Nucleoside-triphosphate binding of the two cytosolic components of the respiratory burst oxidase system: Evidence for its inhibition by the 2′,3′-dialdehyde derivative of NADPH and desensitization in their translocated states

Mizunari, H.; Kakinuma, Katsuyoshi; Suzuki, Kingo; Namiki, Hideo; Kuratsuji, Tadatoshi; Tsunawaki, Shohko

doi:10.1016/0167-4889(93)90092-4

Cited by 10 publications

(13 citation statements)

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“…NADPH oxidase activity was quantified by the rate of superoxide dismutase (SOD)-inhibitable ferricytochrome c reduction by O 2 Ϫ , using a dual-wavelength spectrophotometer recording ⌬A 550-540 (Hitachi 557). The oxidase activity of intact cells was determined by the O 2 Ϫ generation stimulated with 100 ng of PMA/10 6 cells/ml; the oxidase activity of membranes was measured after adding 0.2 mM NADPH (37,55). At maximal velocity, 200 U of SOD/ml was added to determine the net cytochrome c reduced by O 2 Ϫ .…”

Section: Methodsmentioning

confidence: 99%

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Fungal Metabolite Gliotoxin Inhibits Assembly of the Human Respiratory Burst NADPH Oxidase

et al. 2004

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Reactive oxygen species are a critical weapon in the killing of Aspergillus fumigatus by polymorphonuclear leukocytes (PMN), as demonstrated by severe aspergillosis in chronic granulomatous disease. In the present study, A. fumigatus-produced mycotoxins (fumagillin, gliotoxin [GT], and helvolic acid) are examined for their effects on the NADPH oxidase activity in human PMN. Of these mycotoxins, only GT significantly and stoichiometrically inhibits phorbol myristate acetate (PMA)-stimulated O 2 ؊ generation, while the other two toxins are ineffective. The inhibition is dependent on the disulfide bridge of GT, which interferes with oxidase activation but not catalysis of the activated oxidase. Specifically, GT inhibits PMA-stimulated events: p47 phox phosphorylation, its incorporation into the cytoskeleton, and the membrane translocation of p67 phox , p47 phox , and p40 phox , which are crucial steps in the assembly of the active NADPH oxidase. Thus, damage to p47 phox phosphorylation is likely a key to inhibiting NADPH oxidase activation. GT does not inhibit the membrane translocation of Rac2. The inhibition of p47 phox phosphorylation is due to the defective membrane translocation of protein kinase C (PKC) ␤II rather than an effect of GT on PKC ␤II activity, suggesting a failure of PKC ␤II to associate with the substrate, p47 phox , on the membrane. These results suggest that A. fumigatus may confront PMN by inhibiting the assembly of the NADPH oxidase with its hyphal product, GT.The best-known NADPH oxidase occurs in phagocytes and provides large quantities of reactive oxygen species (ROS) to oxidatively modify microbes as a microbicidal mechanism. The phagocyte NADPH oxidase is a highly regulated multisubunit enzyme composed of p67 phox , p47 phox , p40 phox , and Rac2 in the cytosol, as well as flavocytochrome b 558 , which consists of gp91 phox and p22 phox , in the membrane (4,9,15,29,36,39,57). The enzyme is dormant in resting cells, with these phox (for phagocyte oxidase) components being distributed to their cellular compartments. However, once phagocytes encounter invading microbes or soluble stimulants, such as phorbol myristate acetate (PMA), cytosolic phox components and Rac2 migrate to the membrane to assemble with flavocytochrome b 558 , forming the active enzyme. The activated flavocytochrome b 558 works as the catalytic redox center, where electrons are transferred from NADPH to molecular oxygen to generate O 2 Ϫ , a precursor of ROS. Recently, increasing attention has been directed toward the Nox/Duox family in nonphagocytes (33), which is composed of homologues of gp91 phox . Although the rates of ROS production are quite low in nonphagocytes, their ROS are believed to play important roles in cell signaling, hypoxic response, the immune system, etc.Over the past decade, the biochemistry of phagocyte NADPH oxidase has been intensively studied, particularly concerning the roles of Src homology 3 (SH3) domains and proline-rich regions (PRR), as well as those of other domains recently identified in...

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Section: Methodsmentioning

confidence: 99%

“…Detection of the NADPH oxidase components was performed as previously reported (37,54,55). Antibodies against either the C-or N-terminal polypeptides of p67 phox , p47 phox , p40 phox , and p22 phox were raised in rabbits.…”

Section: Methodsmentioning

confidence: 99%

Fungal Metabolite Gliotoxin Inhibits Assembly of the Human Respiratory Burst NADPH Oxidase

et al. 2004

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“…It has been recently reported that p47 binds covalently GTP analogues (Klinger and Aviram, 1992) and that both p47 and p67 have the ability to bind GTP (Mizunari et al, 1993). Experiments were therefore carried out to examine whether oxidase activation could be stimulated with recombinant p47 and p67 loaded with GTP.…”

Section: Compared Efficiencies Of Purified Prenylated and Unprocessedmentioning

confidence: 99%

Activation of the O^–₂‐Generating NADPH Oxidase in a Semi–Recombinant Cell‐Free System

Fuchs

Dagher

Jouan

et al. 1994

European Journal of Biochemistry

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The neutrophil NADPH oxidase activation factors, p47, p67 and the small guanosine-nucleotidebinding regulatory (G) protein Racl , were expressed in a baculovirus/insect cell system and purified. In coinfection experiments in which Sf9 cells overexpressed concomitantly p47, p67 and Racl , the latter was not detected in the p47-p67 complex. The propensity of p47 and p67 to associate together was used to purify recombinant p67 from baculovirus-infected Sf9 cells. 20% of the overexpressed Racl in infected Sf9 cells was prenylated and was extracted with low doses of detergent from membranes. Elicitation of full oxidase activity on crude neutrophil membranes using a cell-free system required addition of recombinant p47 and p67, but not that of Rac. In contrast, in the case of KC1-washed membranes, addition of Rac, prenylated or unprocessed, together with p47 and p67 was found to enhance oxidase activation up to fivefold. In all experiments, the amount of added arachidonic acid was optimized. In contrast to prenylated Rac, non-prenylated Rac had to be loaded with guanosine 5'-(3-thiotriphosphate) (GTP[S]) to exhibit full activation efficiency. In the cell-free system used, Rac was shown to be the mediator of the GTP[S] effect. The results suggest that the plasma membrane of resting neutrophils contains a sufficient amount of prenylated Rac for efficient oxidase activation. We therefore propose that Rac has a membrane-associated role and helps to dock and position p47 and p67 on the flavocytochrome b component of the oxidase complex.Phagocytes, among which neutrophils are the most studied, respond to soluble or particulate stimuli, by a large increase in oxygen consumption with concomitant production of the superoxide anion 0;. The enzyme complex which catalyzes the monoelectronic reduction of 0, to 0; is termed NADPH oxidase and is located in the plasma membrane. Oxidase activation corresponds to the functional assembly of a number of components including an heterodimeric redox protein of hydrophobic nature, the flavocytochrome b,,,, and water soluble proteins referred to as cytosolic factors, p47 and p67. The monomeric guanosine-nucleotide-binding regulatory (G) proteins, Rac and possibly RaplA, are also involved in the modulation of oxidase activation in a manner which is not yet clear (for review see Babior, 1992; Morel et al., 1991 ;Segal and Abo, 1993).The observation that addition of GTP to a cell-free system of oxidase activation significantly enhanced the elicited oxidase activity (Seifert and Schultz, 1987;Gabig et al., 1987;Ligeti et al., 1988) was the beginning of a series of investigations that ended with in vivo experiments in which Correspondence to A. Fuchs,

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“…For this purpose, the isolated membranes and cytosol from GT-treated neutrophils were tested for the ability to reconstitute O 2 Ϫ generation in the cell-free activation assay. In the assay, an anionic amphiphile such as MA or SDS is used as a stimulant (6,44) to provoke the disruption of the autoinhibited state of p47 phox , necessary to allow its association with the p22 phox subunit of flavocytochrome b 558 , as well as with p67 phox , eventually leading to oxidase activation (1,21,23,25,39,53,59). The cell-free activation assay thus allows the verification of the integrity of the oxidase components in reconstituting the enzyme activity.…”

Section: Which Cellular Target Membrane or Cytosolic Components Is mentioning

confidence: 99%

Fungal Metabolite Gliotoxin Targets Flavocytochrome b ₅₅₈ in the Activation of the Human Neutrophil NADPH Oxidase

et al. 2005

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Fungal gliotoxin (GT) is a potent inhibitor of the O 2؊ -generating NADPH oxidase of neutrophils. We reported that GT-treated neutrophils fail to phosphorylate p47 phox , a step essential for the enzyme activation, because GT prevents the colocalization of protein kinase C ␤II with p47 phox on the membrane. However, it remains unanswered whether GT directly affects any of NADPH oxidase components. Here, we examine the effect of GT on the NADPH oxidase components in the cell-free activation assay. The O 2 ؊ -generating ability of membranes obtained from GT-treated neutrophils is 40.0 and 30.6% lower, respectively, than the untreated counterparts when assayed with two distinct electron acceptors, suggesting that flavocytochrome b 558 is affected in cells by GT. In contrast, the corresponding cytosol remains competent for activation. Next, GT addition in vitro to the assay consisting of flavocytochrome b 558 and cytosolic components (native cytosol or recombinant p67 phox , p47 phox , and Rac2) causes a striking inhibition (50% inhibitory concentration ‫؍‬ 3.3 M) when done prior to the stimulation with myristic acid. NADPH consumption is also prevented by GT, but the in vitro assembly of p67 phox , p47 phox , and Rac2 with flavocytochrome b 558 is normal. Posterior addition of GT to the activated enzyme is ineffective. The separate treatment of membranes with GT also causes a marked loss of flavocytochrome b 558 's ability to reconstitute O 2 ؊ generation, supporting the conclusion at the cellular level. The flavocytochrome b 558 heme spectrum of the GT-treated membranes stays, however, unchanged, showing that hemes remain intact. These results suggest that GT directly harms site(s) crucial for electron transport in flavocytochrome b 558 , which is accessible only before oxidase activation.Host defense against Aspergillus spp. depends mainly on the oxidative killing by superoxide anion (O 2 Ϫ )-derived reactive oxygen species (ROS) of neutrophils, as demonstrated by severe aspergillosis in patients with chronic granulomatous disease (9, 27, 51). The enzyme responsible for O 2 Ϫ generation is the multicomponent NADPH oxidase comprised of the membrane-integrated flavocytochrome b 558 (a heterodimer of gp91 phox and p22 phox ) and four cytosolic regulatory proteins: p67 phox , p47 phox , p40 phox , and the small G-protein Rac2 (for reviews, see references 3, 14, 29, and 59). The N-terminal half of gp91 phox contains four histidines coordinating two hemes according to a bis-heme motif (His101 and His209; His115 and His222) (5, 20), which is conserved in all members of the Nox (for NADPH oxidase)/Duox (for dual oxidase) family (for reviews, see references 35, 36, and 59). The C-terminal half of gp91 phox has a reductase domain with binding sites for NADPH and FAD, which are also shared within the Nox/Duox members (for reviews, see references 35, 36, and 59).In unstimulated neutrophils, the NADPH oxidase is kept dormant through maintaining its membrane and cytosolic components in the respective cellular locations under i...

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Nucleoside-triphosphate binding of the two cytosolic components of the respiratory burst oxidase system: Evidence for its inhibition by the 2′,3′-dialdehyde derivative of NADPH and desensitization in their translocated states

Cited by 10 publications

References 32 publications

Fungal Metabolite Gliotoxin Inhibits Assembly of the Human Respiratory Burst NADPH Oxidase

Fungal Metabolite Gliotoxin Inhibits Assembly of the Human Respiratory Burst NADPH Oxidase

Activation of the O^–₂‐Generating NADPH Oxidase in a Semi–Recombinant Cell‐Free System

Fungal Metabolite Gliotoxin Targets Flavocytochrome b ₅₅₈ in the Activation of the Human Neutrophil NADPH Oxidase

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Nucleoside-triphosphate binding of the two cytosolic components of the respiratory burst oxidase system: Evidence for its inhibition by the 2′,3′-dialdehyde derivative of NADPH and desensitization in their translocated states

Cited by 10 publications

References 32 publications

Fungal Metabolite Gliotoxin Inhibits Assembly of the Human Respiratory Burst NADPH Oxidase

Fungal Metabolite Gliotoxin Inhibits Assembly of the Human Respiratory Burst NADPH Oxidase

Activation of the O–2‐Generating NADPH Oxidase in a Semi–Recombinant Cell‐Free System

Fungal Metabolite Gliotoxin Targets Flavocytochrome b 558 in the Activation of the Human Neutrophil NADPH Oxidase

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Activation of the O^–₂‐Generating NADPH Oxidase in a Semi–Recombinant Cell‐Free System

Fungal Metabolite Gliotoxin Targets Flavocytochrome b ₅₅₈ in the Activation of the Human Neutrophil NADPH Oxidase