RESUMEN
RATIONALE: Using a proteomic-based approach we have investigated possible altered expression of a range of cerebral spinal fluid (CSF) proteins following exposure to the neurotoxicant carbonyl sulfide (COS). CSF is ideal for the investigation of markers of brain injury or disease since it is secreted from several central nervous system structures and changes in the CSF composition may reflect brain insult and many pathological processes. METHODS: Animals were placed in exposure chambers and were exposed to 0 ppm or 500 ppm COS for 1, 2 or 3 days, 6 h per day. After the last inhalation exposure, 50-70 µL CSF sample was obtained by lumbar puncture. CSF samples were analyzed by electrospray ionization mass spectrometry (ESI-MS) on either a Premier quadrupole time-of-flight (QTOF) or an Agilent 6340 ion trap and by matrix-assisted laser desorption/ionization (MALDI)-MS on a 4800 MALDI-TOF/TOF analyzer. RESULTS: The dynamic range of abundance of the identified proteins spanned over more than three orders of magnitude. The four most abundant proteins identified (albumin, cystatin C, serotransferrin, transthyretin) are major proteins that are present in both CSF and blood at high levels but the fifth most abundant protein identified (prostaglandin H2D isomerase) is the second most abundant protein in human CSF and is secreted and synthesized in the rat central nervous system. No significant differences were observed between COS-treated CSF samples and the control CSF samples because of blood contamination. CONCLUSIONS: Quantitative MS protein analyses of rat CSF is limited by the low sample volumes that can practicably be obtained from rats and the low protein concentrations in rat CSF. Results of this work suggest a clear need for CSF collection that would minimize blood contamination. Published in 2014. This article is a U.S. Government work and is in the public domain in the USA.
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Síndromes de Neurotoxicidad/líquido cefalorraquídeo , Síndromes de Neurotoxicidad/etiología , Proteoma/análisis , Espectrometría de Masa por Ionización de Electrospray/métodos , Óxidos de Azufre/toxicidad , Animales , Proteínas del Líquido Cefalorraquídeo/análisis , Exposición por Inhalación , Masculino , Análisis de Componente Principal , Proteoma/química , Proteómica , RatasRESUMEN
BACKGROUND: Recent immunological data demonstrated that dendritic cells preferentially recognize advanced glycation end product (AGE)-modified proteins, upregulate expression of the receptor for AGE (RAGE), and consequently bias the immune response toward allergy. METHODS: Peanut extract was characterized by mass spectrometry (MS) to elucidate the specific residues and specific AGE modifications found in raw and roasted peanuts and on rAra h 1 that was artificially glycated by incubation with glucose or xylose. The binding of the RAGE-V1C1 domain to peanut allergens was assessed by PAGE and Western analysis with anti-Ara h 1, 2, and 3 antibodies. IgE binding to rAra h 1 was also assessed using the same methods. RESULTS: AGE modifications were found on Ara h 1 and Ara h 3 in both raw and roasted peanut extract. No AGE modifications were found on Ara h 2. Mass spectrometry and Western blot analysis demonstrated that RAGE binds selectively to Ara h 1 and Ara h 3 derived from peanut extract, whereas the analysis failed to demonstrate Ara h 2 binding to RAGE. rAra h 1 with no AGE modifications did not bind RAGE; however, after AGE modification with xylose, rAra h 1 bound to RAGE. CONCLUSIONS: AGE modifications to Ara h 1 and Ara h 3 can be found in both raw and roasted peanuts. Receptor for AGE was demonstrated to selectively interact with AGE-modified rAra h 1. If sensitization to peanut allergens occurs in dendritic cells via RAGE interactions, these cells are likely interacting with modified Ara h 1 and Ara h 3, but not Ara h 2.
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Alérgenos/química , Arachis/química , Productos Finales de Glicación Avanzada/metabolismo , Reacción de Maillard , Alérgenos/inmunología , Secuencia de Aminoácidos , Antígenos de Plantas/química , Antígenos de Plantas/inmunología , Antígenos de Plantas/metabolismo , Arachis/inmunología , Productos Finales de Glicación Avanzada/química , Productos Finales de Glicación Avanzada/inmunología , Glicoproteínas/inmunología , Glicoproteínas/metabolismo , Glicosilación , Humanos , Inmunoglobulina E/inmunología , Inmunoglobulina E/metabolismo , Proteínas de la Membrana , Modelos Moleculares , Proteínas de Plantas/química , Proteínas de Plantas/inmunología , Proteínas de Plantas/metabolismo , Unión Proteica , Conformación Proteica , Espectrometría de Masas en TándemRESUMEN
Liver tumors from a previous National Toxicology Program study were examined using global gene expression and mutation analysis to define the mechanisms of carcinogenesis in mice exposed to oxazepam. Five hepatocellular adenomas and 5 hepatocellular carcinomas from male B6C3F1 mice exposed to 5000 ppm oxazepam and 6 histologically normal liver samples from control animals were examined. One of the major findings in the study was upregulation of the Wnt/ß-catenin signaling pathway. Genes that activate ß-catenin, such as Sox4, were upregulated, whereas genes that inhibit Wnt signaling, such as APC and Crebbp, were downregulated. In addition, liver tumors from oxazepam-exposed mice displayed ß-catenin mutations and increased protein expression of glutamine synthetase, a downstream target in the Wnt signaling pathway. Another important finding in this study was the altered expression of oxidative stress-related genes, specifically increased expression of cytochrome p450 genes, including Cyp1a2 and Cyp2b10, and decreased expression of genes that protect against oxidative stress, such as Sod2 and Cat. Increased oxidative stress was confirmed by measuring isoprostane expression using mass spectrometry. Furthermore, global gene expression identified altered expression of genes that are associated with epigenetic mechanisms of cancer. There was decreased expression of genes that are hypermethylated in human liver cancer, including tumor suppressors APC and Pten. Oxazepam-induced tumors also exhibited decreased expression of genes involved in DNA methylation (Crebbp, Dnmt3b) and histone modification (Sirt1). These data suggest that formation of hepatocellular adenomas and carcinomas in oxazepam-exposed mice involves alteration of the Wnt signaling pathway, oxidative stress, and potential epigenetic alterations.
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Carcinógenos/toxicidad , Epigénesis Genética/fisiología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Neoplasias Hepáticas Experimentales/inducido químicamente , Oxazepam/toxicidad , Animales , Femenino , Genoma , Neoplasias Hepáticas Experimentales/genética , Neoplasias Hepáticas Experimentales/patología , Masculino , Ratones , Ratones Endogámicos , Mutación , Estrés Oxidativo , Reacción en Cadena de la Polimerasa/métodos , Análisis por Matrices de Proteínas , Reproducibilidad de los Resultados , Transducción de Señal , Proteínas Wnt/genética , Proteínas Wnt/metabolismoRESUMEN
Previous studies have shown that short-wavelength blue visible light induces retinal injury and may be a risk factor for age related macular degeneration. A2E is a blue light absorbing retinal chromophore that accumulates with age. Our previous in vitro studies have determined that, although A2E itself has a low phototoxic efficiency, the oxidation products of A2E that are formed in the presence of visible light can contribute to observed retinal pigment epithelial photodamage. The purpose of this study was to investigate the effects of blue light on retinal phototoxicity and its relationship to A2E, oxidized A2E and its isomers. Sprague-Dawley albino rats were dark adapted for 24 h. Control rats remained in the dark while experimental rats were exposed to blue light (λ = 450 nm, 3.1 mW cm(-2)) for 6 h. Isolated retinas were homogenized in Folch extraction mixture and then in chloroform. The dried extracts were reconstituted and divided for determination of organic soluble compound. Esters of fatty acids were determined with GC-MS, A2E and other chromophores using HPLC, and A2E oxidation products with LC-MS. Exposure of rat eyes to blue light did not significantly change the fatty acid composition of the retina. The A2E concentration (normalized to fatty acid content) in blue light exposed animals was found to be lower than the A2E concentration in control rats. The concentrations of all-trans-retinal-ethanolamine adduct and iso-A2E a precursor and an isomer of A2E respectively, were also lower after blue-light exposure than in the retinas of rats housed in the dark. On the other hand, the amount of oxidized forms of A2E was higher in the animals exposed to blue light. We conclude that in the rat eye, blue-light exposure promotes oxidation of A2E and iso-A2E to the products that are toxic to retinal tissue. Although high concentrations of A2E may be cytotoxic to the retina, the phototoxicity associated with blue light damage to the retina is in part a result of the formation of toxic A2E oxides. This effect may partially explain the association between blue light induced retinal injury and macular degeneration.
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Modelos Animales de Enfermedad , Luz/efectos adversos , Degeneración Macular/etiología , Compuestos de Piridinio/efectos adversos , Retinoides/efectos adversos , Animales , Colesterol/metabolismo , Ácidos Grasos/metabolismo , Degeneración Macular/patología , Masculino , Oxidación-Reducción , Compuestos de Piridinio/metabolismo , Ratas , Ratas Sprague-Dawley , Retina/patología , Retina/efectos de la radiación , Retinoides/metabolismoRESUMEN
BACKGROUND: Dietary (n-6)-polyunsaturated fatty acids influence cancer development, but the mechanisms have not been well characterised in gastric carcinoma. METHODS: We used two in vivo models to investigate the effects of these common dietary components on tumour metastasis. In a model of experimental metastasis, immunocompromised mice were fed diets containing linoleic acid (LA) at 2% (LLA), 8% (HLA) or 12% (VHLA) by weight and inoculated intraperitoneally (i.p.) with human gastric carcinoma cells (OCUM-2MD3). To model spontaneous metastasis, OCUM-2MD3 tumours were grafted onto the stomach walls of mice fed with the different diets. In in vitro assays, we investigated invasion and ERK phosphorylation of OCUM-2MD3 cells in the presence or absence of LA. Finally, we tested whether a cyclooxygenase (COX) inhibitor, indomethacin, could block peritoneal metastasis in vivo. RESULTS: Both the HLA and VHLA groups showed increased incidence of tumour nodules (LA: 53%; HLA: 89%; VHLA: 100%; P<0.03); the VHLA group also displayed increased numbers of tumour nodules and higher total volume relative to LLA group in experimental metastasis model. Both liver invasion (78%) and metastasis to the peritoneal cavity (67%) were more frequent in VHLA group compared with the LLA group (22% and 11%, respectively; P<0.03) in spontaneous metastasis model. We also found that the invasive ability of these cells is greatly enhanced when exposed to LA in vitro. Linoleic acid also increased invasion of other scirrhous gastric carcinoma cells, OCUM-12, NUGC3 and MKN-45. Linoleic acid effect on OCUM-2MD3 cells seems to be dependent on phosphorylation of ERK. The data suggest that invasion and phosphorylation of ERK were dependent on COX. Indomethacin decreased the number of tumours and total tumour volume in both LLA and VHLA groups. Finally, COX-1, which is known to be an important enzyme in the generation of bioactive metabolites from dietary fatty acids, appears to be responsible for the increased metastatic behaviour of OCUM-2MD3 cells in the mouse model. CONCLUSION: Dietary LA stimulates invasion and peritoneal metastasis of gastric carcinoma cells through COX-catalysed metabolism and activation of ERK, steps that compose pathway potentially amenable to therapeutic intervention.
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Carcinoma/patología , Movimiento Celular/efectos de los fármacos , Grasas Insaturadas en la Dieta/farmacología , Ácido Linoleico/efectos adversos , Ácido Linoleico/farmacología , Neoplasias Gástricas/patología , Animales , Grasas Insaturadas en la Dieta/efectos adversos , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Ratones , Ratones Desnudos , Invasividad Neoplásica , Metástasis de la Neoplasia , Trasplante de Neoplasias , Trasplante Heterólogo , Células Tumorales CultivadasRESUMEN
Oxidation products of lipids, proteins, and DNA in the blood, plasma, and urine of rats were measured as part of a comprehensive, multilaboratory validation study searching for noninvasive biomarkers of oxidative stress. This article is the second report of the nationwide Biomarkers of Oxidative Stress Study using acute CCl4 poisoning as a rodent model for oxidative stress. The time-dependent (2, 7, and 16 h) and dose-dependent (120 and 1200 mg/kg i.p.) effects of CCl4 on concentrations of lipid hydroperoxides, TBARS, malondialdehyde (MDA), isoprostanes, protein carbonyls, methionine sulfoxidation, tyrosine products, 8-hydroxy-2'-deoxyguanosine (8-OHdG), leukocyte DNA-MDA adducts, and DNA-strand breaks were investigated to determine whether the oxidative effects of CCl4 would result in increased generation of these oxidation products. Plasma concentrations of MDA and isoprostanes (both measured by GC-MS) and urinary concentrations of isoprostanes (measured with an immunoassay or LC/MS/MS) were increased in both low-dose and high-dose CCl4-treated rats at more than one time point. The other urinary markers (MDA and 8-OHdG) showed significant elevations with treatment under three of the four conditions tested. It is concluded that measurements of MDA and isoprostanes in plasma and urine as well as 8-OHdG in urine are potential candidates for general biomarkers of oxidative stress. All other products were not changed by CCl4 or showed fewer significant effects.
Asunto(s)
Intoxicación por Tetracloruro de Carbono/metabolismo , Tetracloruro de Carbono/toxicidad , ADN/metabolismo , Desoxiguanosina/análogos & derivados , Metabolismo de los Lípidos , Estrés Oxidativo , 8-Hidroxi-2'-Desoxicoguanosina , Animales , Ensayo Cometa , Daño del ADN , Desoxiguanosina/farmacología , Radicales Libres , Cromatografía de Gases y Espectrometría de Masas , Peróxido de Hidrógeno/metabolismo , Inmunoensayo , Immunoblotting , Hígado/metabolismo , Masculino , Malondialdehído/farmacología , Metionina/metabolismo , Oxígeno/metabolismo , Ratas , Ratas Endogámicas F344 , Espectrofotometría , Sustancias Reactivas al Ácido Tiobarbitúrico , Factores de Tiempo , Tirosina/química , Tirosina/metabolismoRESUMEN
Plasma and urinary levels of malondialdehyde-like products (MDA) and isoprostanes were identified as markers of in vivo lipid peroxidation in an animal model of CCl4 poisoning. We sought to determine the extent to which the formation of these oxidation products is influenced by inhibition of the cyclooxygenase enzymes which catalytically generate proinflammatory lipid peroxidation products known as prostaglandins and thromboxane. In the present studies, after induction of oxidant stress in rats with CCl4, lipid peroxidation products measured in plasma and urine demonstrate that isoprostanes and MDA can be partially inhibited by cyclooxygenase inhibitors, albeit to different extents. The lowering of isoprostane and MDA formation, however, may not to due primarily to the diminution of catalytic generation of isoprostanes or MDA by the cyclooxygenases but, rather, may be the result of the suppression of nonenzymatic lipid peroxidation. This is suggested since 8,12-iso-iPF2alpha-VI is also reduced by indomethacin, yet, unlike other isoprostanes and MDA, it is not generated catalytically by the cyclooxygenase. Thus, although the two cyclooxygenase inhibitors we tested have statistically significant effects on the measurements of both isoprostanes and MDA in this study, the results provide evidence that these lipid-degradation products primarily constitute markers of oxidative stress.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Biomarcadores/metabolismo , Intoxicación por Tetracloruro de Carbono/tratamiento farmacológico , Tetracloruro de Carbono/toxicidad , Indometacina/farmacología , Metabolismo de los Lípidos , Ácido Meclofenámico/farmacología , Estrés Oxidativo , Animales , Cromatografía Líquida de Alta Presión , Radicales Libres , Cromatografía de Gases y Espectrometría de Masas , Inmunoensayo , Indometacina/metabolismo , Inflamación , Peroxidación de Lípido , Espectrometría de Masas , Oxígeno/metabolismo , Prostaglandinas/metabolismo , Isoformas de Proteínas , Ratas , Ratas Endogámicas F344 , Tromboxano A2/metabolismo , Factores de TiempoRESUMEN
BACKGROUND: Although alcohol intake has been positively associated with breast cancer risk in epidemiologic studies, the mechanisms mediating this association are speculative. OBJECTIVE: The Postmenopausal Women's Alcohol Study was designed to explore the effects of moderate alcohol consumption on potential risk factors for breast cancer. In the present analysis, we evaluated the relationship of alcohol consumption with antioxidant nutrients and a biomarker of oxidative stress. DESIGN: Participants (n=53) consumed a controlled diet plus each of three treatments (15 or 30 g alcohol/day or a no-alcohol placebo beverage), during three 8-week periods in random order. We measured the antioxidants, vitamin E (alpha (alpha)- and gamma (gamma)-tocopherols), selenium, and vitamin C in fasting blood samples which were collected at the end of diet periods, treated and frozen for assay at the end of the study. We also measured 15-F(2t)-IsoP isoprostane, produced by lipid peroxidation, which serves as an indicator of oxidative stress and may serve as a biomarker for conditions favorable to carcinogenesis. RESULTS: After adjusting for BMI (all models) and total serum cholesterol (tocopherol and isoprostane models) we observed a significant 4.6% decrease (P=0.02) in alpha-tocopherol and a marginally significant 4.9% increase (P=0.07) in isoprostane levels when women consumed 30 g alcohol/day (P=0.06 and 0.05 for overall effect of alcohol on alpha-tocopherol and isoprostanes, respectively). The other antioxidants were not significantly modified by the alcohol treatment. CONCLUSIONS: These results suggest that moderate alcohol consumption increases some biomarkers of oxidative stress in postmenopausal women.
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Consumo de Bebidas Alcohólicas , Antioxidantes/metabolismo , Neoplasias de la Mama/epidemiología , Etanol/administración & dosificación , Isoprostanos/sangre , Estrés Oxidativo/efectos de los fármacos , Posmenopausia/sangre , Consumo de Bebidas Alcohólicas/efectos adversos , Ácido Ascórbico/sangre , Biomarcadores/sangre , Estudios Cruzados , Femenino , Humanos , Peroxidación de Lípido/efectos de los fármacos , Peroxidación de Lípido/fisiología , Persona de Mediana Edad , Estrés Oxidativo/fisiología , Factores de Riesgo , Selenio/sangre , Vitamina E/sangreRESUMEN
Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS), in combination with proteolytic protection assays, has been used to identify the functional epitope on human immunodeficiency virus envelope glycoprotein gp41 for the broadly neutralizing anti-gp41 human monoclonal antibody 2F5. In this protection assay-based procedure, a soluble gp140 protein with a stabilizing intermolecular disulfide bond between the gp120 and gp41 subunits (SOS gp140) was affinity bound to immobilized 2F5 under physiological conditions. A combination of proteolytic enzymatic cleavages was then performed to remove unprotected residues. Residues of SOS gp140 protected by their binding to 2F5 were then identified based on their molecular weights as determined by direct MALDI-MS of the immobilized antibody beads. The epitope, NEQELLELDKWASLWN, determined by this MALDI-MS protection assay approach consists of 16 amino acid residues near the C terminus of gp41. It is significantly longer than the ELDKWA core epitope previously determined for 2F5 by peptide enzyme-linked immunosorbent assay. This new knowledge of the structure of the 2F5 epitope may facilitate the design of vaccine antigens intended to induce antibodies with the breadth and potency of action of the 2F5 monoclonal antibody.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Anti-VIH/inmunología , Proteína gp41 de Envoltorio del VIH/inmunología , VIH-1/inmunología , Vacunas contra el SIDA/inmunología , Secuencia de Aminoácidos , Animales , Células CHO , Cricetinae , Mapeo Epitopo , Proteína gp41 de Envoltorio del VIH/química , Humanos , Espectrometría de Masas , Datos de Secuencia Molecular , Pruebas de NeutralizaciónRESUMEN
The combination of limited proteolysis and MALDI-TOF mass spectrometry has become an important tool for the determination of epitopes but works best with highly purified antibodies. Here we report the use of capture antibodies to reduce the need for purification of the antibody in the mass spectrometric determination of the epitope. In this new method, a secondary Fc-specific antibody, covalently bound to Sepharose beads, is used to capture the primary antibody (the antibody of interest). After capture, the two antibodies are cross-linked. The antigen is then bound to the immobilized antibodies and subjected to proteolysis using several successive proteinases. In this study, this strategy is demonstrated with a crude mouse anti-ACTH IgG solution and adrenocorticotropin (ACTH). Comparing this strategy with previous methods where the antibody is bound directly to activated beads, the new method (1) results in a higher binding capacity of the bound antibody to ACTH, (2) does not require purification of the antibody of interest, and (3) dramatically reduces the chemical background in the MALDI mass spectra.
Asunto(s)
Anticuerpos/química , Mapeo Epitopo/métodos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Hormona Adrenocorticotrópica/química , Hormona Adrenocorticotrópica/inmunología , Secuencia de Aminoácidos , Animales , Cromatografía Líquida de Alta Presión , Ratones , Datos de Secuencia MolecularRESUMEN
We have previously shown a connection between histone H1 phosphorylation and the transcriptional competence of the hormone inducible mouse mammary tumor virus (MMTV) promoter. Prolonged exposure of mouse cells to dexamethasone concurrently dephosphorylated histone H1 and rendered the MMTV promoter refractory to hormonal stimulation and, therefore, transcriptionally unresponsive. Using electrospray mass spectrometry, we demonstrate here that prolonged dexamethasone treatment differentially effects a subset of the six somatic H1 isoforms in mouse cells. H1 isoforms H1.0, H1.1, and H1.2 are non-responsive to hormone whereas prolonged dexamethasone treatment effectively dephosphorylated the H1.3, H1.4, and H1.5 isoforms. The protein kinase inhibitor staurosporine, shown to dephosphorylate histone H1 and down-regulate MMTV in cultured cells, appears only to completely dephosphorylate the H1.3 isoform. These results suggest that dephosphorylation of specific histone H1 isoforms may contribute to the previously observed decrease in transcriptional competence of the MMTV promoter through the modulation of chromatin structure. In a broader sense, this work advances the hypothesis that post-translational modifications of individual histone H1 isoforms directly influence the transcriptional activation/repression of specific genes.
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Histonas/química , Histonas/metabolismo , Hormonas/farmacología , Animales , Antineoplásicos Hormonales/farmacología , Western Blotting , Línea Celular , Células Cultivadas , Cromatografía Líquida de Alta Presión , Dexametasona/farmacología , Regulación hacia Abajo , Electroforesis en Gel de Poliacrilamida , Inhibidores Enzimáticos/farmacología , Ratones , Fosforilación , Regiones Promotoras Genéticas , Isoformas de Proteínas , Espectrometría de Masa por Ionización de Electrospray , Estaurosporina/farmacología , Transcripción GenéticaRESUMEN
This article describes a procedure for the quantitation of the isoprostane 15-F2t-IsoP (9a,11a,15S-trihydroxy-(8b)-prosta-5Z,13E-dien-1-oic acid [CAS#27415-26-5] formerly known as 8-epi-PGF2a or 8-iso-PGF2a, and also as iPF2a-III). We have combined features from several earlier methods for 15-F2t-IsoP and prostaglandins, and identified and modified those steps that may lead to poor recoveries. The resulting protocol is precise and reliable, and was validated by a blind time-course study of plasma levels in rats treated with 120 and 1200 mg CCl4/kg body weight. Plasma levels of 15-F2t-IsoP, as measured according to the procedure described above, are good indicators of acute oxidative stress as induced by CCl4. The precision of the measurements allows detection of elevated plasma 15-F2t-IsoP levels as long as 16 h after an acute exposure of 120 mg CCl4/kg body weight, and 2 h after an exposure of 1 mg CCl4/kg body weight. The results of this low-dose, pilot study suggest that this method has sufficient analytical precision to allow the detection of the small changes in plasma isoprostane levels, which result from chronic and/or lower-level exposures to agents causing oxidative stress.
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Dinoprost/análogos & derivados , Dinoprost/sangre , Dinoprost/química , Cromatografía de Gases y Espectrometría de Masas/métodos , Animales , Peso Corporal , Tetracloruro de Carbono/farmacología , Dinoprost/metabolismo , Relación Dosis-Respuesta a Droga , Ácidos Grasos Monoinsaturados/farmacología , Hidrólisis , Masculino , Estrés Oxidativo/efectos de los fármacos , Proyectos Piloto , Aceite de Brassica napus , Ratas , Ratas Endogámicas F344 , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Factores de TiempoRESUMEN
Human recombinant p53 (r-p53) protein was studied by mass spectrometry (MS) to determine site-specific posttranslational differences between basal and hyperphosphorylated r-p53. Wild-type p53 was basally expressed after baculovirus infection while a parallel preparation was treated with the phosphatase inhibitor okadaic acid during the terminal stages of expression to create a hyperphosphorylated form of p53 known for its higher DNA binding and transcriptional activation. After immunoaffinity and HPLC purification, MALDI/MS measured a higher molecular mass for r-p53 from okadaic acid treatment relative to control, suggesting a higher phosphorylation state. This was supported by an acidic shift of r-p53 isoforms separated by gel isoelectric focusing. Employing a variety of mass spectrometric analyses combined with separation and affinity techniques, six specific phosphorylation sites of p53 were identified. The MS data indicated that hyperphosphorylated p53 showed a higher degree of phosphorylation than basal p53 at specific amino- and carboxy-terminal sites. In particular, ESI-MS demonstrated that Ser(315) was entirely phosphorylated after okadaic acid treatment, as confirmed biochemically by CDK2 kinase assay and by isoelectric focusing. In summary, MS analysis uniquely revealed increased, site-specific phosphorylations on p53 after phosphatase inhibition, particularly at Ser(315), which may be critical molecular events in defining p53 activity.
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Proteína p53 Supresora de Tumor/metabolismo , Secuencia de Aminoácidos , Animales , Baculoviridae/genética , Cromatografía Líquida de Alta Presión , Cromatografía Liquida , Inhibidores Enzimáticos/farmacología , Humanos , Hidrólisis , Datos de Secuencia Molecular , Ácido Ocadaico/farmacología , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Fosfopéptidos/genética , Fosfopéptidos/metabolismo , Fosforilación/efectos de los fármacos , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/metabolismo , Serina/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Spodoptera/genética , Tripsina , Proteína p53 Supresora de Tumor/biosíntesis , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Cytosolic sulfotransferases sulfate steroids such as estrogens and hydroxysteroids. The enzymes, including human estrogen sulfotransferase (hEST) and hydroxysteroid sulfotransferase (hHST), are generally homodimers in solution with mouse estrogen sulfotransferase (mEST) being one of few exceptions. To identify the amino acid residues responsible for the dimerization, eight residues on the surface of hEST were mutated to their counterparts in mEST and mutated hESTs were then analyzed by gel filtration chromatography. A single mutation of Val(269) to Glu was sufficient to convert hEST to a monomer and the corresponding mutation of Val(260) also altered hHST to a monomer. The hHST crystal structure revealed a short stretch of peptide with the side-chains from two hHST monomers forming a hydrophobic zipper-like structure enforced by ion pairs at both ends. This peptide consisted of 10 residues near the C-terminus that, including the critical Val residue, is conserved as KXXXTVXXXE in nearly all cytosolic sulfotransferases. When mEST underwent the double mutations Pro269Thr/Glu270Val dimerization resulted. Thus, the KXXXTVXXXE sequence appears to be the common protein-protein interaction motif that mediates the homo- as well as heterodimerization of cytosolic sulfotransferases.
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Citosol/enzimología , Dimerización , Sulfotransferasas/química , Sulfotransferasas/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Cromatografía en Gel , Reactivos de Enlaces Cruzados/metabolismo , Cristalografía por Rayos X , Análisis Mutacional de ADN , Electroforesis en Gel de Poliacrilamida , Glutamina/química , Glutatión/metabolismo , Humanos , Espectrometría de Masas , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación , Plásmidos/metabolismo , Unión Proteica , Sefarosa/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Valina/químicaRESUMEN
An analytical approach is reported for the characterization of the specific glycans found on highly glycosylated proteins based on a combination of specific proteolysis and deglycosylation combined with two different mass spectrometric approaches, matrix-assisted laser desorption/ionization mass spectrometry, and nanoelectrospray mass spectrometry/tandem mass spectrometry using a hybrid quadrupole-time-of-flight tandem mass spectrometer. The high resolution and mass accuracy of the mass spectrometric data obtained on the hybrid instrument combined with the high parent mass capabilities are shown to be extremely useful in the site-specific assignment of heterogeneous glycans. Using this methodology, 25 of 26 consensus glycosylation sites on HIV-1(SF2) gp120, expressed in Chinese hamster ovary cells, could be assigned. Good correlations between the relative abundances of members of heterogeneous series in the matrix-assisted laser desorption/ionization mass spectra and the nanoelectrospray mass spectra were observed, indicating that the mass spectrometric data reflected the actual abundances of the members of the series. These data were incorporated with molecular modeling based on the solved structure of a mutant truncated, highly deglycosylated gp120 to propose a structural model for the completely glycosylated form.
Asunto(s)
Células CHO/metabolismo , Proteína gp120 de Envoltorio del VIH/genética , Proteína gp120 de Envoltorio del VIH/metabolismo , Proteínas Recombinantes/metabolismo , Secuencia de Aminoácidos , Animales , Cricetinae , Cristalización , Glicopéptidos/metabolismo , Glicosilación , Proteína gp120 de Envoltorio del VIH/química , VIH-1/metabolismo , Manosa/metabolismo , Espectrometría de Masas , Modelos Moleculares , Datos de Secuencia Molecular , Polisacáridos/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónAsunto(s)
Mapeo Epitopo/métodos , Productos del Gen gag/inmunología , Antígenos VIH/inmunología , VIH-1/inmunología , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Secuencia de Aminoácidos , Animales , Productos del Gen gag/química , Antígenos VIH/química , Humanos , Datos de Secuencia Molecular , Productos del Gen gag del Virus de la Inmunodeficiencia HumanaRESUMEN
The fusion of the human immunodeficiency virus (HIV) with the target cell was assisted by the interaction between the viral envelope glycoprotein HIV-1 gp120 and a chemokine receptor. Studies have shown that the efficiency of the binding depends on the presence of the V3 loop of the gp120 which is known to interact with polyanions, such as phosphorothioate oligodeoxynucleotides (Sd, potential anti-HIV drugs). In this study, capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) was used to systematically evaluate binding between Sd and HIV-1 gp120. A 25-mer fluorescently tagged phosphorothioate oligodeoxynucleotide (GEM) was employed as a probe to study this interaction. The dissociation constant (K(d)) between GEM and gp120 was determined to be 0.98 nM by Scatchard analysis. The competition constants (K(c)) of a set of Sd that compete with GEM for binding to gp120 were also determined. The results showed that the interaction had a strong dependence on the sulfur phosphorothioate backbone. Chain length and the sequence of Sd also affect the ability of binding to gp120. The ability to study the protein-drug binding in the solution with minimal sample consumption makes CE-LIF very attractive for biological studies.
Asunto(s)
Fármacos Anti-VIH/metabolismo , Electroforesis Capilar/métodos , Proteína gp120 de Envoltorio del VIH/metabolismo , Secuencia de Bases , Unión Competitiva , Cartilla de ADN , Estudios de Evaluación como Asunto , VIH-1/metabolismo , Rayos Láser , Unión Proteica , Espectrometría de FluorescenciaRESUMEN
The characterization of sequence-specific noncovalent complexes of the GCN4 peptides and dsDNA using mass spectrometry is reported. The GCN4 peptides belong to a class of proteins which bind to sequence-specific dsDNA and are important in the regulation of gene transcription in yeast. These proteins contain a bZIP structural motif which consists of a basic DNA-binding domain and a leucine zipper dimerization domain. The protein dimers specifically bind double-stranded DNA containing the binding element 5'-ATGA(C/G)TCAT-3' to form a tetramolecular noncovalent complex. Using electrospray ionization, we report the detection of such a specific tetramolecular complex using mass spectrometry. Under conditions necessary for observation of the tetramolecular complex, no ions were detected for the GCN4 peptide dimer or the GCN4 monomer with dsDNA. These observations indicate that the specific interaction of the dsDNA with the protein dimer stabilizes the biologically significant noncovalent complex in the gas phase. Complexes were observed for various lengths of both blunt-ended and cohesive-ended double-stranded DNA containing the specific recognition sequence. The binding specificity of the complex was verified with the use of control DNA not containing the recognition sequence and control peptides not known to bind DNA specifically. Additionally, combining limited proteolysis of GCN4 peptide-DNA complexes with mass spectrometric determination of the products compared to identical experiments with noncomplexed peptides was used to probe interactions of specific amino acids with the DNA. The ability to observe these complexes by mass spectrometry and to probe the specific interactions involved opens the door for utilizing this analytical technique to other structural biological problems including the study of transcription processes and determining the specific binding regions between dsDNA and proteins.