RESUMO
RATIONALE: The efflux capacity of high-density lipoprotein (HDL) with cultured macrophages associates strongly and negatively with coronary artery disease status, indicating that impaired sterol efflux capacity might be a marker-and perhaps mediator-of atherosclerotic burden. However, the mechanisms that contribute to impaired sterol efflux capacity remain poorly understood. OBJECTIVE: Our aim was to determine the relationship between myeloperoxidase-mediated oxidative damage to apolipoprotein A-I, the major HDL protein, and the ability of HDL to remove cellular cholesterol by the ATP-binding cassette transporter A1 (ABCA1) pathway. METHODS AND RESULTS: We quantified both site-specific oxidation of apolipoprotein A-I and HDL's ABCA1 cholesterol efflux capacity in control subjects and subjects with stable coronary artery disease or acute coronary syndrome. Subjects with coronary artery disease and acute coronary syndrome had higher levels of chlorinated tyrosine 192 and oxidized methionine 148 compared with control subjects. In contrast, plasma levels of myeloperoxidase did not differ between the groups. HDL from the subjects with coronary artery disease and acute coronary syndrome was less able to accept cholesterol from cells expressing ABCA1 compared with HDL from control subjects. Levels of chlorinated tyrosine and oxidized methionine associated inversely with ABCA1 efflux capacity and positively with atherosclerotic disease status. These differences remained significant after adjusting for HDL-cholesterol levels. CONCLUSIONS: Our observations indicate that myeloperoxidase may contribute to the generation of dysfunctional HDL with impaired ABCA1 efflux capacity in humans with atherosclerosis. Quantification of chlorotyrosine and oxidized methionine in circulating HDL might be useful indicators of the risk of cardiovascular disease that are independent of HDL-cholesterol.
Assuntos
Transportador 1 de Cassete de Ligação de ATP/metabolismo , Aterosclerose/metabolismo , Colesterol/metabolismo , Lipoproteínas HDL/metabolismo , Peroxidase/metabolismo , Transdução de Sinais/fisiologia , Síndrome Coronariana Aguda/metabolismo , Síndrome Coronariana Aguda/fisiopatologia , Idoso , Apolipoproteína A-I/metabolismo , Aterosclerose/fisiopatologia , Biomarcadores/metabolismo , Proteína C-Reativa/metabolismo , Estudos de Casos e Controles , Células Cultivadas , Doença da Artéria Coronariana/metabolismo , Doença da Artéria Coronariana/fisiopatologia , Feminino , Humanos , Masculino , Metionina/metabolismo , Pessoa de Meia-Idade , OxirreduçãoRESUMO
The antioxidant enzyme methionine sulfoxide reductase A (MsrA) is highly expressed in the retinal pigment epithelium (RPE), a support tissue for neighboring photoreceptors. MsrA protein levels correlate with sensitivity of RPE in culture to experimental oxidative stress. To investigate whether and how MsrA affects RPE functionality regardless of oxidative stress, we tested the effects of acute silencing or overexpression of MsrA on the phagocytosis of photoreceptor outer segment fragments (POS), a demanding, daily function of the RPE that is essential for vision. Endogenous MsrA localized to mitochondria and cytosol of rat RPE in culture. RPE cells manipulated to express higher or lower levels of MsrA than control cells showed no signs of cell death but increased or decreased, respectively, POS binding as well as engulfment. These effects of altered MsrA protein concentration on phagocytosis were independent of the levels of oxidative stress. However, altering MsrA expression had no effect on phagocytosis when mitochondrial respiration was inhibited. Furthermore, ATP content directly correlated with MsrA protein levels in RPE cells that used mitochondrial oxidative phosphorylation for ATP synthesis but not in RPE cells that relied on glycolysis alone. Overexpressing MsrA was sufficient to increase specifically the activity of complex IV of the respiratory chain, whereas activity of complex II and mitochondrial content were unaffected. Thus, MsrA probably enhances ATP synthesis by increasing complex IV activity. Such contribution of MsrA to energy metabolism is independent of its function in protection from elevated oxidative stress but contributes to routine but vital photoreceptor support by RPE cells.
Assuntos
Trifosfato de Adenosina/biossíntese , Mitocôndrias/metabolismo , Oxirredutases/imunologia , Fagocitose/imunologia , Epitélio Pigmentado da Retina/metabolismo , Animais , Antioxidantes , Linhagem Celular , Complexo II de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/genética , Metabolismo Energético/genética , Metabolismo Energético/imunologia , Células Epiteliais/citologia , Glicólise , Fosforilação Oxidativa , Estresse Oxidativo/imunologia , Oxirredutases/biossíntese , Oxirredutases/genética , Fagocitose/genética , Ligação Proteica/imunologia , Interferência de RNA , RNA Interferente Pequeno , Ratos , Segmento Externo das Células Fotorreceptoras da Retina/imunologia , Epitélio Pigmentado da Retina/citologiaRESUMO
Ozone exposure effect on free radical-catalyzed oxidation products of lipids, proteins, and DNA in the plasma and urine of rats was studied as a continuation of the international Biomarker of Oxidative Stress Study (BOSS) sponsored by NIEHS/NIH. The goal was to identify a biomarker for ozone-induced oxidative stress and to assess whether inconsistent results often reported in the literature might be due to the limitations of the available methods for measuring the various types of oxidative products. The time- and dose-dependent effects of ozone exposure on rat plasma lipid hydroperoxides, malondialdehyde, F2-isoprostanes, protein carbonyls, methionine oxidation, and tyrosine- and phenylalanine oxidation products, as well as urinary malondialdehyde and F2-isoprostanes were investigated with various techniques. The criterion used to recognize a marker in the model of ozone exposure was that a significant effect could be identified and measured in a biological fluid seen at both doses at more than one time point. No statistically significant differences between the experimental and the control groups at either ozone dose and time point studied could be identified in this study. Tissue samples were not included. Despite all the work accomplished in the BOSS study of ozone, no available product of oxidation in biological fluid has yet met the required criteria of being a biomarker. The current negative findings as a consequence of ozone exposure are of great importance, because they document that in complex systems, as the present in vivo experiment, the assays used may not provide meaningful data of ozone oxidation, especially in human studies.
Assuntos
DNA/análise , Lipídeos/análise , Estresse Oxidativo , Ozônio/toxicidade , Proteínas/análise , Animais , Biomarcadores/análise , DNA/sangue , DNA/urina , Dinoprosta/análogos & derivados , Dinoprosta/análise , Peróxidos Lipídicos/análise , Lipídeos/sangue , Lipídeos/urina , Masculino , Malondialdeído/análise , Metionina/metabolismo , Oxirredução , Ratos , Ratos Endogâmicos F344RESUMO
Sulindac is a nonsteroidal, anti-inflammatory drug (NSAID) that has also been studied for its anticancer activity. Recent studies suggest that sulindac and its metabolites act by sensitizing cancer cells to oxidizing agents and drugs that affect mitochondrial function, resulting in the production of reactive oxygen species and death by apoptosis. In contrast, normal cells are not killed under these conditions and, in some instances, are protected against oxidative stress. Sulindac has a methyl sulfoxide moiety with a chiral center and was used in all of the previous studies as a mixture of the R- and S-epimers. Because epimers of a compound can have very different chemical and biological properties, we have separated the R- and S-epimers of sulindac, studied their individual metabolism, and performed preliminary experiments on their effect on normal and lung cancer cells exposed to oxidative stress. Previous results had indicated that the reduction of (S)-sulindac to sulindac sulfide, the active NSAID, was catalyzed by methionine sulfoxide reductase (Msr) A. In the present study, we purified an enzyme that reduces (R)-sulindac and resembles MsrB in its substrate specificity. The oxidation of both epimers to sulindac sulfone is catalyzed primarily by the microsomal cytochrome P450 (P450) system, and the individual enzymes responsible have been identified. (S)-Sulindac increases the activity of the P450 system better than (R)-sulindac, but both epimers increase primarily the enzymes that oxidize (R)-sulindac. Both epimers can protect normal lung cells against oxidative damage and enhance the killing of lung cancer cells exposed to oxidative stress.
Assuntos
Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Sistema Enzimático do Citocromo P-450/metabolismo , Metionina Sulfóxido Redutases/metabolismo , Sulindaco/metabolismo , Sulindaco/farmacologia , Animais , Antineoplásicos/química , Antineoplásicos/farmacocinética , Western Blotting , Cromatografia Líquida de Alta Pressão , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/genética , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Células Hep G2 , Humanos , Metionina Sulfóxido Redutases/química , Metionina Sulfóxido Redutases/genética , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/enzimologia , Microssomos Hepáticos/metabolismo , Estrutura Molecular , Oxirredução , Estresse Oxidativo/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Estereoisomerismo , Sulindaco/química , Sulindaco/farmacocinéticaRESUMO
Acute lung injury (ALI) is a life-threatening condition in critically ill patients. Injury to the alveolar epithelium is a critical event in ALI, and accumulating evidence suggests that it is linked to proapoptotic Fas/FasL signals. Active soluble FasL (sFasL) is detectable in the bronchoalveolar lavage (BAL) fluid of patients with ALI, but the mechanisms controlling its bioactivity are unclear. We therefore investigated how the structure of sFasL influences cellular activation in human and mouse lungs and the role of oxidants and proteases in modifying sFasL activity. The sFasL in BAL fluid from patients with ALI was bioactive and present in high molecular weight multimers and aggregates. Oxidants generated from neutrophil myeloperoxidase in BAL fluid promoted aggregation of sFasL in vitro and in vivo. Oxidation increased the biological activity of sFasL at low concentrations but degraded sFasL at high concentrations. The amino-terminal extracellular stalk region of human sFasL was required to induce lung injury in mice, and proteolytic cleavage of the stalk region by MMP-7 reduced the bioactivity of sFasL in human cells in vitro. The sFasL recovered from the lungs of patients with ALI contained both oxidized methionine residues and the stalk region. These data provide what we believe to be new insights into the structural determinants of sFasL bioactivity in the lungs of patients with ALI.
Assuntos
Proteína Ligante Fas/metabolismo , Pulmão/metabolismo , Animais , Apoptose , Lavagem Broncoalveolar , Líquido da Lavagem Broncoalveolar , Dano ao DNA , Humanos , Peróxido de Hidrogênio/química , Metaloproteinase 7 da Matriz/metabolismo , Metionina/metabolismo , Camundongos , Oxidantes/metabolismo , Oxigênio/metabolismo , Estrutura Terciária de ProteínaRESUMO
The methionine sulfoxide reductase (Msr) system has been shown to play an important role in protecting cells against oxidative damage. This family of enzymes can repair damage to proteins resulting from the oxidation of methionine residues to methionine sulfoxide, caused by reactive oxygen species. Previous genetic studies in animals have shown that increased levels of methionine sulfoxide reductase enzyme A (MsrA), an important member of the Msr family, can protect cells against oxidative damage and increase life span. A high-throughput screening (HTS) compatible assay has been developed to search for both activators and inhibitors of MsrA. The assay involves a coupled reaction in which the oxidation of NADPH is measured by either spectrophotometric or fluorometric analysis. Previous studies had shown that MsrA has a broad substrate specificity and can reduce a variety of methyl sulfoxide compounds, including dimethylsulfoxide (DMSO). Since the chemicals in the screening library are dissolved in DMSO, which would compete with any of the standard substrates used for the determination of MsrA activity, an assay has been developed that uses the DMSO that is the solvent for the compounds in the library as the substrate for the MsrA enzyme. A specific activator of MsrA could have important therapeutic value for diseases that involve oxidative damage, especially age-related diseases, whereas a specific inhibitor of MsrA would have value for a variety of research studies.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Ativadores de Enzimas/farmacologia , Inibidores Enzimáticos/farmacologia , Ensaios de Triagem em Larga Escala , Metionina Sulfóxido Redutases/antagonistas & inibidores , Metionina Sulfóxido Redutases/metabolismo , Animais , Bovinos , Dimetil Sulfóxido/metabolismo , Metionina Sulfóxido Redutases/química , NADP/metabolismo , Oxirredução , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Proteínas Recombinantes/metabolismo , Tiorredoxina Dissulfeto Redutase/metabolismo , Tiorredoxinas/metabolismoRESUMO
Reactive oxygen intermediates generated by neutrophils kill bacteria and are implicated in inflammatory tissue injury, but precise molecular targets are undefined. We demonstrate that neutrophils use myeloperoxidase (MPO) to convert methionine residues of ingested Escherichia coli to methionine sulfoxide in high yield. Neutrophils deficient in individual components of the MPO system (MPO, H(2)O(2), chloride) exhibited impaired bactericidal activity and impaired capacity to oxidize methionine. HOCl, the principal physiologic product of the MPO system, is a highly efficient oxidant for methionine, and its microbicidal effects were found to correspond linearly with oxidation of methionine residues in bacterial cytosolic and inner membrane proteins. In contrast, outer envelope proteins were initially oxidized without associated microbicidal effect. Disruption of bacterial methionine sulfoxide repair systems rendered E. coli more susceptible to killing by HOCl, whereas over-expression of a repair enzyme, methionine sulfoxide reductase A, rendered them resistant, suggesting a direct role for methionine oxidation in bactericidal activity. Prominent among oxidized bacterial proteins were those engaged in synthesis and translocation of peptides to the cell envelope, an essential physiological function. Moreover, HOCl impaired protein translocation early in the course of bacterial killing. Together, our findings indicate that MPO-mediated methionine oxidation contributes to bacterial killing by neutrophils. The findings further suggest that protein translocation to the cell envelope is one important pathway targeted for damage.
Assuntos
Escherichia coli/citologia , Metionina/metabolismo , Viabilidade Microbiana , Neutrófilos/enzimologia , Peroxidase/metabolismo , Proteínas de Bactérias/metabolismo , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Citoplasma/efeitos dos fármacos , Citoplasma/metabolismo , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Humanos , Peróxido de Hidrogênio/farmacologia , Ácido Hipocloroso/farmacologia , Metionina Sulfóxido Redutases , Viabilidade Microbiana/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Oxirredução/efeitos dos fármacos , Oxirredutases/metabolismo , Transporte Proteico/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacosRESUMO
Methionine sulphoxide reductases (Msr) reduce methionine sulphoxide to methionine and protect bacteria against reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI). Many organisms express both MsrA, active against methionine-(S)-sulphoxide, and MsrB, active against methionine-(R)-sulphoxide. Mycobacterium tuberculosis (Mtb) expresses MsrA, which protects DeltamsrA-Escherichia coli from ROI and RNI. However, the function of MsrA in Mtb has not been defined, and it is unknown whether Mtb expresses MsrB. We identified MsrB as the protein encoded by Rv2674 in Mtb and confirmed the distinct stereospecificities of recombinant Mtb MsrA and MsrB. We generated strains of Mtb deficient in MsrA, MsrB or both and complemented the mutants. Lysates of singly deficient strains displayed half as much Msr activity as wild type against N-acetyl methionine sulphoxide. However, in contrast to other bacteria, single mutants were no more vulnerable than wild type to killing by ROI/RNI. Only Mtb lacking both MsrA and MsrB was more readily killed by nitrite or hypochlorite. Thus, MsrA and MsrB contribute to the enzymatic defences of Mtb against ROI and RNI.
Assuntos
Proteínas de Bactérias/metabolismo , Ácido Hipocloroso/metabolismo , Mycobacterium tuberculosis/enzimologia , Nitritos/metabolismo , Oxirredutases/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Teste de Complementação Genética , Metionina Sulfóxido Redutases , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Mutagênese Insercional , Mycobacterium tuberculosis/genética , Oxirredutases/genética , Espécies Reativas de Nitrogênio/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Especificidade por SubstratoRESUMO
Methionine sulphoxide reductase A (MSRA) that reduces methionine-S-sulphoxide back to methionine constitutes a catalytic antioxidant mechanism to prevent oxidative damage at multiple sub-cellular loci. This study examined the relative importance of protection of the cytoplasm and mitochondria by MSRA using A-10 vascular smooth muscle cells, a cell type that requires a low level of reactive oxygen species (ROS) for normal function but is readily damaged by higher concentrations of ROS. Adenoviral over-expression of human MSRA variants, targeted to either mitochondria or the cytoplasm, did not change basal viability of non-stressed cells. Oxidative stress caused by treatment with the methionine-preferring oxidizing reagent chloramine-T decreased cell viability in a concentration-dependent manner. Cytoplasmic MSRA preserved cell viability more effectively than mitochondrial MSRA and co-application of S-methyl-L-cysteine, an amino acid that acts as a substrate for MSRA when oxidized, further increased the extent of protection. This suggests an important role for an MSRA catalytic antioxidant cycle for protection of the cytoplasmic compartment against oxidative damage.
Assuntos
Cisteína/farmacologia , Regulação Enzimológica da Expressão Gênica , Músculo Liso Vascular/citologia , Músculo Liso Vascular/metabolismo , Oxirredutases/metabolismo , Animais , Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Cloraminas/farmacologia , Cisteína/análogos & derivados , Regulação Enzimológica da Expressão Gênica/genética , Humanos , Metionina Sulfóxido Redutases , Estresse Oxidativo , Oxirredutases/genética , Ratos , Compostos de Tosil/farmacologiaRESUMO
HDL protects against vascular disease by accepting free cholesterol from macrophage foam cells in the artery wall. This pathway is critically dependent on lecithin:cholesterol acyltransferase (LCAT), which rapidly converts cholesterol to cholesteryl ester. The physiological activator of LCAT is apolipoprotein A-I (apoA-I), the major HDL protein. However, cholesterol removal is compromised if apoA-I is exposed to reactive intermediates. In humans with established cardiovascular disease, myeloperoxidase (MPO) oxidizes HDL, and oxidation by MPO impairs apoA-I's ability to activate LCAT in vitro. Because a single methionine residue in apoA-I, Met-148, resides near the center of the protein's LCAT activation domain, we determined whether its oxidation by MPO could account for the loss of LCAT activity. Mass spectrometric analysis demonstrated that oxidation of Met-148 to methionine sulfoxide associated quantitatively with loss of LCAT activity in both discoidal HDL and HDL(3), the enzyme's physiological substrates. Reversing oxidation with methionine sulfoxide reductase restored HDL's ability to activate LCAT. Discoidal HDL prepared with apoA-I containing a Met-148-->Leu mutation was significantly resistant to inactivation by MPO. Based on structural data in the literature, we propose that oxidation of Met-148 disrupts apoA-I's central loop, which overlaps the LCAT activation domain. These observations implicate oxidation of a single Met in apoA-I in impaired LCAT activation, a critical early step in reverse cholesterol transport.
Assuntos
Apolipoproteína A-I/metabolismo , Colesterol/metabolismo , Metionina/metabolismo , Adulto , Apolipoproteína A-I/química , Transporte Biológico , Humanos , Lipoproteínas HDL/metabolismo , Metionina/análogos & derivados , Oxirredução , Fosfatidilcolina-Esterol O-Aciltransferase/metabolismoRESUMO
Parkinson's disease (PD), a common neurodegenerative disease, is caused by loss of dopaminergic neurons in the substantia nigra. Although the underlying cause of the neuronal loss is unknown, oxidative stress is thought to play a major role in the pathogenesis of PD. The amino acid methionine is readily oxidized to methionine sulfoxide, and its reduction is catalyzed by a family of enzymes called methionine sulfoxide reductases (MSRs). The reversible oxidation-reduction cycle of methionine involving MSRs has been postulated to act as a catalytic antioxidant system protecting cells from oxidative damage. Here, we show that one member of the MSR family, MSRA, inhibits development of the locomotor and circadian rhythm defects caused by ectopic expression of human alpha-synuclein in the Drosophila nervous system. Furthermore, we demonstrate that one way to enhance the MSRA antioxidant system is dietary supplementation with S-methyl-L-cysteine (SMLC), found abundantly in garlic, cabbage, and turnips. SMLC, a substrate in the catalytic antioxidant system mediated by MSRA, prevents the alpha-synuclein-induced abnormalities. Therefore, interventions focusing on the enzymatic reduction of oxidized methionine catalyzed by MSRA represent a new prevention and therapeutic approach for PD and potentially for other neurodegenerative diseases involving oxidative stress.
Assuntos
Cisteína/análogos & derivados , Suplementos Nutricionais , Oxirredutases/administração & dosagem , Doença de Parkinson/prevenção & controle , Animais , Cisteína/administração & dosagem , Drosophila , Humanos , Metionina Sulfóxido Redutases , Atividade Motora/fisiologia , Doença de Parkinson/genética , Doença de Parkinson/metabolismoRESUMO
The reduction of methionine sulfoxide (MetO) is mediated by methionine sulfoxide reductases (Msr). The MsrA and MsrB families can reduce free MetO and MetO within a peptide or protein context. This process is stereospecific with the S- and R-forms of MetO repaired by MsrA and MsrB, respectively. Cell extracts from an MsrA(-)B(-) knockout of Escherichia coli have several remaining Msr activities. This study has identified an enzyme specific for the free form of Met-(R)-O, fRMsr, through proteomic analysis. The recombinant enzyme exhibits the same substrate specificity and is as active as MsrA family members. E. coli fRMsr is, however, 100- to 1,000-fold more active than non-selenocysteine-containing MsrB enzymes for free Met-(R)-O. The crystal structure of E. coli fRMsr was previously determined, but no known function was assigned. Thus, the function of this protein has now been determined. The structural similarity of the E. coli and yeast proteins suggests that most fRMsrs use three cysteine residues for catalysis and the formation of a disulfide bond to enclose a small active site cavity. This latter feature is most likely a key determinant of substrate specificity. Moreover, E. coli fRMsr is the first GAF domain family member to show enzymatic activity. Other GAF domain proteins substitute the Cys residues and others to specifically bind cyclic nucleotides, chromophores, and many other ligands for signal potentiation. Therefore, Met-(R)-O may represent a signaling molecule in response to oxidative stress and nutrients via the TOR pathway in some organisms.
Assuntos
Escherichia coli/enzimologia , Modelos Moleculares , Oxirredutases/metabolismo , Proteínas Recombinantes/metabolismo , Sequência de Aminoácidos , Biologia Computacional , Cinética , Espectrometria de Massas , Metionina/análogos & derivados , Metionina/metabolismo , Metionina Sulfóxido Redutases , Dados de Sequência Molecular , Oxirredutases/genética , Estrutura Terciária de Proteína , Proteínas Recombinantes/genética , Análise de Sequência de DNA , Especificidade por SubstratoRESUMO
In a recent study on the reducing requirement for the methionine sulfoxide reductases (Msr) (Sagher, D., Brunell, D., Hejtmancik, J. F., Kantorow, M., Brot, N. & Weissbach, H. (2006) Proc. Natl. Acad. Sci. U. S. A. 103, 8656-8661), we have shown that thioredoxin, although an excellent reducing system for Escherichia coli MsrA and MsrB and bovine MsrA, is not an efficient reducing agent for either human MsrB2 (hMsrB2) or human MsrB3 (hMsrB3). In a search for another reducing agent for hMsrB2 and hMsrB3, it was recently found that thionein, the reduced, metal-free form of metallothionein, could function as a reducing system for hMsrB3, with weaker activity using hMsrB2. In the present study, we provide evidence that some selenium compounds are potent reducing agents for both hMsrB2 and hMsrB3.
Assuntos
Oxirredutases/química , Selenoproteínas/química , Animais , Bovinos , Colorimetria , Relação Dose-Resposta a Droga , Escherichia coli/metabolismo , Humanos , Metalotioneína/química , Metionina Sulfóxido Redutases , Modelos Químicos , Oxirredutases/metabolismo , Selênio/químicaRESUMO
The PilB protein from Neisseria gonorrhoeae is located in the periplasm and made up of three domains. The N-terminal, thioredoxin-like domain (NT domain) is fused to tandem methionine sulfoxide reductase A and B domains (MsrA/B). We show that the alpha domain of Escherichia coli DsbD is able to reduce the oxidized NT domain, which suggests that DsbD in Neisseria can transfer electrons from the cytoplasmic thioredoxin to the periplasm for the reduction of the MsrA/B domains. An analysis of the available complete genomes provides further evidence for this proposition in other bacteria where DsbD/CcdA, Trx, MsrA, and MsrB gene homologs are all located in a gene cluster with a common transcriptional direction. An examination of wild-type PilB and a panel of Cys to Ser mutants of the full-length protein and the individually expressed domains have also shown that the NT domain more efficiently reduces the MsrA/B domains when in the polyprotein context. Within this frame-work there does not appear to be a preference for the NT domain to reduce the proximal MsrA domain over MsrB domain. Finally, we report the 1.6A crystal structure of the NT domain. This structure confirms the presence of a surface loop that makes it different from other membrane-tethered, Trx-like molecules, including TlpA, CcmG, and ResA. Subtle differences are observed in this loop when compared with the Neisseria meningitidis NT domain structure. The data taken together supports the formation of specific NT domain interactions with the MsrA/B domains and its in vivo recycling partner, DsbD.
Assuntos
Elétrons , Proteínas de Escherichia coli/química , Neisseria gonorrhoeae/química , Oxirredutases/química , Oxirredutases/metabolismo , Tiorredoxinas/química , Sítios de Ligação , Cristalografia por Raios X , Cisteína/química , Metionina Sulfóxido Redutases , Modelos Químicos , Modelos Moleculares , Oxirredução , Estrutura Terciária de Proteína , Tiorredoxinas/isolamento & purificaçãoRESUMO
It has been generally accepted, primarily from studies on methionine sulfoxide reductase (Msr) A, that the biological reducing agent for the members of the Msr family is reduced thioredoxin (Trx), although high levels of DTT can be used as the reductant in vitro. Preliminary experiments using both human recombinant MsrB2 (hMsrB2) and MsrB3 (hMsrB3) showed that although DTT can function in vitro as the reducing agent, Trx works very poorly, prompting a more careful comparison of the ability of DTT and Trx to function as reducing agents with the various members of the Msr family. Escherichia coli MsrA and MsrB and bovine MsrA efficiently use either Trx or DTT as reducing agents. In contrast, hMsrB2 and hMsrB3 show <10% of the activity with Trx as compared with DTT, raising the possibility that, in animal cells, Trx may not be the direct hydrogen donor or that there may be a Trx-independent reducing system required for MsrB2 and MsrB3 activity. A heat-stable protein has been detected in bovine liver that, in the presence of EDTA, can support the Msr reaction in the absence of either Trx or DTT. This protein has been identified as a zinc-containing metallothionein (Zn-MT). The results indicate that thionein (T), which is formed when the zinc is removed from Zn-MT, can function as a reducing system for the Msr proteins because of its high content of cysteine residues and that Trx can reduce oxidized T.
Assuntos
Ergotioneína/metabolismo , Oxirredutases/metabolismo , Animais , Bovinos , Ditiotreitol/farmacologia , Ácido Edético/farmacologia , Ergotioneína/farmacologia , Humanos , Fígado/enzimologia , Metalotioneína/metabolismo , Metionina Sulfóxido Redutases , Oxirredução/efeitos dos fármacos , Oxirredutases/isolamento & purificação , Ratos , Proteínas Recombinantes/metabolismo , Tiorredoxinas/metabolismo , Zinco/metabolismoRESUMO
High density lipoprotein (HDL) isolated from human atherosclerotic lesions and the blood of patients with established coronary artery disease contains elevated levels of 3-chlorotyrosine. Myeloperoxidase (MPO) is the only known source of 3-chlorotyrosine in vivo, indicating that MPO oxidizes HDL in humans. We previously reported that Tyr-192 is the major site that is chlorinated in apolipoprotein A-I (apoA-I), the chief protein in HDL, and that chlorinated apoA-I loses its ability to promote cholesterol efflux from cells by the ATP-binding cassette transporter A1 (ABCA1) pathway. However, the pathways that promote the chlorination of specific Tyr residues in apoA-I are controversial, and the mechanism for MPO-mediated loss of ABCA1-dependent cholesterol efflux of apoA-I is unclear. Using site-directed mutagenesis, we now demonstrate that lysine residues direct tyrosine chlorination in apoA-I. Importantly, methionine residues inhibit chlorination, indicating that they can act as local, protein-bound antioxidants. Moreover, we observed near normal cholesterol efflux activity when Tyr-192 of apoA-I was mutated to Phe and the oxidized protein was incubated with methionine sulfoxide reductase. Thus, a combination of Tyr-192 chlorination and methionine oxidation is necessary for depriving apoA-I of its ABCA1-dependent cholesterol transport activity. Our observations suggest that biologically significant oxidative damage of apoA-I involves modification of a limited number of specific amino acids, raising the feasibility of producing oxidation-resistant forms of apoA-I that have enhanced anti-atherogenic activity in vivo.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Apolipoproteína A-I/metabolismo , HDL-Colesterol/metabolismo , Peroxidase/metabolismo , Transportador 1 de Cassete de Ligação de ATP , Aterosclerose/fisiopatologia , Células Espumosas , Humanos , Lisina/metabolismo , Metionina/metabolismo , Metionina Sulfóxido Redutases , Mutagênese Sítio-Dirigida , Oxirredução , Oxirredutases/metabolismo , Ligação Proteica , Tirosina/metabolismoRESUMO
PURPOSE: Methionine-sulfoxide reductases are unique, in that their ability to repair oxidized proteins and MsrA, which reduces S-methionine sulfoxide, can protect lens cells against oxidative stress damage. To date, the roles of MsrB1, -B2 and -B3 which reduce R-methionine sulfoxide have not been established for any mammalian system. The present study was undertaken to identify those MsrBs expressed by the lens and to evaluate the enzyme activities, expression patterns, and abilities of the identified genes to defend lens cells against oxidative stress damage. METHODS: Enzyme activities were determined with bovine lens extracts. The identities and spatial expression patterns of MsrB1, -B2, and -B3 transcripts were examined by RT-PCR in human lens and 21 other tissues. Oxidative stress resistance was measured using short interfering (si)RNA-mediated gene-silencing in conjunction with exposure to tert-butyl hydroperoxide (tBHP) and MTS viability measurements in SRA04/01 human lens epithelial cells. RESULTS: Forty percent of the Msr enzyme activity present in the lens was MsrB, whereas the remaining enzyme activity was MsrA. MsrB1 (selenoprotein R, localized in the cytosol and nucleus), MsrB2 (CBS-1, localized in the mitochondria), and MsrB3 (localized in the endoplasmic reticulum and mitochondria) were all expressed by the lens. These genes exhibit asymmetric expression patterns between different human tissues and different lens sublocations, including lens fibers. All three genes are required for lens cell viability, and their silencing in lens cells results in increased oxidative-stress-induced cell death. CONCLUSIONS: The present data suggest important roles for both MsrA and -Bs in lens cell viability and oxidative stress protection. The differential tissue distribution and lens expression patterns of these genes, coupled with increased oxidative-stress-induced cell death on their deletion provides evidence that they are important for lens cell function, resistance to oxidative stress, and, potentially, cataractogenesis.
Assuntos
Regulação Enzimológica da Expressão Gênica/fisiologia , Cristalino/enzimologia , Estresse Oxidativo , Oxirredutases/genética , Oxirredutases/metabolismo , Sequência de Aminoácidos , Animais , Bovinos , Sobrevivência Celular , Células Cultivadas , Células Epiteliais/enzimologia , Inativação Gênica/fisiologia , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Cristalino/citologia , Metionina Sulfóxido Redutases , Proteínas dos Microfilamentos , Dados de Sequência Molecular , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Distribuição Tecidual , Fatores de Transcrição , terc-Butil Hidroperóxido/farmacologiaRESUMO
An enzyme that can reduce methionine sulfoxide in proteins was first discovered in Escherichia coli about 25 years ago. It is now apparent that there is a family of enzymes, referred to as methionine sulfoxide reductases (Msr), and in recent years there has been considerable interest in one of the members of the Msr family, MsrA. This enzyme has been shown to protect cells against oxidative damage, which suggests a possible role in a large number of age-related diseases. This review summarizes the history of the discovery of MsrA, properties of the enzyme and its role in protecting cells against oxidative damage. Other members of the Msr family that differ in substrate specificity and localization are described as well as a possible role for the Msr system in drug metabolism. The concept that the Msr system can be used to develop novel drugs that could be catalytic anti-oxidants is discussed.
Assuntos
Estresse Oxidativo , Oxirredutases/metabolismo , Metionina Sulfóxido Redutases , Oxirredutases/químicaRESUMO
Hypoxia/reoxygenation induces cellular injury by promoting oxidative stress. Reversible oxidation of methionine in proteins involving the enzyme peptide methionine sulfoxide reductase type A (MSRA) is postulated to serve a general antioxidant role. Therefore, we examined whether overexpression of MSRA protected cells from hypoxia/reoxygenation injury. Brief hypoxia increased the intracellular reactive oxygen species (ROS) level in PC12 cells and promoted apoptotic cell death. Adenovirus-mediated overexpression of MSRA significantly diminished the hypoxia-induced increase in ROS and facilitated cell survival. Measurements of the membrane potentials of intact mitochondria in PC12 cells and of isolated rat liver mitochondria showed that hypoxia induced depolarization of the mitochondrial membrane. The results demonstrate that MSRA plays a protective role against hypoxia/reoxygenation-induced cell injury and suggest the therapeutic potential of MSRA in ischemic heart and brain disease.
Assuntos
Hipóxia Celular , Citoproteção , Metionina/análogos & derivados , Neurônios/metabolismo , Oxirredutases/fisiologia , Animais , Apoptose , Potenciais da Membrana , Metionina/análise , Metionina Sulfóxido Redutases , Mitocôndrias/fisiologia , Neurônios/patologia , Células PC12 , Ratos , Espécies Reativas de OxigênioRESUMO
Sulindac is a known anti-inflammatory drug that functions by inhibition of cyclooxygenases 1 and 2 (COX). There has been recent interest in Sulindac and other non-steroidal anti-inflammatory drugs (NSAID) because of their anti-tumor activity against colorectal cancer. Studies with sulindac have indicated that it may also function as an anti-tumor agent by stimulating apoptosis. Sulindac is a pro-drug, containing a methyl sulfoxide group, that must be reduced to sulindac sulfide to be active as a COX inhibitor. In the present studies we have developed a simple assay to measure sulindac reduction and tested sulindac as a substrate for 6 known members of the methionine sulfoxide reductase (Msr) family that have been identified in Escherichia coli. Only MsrA and a membrane associated Msr can reduce sulindac to the active sulfide. The reduction of sulindac also has been demonstrated in extracts of calf liver, kidney, and brain. Sulindac reductase activity is also present in mitochondria and microsomes.