RESUMEN
BACKGROUND: An increasing amount of research has led to the positioning of nucleoside diphosphate kinases (NDPK/NDK) as key metabolic enzymes among all organisms. They contribute to the maintenance the intracellular di- and tri- phosphate nucleoside homeostasis, but they also are involved in widely diverse processes such as gene regulation, apoptosis, signal transduction and many other regulatory roles. OBJETIVE: Examine in depth the NDPKs of trypanosomatid parasites responsible for devastating human diseases (e.g., Trypanosoma cruzi, Trypanosoma brucei and Leishmania spp.) which deserve special attention. METHODS: The earliest and latest advances in the topic were explored, focusing on trypanosomatid NDPK features, multifunctionality and suitability as molecular drug targets. FINDINGS: Trypanosomatid NDPKs appear to play functions different from their host counterparts. Evidences indicate that they would perform key roles in the parasite metabolism such as nucleotide homeostasis, drug resistance, DNA damage responses and gene regulation, as well as host-parasite interactions, infection, virulence and immune evasion, placing them as attractive pharmacological targets. MAIN CONCLUSIONS: NDPKs are very interesting multifunctional enzymes. In the present review, the potential of trypanosomatid NDPKs was highlighted, raising awareness of their value not only with respect to parasite biology but also as molecular targets.
Asunto(s)
Nucleósido-Difosfato Quinasa , Trypanosoma brucei brucei , Trypanosoma cruzi , Interacciones Huésped-Parásitos , Humanos , Nucleósido-Difosfato Quinasa/genética , Nucleótidos , Trypanosoma brucei brucei/genéticaRESUMEN
Trypanosoma cruzi is a flagellated protozoan parasite that causes Chagas disease, which represents a serious health problem in the Americas. Nucleoside diphosphate kinases (NDPKs) are key enzymes that are implicated in cellular energy management. TcNDPK1 is the canonical isoform in the T. cruzi parasite. TcNDPK1 has a cytosolic, perinuclear and nuclear distribution. It is also found in non-membrane-bound filaments adjacent to the nucleus. In the present work, X-ray diffraction and in vivo studies of TcNDPK1 are described. The structure reveals a novel, multi-hexameric, left-handed helical oligomer structure. The results of directed mutagenesis studies led to the conclusion that the microscopic TcNDPK1 granules observed in vivo in T. cruzi parasites are made up by the association of TcNDPK1 oligomers. In the absence of experimental data, analysis of the interactions in the X-ray structure of the TcNDPK1 oligomer suggests the probable assembly and disassembly steps: dimerization, assembly of the hexamer as a trimer of dimers, hexamer association to generate the left-handed helical oligomer structure and finally oligomer association in a parallel manner to form the microscopic TcNDPK1 filaments that are observed in vivo in T. cruzi parasites. Oligomer disassembly takes place on the binding of substrate in the active site of TcNDPK1, leading to dissociation of the hexamers. This study constitutes the first report of such a protein arrangement, which has never previously been seen for any protein or NDPK. Further studies are needed to determine its physiological role. However, it may suggest a paradigm for protein storage reflecting the complex mechanism of action of TcNDPK1.
Asunto(s)
Nucleósido-Difosfato Quinasa/química , Trypanosoma cruzi/química , Difracción de Rayos X/métodos , Secuencia de Aminoácidos , Animales , Dominio Catalítico , Clonación Molecular , Modelos Moleculares , Estructura Molecular , Mutagénesis , Nucleósido-Difosfato Quinasa/genética , Proteínas Protozoarias , Trypanosoma cruzi/genéticaRESUMEN
Schistosomiasis, caused by Schistosoma mansoni trematode worm, affects more than 1.5 million people in Brazil. The current treatment consists in the administration of Praziquantel, the only medicine used for treatment for more than 40 years. Some of the limitations of this drug consist in its inactivity against schistosomula and parasite eggs, the appearance of resistant strains and non-prevention against reinfection. Thus, the objective of this study was to evaluate the effect of immunization with recombinant functional enzymes of the purine salvage pathway of S. mansoni, Nucleoside Diphosphate Kinase (NDPK) and Adenylosuccinate Lyase (ADSL), to evaluate the host immune response, as well as the parasite load after vaccination. For this, Balb/c mice were divided into 5 groups: control (uninfected and untreated), non-immunized/infected, NDPK infected, ADSL infected, and NDPK + ADSL infected. Immunized groups received three enzyme dosages, with a 15-day interval between each dose, and after 15 days of the last application the animals were infected with 80 cercariae of S. mansoni. On the 47th day after the infection, fecal eggs were counted and, on the 48th day after the infection, the evaluation of leukocyte response, parasite load, antibody production, cytokines quantification, and histopathological analysis were performed. The results showed that immunizations with NDPK, ADSL or NDPK + ADSL promoted a discreet reduction in eosinophil counts in lavage of peritoneal cavity. All immunized animals showed increased production and secretion of IgG1, IgG2a, and IgE antibodies. Increased production of IL-4 was observed in the group immunized with the combination of both enzymes (NDPK + ADSL). In addition, in all immunized groups there were reductions in egg counts in the liver and intestine, such as reductions in liver granulomas. Thus, we suggest that immunizations with these enzymes could contribute to the reduction of schistosomiasis transmission, besides being important in immunopathogenesis control of the disease.
Asunto(s)
Adenilosuccinato Liasa/inmunología , Antígenos Helmínticos/inmunología , Nucleósido-Difosfato Quinasa/inmunología , Schistosoma mansoni/enzimología , Schistosoma mansoni/inmunología , Esquistosomiasis mansoni/inmunología , Esquistosomiasis mansoni/parasitología , Animales , Antígenos Helmínticos/administración & dosificación , Biomarcadores , Citocinas/sangre , Eosinófilos , Femenino , Inmunización , Esquemas de Inmunización , Recuento de Leucocitos , Hígado/metabolismo , Hígado/parasitología , Hígado/patología , Ratones , Carga de Parásitos , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/inmunología , Esquistosomiasis mansoni/patología , Esquistosomiasis mansoni/prevención & controlRESUMEN
BACKGROUND: NME23/NDPKs are well conserved proteins found in all living organisms. In addition to being nucleoside diphosphate kinases (NDPK), they are multifunctional enzymes involved in different processes such as DNA stability, gene regulation and DNA repair among others. TcNDPK1 is the canonical NDPK isoform present in Trypanosoma cruzi, which has nuclease activity and DNA-binding properties in vitro. OBJECTIVES: In the present study we explored the role of TcNDPK1 in DNA damage responses. METHODS: TcNDPK1 was expressed in mutant bacteria and yeasts and over-expressed in epimastigotes. Mutation frequencies, tolerance to genotoxic agents and activity of DNA repair enzymes were evaluated. FINDINGS: Bacteria decreased about 15-folds the spontaneous mutation rate and yeasts were more resistant to hydrogen peroxide and to UV radiation than controls. Parasites overexpressing TcNDPK1 were able to withstand genotoxic stresses caused by hydrogen peroxide, phleomycin and hidroxyurea. They also presented less genomic damage and augmented levels of poly(ADP)ribose and poly(ADP)ribose polymerase, an enzyme involved in DNA repair. MAIN CONCLUSION: These results strongly suggest a novel function for TcNDPK1; its involvement in the maintenance of parasite's genome integrity.
Asunto(s)
Daño del ADN , Nucleósido-Difosfato Quinasa/metabolismo , Trypanosoma cruzi/enzimología , Reparación del ADN , Nucleósido-Difosfato Quinasa/genética , Poli(ADP-Ribosa) Polimerasas , Trypanosoma cruzi/genéticaRESUMEN
BACKGROUND NME23/NDPKs are well conserved proteins found in all living organisms. In addition to being nucleoside diphosphate kinases (NDPK), they are multifunctional enzymes involved in different processes such as DNA stability, gene regulation and DNA repair among others. TcNDPK1 is the canonical NDPK isoform present in Trypanosoma cruzi, which has nuclease activity and DNA-binding properties in vitro. OBJECTIVES In the present study we explored the role of TcNDPK1 in DNA damage responses. METHODS TcNDPK1 was expressed in mutant bacteria and yeasts and over-expressed in epimastigotes. Mutation frequencies, tolerance to genotoxic agents and activity of DNA repair enzymes were evaluated. FINDINGS Bacteria decreased about 15-folds the spontaneous mutation rate and yeasts were more resistant to hydrogen peroxide and to UV radiation than controls. Parasites overexpressing TcNDPK1 were able to withstand genotoxic stresses caused by hydrogen peroxide, phleomycin and hidroxyurea. They also presented less genomic damage and augmented levels of poly(ADP)ribose and poly(ADP)ribose polymerase, an enzyme involved in DNA repair. MAIN CONCLUSION These results strongly suggest a novel function for TcNDPK1; its involvement in the maintenance of parasite's genome integrity.
Asunto(s)
Trypanosoma cruzi/enzimología , Daño del ADN , Nucleósido-Difosfato Quinasa/metabolismo , Trypanosoma cruzi/genética , Poli(ADP-Ribosa) Polimerasas , Nucleósido-Difosfato Quinasa/genética , Reparación del ADNRESUMEN
Nucleoside diphosphate kinases (NDPKs) are crucial to keep the high triphosphate nucleotide levels in the biological process. The enzymatic mechanism has been extensively described; however, the structural characteristics and kinetic parameters have never been fully determined. In Schistosoma mansoni, NDPK (SmNDPK) is directly involved in the pyrimidine and purine salvage pathways, being essential for nucleotide metabolism. The SmNDPK enzymatic activity is the highest of the known purine metabolisms when compared to the mammalian NDPKs, suggesting the importance of this enzyme in the worm metabolism. Here, we report the recombinant expression of SmNDPK that resulted in 1.7 and 1.9 Å apo-form structure in different space-groups, as well as the 2.1 Å SmNDPK.ADP complex. The binding and kinetic assays reveal the ATP-dependence for enzyme activation. Moreover, in situ hybridization showed that SmNDPK transcripts are found in reproductive organs and in the esophagus gland of adult worms, which can be intrinsically related with the oviposition and digestive processes. These results will help us fully understand the crucial participation of this enzyme in Schistosoma mansoni and its importance for the pathology of the disease.
Asunto(s)
Proteínas del Helminto/química , Proteínas del Helminto/metabolismo , Nucleósido-Difosfato Quinasa/química , Nucleósido-Difosfato Quinasa/metabolismo , Schistosoma mansoni/enzimología , Esquistosomiasis mansoni/parasitología , Adenosina Difosfato/química , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/química , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Animales , Dominio Catalítico , Esófago/química , Esófago/enzimología , Femenino , Tracto Gastrointestinal/química , Tracto Gastrointestinal/enzimología , Proteínas del Helminto/genética , Humanos , Cinética , Masculino , Modelos Moleculares , Nucleósido-Difosfato Quinasa/genética , Schistosoma mansoni/genética , Schistosoma mansoni/metabolismo , Alineación de SecuenciaRESUMEN
Nucleoside diphosphate kinases (NDKs) are key enzymes in the purine-salvage pathway of trypanosomatids and have been associated with the maintenance of host-cell integrity for the benefit of the parasite, being potential targets for rational drug discovery and design. The NDK from Leishmania major (LmNDK) and mutants were expressed and purified to homogeneity. Thermal shift assays were employed to identify potential inhibitors for LmNDK. Calorimetric experiments, site-directed mutagenesis and molecular docking analysis were performed to validate the interaction and to evaluate the structural basis of ligand recognition. Furthermore, the anti-leishmanial activity of the newly identified and validated compound was tested in vitro against different Leishmania species. The molecule SU11652, a Sunitinib analog, was identified as a potential inhibitor for LmNDK and structural studies indicated that this molecule binds to the active site of LmNDK in a similar conformation to nucleotides, mimicking natural substrates. Isothermal titration calorimetry experiments combined with site-directed mutagenesis revealed that the residues H50 and H117, considered essential for catalysis, play an important role in ligand binding. In vitro cell studies showed that SU11652 had similar efficacy to Amphotericin b against some Leishmania species. Together, our results indicate the pyrrole-indolinone SU11652 as a promising scaffold for the rational design of new drugs targeting the enzyme NDK from Leishmania parasites.
Asunto(s)
Antiprotozoarios/farmacología , Indoles/farmacología , Leishmania major/enzimología , Nucleósido-Difosfato Quinasa/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Pirroles/farmacología , Calorimetría , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Leishmania major/efectos de los fármacos , Simulación del Acoplamiento Molecular , Mutagénesis Sitio-Dirigida , Nucleósido-Difosfato Quinasa/genética , Nucleósido-Difosfato Quinasa/metabolismo , Pruebas de Sensibilidad Parasitaria , Inhibidores de Proteínas Quinasas/química , Relación Estructura-ActividadRESUMEN
Nucleoside diphosphate kinases (NDPK) are key enzymes involved in the intracellular nucleotide maintenance in all living organisms, especially in trypanosomatids which are unable to synthesise purines de novo. Four putative NDPK isoforms were identified in the Trypanosoma cruzi Chagas, 1909 genome but only two of them were characterised so far. In this work, we studied a novel isoform from T. cruzi called TcNDPK3. This enzyme presents an atypical N-terminal extension similar to the DM10 domains. In T. cruzi, DM10 sequences targeted other NDPK isoform (TcNDPK2) to the cytoskeleton, but TcNDPK3 was localised in glycosomes despite lacking a typical peroxisomal targeting signal. In addition, TcNDPK3 was found only in the bloodstream trypomastigotes where glycolytic enzymes are very abundant. However, TcNDPK3 mRNA was also detected at lower levels in amastigotes suggesting regulation at protein and mRNA level. Finally, 33 TcNDPK3 gene orthologs were identified in the available kinetoplastid genomes. The characterisation of new glycosomal enzymes provides novel targets for drug development to use in therapies of trypanosomatid associated diseases.
Asunto(s)
Enfermedad de Chagas/parasitología , Metabolismo Energético , Nucleósido-Difosfato Quinasa/genética , Trypanosoma cruzi/enzimología , Isoenzimas , Estadios del Ciclo de Vida , Microcuerpos/enzimología , Filogenia , Dominios Proteicos , Proteínas Protozoarias/genética , Trypanosoma cruzi/genética , Trypanosoma cruzi/fisiologíaRESUMEN
Nucleoside diphosphate kinase (NDK) is a housekeeping enzyme that plays key roles in nucleotide recycling and homeostasis in trypanosomatids. Moreover, it is secreted by the intracellular parasite Leishmania to modulate the host response. These functions make NDK an attractive target for drug design and for studies aiming at a better understanding of the mechanisms mediating host-pathogen interactions. Here, we report the crystal structures of three mutants of the NDK from Leishmania major (LmNDK) that affects the stability of the hexameric biological assembly including P95S, Δ5Ct (lacking the last five residues) and the double mutant P100S/Δ5Ct. Although P95S and Δ5Ct variants conserve the hexameric structure of the wild-type protein, the double mutant becomes a dimer as shown by in solution studies. Free energy calculation of dimer-dimer interfaces and enzymatic assays indicate that P95S, Δ5Ct and P100S/Δ5Ct mutations progressively decrease the hexamer stability and enzyme activity. These results demonstrate that the mutated regions play a role in protein function through stabilizing the quaternary arrangement.
Asunto(s)
Leishmania major/enzimología , Nucleósido-Difosfato Quinasa/genética , Nucleósido-Difosfato Quinasa/ultraestructura , Estructura Cuaternaria de Proteína/genética , Secuencia de Aminoácidos , Cristalografía por Rayos X , Interacciones Huésped-Patógeno , Modelos MolecularesRESUMEN
BACKGROUND: Nucleoside diphosphate kinase (NDK) is a housekeeping enzyme that plays key roles in nucleotide recycling and homeostasis in trypanosomatids. It is also secreted by the intracellular parasite Leishmania to modulate the host response. These functions make NDK an attractive target for drug design and for studies aiming at a better understanding of the mechanisms mediating host-pathogen interactions. RESULTS: We report the crystal structure and biophysical characterization of the NDK from Leishmania braziliensis (LbNDK). The subunit consists of six α-helices along with a core of four ß-strands arranged in a ß2ß3ß1ß4 antiparallel topology order. In contrast to the NDK from L. major, the LbNDK C-terminal extension is partially unfolded. SAXS data showed that LbNDK forms hexamers in solution in the pH range from 7.0 to 4.0, a hydrodynamic behavior conserved in most eukaryotic NDKs. However, DSF assays show that acidification and alkalization decrease the hexamer stability. CONCLUSIONS: Our results support that LbNDK remains hexameric in pH conditions akin to that faced by this enzyme when secreted by Leishmania amastigotes in the parasitophorous vacuoles (pH 4.7 to 5.3). The unusual unfolded conformation of LbNDK C-terminus decreases the surface buried in the trimer interface exposing new regions that might be explored for the development of compounds designed to disturb enzyme oligomerization, which may impair the important nucleotide salvage pathway in these parasites.
Asunto(s)
Leishmania braziliensis/enzimología , Nucleósido-Difosfato Quinasa/química , Proteínas Protozoarias/química , Cristalografía por Rayos X , Estabilidad de Enzimas , Concentración de Iones de Hidrógeno , Leishmania braziliensis/química , Modelos Moleculares , Nucleósido-Difosfato Quinasa/genética , Multimerización de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Desplegamiento Proteico , Proteínas Protozoarias/genética , Dispersión del Ángulo PequeñoRESUMEN
Nucleoside diphosphate kinase (NDK; EC 2.7.4.6) is an enzyme that catalyzes the third phosphorylation of nucleoside diphosphates, leading to nucleoside triphosphates for DNA replication. Expression of the NDK from Litopenaeus vannamei (LvNDK) is known to be regulated under viral infection. Also, as determined by isothermal titration calorimetry, LvNDK binds both purine and pyrimidine deoxynucleoside diphosphates with high binding affinity for dGDP and dADP and with no heat of binding interaction for dCDP [Quintero-Reyes et al. (2012), J. Bioenerg. Biomembr. 44, 325-331]. In order to investigate the differences in selectivity, LvNDK was crystallized as binary complexes with both acceptor (dADP and dCDP) and donor (ADP) phosphate-group nucleoside diphosphate substrates and their structures were determined. The three structures with purine or pyrimidine nucleotide ligands are all hexameric. Also, the binding of deoxy or ribonucleotides is similar, as in the former a water molecule replaces the hydrogen bond made by Lys11 to the 2'-hydroxyl group of the ribose moiety. This allows Lys11 to maintain a catalytically favourable conformation independently of the kind of sugar found in the nucleotide. Because of this, shrimp NDK may phosphorylate nucleotide analogues to inhibit the viral infections that attack this organism.
Asunto(s)
Crustáceos/enzimología , Nucleósido-Difosfato Quinasa/metabolismo , Nucleósidos de Purina/metabolismo , Nucleósidos de Pirimidina/metabolismo , Animales , Cristalización , Cristalografía por Rayos X , Modelos Moleculares , Nucleósido-Difosfato Quinasa/química , Conformación ProteicaRESUMEN
Nucleoside diphosphate kinase (NDPK) is a key enzyme in the control of cellular concentrations of nucleoside triphosphates, and has been shown to play important roles in many cellular processes. In this work we investigated the subcellular localization of the canonical NDPK1 from Trypanosoma cruzi (TcNDPK1), the etiological agent Chagas's Disease, and evaluated the effect of adding an additional weak protein-protein interaction domain from the green fluorescent protein (GFP). Immunofluorescence microscopy revealed that the enzyme from wild-type and TcNDPK1 overexpressing parasites has a cytosolic distribution, being the signal more intense around the nucleus. However, when TcNDPK1 was fused with dimeric GFP it relocalizes in non-membrane bounded granules also located adjacent to the nucleus. In addition, these granular structures were dependent on the quaternary structure of TcNDPK1 and GFP since mutations in residues involved in their oligomerization dramatically decrease the amount of granules. This phenomenon seems to be specific for TcNDPK1 since other cytosolic hexameric enzyme from T. cruzi, such as the NADP(+)-linked glutamate dehydrogenase, was not affected by the fusion with GFP. In addition, in parasites without GFP fusions granules could be observed in a subpopulation of epimastigotes under metacyclogenesis and metacyclic trypomastigotes. Organization into higher protein arrangements appears to be a singular feature of canonical NDPKs; however the physiological function of such structures requires further investigation.
Asunto(s)
Nucleósido-Difosfato Quinasa/metabolismo , Trypanosoma cruzi/enzimología , Animales , Anticuerpos Antiprotozoarios/inmunología , Western Blotting , Gránulos Citoplasmáticos/química , Citosol/enzimología , Digitonina , Regulación Enzimológica de la Expresión Génica , Proteínas Fluorescentes Verdes , Indicadores y Reactivos , Sustancias Luminiscentes , Ratones , Microscopía Fluorescente , Nucleósido-Difosfato Quinasa/química , Nucleósido-Difosfato Quinasa/inmunología , Estructura Cuaternaria de ProteínaRESUMEN
The Ca(2+)-calcineurin pathway affects virulence and morphogenesis in filamentous fungi. Here, we identified 37 CalA-interacting proteins that interact with the catalytic subunit of calcineurin (CalA) in Aspergillus fumigatus, including the nucleoside diphosphate kinase (SwoH). The in vivo interaction between CalA and SwoH was validated by bimolecular fluorescence complementation. A. fumigatus swoH is an essential gene. Therefore, a temperature-sensitive conditional mutant strain with a point mutation in the active site, SwoH(V83F), was constructed, which demonstrated reduced growth and increased sensitivity to elevated temperatures. The SwoH(V83F) mutation did not cause a loss in virulence in the Galleria mellonella infection model. Taken together these results imply that CalA interacts with SwoH.
Asunto(s)
Aspergillus fumigatus/enzimología , Calcineurina/metabolismo , Proteínas Fúngicas/metabolismo , Nucleósido-Difosfato Quinasa/metabolismo , Aspergillus fumigatus/genética , Calcineurina/química , Calcineurina/genética , Calcio/metabolismo , Dominio Catalítico , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Mutación , Nucleósido-Difosfato Quinasa/química , Nucleósido-Difosfato Quinasa/genética , TemperaturaRESUMEN
Nucleoside diphosphate kinases (NDPKs) are enzymes required to preserve the intracellular nucleoside phosphate equilibrium. Trypanosoma cruzi has four putative nucleoside diphosphate kinases with unidentified biological roles and subcellular localization. TcNDPK2 has an N-terminal domain (DM10) with unknown function, which defines a subgroup of NDPKs distributed in a wide variety of organisms. Digitonin extraction demonstrated that this isoform is distributed in detergent soluble and insoluble fractions. Fluorescence microscopy showed that TcNDPK2 alone or fused to GFP was localized in cytoskeleton and flagella. TcNDPK2 was also detected by Western blot in purified polymerized tubulin and flagellar samples. In parasites expressing DM10 fused with GFP, the fluorescence was localized in cytoskeleton and flagellum with an identical pattern to TcNDPK2. This constitutes the first report that could give insights on the role of DM10 domains in NDPKs and also the identification of the first T. cruzi peptide that contains a microtubule association domain.
Asunto(s)
Microtúbulos/metabolismo , Nucleósido-Difosfato Quinasa/metabolismo , Trypanosoma cruzi/enzimología , Western Blotting , Citoesqueleto/química , Flagelos/química , Microscopía Fluorescente , Unión Proteica , Tubulina (Proteína)/químicaRESUMEN
The causative agent of tuberculosis (TB), Mycobacterium tuberculosis, infects one-third of the world population. TB remains the leading cause of mortality due to a single bacterial pathogen. The worldwide increase in incidence of M. tuberculosis has been attributed to the high proliferation rates of multi and extensively drug-resistant strains, and to co-infection with the human immunodeficiency virus. There is thus a continuous requirement for studies on mycobacterial metabolism to identify promising targets for the development of new agents to combat TB. Singular characteristics of this pathogen, such as functional and structural features of enzymes involved in fundamental metabolic pathways, can be evaluated to identify possible targets for drug development. Enzymes involved in the pyrimidine salvage pathway might be attractive targets for rational drug design against TB, since this pathway is vital for all bacterial cells, and is composed of enzymes considerably different from those present in humans. Moreover, the enzymes of the pyrimidine salvage pathway might have an important role in the mycobacterial latent state, since M. tuberculosis has to recycle bases and/or nucleosides to survive in the hostile environment imposed by the host. The present review describes the enzymes of M. tuberculosis pyrimidine salvage pathway as attractive targets for the development of new antimycobacterial agents. Enzyme functional and structural data have been included to provide a broader knowledge on which to base the search for compounds with selective biological activity.
Asunto(s)
Mycobacterium tuberculosis/enzimología , Pirimidinas/metabolismo , Citidina Desaminasa/metabolismo , Mycobacterium tuberculosis/metabolismo , Nucleósido-Difosfato Quinasa/metabolismo , Nucleósido-Fosfato Quinasa/metabolismo , Nucleótido Desaminasas/metabolismo , Pentosiltransferasa/metabolismo , Pirimidina Fosforilasas/metabolismo , Pirofosfatasas/metabolismo , Timidilato Sintasa/metabolismoRESUMEN
The flagellated protozoan parasite Trypanosoma cruzi is the aetiological agent of Chagas disease. Nucleoside diphosphate kinases (NDPKs) are enzymes that are involved in energy management and nucleoside balance in the cell. T. cruzi TcNDPK1, a canonical isoform, was overexpressed in Escherichia coli as an N-terminally poly-His-tagged fusion protein and crystallized. Crystals grew after 72 h in 0.2 M MgCl(2), 20% PEG 3350. Data were collected to 3.5 A resolution using synchrotron X-ray radiation at the National Synchrotron Light Laboratory (Campinas, Brazil). The crystals belonged to the trigonal space group P3, with unit-cell parameters a = b = 127.84, c = 275.49 A. Structure determination is under way and will provide relevant information that may lead to the first step in rational drug design for the treatment of Chagas disease.
Asunto(s)
Nucleósido-Difosfato Quinasa/química , Trypanosoma cruzi/enzimología , Cristalización , Cristalografía por Rayos XRESUMEN
Here, we present the characterization of a trypanosomatid nucleoside diphosphate kinase (TcNDPK1) exhibiting nuclease activity. This is the first identification of a NDPK with this property in trypanosomatid organisms. The recombinant TcNDPK1 protein cleaves not only linear DNA, but also supercoiled plasmid DNA. Additionally, TcNDPK1 is capable of degrading Trypanosoma cruzi genomic DNA. ATP or ADP did not affect the nuclease activity, while the absence of Mg2+ completely inhibits this activity. NDPK and nuclease activities were inhibited at the same temperature, suggesting the presence of related catalytic sites. Furthermore, phenogram analysis showed that TcNDPK1 is close to Drosophila melanogaster and human NDPKs. The unspecific nuclease activity could suggest a participation in cellular processes such as programmed cell death.
Asunto(s)
Nucleósido-Difosfato Quinasa/metabolismo , Trypanosoma cruzi/enzimología , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia Conservada , ADN Protozoario/metabolismo , ADN Superhelicoidal/metabolismo , Calor , Magnesio/metabolismo , Nucleósido-Difosfato Quinasa/química , Nucleósido-Difosfato Quinasa/genética , Filogenia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Factores de Tiempo , Trypanosoma cruzi/clasificación , Trypanosoma cruzi/genéticaRESUMEN
Nucleoside diphosphate kinases (NDPKs) are multifunctional enzymes involved mainly in the conservation of nucleotides and deoxynucleotides at intracellular levels. Here we report the characterization of two NDPKs from the protozoan parasite Trypanosoma cruzi, the etiological agent of Chagas disease. TcNDPK1 and TcNDPK2 were biochemically characterized presenting different kinetic parameters and regulation mechanisms. NDPK activity was mainly detected in soluble fractions according to the digitonin extraction technique; however 20% of the activity remains insoluble at digitonin concentrations up to 5 mg ml(-1). TcNDPK1 is a short enzyme isoform, whereas TcNDPK2 is a long one containing a DM10 motif. In addition, two other putative NDPK genes (TcNPDK3 and TcNDPK4) were detected by data mining at the T. cruzi genome database. The large number and diversity of NDPK isoforms are in agreement with those previously observed for other T. cruzi phosphotransferases, such as adenylate kinases.
Asunto(s)
Nucleósido-Difosfato Quinasa/análisis , Trypanosoma cruzi/enzimología , Animales , Clonación Molecular , Digitonina , Regulación Enzimológica de la Expresión Génica , Indicadores y Reactivos , Isoenzimas/análisis , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/aislamiento & purificación , Ratones , Ratones Endogámicos BALB C , Nucleósido-Difosfato Quinasa/química , Nucleósido-Difosfato Quinasa/genética , Nucleósido-Difosfato Quinasa/aislamiento & purificación , Trypanosoma cruzi/genéticaRESUMEN
The first proteomic analysis of Trypanosoma cruzi resistance to Benznidazole (BZ) is presented. The differential proteome of T. cruzi with selected in vivo resistance to Benznidazole (BZR and Clone27R), its susceptible pairs (BZS and Clone9S), and a pair from a population with Benznidazole- in vitro-induced resistance (17LER) and the susceptible pair 17WTS were analyzed by two-dimensional gel electrophoresis (2-DE) followed by mass spectrometry (MS) for protein identification. Out of 137 spots analyzed through MS, 110 were identified as 56 distinct proteins. Out of the 56 distinct proteins, 36 were present in resistant, 9 in susceptible, and 11 in both phenotypes. Among the proteins identified in resistant samples, 5 were found in Cl 27R and in BZR (calpain-like cysteine peptidase, hypothetical protein conserved 26 kDa, putative peptidase, peroxiredoxin and tyrosine amino transferase) and 4 in Cl 27R and 17LER (cyclophilin A, glutamate dehydrogenase, iron superoxide dismutase and nucleoside diphosphate kinase). As for the proteins identified in Benznidazole-susceptible samples, PGF-2a was found in BZS and 17WTS. A functional category analysis showed that the proteins involved with transcription and protein destination were overexpressed for the Benznidazole-resistant phenotype. Thus, the present study provides large-scale, protein-related information for investigation of the mechanism of T. cruzi resistance to Benznidazole.
Asunto(s)
Resistencia a Medicamentos , Nitroimidazoles/farmacología , Proteoma/metabolismo , Proteínas Protozoarias/metabolismo , Trypanosoma cruzi/metabolismo , Animales , Ciclofilina A/análisis , Ciclofilina A/metabolismo , Cisteína Endopeptidasas/análisis , Cisteína Endopeptidasas/metabolismo , Electroforesis en Gel Bidimensional , Glutamato Deshidrogenasa/análisis , Glutamato Deshidrogenasa/metabolismo , Hidroxiprostaglandina Deshidrogenasas/análisis , Hidroxiprostaglandina Deshidrogenasas/metabolismo , Nucleósido-Difosfato Quinasa/análisis , Nucleósido-Difosfato Quinasa/metabolismo , Péptido Hidrolasas/análisis , Péptido Hidrolasas/metabolismo , Peroxirredoxinas/análisis , Peroxirredoxinas/metabolismo , Proteoma/análisis , Proteínas Protozoarias/análisis , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Superóxido Dismutasa/análisis , Superóxido Dismutasa/metabolismo , Espectrometría de Masas en Tándem , Tripanocidas/farmacología , Trypanosoma cruzi/efectos de los fármacos , Tirosina Transaminasa/análisis , Tirosina Transaminasa/metabolismoRESUMEN
The NM23 protein was shown to be associated with metastasis suppression in human malignancies with various tissue origins. However, its association with the metastatic phenotype of salivary gland neoplasms (SGN) remains unknown. To evaluate the role of NM23 in SGN, the expression patterns of NM23 in the following were compared: benign (pleomorphic adenoma) vs malignant (adenoid cystic carcinoma and mucoepidermoid carcinoma) SGN, and primary malignancies with/without evidence of metastasis vs their metastatic implants (MI). The lesions were studied immunohistochemically. NM23 protein was found in the cytoplasm of 75% of benign SGN, 73.3% of primary SGN malignancies with no evidence of metastasis, 86.6% of primary SGN malignancies with evidence of metastasis, and 60% of MI. There was no statistically significant difference in the frequency of NM23-positive cells between benign and primary malignant tumors (p = 0.79), nor between primary malignancies with/without evidence of metastasis and MI (p = 0.51). However, nuclear NM23 protein was restricted to primary SGN malignancies with evidence of metastasis and MI. The presence of nuclear NM23 protein may be a good marker for predicting the metastatic potential of SGN malignancies.