RESUMO
The NADPH oxidase of phagocytes catalyzes the conversion of oxygen to O2(-). This multicomponent enzyme complex contains five essential protein components, two in the membrane and three in the cytosol. Unassembled and inactive in resting phagocytes, the oxidase becomes active after translocation of cytosolic components to the membrane to assemble a functional oxidase. Multiple factors regulate its assembly and activity, thus serving to maintain this highly reactive system under spatial and temporal control until recruited for antimicrobial or proinflammatory events. The recent identification of homologs of one of the membrane components in nonphagocytic cells will expand understanding of the biological contexts in which this system may function.
Assuntos
NADPH Oxidases/metabolismo , Neutrófilos/enzimologia , Grupo dos Citocromos b/metabolismo , Regulação Enzimológica da Expressão Gênica , Fosfoproteínas/metabolismoRESUMO
In this work, we demonstrate that the phosphatidylinositol 3,4,5-trisphosphate-binding protein JFC1 is an ATP-binding protein with magnesium-dependent ATPase activity. We show that JFC1 specifically binds to the ATP analog 8-azido-[alpha-(32)P]ATP. The affinity of JFC1 for [alpha-(32)P]ATP was 10x greater than its affinity for [alpha-(32)P]ADP; the protein did not appear to bind to [alpha-(32)P]GTP. JFC1 hydrolyzed [alpha-(32)P]ATP in a Mg(2+)-dependent manner. JFC1, which also hydrolyzed dATP, has a relatively high affinity for ATP, with a K(M) value of 58 microM, and a k(cat) value of 2.27 per min. The predicted amino acid sequence of JFC1 denotes a putative nucleotide-binding site similar to those in the GHKL ATPase/kinase superfamily. However, a truncation of JFC1 that contains boxes G2 and G3 but not boxes N and G1 of the Bergerat-binding site showed residual ATPase activity. Secondly, the antitumor ATP-mimetic agent geldanamycin, which inhibits the ATPase activity of Hsp-90, did not affect JFC1 ATPase. Therefore, the characteristics of the ATP-binding site of JFC1 are unique. Phosphatidylinositol 3,4,5-trisphosphate, a high-affinity ligand of JFC1 did not affect its ATPase kinetics parameters, suggesting that the phosphoinositide have a different role in JFC1 function.
Assuntos
Difosfato de Adenosina/metabolismo , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/metabolismo , Azidas/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Marcadores de Afinidade , Sequência de Aminoácidos , Animais , Benzoquinonas , Sítios de Ligação , Reagentes de Ligações Cruzadas , Nucleotídeos de Desoxiadenina/metabolismo , Glutationa Transferase/metabolismo , Guanosina Trifosfato/metabolismo , Cinética , Lactamas Macrocíclicas , Magnésio/farmacologia , Dados de Sequência Molecular , Quinonas/farmacologia , Proteínas Recombinantes de Fusão/metabolismo , Deleção de Sequência , Especificidade por SubstratoRESUMO
Fish oils are known for their anti-inflammatory effects. In this paper we investigated the influence of eicosapentaenoic acid and docosahexaenoic acid (omega-3 fatty acids), as well as docosapentaenoic acid, a metabolic product of omega-3 fatty acid metabolism, on O2(-)-production catalyzed by the NADPH oxidase in whole neutrophils and in a cell-free system consisting of neutrophil membranes and cytosol. As a standard we used arachidonic acid (an omega-6 fatty acid) found in a high proportion in the Western diet, and known as an effective activator of the oxidase in both systems. Our data show that with omega-3 fatty acids, the O2(-)-production in both systems is reduced as compared to the effect of arachidonic acid. The effects are more pronounced with increasing carbon chain length and increasing numbers of double bonds. Our results suggest another mechanism besides the inhibition of eicosanoid and cytokine production to explain the beneficial effects of fish oils in reducing inflammation.
Assuntos
Ácidos Docosa-Hexaenoicos/farmacologia , Ácido Eicosapentaenoico/farmacologia , Ácidos Graxos Insaturados/farmacologia , NADPH Oxidases/metabolismo , Neutrófilos/efeitos dos fármacos , Adulto , Ácido Araquidônico/farmacologia , Membrana Celular/enzimologia , Sistema Livre de Células , Citosol/enzimologia , Gorduras na Dieta/farmacologia , Ativação Enzimática , Ácidos Graxos Ômega-3/farmacologia , Óleos de Peixe/farmacologia , Humanos , Mediadores da Inflamação/metabolismo , Neutrófilos/enzimologia , Explosão Respiratória/efeitos dos fármacos , Superóxidos/metabolismoRESUMO
We have employed a yeast two-hybrid system to screen a B lymphoblast-derived cDNA library, searching for regulatory components of the NADPH oxidase. Using as bait the C-terminal half of p67(phox), which contains both Src homology 3 domains, we have cloned JFC1, a novel human 62-kDa protein. JFC1 possesses two C2 domains in tandem. The C2A domain shows homology with the C2B domain of synaptotagmins. JFC1 mRNA was abundantly expressed in bone marrow and leukocytes. The expression of JFC1 in neutrophils was restricted to the plasma membrane/secretory vesicle fraction. We confirmed JFC1-p67(phox) association by affinity chromatography. JFC1-containing beads pulled down both p67(phox) and p47(phox) subunits from neutrophil cytosol, but when the recombinant proteins were used, only p67(phox) bound to JFC1, indicating that JFC1 binds to the cytosolic complex via p67(phox) without affecting the interaction between p67(phox) and p47(phox). In contrast to synaptotagmins, JFC1 was unable to bind to inositol 1,3,4,5-tetrakisphosphate but did bind to phosphatidylinositol 3,4,5-trisphosphate and to a lesser extent to phosphatidylinositol 3,4-diphosphate. From the data presented here, it is proposed that JFC1 is acting as an adaptor protein between phosphatidylinositol 3-kinase products and the oxidase cytosolic complex.
Assuntos
Proteínas de Ligação ao Cálcio , Leucócitos/enzimologia , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , NADPH Oxidases/química , NADPH Oxidases/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Northern Blotting , Membrana Celular/metabolismo , Cromatografia de Afinidade , Citosol/metabolismo , DNA Complementar/metabolismo , Biblioteca Gênica , Humanos , Fosfatos de Inositol/metabolismo , Leucócitos/metabolismo , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Neutrófilos/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Fosfoproteínas/metabolismo , Ligação Proteica , Biossíntese de Proteínas , Estrutura Terciária de Proteína , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Sinaptotagminas , Acetato de Tetradecanoilforbol/metabolismo , Distribuição Tecidual , Técnicas do Sistema de Duplo-Híbrido , Domínios de Homologia de srcRESUMO
Activation of the phagocyte NADPH oxidase complex requires the assembly of the cytosolic factors p47(PHOX), p67(PHOX), p40(PHOX), and Rac1 or Rac2, with the membrane-bound cytochrome b(558). Whereas the interaction of p47(PHOX) with cytochrome b(558) is well established, an interaction between p67(PHOX) and cytochrome b(558) has never been investigated. We report here a direct interaction between p67(PHOX) and cytochrome b(558). First, labeled p67(PHOX) recognizes a 91-kDa band in specific granules from a normal patient but not from a cytochrome b(558)-deficient patient. Second, p67(PHOX) binds to cytochrome b(558) that has been bound to nitrocellulose. Third, GTP-p67(PHOX) bound to glutathione agarose is able to pull down cytochrome b(558.) Rac1-GTP or Rac1-GDP increased the binding of p67(PHOX) to cytochrome b(558), suggesting that at least one of the oxidase-related functions of Rac1 is to promote the interaction between p67(PHOX) and cytochrome b(558).
Assuntos
Grupo dos Citocromos b/metabolismo , Glicoproteínas de Membrana/metabolismo , NADPH Oxidases/metabolismo , Neutrófilos/enzimologia , Fosfoproteínas/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Humanos , NADH NADPH Oxirredutases/metabolismo , NADPH Oxidase 2 , NADPH Oxidases/genética , Fosfoproteínas/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Explosão Respiratória , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
L-Carnitine facilitates the transport of fatty acids into the mitochondrial matrix where they are used for energy production. Recent studies have shown that L-carnitine is capable of protecting the heart against ischemia/reperfusion injury and has beneficial effects against Alzheimer's disease and AIDS. The mechanism of action, however, is not yet understood. In the present study, we found that in Jurkat cells, L-carnitine inhibited apoptosis induced by Fas ligation. In addition, 5 mM carnitine potently inhibited the activity of recombinant caspases 3, 7 and 8, whereas its long-chain fatty acid derivative palmitoylcarnitine stimulated the activity of all the caspases. Palmitoylcarnitine reversed the inhibition mediated by carnitine. Levels of carnitine and palmitoyl-CoA decreased significantly during Fas-mediated apoptosis, while palmitoylcarnitine formation increased. These alterations may be due to inactivation of beta-oxidation or to an increase in the activity of the enzyme that converts carnitine to palmitoylcarnitine, carnitine palmitoyltransferase I (CPT I). In support of the latter possibility, fibroblasts deficient in CPT I activity were relatively resistant to staurosporine-induced apoptosis. These observations suggest that caspase activity may be regulated in part by the balance of carnitine and palmitoylcarnitine.
Assuntos
Apoptose/efeitos dos fármacos , Carnitina/farmacologia , Caspases/metabolismo , Palmitoilcarnitina/farmacologia , Receptor fas/fisiologia , Acilação , Carnitina/análogos & derivados , Carnitina/antagonistas & inibidores , Carnitina/metabolismo , Carnitina O-Palmitoiltransferase/deficiência , Carnitina O-Palmitoiltransferase/genética , Carnitina O-Palmitoiltransferase/metabolismo , Caspase 3 , Caspase 7 , Caspase 8 , Caspase 9 , Inibidores de Caspase , Linhagem Celular , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Fibroblastos , Humanos , Células Jurkat , Palmitoil Coenzima A/metabolismo , Palmitoilcarnitina/antagonistas & inibidores , Palmitoilcarnitina/metabolismo , Estaurosporina/farmacologiaRESUMO
Neutrophils and other phagocytes manufacture O(2)(-) (superoxide) by the one-electron reduction of oxygen at the expense of NADPH. Most of the O(2)(-) reacts with itself to form H(2)O(2) (hydrogen peroxide). From these agents a large number of highly reactive microbicidal oxidants are formed, including HOCl (hypochlorous acid), which is produced by the myeloperoxidase-catalyzed oxidation of Cl(-) by H(2)O(2); OH(*) (hydroxyl radical), produced by the reduction of H(2)O(2) by Fe(++) or Cu(+); ONOO(-) (peroxynitrite), formed by the reaction between O(2)(-) and NO(*); and many others. These reactive oxidants are manufactured for the purpose of killing invading microorganisms, but they also inflict damage on nearby tissues, and are thought to be of pathogenic significance in a large number of diseases. Included among these are emphysema, acute respiratory distress syndrome, atherosclerosis, reperfusion injury, malignancy and rheumatoid arthritis.
Assuntos
Estresse Oxidativo , Fagócitos/fisiologia , Fagocitose/fisiologia , Humanos , Óxido Nítrico/metabolismo , Oxidantes/metabolismo , Estresse Oxidativo/fisiologia , Superóxidos/metabolismoRESUMO
The nicotinamide adenine dinucleotide phosphate (NADPH) binding site of the NADPH oxidase complex is believed to be located on the beta, subunit of cytochrome b558. However, our previous studies showed that p67PHOX also contains an NADPH binding site that is essential for normal oxidase activity and that p67PHOX is able to mediate a slow electron transfer from a reduced pyridine nucleotide to an artificial electron acceptor. Using both affinity labeling and fluorescence quenching, we have obtained further evidence that p67PHOX is able to bind NADPH. We have used a number of truncated forms of p67PHOX, including p67PHOX(1-243), p67PHOX(1-210), p67PHOX(1-199), and p67PHOX(244-526) (where the numbers represent the initial and final amino acids in the truncated p67PHOX) in order to localize the binding site. We found that NADPH could bind to p67PHOX(1-243), p67PHOX(1-210), and p67PHOX(1-199) but not to p67PHOX(244-526). The p67PHOX(1-199) fragment consists largely of four tetratricopeptide (TPR) domains. We showed further that Rac2-GTP gamma S and to a lesser extent Rac2-GDP beta S could modulate the binding of NADPH to p67PHOX.
Assuntos
NADPH Oxidases/metabolismo , NADP/metabolismo , Neutrófilos/enzimologia , Fragmentos de Peptídeos/metabolismo , Fosfoproteínas/metabolismo , Sequências Repetitivas de Aminoácidos , Marcadores de Afinidade/metabolismo , Sítios de Ligação/genética , Ativação Enzimática/efeitos dos fármacos , Humanos , Mutagênese Insercional , NADP/análogos & derivados , NADP/antagonistas & inibidores , NADP/farmacologia , NADPH Oxidases/antagonistas & inibidores , Fragmentos de Peptídeos/antagonistas & inibidores , Fragmentos de Peptídeos/genética , Fosfoproteínas/antagonistas & inibidores , Fosfoproteínas/genética , Estrutura Terciária de Proteína , Proteínas Recombinantes/síntese química , Proteínas Recombinantes/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo , Proteína RAC2 de Ligação ao GTPRESUMO
The best known NADPH oxidase is that of phagocytes-neutrophils and monocytes. In these cells, the enzyme manufactures large quantities of O2- and other reactive oxidants that are used for the purpose of killing invading microorganisms. Recent studies, however, have suggested that a number of other tissues contain NADPH oxidases. In contrast to the very vigorous production of oxidants by phagocytes, rates of oxidant production by these other cell types are quite low. Oxidant production by these cells is generally thought to serve a signaling function.
Assuntos
Endotélio Vascular/enzimologia , NADPH Oxidases/metabolismo , Animais , Anticolesterolemiantes/farmacologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Humanos , Fagócitos/enzimologia , Espécies Reativas de Oxigênio/metabolismo , Superóxidos/metabolismoRESUMO
Targeted mutation of the myeloid transcription factor C/EBPepsilon in mice results in gram-negative septic death at 3 to 5 months of age. This study defines the underlying molecular defects in their terminal granulocytic differentiation. The mRNA for the precursor protein of the cathelin-related antimicrobial peptides was almost completely absent in the bone marrow cells of C/EBPepsilon-/- mice. This finding may help explain their susceptibility to gram-negative sepsis, because both are bacteriocidal peptides with potent activity against gram-negative bacteria. Superoxide production was found to be reduced in both granulocytes and monocytes of C/EBPepsilon-/- mice. While gp91 phox protein levels were normal, p47phox protein levels were considerably reduced in C/EBPepsilon -/- granulocytes/monocytes, possibly limiting the assembly of the NADPH oxidase. In addition, expression of mRNA of the secondary and tertiary granule proteins, lactoferrin and gelatinase, were not detected, and levels of neutrophil collagenase mRNA were reduced in bone marrow cells of the knock-out mice. The murine lactoferrin promoter has a putative C/EBP site close to the transcription start site. C/EBPepsilon bound to this site in electromobility shift assay studies and mutation of this site abrogated binding to it. A mutation in the C/EBP site reduced the activity of the promoter by 35%. Furthermore, overexpression of C/EBPepsilon in U937 cells increased the activity of the wild-type lactoferrin promoter by 3-fold. In summary, our data implicate C/EBPepsilon as a critical factor of host antimicrobial defense and suggests that it has a direct role as a positive regulator of expression of lactoferrin in vivo.
Assuntos
Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica , Lactoferrina/genética , Neutrófilos/fisiologia , Proteínas Nucleares/genética , Animais , Proteínas Estimuladoras de Ligação a CCAAT , Camundongos , Camundongos Knockout , Microscopia Eletrônica , Mutação , Neutrófilos/ultraestrutura , Regiões Promotoras Genéticas , RNA Mensageiro/análise , RNA Mensageiro/genéticaRESUMO
The leukocyte NADPH oxidase catalyzes the one-electron reduction of oxygen to O2- at the expense of NADPH. It is a multicomponent enzyme comprising a membrane-bound flavocytochrome (cytochrome b558) and at least four cytosolic components: p47PHOX, p67PHOX, p40PHOX, and Rac, a small GTPase. All the oxidase components except p40PHOX are required for enzyme activity. Many aspects of their function, however, are unclear. Using the electron acceptor ferricyanide, we found that recombinant p67PHOX from baculovirus-infected Sf9 cells could mediate the dehydrogenation of NADPH. NADPH dehydrogenation was not dependent on FAD and was insensitive to superoxide dismutase. Several control experiments showed that NADPH dehydrogenation was accomplished by p67PHOX, not by a trace contaminant in the p67PHOX preparation. The NADPH dehydrogenase activity of p67PHOX was proportional to enzyme concentration, and showed saturation kinetics with NADPH (Km 92 +/- 5 microM), but was inhibited at high concentrations of ferricyanide. NADH was also used as a substrate by p67PHOX (Km 123 +/- 38 microM). Taken together, these results show that p67PHOX is able to mediate pyridine nucleotide dehydrogenation. These findings raise the possibility that p67PHOX might participate directly in electron transfer between NADPH and the oxidase flavin.
Assuntos
Citosol/enzimologia , NADPH Desidrogenase/metabolismo , Neutrófilos/enzimologia , Fosfoproteínas/metabolismo , Animais , Baculoviridae/genética , Ativação Enzimática , Ferricianetos/química , Ferricianetos/metabolismo , NADP/química , NADP/metabolismo , NADPH Desidrogenase/química , Oxirredução , Fosfoproteínas/química , Fosfoproteínas/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Spodoptera/genéticaRESUMO
The leukocyte NADPH oxidase is an enzyme present in phagocytes and B lymphocytes that when activated catalyzes the production of O-2 from oxygen at the expense of NADPH. A correlation between the activation of the oxidase and the phosphorylation of p47(PHOX), a cytosolic oxidase component, is well recognized in whole cells, and direct evidence for a relationship between the phosphorylation of this oxidase component and the activation of the oxidase has been obtained in a number of cell-free systems containing neutrophil membrane and cytosol. Using superoxide dismutase-inhibitable cytochrome c reduction to quantify O-2 production, we now show that p47(PHOX) phosphorylated by protein kinase C activates the NADPH oxidase not only in a cell-free system containing neutrophil membrane and cytosol, but also in a system in which the cytosol is replaced by the recombinant proteins p67(PHOX), Rac2, and phosphorylated p47(PHOX), suggesting that neutrophil plasma membrane plus those three cytosolic proteins are both necessary and sufficient for oxidase activation. In both the cytosol-containing and recombinant cell-free systems, however, activation by SDS yielded greater rates of O-2 production than activation by protein kinase C-phosphorylated p47(PHOX), indicating that a system that employs protein kinase C-phosphorylated p47(PHOX) as the sole activating agent, although more physiological than the SDS-activated system, is nevertheless incomplete.
Assuntos
Leucócitos/enzimologia , NADPH Oxidases/metabolismo , Proteína Quinase C/metabolismo , Sistema Livre de Células , Grupo dos Citocromos b/metabolismo , Grupo dos Citocromos c/metabolismo , Citosol/metabolismo , Ativação Enzimática , Proteínas de Ligação ao GTP/metabolismo , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Humanos , Neutrófilos/enzimologia , Neutrófilos/metabolismo , Fosfoproteínas/metabolismo , Fosforilação , Espécies Reativas de Oxigênio/metabolismo , Proteínas Recombinantes/metabolismo , Dodecilsulfato de Sódio/farmacologia , Proteínas rac de Ligação ao GTPRESUMO
The leukocyte NADPH oxidase catalyzes the reduction of oxygen to superoxide (O-2) at the expense of NADPH in phagocytes and B lymphocytes. The enzyme is dormant in resting cells but becomes active when the cells are exposed to appropriate stimuli. During oxidase activation, the highly basic cytosolic oxidase component p47(PHOX) becomes phosphorylated on several serines and migrates to the plasma membrane. We report here that p47(PHOX)-deficient B lymphoblasts expressing the p47(PHOX) S359A/S370A or p47(PHOX) S359K/S370K double mutation show dramatically reduced levels of enzyme activity and phosphorylation of p47(PHOX) as compared with the same cells expressing wild type p47(PHOX). In addition, these mutant p47(PHOX) proteins fails to translocate to the plasma membrane when the cells are stimulated. In contrast, normal phosphorylation and translocation are seen in mutants containing aspartate or glutamate at positions 359 and 370, but oxidase activity is still greatly reduced. These results imply that a negative charge at position 359 and/or 370 is sufficient to allow the phosphorylation and translocation of p47(PHOX) to take place but that features unique to a phosphorylated hydroxyamino acid are required to support O-2 production. These findings, plus those from an earlier study (Inanami, O., Johnson, J. L., McAdara, J. K., El Benna, J., Faust, L. P., Newburger, P. E., and Babior, B. M. (1998) J. Biol. Chem. 273, 9539-9543), suggest that oxidase activation requires 1) the sequential phosphorylation of at least two serines on p47(PHOX): Ser-359 or Ser-370, followed by Ser-303 or Ser-304; and 2) the translocation of p47(PHOX) to the membrane at some point after the first phosphorylation takes place.
Assuntos
Citosol/enzimologia , Leucócitos/enzimologia , NADPH Oxidases/metabolismo , Fosfoproteínas/metabolismo , Ácido Araquidônico/farmacologia , Transporte Biológico , Ativação Enzimática , Células-Tronco Hematopoéticas/enzimologia , Linfócitos/enzimologia , Mutação , NADPH Oxidases/efeitos dos fármacos , NADPH Oxidases/genética , Fosfoproteínas/efeitos dos fármacos , Fosfoproteínas/genética , Fosforilação , Proteínas Recombinantes/metabolismo , Serina/metabolismoRESUMO
Several groups have reported that during apoptosis, cytochrome c is released from the mitochondria into the cytosol, but we have found that in apoptotic cells the cytochrome appears to remain with the mitochondria. In hopes of reconciling these findings, we compared the results obtained from cells disrupted by our method (nitrogen cavitation) with those obtained using the cell-disruption method employed by others (homogenization). We observed that at 2 h, cytochrome c levels in apoptotic cytosols from homogenized cells exceeded control levels, whereas cytochrome c levels in apoptotic cytosols from cavitated cells were similar to control. Outer membranes of homogenized mitochondria appeared damaged because the mitochondria had become permeable to cytochrome c, whereas outer membranes of cavitated mitochondria excluded the cytochrome. 4 h after Fas ligation, both cavitated and homogenized mitochondria had released small amounts of cytochrome c into the cytosol, whereas after 6 h the cytochrome had disappeared from the cell lysate. We believe that the differences between our results and those reported by others were due to 1) our examining the cells after a short (2 h) incubation with the anti-Fas antibody, and 2) our use of nitrogen cavitation instead of homogenization to disrupt the cells.
Assuntos
Apoptose/fisiologia , Grupo dos Citocromos c/metabolismo , Mitocôndrias/fisiologia , Receptor fas/imunologia , Anticorpos/farmacologia , Fracionamento Celular/métodos , Citosol/metabolismo , Digitonina/farmacologia , Humanos , Membranas Intracelulares/fisiologia , Células Jurkat , Proteínas de Membrana/metabolismo , Oxirredução , Permeabilidade , Ligação Proteica/fisiologiaRESUMO
The leukocyte NADPH oxidase is an enzyme in phagocytes and B lymphocytes that when activated catalyzes the production of O-2 from oxygen and NADPH. During oxidase activation, serine residues in the C-terminal quarter of the oxidase component p47(PHOX) become extensively phosphorylated, the protein acquiring as many as 9 phosphate residues. In a study of 11 p47(PHOX) mutants, each containing an alanine instead of a serine at a single potential phosphorylation site, we found that all but S379A corrected the defect in O-2 production in Epstein-Barr virus (EBV)-transformed p47(PHOX)-deficient B cells (Faust, L. P., El Benna, J., Babior, B. M., and Chanock, S. J. (1995) J. Clin. Invest. 96, 1499-1505). In particular, O-2 production was restored to these cells by the mutants S303A and S304A. Therefore, apart from serine 379, whose state of phosphorylation in the activated oxidase is unclear, no single potential phosphorylation site appeared to be essential for oxidase activation. We now report that the double mutant p47(PHOX) S303A/S304A was almost completely inactive when expressed in EBV-transformed p47(PHOX)-deficient B cells, even though it was expressed in normal amounts in the transfected cells and was able to translocate to the plasma membrane when the cells were stimulated. In contrast, the double mutant p47(PHOX) S303E/S304E was able to support high levels of O-2 production by EBV-transformed p47(PHOX)-deficient B cells. The surprising discovery that the double mutant S303K/S304K was also able to support considerable O-2 production suggests either that the effect of phosphorylation is related to the increase in hydrophilicity around serines 303 and 304 or that activation involves the formation of a metal bridge between the phosphorylated serines and another region of the protein.
Assuntos
Leucócitos/metabolismo , NADPH Oxidases/sangue , Fosfoproteínas/sangue , Serina , Acetato de Tetradecanoilforbol/farmacologia , Linfócitos B/metabolismo , Sequência de Bases , Células Cultivadas , Primers do DNA , Ativação Enzimática , Humanos , Medições Luminescentes , Mutagênese Sítio-Dirigida , Fosfoproteínas/química , Fosforilação , Fosfosserina/metabolismo , Mutação Puntual , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Superóxidos/metabolismo , TransfecçãoRESUMO
The leukocyte NADPH oxidase is a multi-subunit enzyme that catalyzes the reduction of oxygen to O2- at the expense of a reduced pyridine nucleotide. We have used site-directed mutagenesis to examine the functional role of the four cysteines in p47PHOX, one of the subunits of the oxidase. For these experiments, mutant proteins in which a single cysteine was replaced with alanine were expressed in p47PHOX-deficient Epstein-Barr virus-transformed B lymphoblasts, and O2- production by these transfected cells was measured. The activity of the mutant C98A was similar to that of wild type, but the maximum rate of O2- production by C196A was significantly larger than seen with wild type. The other two mutants (i.e., C111A and C378A) differed from wild type not only in maximum O2- production, but also in the time required for activation, which was considerably delayed with both of these mutants. The similarity in the time courses of oxidase activation with the C111A and C378A mutants, and the finding that C378A occurs in the sequence CSE, raises the possibility that these cysteines may be involved in redox regulation of oxidase activity.
Assuntos
Linfócitos B/enzimologia , Cisteína/metabolismo , NADPH Desidrogenase/metabolismo , Fosfoproteínas/metabolismo , Cisteína/genética , Ativação Enzimática , Células-Tronco Hematopoéticas/enzimologia , Mutagênese Sítio-Dirigida , NADPH Oxidases , Oxirredução , Espécies Reativas de OxigênioRESUMO
We examined the ability of C-terminal deletion mutants of p47PHOX, a cytosolic subunit of the leukocyte NADPH oxidase, to support the activity of the oxidase in two different cell-free systems, one using protein kinase C and the other an anionic amphiphile (SDS or arachidonic acid) as the oxidase-activating agent. Two deletion mutants were studied: p47PHOXdelta330 and p47PHOXdelta348, each named according to the first residue of the deleted polypeptide. Wild-type (WT) p47PHOX and both mutants were phosphorylated by protein kinase C, but the WT protein was the most heavily phosphorylated, containing 6.0 +/- 0.5 mol phosphate/mol protein. Of the two deletion mutants, only p47PHOXdelta348 could support oxidase activity, and then only in the amphiphile-activated system; neither of the mutants supported oxidase activity in the system activated by protein kinase C. Translocation correlated with activity: WT p47PHOX translocated to the membrane in response to both protein kinase C and amphiphile, but p47PHOXdelta348 translocated only in the amphiphile-activated system. Comparison of these findings with the results of earlier studies suggests that the phosphorylation of p47PHOX is an important component of oxidase activation. The findings provide no information, however, about whether amphiphiles participate in the activation process in intact cells. Consequently, a mechanism of in vivo oxidase activation involving both phosphorylation and the generation of an amphiphile remains a distinct possibility.