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1.
Sci Rep ; 9(1): 2651, 2019 02 25.
Artículo en Inglés | MEDLINE | ID: mdl-30804377

RESUMEN

Dengue is an important mosquito-borne disease and a global public health problem. The disease is caused by dengue virus (DENV), which is a member of the Flaviviridae family and contains a positive single-stranded RNA genome that encodes a single precursor polyprotein that is further cleaved into structural and non-structural proteins. Among these proteins, the non-structural 3 (NS3) protein is very important because it forms a non-covalent complex with the NS2B cofactor, thereby forming the functional viral protease. NS3 also contains a C-terminal ATPase/helicase domain that is essential for RNA replication. Here, we identified 47 NS3-interacting partners using the yeast two-hybrid system. Among those partners, we highlight several proteins involved in host energy metabolism, such as apolipoprotein H, aldolase B, cytochrome C oxidase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). GAPDH directly binds full-length NS3 and its isolated helicase and protease domains. Moreover, we observed an intense colocalization between the GAPDH and NS3 proteins in DENV2-infected Huh7.5.1 cells, in NS3-transfected BHK-21 cells and in hepatic tissue from a fatal dengue case. Taken together, these results suggest that the human GAPDH-DENV NS3 interaction is involved in hepatic metabolic alterations, which may contribute to the appearance of steatosis in dengue-infected patients. The interaction between GAPDH and full-length NS3 or its helicase domain in vitro as well as in NS3-transfected cells resulted in decreased GAPDH glycolytic activity. Reduced GAPDH glycolytic activity may lead to the accumulation of metabolic intermediates, shifting metabolism to alternative, non-glycolytic pathways. This report is the first to identify the interaction of the DENV2 NS3 protein with the GAPDH protein and to demonstrate that this interaction may play an important role in the molecular mechanism that triggers hepatic alterations.

2.
Gene ; 671: 152-160, 2018 Sep 10.
Artículo en Inglés | MEDLINE | ID: mdl-29859286

RESUMEN

High-mobility group B (HMGB) proteins have highly conserved, unique DNA-binding domains, HMG boxes, that can bind non-B-type DNA structures, such as bent, kinked and unwound structures, with high affinity. HMGB proteins also promote DNA bending, looping and unwinding. In this study, we determined the role of the Aedes aegypti single HMG-box domain protein AaHMGB; characterized its structure, spatiotemporal expression levels, subcellular localization, and nucleic acid binding activities; and compared these properties with those of its double-HMG-box counterpart protein, AaHMGB1. Via qRT-PCR, we showed that AaHMGB is expressed at much higher levels than AaHMGB1 throughout mosquito development. In situ hybridization results suggested a role for AaHMGB and AaHMGB1 during embryogenesis. Immunolocalization in the midgut revealed that AaHMGB is exclusively nuclear. Circular dichroism and fluorescence spectroscopy analyses showed that AaHMGB exhibits common features of α-helical structures and is more stably folded than AaHMGB1, likely due to the presence of one or two HMG boxes. Using several DNA substrates or single-stranded RNAs as probes, we observed significant differences between AaHMGB and AaHMGB1 in terms of their binding patterns, activity and/or specificity. Importantly, we showed that the phosphorylation of AaHMGB plays a critical role in its DNA-binding activity. Our study provides additional insight into the roles of single- versus double-HMG-box-containing proteins in nucleic acid interactions for better understanding of mosquito development, physiology and homeostasis.


Asunto(s)
Aedes/crecimiento & desarrollo , Proteínas HMGB/química , Proteínas HMGB/genética , Proteínas HMGB/metabolismo , Aedes/genética , Aedes/metabolismo , Animales , Núcleo Celular/metabolismo , Dicroismo Circular , Regulación del Desarrollo de la Expresión Génica , Proteínas de Insectos/química , Proteínas de Insectos/genética , Proteínas de Insectos/metabolismo , Modelos Moleculares , Fosforilación , Estructura Secundaria de Proteína , Distribución Tisular
3.
Development ; 144(16): 2907-2913, 2017 08 15.
Artículo en Inglés | MEDLINE | ID: mdl-28705899

RESUMEN

The evolutionarily conserved Toll signaling pathway controls innate immunity across phyla and embryonic patterning in insects. In the Drosophila embryo, Toll is required to establish gene expression domains along the dorsal-ventral axis. Pathway activation induces degradation of the IκB inhibitor Cactus, resulting in a ventral-to-dorsal nuclear gradient of the NFκB effector Dorsal. Here, we investigate how cactus modulates Toll signals through its effects on the Dorsal gradient and on Dorsal target genes. Quantitative analysis using a series of loss- and gain-of-function conditions shows that the ventral and lateral aspects of the Dorsal gradient can behave differently with respect to Cactus fluctuations. In lateral and dorsal embryo domains, loss of Cactus allows more Dorsal to translocate to the nucleus. Unexpectedly, cactus loss-of-function alleles decrease Dorsal nuclear localization ventrally, where Toll signals are high. Overexpression analysis suggests that this ability of Cactus to enhance Toll stems from the mobilization of a free Cactus pool induced by the Calpain A protease. These results indicate that Cactus acts to bolster Dorsal activation, in addition to its role as a NFκB inhibitor, ensuring a correct response to Toll signals.


Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Embrión no Mamífero/metabolismo , Fosfoproteínas/metabolismo , Alelos , Animales , Calpaína/genética , Calpaína/metabolismo , Proteínas de Unión al ADN/genética , Drosophila/genética , Proteínas de Drosophila/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosfoproteínas/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo
4.
J Proteomics ; 80: 34-42, 2013 Mar 27.
Artículo en Inglés | MEDLINE | ID: mdl-23159400

RESUMEN

Trypanosoma cruzi proteins with molecular weight between 30 and 34 kDa have shown high reactivity in western blot assays with serum samples from chagasic individuals. However, in-depth analysis of the constituents of these protein fractions has not been performed. This is the first report of an immunoaffinity proteomic approach to identify the immunodominant 30-34 kDa proteins of T. cruzi that could eventually be used for the diagnosis of Chagas disease. We used two different sample preparation protocols for protein digestion coupled to mass spectrometry to identify proteins in the protein fraction. The immunodominant proteins and their respective epitopes were then identified by co-immunoprecipitation and excision-epitope mapping/mass spectrometry, using human sera followed by the prediction and three-dimensional structural modeling of reactive epitopes. The use of different sample preparation methods allowed the identification of a relatively high number of proteins, some of which were only identified after one or multiple sample preparation and digestion protocols. Seven immunodominant proteins were identified by co-immunoprecipitation with purified IgGs from chagasic serum samples. Moreover, six reactive peptide epitopes were detected in four of these proteins by excision-epitope mapping/mass spectrometry. Three-dimensional structural models were obtained for the immunoreactive peptides, which correlated well with the linear B-cell epitope prediction tools.


Asunto(s)
Anticuerpos/química , Enfermedad de Chagas/sangre , Enfermedad de Chagas/parasitología , Epítopos/química , Trypanosoma cruzi/metabolismo , Anticuerpos Antiprotozoarios/inmunología , Antígenos de Protozoos/inmunología , Electroforesis en Gel de Poliacrilamida , Mapeo Epitopo , Epítopos de Linfocito B/química , Humanos , Inmunoglobulina G/química , Inmunoprecipitación , Espectrometría de Masas , Peso Molecular , Péptidos/química , Conformación Proteica , Proteoma , Proteómica/métodos
5.
J Mol Graph Model ; 39: 29-38, 2013 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-23220279

RESUMEN

Zinc finger proteins are widely found in eukaryotes, representing an important class of DNA-binding proteins frequently involved in transcriptional regulation. Zinc finger motifs are composed by two antiparallel ß-strands and one α-helix, stabilized by a zinc ion coordinated by conserved histidine and cysteine residues. In Schistosoma mansoni, these regulatory proteins are known to modulate morphological and physiological changes, having crucial roles in parasite development. A previously described C(2)H(2) zinc finger protein, SmZF1, was shown to be present in cell nuclei of different life stages of S. mansoni and to activate gene transcription in a heterologous system. A high-quality SmZF1 tridimensional structure was generated using comparative modeling. Molecular dynamics simulations of the obtained structure revealed stability of the zinc fingers motifs and high flexibility on the terminals, comparable to the profile observed on the template X-ray structure based on thermal b-factors. Based on the protein tridimensional features and amino acid composition, we were able to characterize four C(2)H(2) zinc finger motifs, the first involved in protein-protein interactions while the three others involved in DNA binding. We defined a consensus DNA binding sequence using three distinct algorithms and further carried out docking calculations, which revealed the interaction of fingers 2-4 with the predicted DNA. A search for S. mansoni genes presenting putative SmZF1 binding sites revealed 415 genes hypothetically under SmZF1 control. Using an automatic annotation and GO assignment approach, we found that the majority of those genes code for proteins involved in developmental processes. Taken together, these results present a consistent base to the structural and functional characterization of SmZF1.


Asunto(s)
Proteínas del Helminto/química , Modelos Moleculares , Factores de Transcripción/química , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Proteínas del Helminto/genética , Proteínas del Helminto/metabolismo , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Datos de Secuencia Molecular , Unión Proteica , Conformación Proteica , Estabilidad Proteica , Factores de Transcripción/genética , Factores de Transcripción/metabolismo
6.
PLoS One ; 7(10): e48170, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-23118944

RESUMEN

Phytomonas species are plant parasites of the family Trypanosomatidae, which are transmitted by phytophagous insects. Some Phytomonas species cause major agricultural damages. The hemipteran Oncopeltus fasciatus is natural and experimental host for several species of trypanosomatids, including Phytomonas spp. The invasion of the insect vectors' salivary glands is one of the most important events for the life cycle of Phytomonas species. In the present study, we show the binding of Phytomonas serpens at the external face of O. fasciatus salivary glands by means of scanning electron microscopy and the in vitro interaction of living parasites with total proteins from the salivary glands in ligand blotting assays. This binding occurs primarily through an interaction with a 130 kDa salivary gland protein. The mass spectrometry of the trypsin-digest of this protein matched 23% of human laminin-5 ß3 chain precursor sequence by 16 digested peptides. A protein sequence search through the transcriptome of O. fasciatus embryo showed a partial sequence with 51% similarity to human laminin ß3 subunit. Anti-human laminin-5 ß3 chain polyclonal antibodies recognized the 130 kDa protein by immunoblotting. The association of parasites with the salivary glands was strongly inhibited by human laminin-5, by the purified 130 kDa insect protein, and by polyclonal antibodies raised against the human laminin-5 ß3 chain. This is the first report demonstrating that a laminin-like molecule from the salivary gland of O. fasciatus acts as a receptor for Phytomonas binding. The results presented in this investigation are important findings that will support further studies that aim at developing new approaches to prevent the transmission of Phytomonas species from insects to plants and vice-versa.


Asunto(s)
Heterópteros/parasitología , Proteínas de Insectos/metabolismo , Insectos Vectores/parasitología , Laminina/metabolismo , Glándulas Salivales/metabolismo , Trypanosomatina/fisiología , Secuencia de Aminoácidos , Animales , Anticuerpos/farmacología , Moléculas de Adhesión Celular/inmunología , Moléculas de Adhesión Celular/farmacología , Interacciones Huésped-Parásitos/efectos de los fármacos , Humanos , Proteínas de Insectos/antagonistas & inhibidores , Proteínas de Insectos/química , Laminina/antagonistas & inhibidores , Laminina/química , Datos de Secuencia Molecular , Enfermedades de las Plantas/parasitología , Unión Proteica , Glándulas Salivales/parasitología , Glándulas Salivales/ultraestructura , Alineación de Secuencia , Homología de Secuencia de Aminoácido
7.
Infect Genet Evol ; 12(7): 1397-404, 2012 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-22579479

RESUMEN

The polymorphism of species A rotavirus genotype G1 strains (RVA-G1) circulating in Rio de Janeiro between 1996 and 2004 was evaluated. The VP7 encoding gene of 36 G1 isolates was sequenced and compared to references strains. The deduced amino acid sequences were used as basis for in silico analysis of the VP7 protein. We observed the circulation of two major G1 lineages and five sublineages during the studied period. Comparison between the VP7 trimeric structures of a rotavirus vaccine strain and Brazilian G1 strains showed mutations at amino acid residues located at the calcium binding site and at several neutralizing antibody recognition sites. Although the rotavirus vaccine program has clearly been successful in Brazil, these results suggest the possibility of the emergence of G1 strains that could evade the immune response elicited by a RVA vaccine and cause a vaccine breakthrough. Consequently, continuous monitoring of rotavirus intragenotypes diversity is critical to understand how it could affect vaccine effectiveness.


Asunto(s)
Diarrea/virología , Polimorfismo Genético , Infecciones por Rotavirus/virología , Rotavirus/genética , Secuencia de Aminoácidos , Antígenos Virales/química , Antígenos Virales/genética , Brasil , Proteínas de la Cápside/química , Proteínas de la Cápside/genética , Preescolar , Simulación por Computador , Diarrea/prevención & control , Evolución Molecular , Humanos , Vacunación Masiva , Modelos Genéticos , Modelos Moleculares , Datos de Secuencia Molecular , Filogenia , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Valores de Referencia , Infecciones por Rotavirus/prevención & control , Análisis de Secuencia de ADN
8.
Braz. j. microbiol ; 42(4): 1547-1559, Oct.-Dec. 2011. graf, tab
Artículo en Inglés | LILACS | ID: lil-614621

RESUMEN

Cylindrospermopsis raciborskii (Woloszynska) Seenaya & Subba Raju is a freshwater cyanobacterium of worldwide distribution. In the North-eastern region of Brazil many eutrophic water reservoirs are characterized by the dominance of C. raciborskii, with recurrent occurrence of blooms. These water bodies have high conductivity due to a high ionic concentration, and are defined as hard (with high values of CaCO3). In this study, we investigated the long-term effect (12 days) of high calcium concentration (8 mM Ca2+) on C. raciborskii (T3 strain) growth, morphology, toxin content, and metabolism. Changes in protein expression profiles were investigated by proteomic analysis using 2D gel electrophoresis and mass spectrometry. A continued exposure to calcium had a pronounced effect on C. raciborskii (T3): it limited growth, decreased thricome length, increased chlorophyll-a content, altered toxin profile (although did not affect PST content, saxitoxin + neosaxitoxin), and inhibited the expression of proteins related to primary metabolism.


Asunto(s)
Toxinas Bacterianas , Cylindrospermopsis/crecimiento & desarrollo , Cylindrospermopsis/aislamiento & purificación , Cylindrospermopsis/metabolismo , Electroforesis en Gel de Agar , Proteoma/análisis , Tanques Elevados/análisis , Microbiología Ambiental , Concentración de Iones de Hidrógeno , Métodos , Técnicas , Muestras de Agua
9.
J Bacteriol ; 193(24): 6929-38, 2011 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-21984792

RESUMEN

The control of Vibrio cholerae phoBR expression by PhoB involves its binding to Pho boxes at -35 (box 1), -60 (box 2), and -80 (box 3) from the putative phoB translation start site. These loci were located in the sense (box 1) and antisense (boxes 2 and 3) strands of the phoBR regulatory region, and PhoB binds to these individual boxes with distinct affinities. Fusions of sequences containing different combinations of these boxes upstream of the lacZ reporter in a plasmid demonstrated that only those carrying boxes 1, 2, and 3, or 1 alone, activated transcription under inorganic phosphate (P(i)) limitation. When a fragment, including only boxes 1 and 2, was fused to lacZ, expression was no longer induced by low P(i), suggesting a repressive role for PhoB~box2 (PhoB bound to box 2) over the transcriptional activity induced by PhoB~box1. The similarity between lacZ expression levels from promoter fragments containing the three boxes or box 1 alone showed that PhoB~box3 eliminated the repressive effect imposed by PhoB~box2 on phoBR transcription. Complementation assays with a phoBR-containing plasmid demonstrated that the 234-bp promoter fragment carrying the three boxes is absolutely required for operon expression in Vibrio cholerae ΔphoBR cells. This was observed under P(i) abundance, when phoBR was expressed at a basal level and, also in low P(i) conditions, when Pho regulon genes were fully expressed. Thus, under P(i) limitation, PhoB exerts dual regulatory functions by binding sequentially distinct Pho boxes to enable the fine-tuning and precise control of phoBR expression in V. cholerae cells.


Asunto(s)
Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Operón , Vibrio cholerae/metabolismo , Proteínas Bacterianas/química , Secuencia de Bases , Sitios de Unión , Datos de Secuencia Molecular , Fosfatos/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , Vibrio cholerae/química , Vibrio cholerae/genética
10.
Microbiology ; 157(Pt 10): 2818-30, 2011 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-21757490

RESUMEN

Crithidia deanei is a trypanosomatid protozoan that harbours a symbiotic bacterium. The partners maintain a mutualistic relationship, thus constituting an excellent model for studying metabolic exchanges between the host and the symbiont, the origin of organelles and cellular evolution. According to molecular analysis, symbionts of different trypanosomatid species share high identity and descend from a common ancestor, a ß-proteobacterium of the genus Bordetella. The endosymbiont is surrounded by two membranes, like Gram-negative bacteria, but its envelope presents special features, since phosphatidylcholine is a major membrane component and the peptidoglycan layer is highly reduced, as described in other obligate intracellular bacteria. Like the process that generated mitochondria and plastids, the endosymbiosis in trypanosomatids depends on pathways that facilitate the intensive metabolic exchanges between the bacterium and the host protozoan. A search of the annotated symbiont genome database identified one sequence with identity to porin-encoding genes of the genus Bordetella. Considering that the symbiont outer membrane has a great accessibility to cytoplasm host factors, it was important to characterize this single porin-like protein using biochemical, molecular, computational and ultrastructural approaches. Antiserum against the recombinant porin-like molecule revealed that it is mainly located in the symbiont envelope. Secondary structure analysis and comparative modelling predicted the protein 3D structure as an 18-domain ß-barrel, which is consistent with porin channels. Electrophysiological measurements showed that the porin displays a slight preference for cations over anions. Taken together, the data presented herein suggest that the C. deanei endosymbiont porin is phylogenetically and structurally similar to those described in Gram-negative bacteria, representing a diffusion channel that might contribute to the exchange of nutrients and metabolic precursors between the symbiont and its host cell.


Asunto(s)
Bacterias/metabolismo , Proteínas Bacterianas/química , Crithidia/microbiología , Porinas/química , Simbiosis , Secuencia de Aminoácidos , Bacterias/clasificación , Bacterias/genética , Bacterias/aislamiento & purificación , Fenómenos Fisiológicos Bacterianos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Crithidia/fisiología , Datos de Secuencia Molecular , Filogenia , Porinas/genética , Porinas/metabolismo , Alineación de Secuencia
11.
Phys Chem Chem Phys ; 13(30): 13709-20, 2011 Aug 14.
Artículo en Inglés | MEDLINE | ID: mdl-21713261

RESUMEN

Cellulases from thermophiles are capable of cleaving sugar chains from cellulose efficiently at high temperatures. The thermo-resistant Cel9A-68 cellulase possesses two important domains: CBM and a catalytic domain connected by a Pro/Ser/Thr rich linker. These domains act cooperatively to allow efficient catalysis. Despite exhaustive efforts to characterize cellulase binding and mechanism of action, a detailed description of the cellulose intrinsic flexibility is still lacking. From computational simulations we studied the temperature influence on the enzyme plasticity, prior to substrate binding. Interestingly, we observed an enhancement of collective motions at high temperatures. These motions are the most representative and describe an intrinsic hinge bending transition. A detailed analysis of these motions revealed an interdomain approximation where D459 and G460, located at the linker region, are the hinge residues. Therefore, we propose a new putative site for mutagenesis targeting the modulation of such conformational transition that may be crucial for activity.


Asunto(s)
Carbohidratos/química , Celulasa/química , Dominio Catalítico , Celulasa/metabolismo , Calor , Simulación de Dinámica Molecular , Unión Proteica , Estructura Terciaria de Proteína , Electricidad Estática , Termodinámica
12.
Braz J Microbiol ; 42(4): 1547-59, 2011 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-24031789

RESUMEN

Cylindrospermopsis raciborskii (Woloszynska) Seenaya & Subba Raju is a freshwater cyanobacterium of worldwide distribution. In the North-eastern region of Brazil many eutrophic water reservoirs are characterized by the dominance of C. raciborskii, with recurrent occurrence of blooms. These water bodies have high conductivity due to a high ionic concentration, and are defined as hard (with high values of CaCO3). In this study, we investigated the long-term effect (12 days) of high calcium concentration (8 mM Ca(2+)) on C. raciborskii (T3 strain) growth, morphology, toxin content, and metabolism. Changes in protein expression profiles were investigated by proteomic analysis using 2D gel electrophoresis and mass spectrometry. A continued exposure to calcium had a pronounced effect on C. raciborskii (T3): it limited growth, decreased thricome length, increased chlorophyll-a content, altered toxin profile (although did not affect PST content, saxitoxin + neosaxitoxin), and inhibited the expression of proteins related to primary metabolism.

13.
Microbiology ; 156(Pt 10): 3011-20, 2010 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-20688821

RESUMEN

The putative phosphoporin encoded by vca1008 of Vibrio cholerae O1 is expressed in vivo during infection and is essential for the intestinal colonization of infant mice. In vitro, its expression is induced under inorganic phosphate (P(i)) limitation in a PhoB/R-dependent manner. In this work we demonstrated that VCA1008 has a strain-specific role in the physiology and pathogenicity of V. cholerae O1. Disruption of vca1008 led to a growth defect, an inability to colonize and a high susceptibility to sodium deoxycholate (DOC; the major bile compound) in the El Tor biotype strain N16961, but did not affect the classical strain O395 in the same way. Furthermore, vca1008 promoter activity was higher in N16961 cells grown under a low P(i) supply in the presence of DOC than in the absence of the detergent. In the P(i)-limited cells, vca1008 was positively regulated by PhoB, but when DOC was added to the medium, it negatively affected the PhoB-mediated activation of the gene, and enhanced vca1008 expression in a ToxR-dependent manner. These findings reveal for the first time a complex strain-specific interplay between ToxR and PhoB/R systems to control porin genes, as well as the influence of DOC on the expression of PhoB- and ToxR-regulated genes and pathogenesis in pandemic strains of V. cholerae.


Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas de Unión al ADN/metabolismo , Ácido Desoxicólico/farmacología , Porinas/metabolismo , Factores de Transcripción/metabolismo , Vibrio cholerae/patogenicidad , Animales , Proteínas Bacterianas/genética , Clonación Molecular , Proteínas de Unión al ADN/genética , Prueba de Complementación Genética , Ratones , Mutación , Fosfatos/metabolismo , Porinas/genética , Regiones Promotoras Genéticas , Factores de Transcripción/genética , Vibrio cholerae/genética , Vibrio cholerae/crecimiento & desarrollo , Vibrio cholerae/metabolismo , Virulencia
14.
Micron ; 41(8): 939-44, 2010 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-20719525

RESUMEN

In many cells, motility is mediated by flagellar beating. Protist parasites are capable of highly coordinated motility which contributes to their pathogenicity in mammalian hosts. Understanding the structural aspects of the flagellum may be important to the identification of novel targets for therapeutic intervention. Our group used atomic force microscopy (AFM) to examine the ultrastructure of Trypanosoma cruzi, obtaining valuable information on the organisation of the flagellar sub-structure. AFM images revealed novel flagellar components such as the presence of periodically-spaced protrusions organised along a flagellar furrow and oriented through the major flagellar axis between the axoneme and the paraflagellar rod. The nature and functional role of this structure are still unknown, although the hypothesis that the furrow might physically separate the two distinct domains of the flagellar membrane that comprise the surface of the axoneme and the paraflagellar rod (PFR) has been raised. To test whether the furrow was present or not only in PFR-bearing flagella, different protists containing or lacking the PFR, were analysed by AFM. Analysis of T. cruzi, Trypanosoma brucei and Herpetomonas megaseliae, which present distinct PFRs, showed similar and equivalent furrows along the main axis of their flagella, whereas Crithidia deanei, Giardia lamblia and Tritrichomonas foetus (in which the PFR is reduced or absent) lacked a furrow. Our results strongly suggest that the flagellar furrow is a characteristic feature of PFR-containing flagella and opens new perspectives for its functional role in the definition of sub-domains on the flagellar membrane.


Asunto(s)
Flagelos/ultraestructura , Trypanosoma cruzi/ultraestructura , Membrana Celular/ultraestructura , Microscopía de Fuerza Atómica
15.
PLoS One ; 5(6): e11407, 2010 Jun 30.
Artículo en Inglés | MEDLINE | ID: mdl-20613980

RESUMEN

BACKGROUND: Trypanosoma cruzi, the agent of Chagas disease, is a protozoan member of the Kinetoplastidae family characterized for the presence of specific and unique structures that are involved in different cell activities. One of them is the paraflagellar rod (PFR), a complex array of filaments connected to the flagellar axoneme. Although the function played by the PFR is not well established, it has been shown that silencing of the synthesis of its major proteins by either knockout of RNAi impairs and/or modifies the flagellar motility. METHODOLOGY/PRINCIPAL FINDINGS: Here, we present results obtained by atomic force microscopy (AFM) and transmission electron microscopy (TEM) of replicas of quick-frozen, freeze-fractured, deep-etched and rotary-replicated cells to obtain detailed information of the PFR structures in regions of the flagellum in straight and in bent state. The images obtained show that the PFR is not a fixed and static structure. The pattern of organization of the PFR filament network differs between regions of the flagellum in a straight state and those in a bent state. Measurements of the distances between the PFR filaments and the filaments that connect the PFR to the axoneme as well as of the angles between the intercrossed filaments supported this idea. CONCLUSIONS/SIGNIFICANCE: Graphic computation based on the information obtained allowed the proposal of an animated model for the PFR structure during flagellar beating and provided a new way of observing PFR filaments during flagellar beating.


Asunto(s)
Flagelos/ultraestructura , Trypanosoma cruzi/fisiología , Animales , Técnica de Fractura por Congelación , Microscopía de Fuerza Atómica , Microscopía Electrónica de Transmisión
16.
Phys Chem Chem Phys ; 12(12): 2850-9, 2010 Mar 28.
Artículo en Inglés | MEDLINE | ID: mdl-20449375

RESUMEN

Protein flexibility is essential for enzymatic function, ligand binding, and protein-protein or protein-nucleic acid interactions. Normal mode analysis has increasingly been shown to be well suited for studying such flexibility, as it can be used to identify favorable structural deformations that correspond to functional motions. However, normal modes are strictly relevant to a single structure, reflecting a particular minimum on a complex energy surface, and are thus susceptible to artifacts. We describe a new theoretical framework for determining "consensus" normal modes from a set of related structures, such as those issuing from a short molecular dynamics simulation. This approach is more robust than standard normal mode analysis, and provides higher collectivity and symmetry properties. In an application to HIV-1 protease, the low-frequency consensus modes describe biologically relevant motions including flap opening and closing that can be used in interpreting structural changes accompanying the binding of widely differing inhibitors.


Asunto(s)
Proteasa del VIH/química , VIH-1 , Simulación de Dinámica Molecular , Humanos , Espectroscopía de Resonancia Magnética , Modelos Moleculares
17.
Microsc Res Tech ; 71(2): 133-9, 2008 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-17992694

RESUMEN

Most advances in atomic force microscopy (AFM) have been accomplished in recent years. Previous attempts to use AFM to analyze the organization of pathogenic protozoa did not significantly contribute with new structural information. In this work, we introduce a new perspective to the study of the ultrastructure of the epimastigote form of Trypanosoma cruzi by AFM. Images were compared with those obtained using field emission scanning electron microscopy of critical point dried cells and transmission electron microscopy of negative stained detergent-extracted and air-dried cells. AFM images of epimastigote forms showed a flagellum furrow separating the axoneme from the paraflagellar rod (PFR) present from the emergence of the flagellar pocket to the tip of the flagellum. At high magnification, a row of periodically organized structures, which probably correspond to the link between the axoneme, the PFR and the flagellar membrane were seen along the furrow. In the origin of the flagellum, two basal bodies were identified. Beyond the basal bodies, small periodically arranged protrusions, positioned at 400 nm from the flagellar basis were seen. This structure was formed by nine substructures distributed around the flagellar circumference and may correspond to the flagellar necklace. Altogether, our results demonstrate the importance of the application of AFM in the structural characterization of the surface components and cytoskeleton on protozoan parasites.


Asunto(s)
Microscopía de Fuerza Atómica , Trypanosoma cruzi/ultraestructura , Animales , Axonema/ultraestructura , Flagelos/ultraestructura , Microscopía Electrónica de Rastreo
18.
Cell Biochem Biophys ; 44(3): 530-8, 2006.
Artículo en Inglés | MEDLINE | ID: mdl-16679541

RESUMEN

The enzyme 2'-aminobiphenyl-2,3-diol-1,2-dioxygenase (CarB), encoded by two genes (carBa and carBb), is an alpha(2)beta(2) heterotetramer that presents meta-cleavage activity toward the hydroxylated aromatic ring in the carbazole degradation pathway from petroleum-degrader bacteria Pseudomonas spp. The 1,082-base pair polymerase chain reaction product corresponding to carBaBb genes from Pseudomonas stutzeri ATCC 31258 was cloned by site-specific recombination and expressed in high levels in Escherichia coli BL21-SI with a histidine-tag and in native form. The CarB activity toward 2,3-dihydroxybiphenyl was similar for these two constructions. The alpha(2)beta(2)-heterotetrameric 3D model of CarB dioxygenase was proposed by homology modeling using the protocatechuate 4,5-dioxygenase (LigAB) structure as template. Accordingly, His12, His53, and Glu230 coordinate the Fe(II) in the catalytic site at the subunit CarBb. The model also indicates that His182 is the catalytic base responsible for deprotonating one of the hydroxyl group of the substrate by a hydrogen bond. The hydrophobic residues Trp257 and Phe258 in the CarB structure substituted the LigAB amino acid residues Ser269 and Asn270. These data could explain why the CarB was active for 2,3-dihydroxybiphenyl and not for protocatechuate.


Asunto(s)
Carbazoles/metabolismo , Dioxigenasas/química , Dioxigenasas/metabolismo , Modelos Moleculares , Pseudomonas stutzeri/enzimología , Secuencia de Aminoácidos , Dominio Catalítico , Clonación Molecular , ADN Bacteriano , Escherichia coli/genética , Escherichia coli/metabolismo , Ácidos Grasos Insaturados/metabolismo , Genes Bacterianos , Hierro/química , Hierro/metabolismo , Mutagénesis Sitio-Dirigida/métodos , Subunidades de Proteína/genética , Pseudomonas stutzeri/genética , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad por Sustrato
19.
Biochim Biophys Acta ; 1764(5): 929-42, 2006 May.
Artículo en Inglés | MEDLINE | ID: mdl-16581319

RESUMEN

Chronic myeloid leukemia is a hematological disorder in which the Ph chromosome is a marker of the disease, detected virtually in all cases. The chimeric transcripts encode a 210-kDa chimeric protein with altered tyrosine kinase activity, responsible for the disease phenotype. In this work, we tried to identify which are the molecular changes common to chronic phase patients, those that represent the chronic phase molecular phenotype. To address this problem we analyzed through a comparative proteomic approach, several CML bone marrow cells protein profile from patients in chronic phase and healthy bone marrow donors. From these results, we identified 31 differentially expressed proteins. Among these proteins, we pointed out c-Myc binding protein 1, 53BP1, Mdm4, OSBP-related protein 3 and Mortalin as putative candidates to BCR-ABL targets in chronic phase. Moreover, we describe for the first time the cytoplasmic protein map from bone marrow cells that helped in the elucidation of the changes we were looking for.


Asunto(s)
Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Proteínas de Neoplasias/biosíntesis , Proteoma/metabolismo , Proteómica , Adulto , Biomarcadores de Tumor/biosíntesis , Femenino , Humanos , Masculino
20.
Proteomics ; 6(5): 1495-511, 2006 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-16447160

RESUMEN

A proteomic analysis of a wild-type and of a phoB mutant showed that Vibrio cholerae expresses genes of two major regulons in response to phosphate starvation. The Pho regulon, expressed by the wild-type, allowed the cells to adapt to the new environment. Induction of the general stress regulon was mainly observed in the phoB mutant as a strategy to resist stress and survive. Some functions of the adaptative and survival responses play roles in the pathogenicity of the bacteria. Among the members of the Pho regulon, we found a porin described as an important factor for the intestinal colonisation. Other functions not obviously related to phosphate metabolism, expressed preferentially by the wild-type cells, have also been implicated in virulence. These findings might explain the lack of virulence of the phoB mutant. The Pho regulon picture of V. cholerae, however, will not be complete until minor members and membrane proteins are identified. Among the phosphate-starvation induced genes we have found 13 hypothetical ones and for some of them functions have been assigned. The majority of the genes identified here have not been described before, thus they could be used to expand the proteomic reference map of V. cholerae El Tor.


Asunto(s)
Adaptación Fisiológica , Proteínas Bacterianas/metabolismo , Fosfatos/metabolismo , Proteoma/análisis , Regulón , Vibrio cholerae O1/fisiología , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Secuencia de Bases , Electroforesis en Gel Bidimensional , Datos de Secuencia Molecular , Operón , Vibrio cholerae O1/genética , Vibrio cholerae O1/patogenicidad
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