressed in thymidylate synthase-deficient Escherichia coli strain, was poorly soluble and with only feeble enzyme activity. The mutated protein, incubated with FdUMP and N(5,10)-methylenetetrahydrofolate, did not form a complex stable under conditions of SDS/polyacrylamide gel electrophoresis. The reaction catalyzed by the R209K enzyme (studied in a crude extract), compared to that catalyzed by purified wild-type recombinant mouse thymidylate synthase, showed the K(m) value for dUMP 571-fold higher and V(max) value over 50-fold (assuming that the mutated enzyme constituted 20% of total crude extract protein) lower. Thus the ratios k(cat, R209K)/k(cat, 'wild') and (k(cat, R209K)/K(m, R209K)(dUMP))/( k(cat, 'wild')/K(m, 'wild')(dUMP)) were 0.019 and 0.000032, respectively, documenting that mouse thymidylate synthase R209, similar to the corresponding L. casei R218, is essential for both dUMP binding and enzyme reaction.
Asunto(s)
Timidilato Sintasa/genética , Timidilato Sintasa/metabolismo , Sustitución de Aminoácidos , Animales , Arginina/genética , Catálisis , Nucleótidos de Desoxiuracil/química , Nucleótidos de Desoxiuracil/metabolismo , Inducción Enzimática/efectos de los fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Cinética , Lacticaseibacillus casei/enzimología , Leucemia L1210/metabolismo , Lisina/genética , Ratones , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Solubilidad , Células Tumorales CultivadasRESUMEN
Synthesis and biological evaluation are described of seven new analogues (3-9) of two potent thymidylate synthase inhibitors, 10-propargyl-5,8-dideazafolate (1) and its 2-methyl-2-deamino congener ICI 198583 (2). While the new compunds 3 and 4 were analogues of 1 and 2, respectively, containing a p-aminobenzenesulfonyl residue in place of the p-aminobenzoic acid residue, the remaining 5 new compounds were analogues of 4 with the L-glutamic acid residue replaced by glycine (5), L-valine (6), L-alanine (7), L-phenylglycine (8) or L-norvaline (9). The new analogues were tested as inhibitors of thymidylate synthases isolated from tumour (Ehrlich carcinoma), parasite (Hymenolepis diminuta) and normal tissue (regenerating rat liver) and found to be weaker inhibitors than the parent 10-propargyl-5,8-dideazafolic acid. Selected new analogues, tested as inhibitors of growth of mouse leukemia L 5178Y cells, were less potent than the parent 10-propargyl-5,8-dideazafolic acid. Substitution of the glutamyl residue in compound 4 with L-norvaline (9) resulted in only a 5-fold stronger thymidylate synthase inhibitor, but a 40-fold weaker cell growth inhibitor.
Asunto(s)
Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/toxicidad , Antagonistas del Ácido Fólico/síntesis química , Antagonistas del Ácido Fólico/toxicidad , Sulfonamidas/síntesis química , Sulfonamidas/farmacología , Timidilato Sintasa/antagonistas & inhibidores , Animales , Carcinoma de Ehrlich/enzimología , Carcinoma de Ehrlich/patología , División Celular/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Antagonistas del Ácido Fólico/farmacología , Cinética , Hígado/enzimología , Ratones , Estructura Molecular , Ratas , Sulfonamidas/toxicidad , Timidilato Sintasa/metabolismoRESUMEN
In order to assess immunological response, induced in guinea-pig lungs by Trichinella spiralis, cellular infiltration into pulmonary alveolar space and production of O(2)(-) and NO in alveolar macrophages obtained from bronchoalveolar lavage fluid (BALF), as well as accumulation of nitric oxide (NO) metabolites in BALF and serum, were evaluated during the early period of primary T. spiralis infection (from 4th to 8th and on 14th day after oral administration of larvae) and on 6th day after secondary infection. Primary infection caused increased infiltration of lymphocytes, macrophages, neutrophils and eosinophils, while secondary infection resulted in raised lymphocyte and eosinophil numbers. In spite of marked cellular infiltration of alveolar space, only very limited activation of effector cells, pointing to a suppressed innate response, was apparent, as (i) BALF supernatant phospholipid/protein concentration ratio, and lung levels of phospholipid peroxidation markers, conjugated dienes and malondialdehyde, did not change during 7 days following infection; (ii) primary, but not secondary, infection caused only a transient increase of superoxide anion production by alveolar macrophages; (iii) despite expression of inducible nitric oxide synthase in macrophages of control, infected and BCG-treated animals, and of interferon (IFN)-gamma-like activity in sera of infected animals, macrophage nitric oxide production was not affected by infection, even after additional stimulation in vitro (lipopolisaccharide + hrIFN-gamma) or in vivo (BCG or secondary T. spiralis infection); and (iv) increased nitrate concentrations were found in BALF supernatant and serum, but not in lung homogenates, of infected animals.
Asunto(s)
Líquido del Lavado Bronquioalveolar/citología , Líquido del Lavado Bronquioalveolar/inmunología , Pulmón/inmunología , Trichinella spiralis/inmunología , Triquinelosis/inmunología , Animales , Cobayas , Pulmón/parasitología , Macrófagos Alveolares/inmunología , Macrófagos Alveolares/metabolismo , Masculino , Ratones , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico Sintasa de Tipo II , Superóxidos/metabolismo , Triquinelosis/parasitologíaRESUMEN
2-Deamino-2-methyl-N10-propargyl-5,8-dideazafolic acid (ICI 198583) is a potent inhibitor of thymidylate synthase. Its analogue, N(alpha)-[4-[N-[(3,4-dihydro-2-methyl-4-oxo-6-quinazolinyl)methyl]-N-propargylamino]phenylacetyl]-L-glutamic acid, containing p-aminophenylacetic acid residue substituting p-aminobenzoic acid residue, was synthesized. The new analogue exhibited a moderately potent thymidylate synthase inhibition, of linear mixed type vs. the cofactor, N(5,10)-methylenetetrahydrofolate. The Ki value of 0.34 microM, determined with a purified recombinant rat hepatoma enzyme, was about 30-fold higher than that reported for inhibition of thymidylate synthase from mouse leukemia L1210 cells by ICI 198583 (Hughes et al., 1990, J. Med. Chem. 33, 3060). Growth of mouse leukemia L5178Y cells was inhibited by the analogue (IC50 = 1.26 mM) 180-fold weaker than by ICI 198583 (IC50 = 6.9 microM).
Asunto(s)
Ácido Fólico/análogos & derivados , Ácido Fólico/síntesis química , Ácido Glutámico , Leucemia/tratamiento farmacológico , Animales , Ácido Glutámico/síntesis química , Ácido Glutámico/química , Ratones , RatasRESUMEN
The effects of cyclosporin A (CsA), a potent immunosuppressive drug with antiparasitic activity, on the innate immunological response in guinea pig lungs during an early period (6th and 14th days) after T. spiralis infection were studied. CsA treatment of T. spiralis-infected guinea pigs caused a significant attenuation of immunological response in lungs by decreasing lymphocyte infiltration into pulmonary alveolar space, inhibiting alveolar macrophage superoxide anion production and lowering both the production of NO metabolites measured in bronchoalveolar lavage fluid and expression of the iNOS protein in lung homogenates, allowing us to speculate that the T. spiralis-dependent immunological response is dependent on lymphocyte T function. Interestingly, CsA itself had a pro-inflammatory effect, promoting leucocyte accumulation and macrophage superoxide production in guinea pig lungs. This observation may have a relevance to the situation in patients undergoing CsA therapy. Macrophage expression of the iNOS protein, evaluated by immunoblotting was not influenced by treatment of animals with CsA or anti-TGF-antibody, indicating different regulation of the guinea pig and murine enzymes.