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1.
J Pharmacol Exp Ther ; 317(2): 752-61, 2006 May.
Artigo em Inglês | MEDLINE | ID: mdl-16424150

RESUMO

We investigated the in vitro metabolism of two (nitrooxy)butyl ester nitric oxide (NO) donor derivatives of flurbiprofen and ferulic acid, [1,1'-biphenyl]-4-acetic acid-2-fluoro-alpha-methyl-4-(nitrooxy)butyl ester (HCT 1026) and 3-(4-hydroxy-3-methoxyphenyl)-2-propenoic acid 4-(nitrooxy)butyl ester (NCX 2057), respectively, in rat blood plasma and liver subcellular fractions compared with (nitrooxy)butyl alcohol (NOBA) and glyceryl trinitrate (GTN). HCT 1026 and NCX 2057 undergo rapid ubiquitous carboxyl ester hydrolysis to their respective parent compounds and NOBA. The nitrate moiety of this latter is subsequently metabolized to inorganic nitrogen oxides (NOx), predominantly in liver cytosol by glutathione S-transferase (GST) and to a lesser extent in liver mitochondria. If, however, in liver cytosol, the carboxyl ester hydrolysis is prevented by an esterase inhibitor, the metabolism at the nitrate moiety level does not occur. In blood plasma, HCT 1026 and NCX 2057 are not metabolized to NOx, whereas a slow but sustained NO generation in deoxygenated whole blood as detected by electron paramagnetic resonance indicates the involvement of erythrocytes in the bioactivation of these compounds. Differently from NOBA, GTN is also metabolized in blood plasma and more quickly metabolized by different GST isoforms in liver cytosol. The cytosolic GST-mediated denitration of these organic nitrates in liver limits their interaction with other intracellular compartments to possible generation of NO and/or their subsequent availability and bioactivation in the systemic circulation and extrahepatic tissues. We show the possibility of modulating the activity of hepatic cytosolic enzymes involved in the metabolism of (nitrooxy)butyl ester compounds, thus increasing the therapeutic potential of this class of compounds.


Assuntos
Butanos/farmacocinética , Flurbiprofeno/análogos & derivados , Fígado/metabolismo , Doadores de Óxido Nítrico/farmacocinética , Óxido Nítrico/metabolismo , Nitrocompostos/farmacocinética , Animais , Biotransformação , Butanos/sangue , Citosol/metabolismo , Flurbiprofeno/sangue , Flurbiprofeno/farmacocinética , Técnicas In Vitro , Fígado/citologia , Masculino , Mitocôndrias Hepáticas/metabolismo , Estrutura Molecular , Doadores de Óxido Nítrico/sangue , Nitrocompostos/sangue , Ratos , Ratos Sprague-Dawley
2.
Proteomics ; 6(3): 817-25, 2006 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-16372262

RESUMO

Using redox proteomics techniques to characterize the thiol status of proteins in human T lymphocytes, we identified cyclophilin A (CypA) as a specifically oxidized protein early after mitogen activation. CypA is an abundantly expressed cytosolic protein, target of the immunosuppressive drug cyclosporin A (CsA), for which a variety of functions has been described. In this study, we could identify CypA as a protein undergoing glutathionylation in vivo. Using MALDI-MS we identified Cys52 and Cys62 as targets of glutathionylation in T lymphocytes, and, using bioinformatic tools, we defined the reasons for the susceptibility of these residues to the modification. In addition, we found by circular dichroism spectroscopy that glutathionylation has an important impact on the secondary structure of CypA. Finally, we suggest that glutathionylation of CypA may have biological implications and that CypA may play a key role in redox regulation of immunity.


Assuntos
Ciclofilina A/metabolismo , Glutationa/metabolismo , Dicroísmo Circular , Biologia Computacional , Cisteína/metabolismo , Eletroforese em Gel Bidimensional , Humanos , Ativação Linfocitária/efeitos dos fármacos , Oxirredução , Proteômica , Proteínas Recombinantes/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Linfócitos T/metabolismo
3.
Proteomics ; 3(7): 1154-61, 2003 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-12872216

RESUMO

Protein glutathionylation is a post-translational modification consisting of the formation of a mixed disulfide between protein cysteines and glutathione (GSH). To identify proteins undergoing glutathionylation in primary rat hepatocytes and in human HepG2 hepatoma cells, we radiolabeled the intracellular GSH pool with L-[(35)S] cysteine. Cells were then exposed to oxidative stress. Proteins were separated by two-dimensional gel electrophoresis under nonreducing conditions, and glutathionylated proteins were located by autoradiography and identified by mass spectrometry after tryptic digestion. Several proteins previously not known to undergo glutathionylation were thus recognized. Among the identified proteins some are the same or belong to the same functional class as those we have already identified in a previous paper on T cell blasts (actin, nucleophosmin, phosphogluconolactonase, myosin, profilin, cyclophilin A, stress 70 protein, ubiquitin in HepG2 cells and actin, peroxiredoxin 5, cytochrome C oxidase, heat shock cognate 70 in hepatocytes) while others are newly recognized (Ran specific GTPase activating protein, histidine triad nucleotide binding protein 2 in HepG2 cells and enoyl CoA hydratase in hepatocytes). The technique described proved equally applicable to a variety of cell types.


Assuntos
Glutationa/química , Estresse Oxidativo , Oxigênio/metabolismo , Animais , Carcinoma Hepatocelular/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Células Cultivadas , Cisteína/química , Dissulfetos/química , Eletroforese em Gel Bidimensional , Glutationa/metabolismo , Hepatócitos/metabolismo , Humanos , Fígado/metabolismo , Ratos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Compostos de Sulfidrila
4.
Proc Natl Acad Sci U S A ; 100(11): 6741-6, 2003 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-12746497

RESUMO

Erythropoietin (EPO) is a tissue-protective cytokine preventing vascular spasm, apoptosis, and inflammatory responses. Although best known for its role in hematopoietic lineages, EPO also affects other tissues, including those of the nervous system. Enthusiasm for recombinant human erythropoietin (rhEPO) as a potential neuroprotective therapeutic must be tempered, however, by the knowledge it also enlarges circulating red cell mass and increases platelet aggregability. Here we examined whether erythropoietic and tissue-protective activities of rhEPO might be dissociated by a variation of the molecule. We demonstrate that asialoerythropoietin (asialoEPO), generated by total enzymatic desialylation of rhEPO, possesses a very short plasma half-life and is fully neuroprotective. In marked contrast with rhEPO, this molecule at doses and frequencies at which rhEPO exhibited erythropoiesis, did not increase the hematocrit of mice or rats. AsialoEPO appeared promptly within the cerebrospinal fluid after i.v. administration; intravenously administered radioiodine-labeled asialoEPO bound to neurons within the hippocampus and cortex in a pattern corresponding to the distribution of the EPO receptor. Most importantly, asialoEPO exhibits a broad spectrum of neuroprotective activities, as demonstrated in models of cerebral ischemia, spinal cord compression, and sciatic nerve crush. These data suggest that nonerythropoietic variants of rhEPO can cross the blood-brain barrier and provide neuroprotection.


Assuntos
Isquemia Encefálica/tratamento farmacológico , Eritropoetina/farmacologia , Fármacos Neuroprotetores/farmacologia , Animais , Eritropoetina/farmacocinética , Eritropoetina/uso terapêutico , Hemoglobinas/análise , Masculino , Camundongos , Fármacos Neuroprotetores/farmacocinética , Fármacos Neuroprotetores/uso terapêutico , Ratos , Ratos Sprague-Dawley
5.
Proc Natl Acad Sci U S A ; 99(15): 9745-9, 2002 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-12119401

RESUMO

To identify proteins undergoing glutathionylation (formation of protein-glutathione mixed disulfides) in human T cell blasts, we radiolabeled the glutathione pool with (35)S, exposed cells to the oxidant diamide, and analyzed cellular proteins by two-dimensional electrophoresis. One of the proteins undergoing glutathionylation was identified by molecular weight, isoelectric point, and immunoblotting as thioredoxin (Trx). Incubation of recombinant human Trx with glutathione disulfide or S-nitrosoglutathione led to the formation of glutathionylated Trx, identified by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry. The glutathionylation site was identified as Cys-72. Glutathionylation of rhTrx abolished its enzymatic activity as insulin disulfide reductase in the presence of NADPH and Trx reductase. Activity was, however, regained with sigmoidal kinetics, indicating a process of autoactivation due to the ability of Trx to de-glutathionylate itself. These data suggest that the intracellular glutathione/glutathione disulfide ratio, an indicator of the redox state of the cell, can regulate Trx functions reversibly through thiol-disulfide exchange reactions.


Assuntos
Glutationa/metabolismo , Tiorredoxinas/química , Tiorredoxinas/metabolismo , Sequência de Aminoácidos , Células Cultivadas , Diamida/farmacologia , Dissulfeto de Glutationa/metabolismo , Humanos , Insulina/metabolismo , Cinética , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Linfócitos T/metabolismo , Tripsina
6.
Proc Natl Acad Sci U S A ; 99(6): 3505-10, 2002 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-11904414

RESUMO

Formation of mixed disulfides between glutathione and the cysteines of some proteins (glutathionylation) has been suggested as a mechanism through which protein functions can be regulated by the redox status. The aim of this study was to identify the proteins of T cell blasts that undergo glutathionylation under oxidative stress. To this purpose, we radiolabeled cellular glutathione with (35)S, exposed T cells to oxidants (diamide or hydrogen peroxide), and performed nonreducing, two-dimensional electrophoresis followed by detection of labeled proteins by phosphorimaging and their identification by mass spectrometry techniques. We detected several proteins previously not recognized to be glutathionylated, including cytoskeletal proteins (vimentin, myosin, tropomyosin, cofilin, profilin, and the already known actin), enzymes (enolase, aldolase, 6-phosphogluconolactonase, adenylate kinase, ubiquitin-conjugating enzyme, phosphoglycerate kinase, triosephosphate isomerase, and pyrophosphatase), redox enzymes (peroxiredoxin 1, protein disulfide isomerase, and cytochrome c oxidase), cyclophilin, stress proteins (HSP70 and HSP60), nucleophosmin, transgelin, galectin, and fatty acid binding protein. Based on the presence of several protein isoforms in control cells, we suggest that enolase and cyclophilin are heavily glutathionylated under basal conditions. We studied the effect of glutathionylation on some of the enzymes identified in the present study and found that some of them (enolase and 6-phosphogluconolactonase) are inhibited by glutathionylation, whereas the enzymatic activity of cyclophilin (peptidylprolyl isomerase) is not. These findings suggest that protein glutathionylation might be a common mechanism for the global regulation of protein functions.


Assuntos
Dissulfetos/metabolismo , Glutationa/metabolismo , Estresse Oxidativo , Proteoma/metabolismo , Linfócitos T/metabolismo , Células Cultivadas , Diamida/farmacologia , Eletroforese em Gel Bidimensional , Humanos , Peróxido de Hidrogênio/farmacologia , Espectrometria de Massas , Peso Molecular , Oxidantes/farmacologia , Oxirredução/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Proteoma/química , Proteoma/efeitos dos fármacos , Corantes de Rosanilina , Coloração e Rotulagem , Linfócitos T/química , Linfócitos T/efeitos dos fármacos , Linfócitos T/enzimologia
7.
Mol Immunol ; 38(10): 773-80, 2002 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-11841837

RESUMO

We show here that exposure to oxidative stress induces glutathione (GSH) modification of protein cysteinyl residues (glutathionylation) in T cell blasts. Treating the cells with the oxidant diamide induces thiolation of a series of proteins that can be detected by 2D electrophoresis when 35S-cysteine is used to label the intracellular GSH pool. This thiolation is reversible, proteins are rapidly dethiolated and GSH is released from proteins once the oxidants are washed and the cells are allowed to recover. Dethiolation is dependent on the availability of GSH and thiols, since it is inhibited by GSH-depleting agents and improved by N-acetyl-L-cysteine (NAC). The capacity of these agents to reverse glutathionylation is diminished in T cell blasts infected in vitro with HIV, which is known to cause oxidative stress. Consistent with these findings, the activity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), an enzyme known to be inhibited by glutathionylation, is inhibited in diamide-treated cells and recovers rapidly when cells are allowed to dethiolate. Further, GAPDH activity is diminished by GSH-depleting agents and augmented by NAC. Thus, reversible glutathionylation of proteins can rapidly shift the activity of a key metabolic enzyme and thereby result in dramatic, reversible changes in cellular metabolism.


Assuntos
Glutationa/metabolismo , Infecções por HIV/sangue , Estresse Oxidativo , Proteínas/metabolismo , Linfócitos T/metabolismo , Acetilcisteína , Diamida/química , Diamida/metabolismo , Glutationa/química , Humanos , Proteínas/química , Compostos de Sulfidrila , Linfócitos T/química
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