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1.
J Proteome Res ; 10(4): 1698-718, 2011 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-21184613

RESUMEN

Many drugs with very different affinity to a large number of receptors are described. Thus, in this work, we selected drug-target pairs (DTPs/nDTPs) of drugs with high affinity/nonaffinity for different targets. Quantitative structure-activity relationship (QSAR) models become a very useful tool in this context because they substantially reduce time and resource-consuming experiments. Unfortunately, most QSAR models predict activity against only one protein target and/or they have not been implemented on a public Web server yet, freely available online to the scientific community. To solve this problem, we developed a multitarget QSAR (mt-QSAR) classifier combining the MARCH-INSIDE software for the calculation of the structural parameters of drug and target with the linear discriminant analysis (LDA) method in order to seek the best model. The accuracy of the best LDA model was 94.4% (3,859/4,086 cases) for training and 94.9% (1,909/2,012 cases) for the external validation series. In addition, we implemented the model into the Web portal Bio-AIMS as an online server entitled MARCH-INSIDE Nested Drug-Bank Exploration & Screening Tool (MIND-BEST), located at http://miaja.tic.udc.es/Bio-AIMS/MIND-BEST.php . This online tool is based on PHP/HTML/Python and MARCH-INSIDE routines. Finally, we illustrated two practical uses of this server with two different experiments. In experiment 1, we report for the first time a MIND-BEST prediction, synthesis, characterization, and MAO-A and MAO-B pharmacological assay of eight rasagiline derivatives, promising for anti-Parkinson drug design. In experiment 2, we report sampling, parasite culture, sample preparation, 2-DE, MALDI-TOF and -TOF/TOF MS, MASCOT search, 3D structure modeling with LOMETS, and MIND-BEST prediction for different peptides as new protein of the found in the proteome of the bird parasite Trichomonas gallinae, which is promising for antiparasite drug targets discovery.


Asunto(s)
Diseño de Fármacos , Evaluación Preclínica de Medicamentos/métodos , Glucosafosfato Deshidrogenasa/metabolismo , Internet , Inhibidores de la Monoaminooxidasa/química , Monoaminooxidasa/metabolismo , Proteínas Protozoarias/metabolismo , Trichomonas , Animales , Antiparasitarios/química , Antiparasitarios/farmacología , Columbidae/microbiología , Descubrimiento de Drogas , Glucosafosfato Deshidrogenasa/química , Indanos/síntesis química , Indanos/química , Modelos Moleculares , Modelos Teóricos , Datos de Secuencia Molecular , Estructura Molecular , Monoaminooxidasa/química , Inhibidores de la Monoaminooxidasa/síntesis química , Péptidos/química , Conformación Proteica , Proteínas Protozoarias/química , Relación Estructura-Actividad Cuantitativa , Trichomonas/química , Trichomonas/efectos de los fármacos , Trichomonas/enzimología
2.
Mol Microbiol ; 69(1): 94-109, 2008 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-18433447

RESUMEN

Frataxin is a small conserved mitochondrial protein; in humans, mutations affecting frataxin expression or function result in Friedreich's ataxia. Much of the current understanding of frataxin function comes from informative studies with yeast models, but considerable debates remain with regard to the primary functions of this ubiquitous protein. We exploit the tractable reverse genetics of Trypanosoma brucei in order to specifically consider the importance of frataxin in an early branching lineage. Using inducible RNAi, we show that frataxin is essential in T. brucei and that its loss results in reduced activity of the marker Fe-S cluster-containing enzyme aconitase in both the mitochondrion and cytosol. Activities of mitochondrial succinate dehydrogenase and fumarase also decreased, but the concentration of reactive oxygen species increased. Trypanosomes lacking frataxin also exhibited a low mitochondrial membrane potential and reduced oxygen consumption. Crucially, however, iron did not accumulate in frataxin-depleted mitochondria, and as T. brucei frataxin does not form large complexes, it suggests that it plays no role in iron storage. Interestingly, RNAi phenotypes were ameliorated by expression of frataxin homologues from hydrogenosomes of another divergent protist Trichomonas vaginalis. Collectively, the data suggest trypanosome frataxin functions primarily only in Fe-S cluster biogenesis and protection from reactive oxygen species.


Asunto(s)
Evolución Molecular , Expresión Génica , Proteínas de Unión a Hierro/metabolismo , Proteínas Mitocondriales/metabolismo , Trichomonas/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Células Eucariotas/clasificación , Células Eucariotas/fisiología , Humanos , Proteínas de Unión a Hierro/química , Proteínas de Unión a Hierro/genética , Proteínas Hierro-Azufre/química , Proteínas Hierro-Azufre/genética , Proteínas Hierro-Azufre/metabolismo , Proteínas Mitocondriales/química , Proteínas Mitocondriales/genética , Datos de Secuencia Molecular , Fenotipo , Filogenia , Células Procariotas/clasificación , Células Procariotas/fisiología , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Interferencia de ARN , Alineación de Secuencia , Trichomonas/química , Trichomonas/clasificación , Trichomonas/genética , Frataxina
3.
Eukaryot Cell ; 5(4): 784-7, 2006 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-16607026

RESUMEN

Hydrogenosomes are found in organisms that lack typical mitochondria. Cardiolipin is a phospholipid located exclusively in bacterial membranes and the inner membrane of mitochondria. Here we show, by cell fractionation, thin-layer chromatography, high-pressure liquid chromatography, and matrix-assisted laser desorption ionization-time of flight mass spectrometry that hydrogenosomes of Tritrichomonas foetus, a cattle vaginal parasite, contain cardiolipin, which is strong evidence for its endosymbiotic origin.


Asunto(s)
Cardiolipinas/análisis , Orgánulos/química , Simbiosis , Trichomonas/química , Animales , Evolución Biológica , Cromatografía Líquida de Alta Presión , Mitocondrias/química , Mitocondrias/metabolismo , Orgánulos/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción
4.
Oral Microbiol Immunol ; 15(6): 355-9, 2000 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-11154431

RESUMEN

An oral protist Trichomonas tenax ATCC 30207 was investigated for the ability to lyse erythrocytes of sheep, rabbits, horses and humans. Five fractions, including intact cells, culture supernatant, culture filtrate, cell debris and lipid-enriched fractions, were prepared from the protozoan cells, and their hemolytic activities were assayed under various conditions. All the samples except culture supernatant had hemolytic activities, which were due to two different kinds of hemolysins. One hemolysin was protein-like and mainly found in cell-free fractions: culture supernatant and culture filtrate. It was heat-labile and inhibited by various cysteine-proteinase inhibitors. The other hemolysin was lipid-like and found in cell-associated fractions: intact cells, cell-debris and lipid-enriched fractions. It was heat-stable, organic solvent-tolerant and unaffected by various proteinase inhibitors and stimulators. These results suggested that T. tenax ATCC 30207 possessed two distinct hemolysins, protein and lipid.


Asunto(s)
Proteínas Hemolisinas/química , Boca/parasitología , Trichomonas/química , Animales , Fraccionamiento Celular , Hemólisis , Caballos , Humanos , Lípidos/química , Proteínas Protozoarias/química , Conejos , Ovinos
5.
Proc Natl Acad Sci U S A ; 93(18): 9651-6, 1996 Sep 03.
Artículo en Inglés | MEDLINE | ID: mdl-8790385

RESUMEN

Trichomonads are among the earliest eukaryotes to diverge from the main line of eukaryotic descent. Keeping with their ancient nature, these facultative anaerobic protists lack two "hallmark" organelles found in most eukaryotes: mitochondria and peroxisomes. Trichomonads do, however, contain an unusual organelle involved in carbohydrate metabolism called the hydrogenosome. Like mitochondria, hydrogenosomes are double-membrane bounded organelles that produce ATP using pyruvate as the primary substrate. Hydrogenosomes are, however, markedly different from mitochondria as they lack DNA, cytochromes and the citric acid cycle. Instead, they contain enzymes typically found in anaerobic bacteria and are capable of producing molecular hydrogen. We show here that hydrogenosomes contain heat shock proteins, Hsp70, Hsp60, and Hsp10, with signature sequences that are conserved only in mitochondrial and alpha-Gram-negative purple bacterial Hsps. Biochemical analysis of hydrogenosomal Hsp60 shows that the mature protein isolated from the organelle lacks a short, N-terminal sequence, similar to that observed for most nuclear-encoded mitochondrial matrix proteins. Moreover, phylogenetic analyses of hydrogenosomal Hsp70, Hsp60, and Hsp10 show that these proteins branch within a monophyletic group composed exclusively of mitochondrial homologues. These data establish that mitochondria and hydrogenosomes have a common eubacterial ancestor and imply that the earliest-branching eukaryotes contained the endosymbiont that gave rise to mitochondria in higher eukaryotes.


Asunto(s)
Evolución Biológica , Mitocondrias , Orgánulos , Trichomonas , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Chaperonina 10/análisis , Chaperonina 60/análisis , Secuencia Conservada , Proteínas HSP70 de Choque Térmico/análisis , Hidrógeno , Mitocondrias/química , Datos de Secuencia Molecular , Orgánulos/química , Homología de Secuencia de Aminoácido , Trichomonas/química
6.
Biochem J ; 299 ( Pt 2): 341-6, 1994 Apr 15.
Artículo en Inglés | MEDLINE | ID: mdl-8172592

RESUMEN

New sialic acid-specific lectin has been isolated from culture supernatant of the protozoan Tritrichomonas mobilensis. It was purified by adsorption by erythrocytes or bovine submaxillary gland mucin (BSM)-Sepharose affinity chromatography. The T. mobilensis lectin (TML) does not require bivalent cations for activity and agglutinates all human erythrocytes. The lectin forms multimeric complexes with molecular mass 556 and 491 kDa as determined by size-exclusion chromatography. SDS/PAGE under reducing conditions disclosed a large band of 343 kDa and three bands of 246, 265 and 286 kDa which, after denaturation with urea, were split into three subunits of 56, 61 and 66 kDa; under non-reducing conditions there were two bands, of 360 and 260 kDa. Western blots performed with anti-TML monoclonal antibodies revealed bands identical with those in the silver-stained gels, suggesting homogeneity of the BSM -Sepharose-purified lectin. TML is a highly glycosylated protein with approx. 8% of N-linked glycosides found by protein-N-glycanase F treatment; the total amount of saccharides revealed by chemical deglycosylation was 20%. Haemagglutination-inhibition studies documented exclusive specificity for sialic acid (NeuAc). Both (alpha 2-->6)- and (alpha 2-->3)-linked and free NeuAc were eight times more potent inhibitors than N-glycolylneuraminic acid. The lectin does not require O-acetyl groups on NeuAc for recognition. A spectrum of mono- and oligo-saccharides other than sialic acid had no inhibitory effect at 200 mM. Anti-TML monoclonal antibodies strongly inhibited the lectin activity. TML was stable at temperatures below 4 degrees C and lyophilized with 3% (w/w) glycerol.


Asunto(s)
Lectinas/química , Lectinas/aislamiento & purificación , Ácidos Siálicos , Trichomonas/química , Adsorción , Aminoácidos/análisis , Animales , Sitios de Unión , Secuencia de Carbohidratos , Carbohidratos/análisis , Bovinos , Cromatografía de Afinidad , Cromatografía en Gel , Disacáridos , Electroforesis en Gel de Poliacrilamida , Eritrocitos/inmunología , Eritrocitos/fisiología , Gangliósidos , Hemaglutinación , Pruebas de Inhibición de Hemaglutinación , Humanos , Datos de Secuencia Molecular , Peso Molecular , Mucinas , Ácido N-Acetilneuramínico , Conformación Proteica
7.
Arch Biochem Biophys ; 309(2): 273-80, 1994 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-8135538

RESUMEN

The lipid moiety of the lipophosphoglycan (LPG)-like glycoconjugates of Trichomonas vaginalis and Trichomonas foetus, parasites of the urogenital tract of human and cattle, respectively, has been isolated and characterized by a combination of enzymatic and chemical degradation, chromatography, and mass spectrometry. The carbohydrate composition of the glycan inositol lipid core is also reported. The glycan inositol core of trichomonad glycoconjugates is unique in having more than one GlcN and is significantly larger than any other glycan core reported so far. T. vaginalis glycoconjugate binds strongly to the lectin RCA-I, which suggest that the macromolecule possesses terminal beta 1,4-linked galactosyl residues. The binding of T. foetus glycoconjugate to the lectin UEA-I suggests the presence of terminal alpha 1,2-linked fucose. Acid hydrolysis of deaminated and reduced LPG products yields a [3H]anhydromannitol-containing product, indicating the presence of unacetylated glucosamine in the trichomonad LPGs. Reductive radiomethylation has been applied to label free amino groups in the hexosamine or other free amine-containing residues of the trichomonad glycoconjugates. Treatment of the LPGs with phosphatidylinositol-specific phospholipase C from Bacillus thuringiensis liberates a ceramide substituent. Treatment of LPGs with nitrous acid releases a phospholipid moiety containing myo-inositol and ceramide, implying that the LPGs are anchored in the membrane via an inositol-phosphate-ceramide. Structural characterization of the ceramide by gas-liquid chromatography (GC) and GC-mass spectrometry indicated the presence of the major long-chain base sphinganine (d 18: 0 dihydrosphingosine) and a C 16:0 N-acyl group. Lipophosphoglycans from both parasites contain ceramide as their only lipid moiety. These results suggest that T. vaginalis and T. foetus anchor their LPG-like glycoconjugates on the cell surface via inositol-phosphoceramide and also the glycan inositol core of the macromolecule appears to be unique in nature.


Asunto(s)
Glicoconjugados/análisis , Glicoesfingolípidos/análisis , Lípidos/análisis , Trichomonas vaginalis/química , Trichomonas/química , Animales , Carbohidratos/análisis , Cromatografía , Cromatografía de Afinidad , Cromatografía de Gases , Cromatografía de Gases y Espectrometría de Masas , Inositol/análisis , Lectinas , Manitol/análisis , Esfingosina/análisis
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