RESUMEN
Therapy for neurodegenerative lysosomal Tay-Sachs (TS) disease requires active hexosaminidase (Hex) A production in the central nervous system and an efficient therapeutic approach that can act faster than human disease progression. We combined the efficacy of a non-replicating Herpes simplex vector encoding for the Hex A alpha-subunit (HSV-T0alphaHex) and the anatomic structure of the brain internal capsule to distribute the missing enzyme optimally. With this gene transfer strategy, for the first time, we re-established the Hex A activity and totally removed the GM2 ganglioside storage in both injected and controlateral hemispheres, in the cerebellum and spinal cord of TS animal model in the span of one month's treatment. In our studies, no adverse effects were observed due to the viral vector, injection site or gene expression and on the basis of these results, we feel confident that the same approach could be applied to similar diseases involving an enzyme defect.
Asunto(s)
Cerebelo/metabolismo , Técnicas de Transferencia de Gen , Médula Espinal/metabolismo , Enfermedad de Tay-Sachs/terapia , beta-N-Acetilhexosaminidasas/genética , Animales , Expresión Génica , Terapia Genética , Vectores Genéticos , Hexosaminidasa A , Cápsula Interna/metabolismo , Ratones , Ratones Endogámicos C57BL , Simplexvirus/genética , Enfermedad de Tay-Sachs/metabolismo , beta-N-Acetilhexosaminidasas/metabolismoRESUMEN
Sandhoff disease is a severe inherited neurodegenerative disorder resulting from deficiency of the beta-subunit of hexosaminidases A and B, lysosomal hydrolases involved in the degradation of G(M2) ganglioside and related metabolites. Currently, there is no viable treatment for the disease. Here, we show that adenovirus-mediated transfer of the beta-subunit of beta-hexosaminidase restored Hex A and Hex B activity after infection of Sandhoff fibroblasts. Gene transfer following intracerebral injection in a murine model of Sandhoff disease resulted in near-normal level of enzymatic activity in the entire brain at the different doses tested. The addition of hyperosmotic concentrations of mannitol to the adenoviral vector resulted in an enhancement of vector diffusion in the injected hemisphere. Adenoviral-induced lesions were found in brains injected with a high dose of the vector, but were not detected in brains injected with 100-fold lower doses, even in the presence of mannitol. Our data underline the advantage of the adjunction of mannitol to low doses of the adenoviral vector, allowing a high and diffuse transduction efficiency without viral cytotoxicity.
Asunto(s)
Adenoviridae/genética , Terapia Genética/métodos , Vectores Genéticos/administración & dosificación , Manitol/administración & dosificación , Enfermedad de Sandhoff/terapia , beta-N-Acetilhexosaminidasas/genética , Animales , Encéfalo/enzimología , Difusión , Fibroblastos/enzimología , Hexosaminidasa A , Hexosaminidasa B , Inyecciones , Ratones , Ratones Mutantes , Modelos Animales , Enfermedad de Sandhoff/enzimología , beta-N-Acetilhexosaminidasas/metabolismoRESUMEN
Glycogenosis type II (GSD II) is a lysosomal disorder affecting skeletal and cardiac muscle. In the infantile form of the disease, patients display cardiac impairment, which is fatal before 2 years of life. Patients with juvenile or adult forms can present diaphragm involvement leading to respiratory failure. The enzymatic defect in GSD II results from mutations in the acid alpha-glucosidase (GAA) gene, which encodes a 76 kDa protein involved in intralysosomal glycogen hydrolysis. We previously reported the use of an adenovirus vector expressing GAA (AdGAA) for the transduction of myoblasts and myotubes cultures from GSD II patients. Transduced cells secreted GAA in the medium, and GAA was internalized by receptor-mediated capture, allowing glycogen hydrolysis in untransduced cells. In this study, using a GSD II mouse model, we evaluated the feasibility of GSD II gene therapy using muscle as a secretary organ. Adenovirus vector encoding AdGAA was injected in the gastrocnemius of neonates. We detected a strong expression of GAA in the injected muscle, secretion into plasma, and uptake by peripheral skeletal muscle and the heart. Moreover, glycogen content was decreased in these tissues. Electron microscopy demonstrated the disappearance of destruction foci, normally present in untreated mice. We thus demonstrate for the first time that muscle can be considered as a safe and easily accessible organ for GSD II gene therapy.
Asunto(s)
Terapia Genética/métodos , Glucano 1,4-alfa-Glucosidasa/genética , Glucano 1,4-alfa-Glucosidasa/metabolismo , Enfermedad del Almacenamiento de Glucógeno Tipo II/genética , Enfermedad del Almacenamiento de Glucógeno Tipo II/terapia , Músculo Esquelético/metabolismo , Adenoviridae/genética , Animales , Vectores Genéticos/farmacología , Glucógeno/metabolismo , Inyecciones Intramusculares , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , Ratones , Ratones Noqueados , Microscopía Electrónica , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/patología , alfa-GlucosidasasRESUMEN
It is believed that the lysosomal glycohydrolase beta-N-acetylhexosaminidase plays a part in several important processes of reproduction and it has been postulated that this enzyme is subject to hormonal regulation. During pregnancy, activity levels of the enzyme are strongly increased in both human and rat serum. However, little is known about the expression of this enzyme in the female reproductive apparatus and there is no evidence linking the production of hexosaminidase alpha- and beta-subunits to pregnancy. To clarify these aspects better, we examined the enzyme activity, isoenzyme subunit composition and distribution, as well as steady state levels of alpha- and beta-subunit mRNAs in the female reproductive organs and in other selected tissues of pregnant and non-pregnant rats. Among the female rat tissues tested, the ovary and kidney had the highest specific activity. Pregnancy modulated the hexosaminidase activity differently in the tissues examined. In pregnant rats, the activity decreased in the ovary but increased significantly in the uterus, liver and to a lesser extent in other tissues. The decreased hexosaminidase activity in the ovary from pregnant rats appeared to be accompanied by a disproportionately large decrease in beta-subunit mRNA abundance, whereas in the uterus and liver, an increased abundance of this transcript was detectable. The abundance of alpha-subunit mRNA was comparable in pregnant and control rat tissues. Hexosaminidase histochemical staining of tissue sections clearly demonstrates that the greatly increased activity of hexosaminidase in the uterus during pregnancy is largely due to the enzyme in the endometrium, and not to the uterus as a whole. The overall results provide evidence that, during pregnancy, a mechanism(s) of regulation of beta-N-acetylhexosaminidase expression is in operation, and that the enzyme is differentially regulated in rat tissues.
Asunto(s)
Hígado/enzimología , Ovario/enzimología , Útero/enzimología , beta-N-Acetilhexosaminidasas/genética , Animales , Northern Blotting , Cromatografía por Intercambio Iónico , Femenino , Regulación del Desarrollo de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Histocitoquímica , Isoenzimas/genética , Embarazo , ARN Mensajero/análisis , Ratas , Ratas Wistar , Transcripción Genética , beta-N-Acetilhexosaminidasas/metabolismoRESUMEN
In the present paper, we investigated the involvement of cryptococcal melanogenesis and macrophage nitric oxide (NO) production in the accomplishment of anticryptococcal activity by microglial effector cells, using the murine cell line BV-2. We demonstrate that the constitutive levels of anticryptococcal activity exerted by BV-2 cells is significantly enhanced upon interferon gamma plus lipopolysaccharide treatment. The phenomenon, which occurs with no enhancement of phagocytic activity, is associated with the production of high levels of NO and is abolished by addition of NG-monomethyl-L-arginine. Comparable patterns of results are observed employing either unopsonized or opsonized microbial targets, the latter microorganisms being markedly more susceptible to BV-2 cell antimicrobial activity. Furthermore, melanization of Cryptococcus neoformans significantly reduces its susceptibility to BV-2 antimicrobial activity, regardless of the fact that activated macrophages or opsonized microorganisms have been employed. In conclusion, our results provide evidence that NO-dependent events are involved in the fulfillment of anticryptococcal activity by activated microglial cells and that fungal melanization is a precious escamotage through which C. neoformans overcomes host defenses.
Asunto(s)
Arginina/análogos & derivados , Cryptococcus neoformans/inmunología , Interferón gamma/farmacología , Microglía/microbiología , Óxido Nítrico/fisiología , Fagocitosis , Animales , Arginina/farmacología , Línea Celular , Genes myc , Ratones , Microglía/efectos de los fármacos , Microglía/inmunología , Proteínas Oncogénicas v-raf , Fagocitosis/efectos de los fármacos , Proteínas Tirosina Quinasas/genética , Proteínas Recombinantes , Retroviridae , Proteínas Oncogénicas de Retroviridae/genética , Transfección , omega-N-MetilargininaAsunto(s)
Antígenos de Diferenciación de Linfocitos T/sangre , Carcinoma de Pulmón de Células no Pequeñas/terapia , Interleucina-2/uso terapéutico , Neoplasias Pulmonares/terapia , Adulto , Biomarcadores/sangre , Antígenos CD4/sangre , Antígenos CD8/sangre , Carcinoma de Pulmón de Células no Pequeñas/sangre , Carcinoma de Pulmón de Células no Pequeñas/inmunología , Femenino , Humanos , Neoplasias Pulmonares/sangre , Neoplasias Pulmonares/inmunología , Masculino , Persona de Mediana Edad , Receptores de Interleucina-2/metabolismo , SolubilidadRESUMEN
The coenzyme-linked fluorescence of aromatic-L-amino-acid decarboxylase decays non-exponentially. The decay of both native and NaBH4 reduced samples can only be fitted by two exponentials each roughly accounting for about half of the total fluorescence. Denaturation of the reduced protein with 8 M urea makes the fluorescence decay mono-exponential, like that observed for the reference compound pyridoxamine-5-phosphate. An extra pyridoxyl moiety can be bound to the enzyme after incubation with excess pyridoxal phosphate and reduction with NaBH4. This sample is almost twice as fluorescent and shows also two lifetimes. After denaturation only one fluorescence lifetime is observed. The presence of two non-equivalent pyridoxal sites in the native enzyme can be postulated. The heterogeneous decay behaviour of the pyridoxyl moiety in the enzyme together with the variability of lifetime shown, makes this fluorophore an even more interesting fluorescent probe for proteins.
Asunto(s)
Descarboxilasas de Aminoácido-L-Aromático/análisis , Fosfato de Piridoxal/análisis , Animales , Sitios de Unión , Fluorescencia , Riñón/enzimología , Desnaturalización Proteica , PorcinosRESUMEN
Pig kidney 3,4-dihydroxyphenylalanine (Dopa) decarboxylase is inactivated by iodoacetamide following pseudo-first order reaction kinetics. The apparent first order rate constant for inactivation is proportional to the concentration of iodoacetamide and a second order rate constant of 37 M-1 min-1 is obtained at pH 6.8 and 25 degrees C. Cyanogen bromide fragmentation of iodo(1-14C)acetamide - modified inactivated Dopa decarboxylase followed by trypsin digestion yields a single radioactive peptide. Automated Edman degradation reveals a heptapeptide sequence which contains labeled carboxyamidomethylcysteine. This finding and the results of the incorporation of the label from ido (1-14C)acetamide into the enzyme clearly indicate that the modification of 1 mol of SH per mol of enzyme dimer is responsible for the inactivation process. The labeled peptide, which was located by means of limited proteolysis on the fragment corresponding to the COOH-terminal third of the enzyme, has been aligned with a 7 amino acid stretch of Drosophila enzyme. Although this region appears highly conserved in the Dopa decarboxylase enzymes, the cysteinyl residue is not conserved. This observation together with the spectral binding properties of the iodoacetamide inactivated enzyme argue against a functional role for the modifiable cysteine in the mechanism of action of pig kidney enzyme. It is suggested that the loss of pig kidney decarboxylase activity produced by iodoacetamide modification might be attributable to steric hindrance. This could be due to the presence of the bulky acetamidic group on a cysteine residue at, or near, the active center or in a site of strategic importance to the maintenance of the active site topography.
Asunto(s)
Inhibidores de Descarboxilasas de Aminoácidos Aromáticos , Yodoacetamida/farmacología , Riñón/enzimología , Oligopéptidos/análisis , Secuencia de Aminoácidos , Animales , Cromatografía Líquida de Alta Presión , Yodoacetamida/metabolismo , Cinética , Datos de Secuencia Molecular , Fragmentos de Péptidos/aislamiento & purificación , Unión Proteica , Porcinos , TripsinaRESUMEN
Pig kidney 3,4-dihydroxyphenylalanine (Dopa) decarboxylase can be nicked by trypsin with complete loss of its catalytic activity. The original dimer of subunit molecular weight of about 52,000 yields fragments of Mr 38,000 and 14,000, as seen on sodium dodecyl sulfate-gel electrophoresis. Though inactive, the nicked protein retains its native molecular weight and its capacity to bind pyridoxal-5'-phosphate (pyridoxal-P), is recognized by an antiserum raised against the native enzyme, and forms Schiff's base intermediates with aromatic amino acids in L and D forms. Thus, the nicked protein appears to be in a conformation--closely resembling that of the original enzyme--which consists of a tight association of the two tryptic fragments. Dissociation and separation of the two fragments can be achieved under denaturing conditions on a reverse-phase HPLC column. The pyridoxal-P binding site is located on the larger fragment. No NH2-terminal residue is detected in either the intact enzyme or the larger fragment, whereas analysis of the smaller fragment yields a sequence of the first 50 amino acid residues. These data indicate that the smaller fragment is located at about one-third from the COOH terminus of Dopa decarboxylase, while the larger fragment constitutes the aminic portion of the molecule. The site of trypsin cleavage seems to be in a region of the enzyme particularly susceptible to proteolysis. The results of these studies contribute to a better understanding of the structural properties of pig kidney Dopa decarboxylase and may constitute an important step toward the elucidation of the enzyme's primary structure.
Asunto(s)
Descarboxilasas de Aminoácido-L-Aromático/metabolismo , Dopa-Decarboxilasa/metabolismo , Riñón/enzimología , Tripsina/metabolismo , 5-Hidroxitriptófano/farmacología , Secuencia de Aminoácidos , Animales , Inhibidores de Descarboxilasas de Aminoácidos Aromáticos , Sitios de Unión , Dihidroxifenilalanina/farmacología , Electroforesis en Gel de Poliacrilamida , Técnicas de Inmunoadsorción , Levodopa/farmacología , Sustancias Macromoleculares , Datos de Secuencia Molecular , Peso Molecular , Fragmentos de Péptidos/metabolismo , Conformación Proteica , Fosfato de Piridoxal/metabolismo , Espectrofotometría , Porcinos , Tripsina/farmacologíaRESUMEN
A simple and rapid procedure, which takes advantage of the effectiveness of conventional and HPLC hydrophobic interaction, for the isolation of highly purified rat liver 3,4-dihydroxyphenylalanine decarboxylase is described in detail. Some of its structural and functional properties are reported and discussed in comparison with those of pig kidney 3,4-dihydroxyphenylalanine decarboxylase.
Asunto(s)
Descarboxilasas de Aminoácido-L-Aromático/aislamiento & purificación , Dopa-Decarboxilasa/aislamiento & purificación , Hígado/enzimología , Aminoácidos/análisis , Animales , Cromatografía , Cromatografía Líquida de Alta Presión , Dopa-Decarboxilasa/metabolismo , Electroforesis en Gel de Poliacrilamida , Inmunodifusión , Riñón/enzimología , Cinética , Peso Molecular , Fosfato de Piridoxal/análisis , Ratas , Espectrofotometría , Especificidad por Sustrato , PorcinosRESUMEN
Sodium boro[3H]hydride reduction of pig kidney 3,4 dihydroxyphenylalanine decarboxylase followed by complete hydrolysis of the enzyme produced epsilon-[3H]pyridoxyllysine. Degradation of this material to 4'-[3H]pyridoxamine and stereochemical analysis with apoaspartate aminotransferase showed that the re side at C-4' of the coenzyme is exposed to solvent. In order to determine the face exposed to the solvent in the external Schiff's base, attempts to trap reaction intermediates were made by reduction with sodium boro [3H]hydride of the holoenzyme in the presence of various substrates or substrate analogs. In all cases, covalently bound radioactive material was found which was identified as epsilon-N-pyridoxyllysine. These results suggest that the internal Schiff's base is in mobile equilibrium with the external Schiff's base and that sodium borohydride reduction displaces this equilibrium, resulting in complete reduction of the internal Schiff's base.
Asunto(s)
Descarboxilasas de Aminoácido-L-Aromático/metabolismo , Borohidruros/farmacología , Dopa-Decarboxilasa/metabolismo , Riñón/enzimología , Animales , Cromatografía Líquida de Alta Presión , Lisina/análogos & derivados , Lisina/metabolismo , Conformación Molecular , Oxidación-Reducción/efectos de los fármacos , Piridoxal/análogos & derivados , Piridoxal/metabolismo , Fosfato de Piridoxal , Bases de Schiff , PorcinosRESUMEN
Diethyl pyrocarbonate inhibits pig kidney holo-3,4-dihydroxyphenylalanine decarboxylase with a second-order rate constant of 1170 M-1 min-1 at pH 6.8 and 25 degrees C, showing a concomitant increase in absorbance at 242 nm due to formation of carbethoxyhistidyl derivatives. Activity can be restored by hydroxylamine, and the pH curve of inactivation indicates the involvement of a residue with a pKa of 6.03. Complete inactivation of 3,4-dihydroxyphenylalanine decarboxylase requires the modification of 6 histidine residues/mol of enzyme. Statistical analysis of the residual enzyme activity and of the extent of modification shows that, among 6 modifiable residues, only one is critical for activity. Protection exerted by substrate analogues, which bind to the active site of the enzyme, suggests that the modification occurs at or near the active site. The modified inactivated 3,4-dihydroxyphenylalanine decarboxylase still retains most of its ability to bind substrates. Thus, it may be suggested that the inactivation of enzyme by diethyl pyrocarbonate is not due to nonspecific steric or conformational changes which prevent substrate binding. However, the modified enzyme fails to produce at high pH either an enzyme-substrate complex or an enzyme-product complex absorbing at 390 nm. Considerations on this peculiar feature of the modified enzyme consistent with a catalytic role for the modified histidyl residue are discussed. The overall conclusion of this study may be that the modification of only one histidyl residue of 3,4-dihydroxyphenylalanine decarboxylase inactivates the enzyme and that this residue plays an essential role in the mechanism of action of the enzyme.
Asunto(s)
Descarboxilasas de Aminoácido-L-Aromático/metabolismo , Dietil Pirocarbonato/farmacología , Dopa-Decarboxilasa/metabolismo , Formiatos/farmacología , Histidina , Riñón/enzimología , Animales , Inhibidores de Descarboxilasas de Aminoácidos Aromáticos , Sitios de Unión , Cinética , Unión Proteica , Espectrofotometría Ultravioleta , PorcinosRESUMEN
Pig kidney 3,4-dihydroxyphenylalanine (Dopa) decarboxylase is inactivated by the arginine-specific reagent phenylglyoxal. Under these experimental conditions, the reaction follows pseudo-first-order kinetics with a second-order rate constant of 25 m-1 min-1. Holo- and apo-enzyme were inactivated at the same rate. However, inactivation seems to be related to modification of 1 and 2 arginyl residues per mol of holo- and apo-enzyme, respectively. Only one of these two residues was essential to decarboxylase activity of the enzyme. Phenylglyoxal-modified apo-Dopa decarboxylase retained the capacity to bind pyridoxal-P. Neither this reconstituted species nor the phenylglyoxal-modified holoenzyme were able to form Schiff base intermediates with aromatic amino acids in L and D forms. These data together with protection experiments suggest that the susceptible arginine residue in holoenzyme may somehow perturb the substrate binding site. However, unlike in other pyridoxal-P enzymes, this critical arginine in Dopa decarboxylase does not seem to behave as an anionic recognition site for the phosphate group of the coenzyme or the carboxy group of the substrate. It is speculated that this guanidyl group could function in hydrogen bonding of substrate side chain.