Opening the black box: lessons from cell-free systems on the phagocyte NADPH-oxidase

The NADPH-oxidase complex of phagocytic cells plays a key role in the defense against invading pathogens, through the release of superoxide anion, precursor of other reactive oxygen species (ROS). NADPH-oxidase deficiency is called Chronic Granulomatous Disease (CGD), in which patients suffer from recurrent infections and from the formation of granulomas in various organs. Research on NADPH-oxidase has much benefited from the discovery of cell-free systems, i.e. reconstitution assays from broken resting (unstimulated) phagocytes, in which activation of the oxidase is elicited in vitro. Cell-free systems were developed in parallel to studies of molecular defects of patients with CGD, both approaches leading to the identification of the major proteins implicated in enzyme activation. Variations around the cell-free system allowed molecular dissection of the mechanism of NADPH-oxidase activation and provided insights into its relationship to phagocytosis.     

Purification and properties of a functional 47-kilodalton cytosolic factor required for NADPH-oxidase activation in bovine neutrophils.

A cytosolic factor of 47 kDa required for activation of the NADPH oxidase, and referred to as p47, has been purified in its functional form from the cytosol of resting bovine neutrophils. The purification was monitored by the determination of the activating potency of p47 in a cell-free system of oxidase activation. The recovery was around 10% and the purification factor greater than 1000. P47 was phosphorylated in vitro by protein kinase A and protein kinase C. [32P] labeled p47 was resolved by isoelectric focusing into two major labeled bands of pI 7.0 and 8.5. Polyclonal antibodies were used to demonstrate that p47 is localized specifically in the cytosol of resting neutrophils, and that, upon activation of neutrophils, p47 is translocated from the cytosol to the membrane.     

Mononuclear phagocyte activation: activation-associated antigens

Mononuclear phagocyte activation is characterized by alterations in cellular metabolism and plasma membrane composition. In rodent and human systems, antibodies (conventional heteroantibodies or monoclonal reagents) that identify plasma membrane antigens selectively expressed by activated macrophages and monocytes have been generated. Among these activation-associated determinants is Mo3e (p50,80), a protease-sensitive antigen that is expressed by human monocytes activated in culture by exposure to bacterial lipopolysaccharide, muramyl dipeptide, or phorbol myristate acetate (PMA) (as well as other biologically active phorbol compounds). Mo3e is also expressed by the monoblastic cell line U-937 after culture in medium containing PMA and other pharmacological activators of protein kinase C (4 beta-phorbol-12,13-dibutyrate, 4 beta-phorbol-12,13-didecanoate, mezerein, and cell-permeable 1,2-diacylglycerol). The human promyelocytic cell line HL-60 becomes Mo3e positive after exposure in vitro to certain inducers of monocytic differentiation (PMA, dibutyryl cyclic AMP, and cholera toxin plus 3-isobutyl-1-methylxanthine). The surface expression of Mo3e is blocked by inhibitors of protein synthesis, N-linked glycosylation, and protein kinase activation, as well as by ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid and calcium antagonists. These data suggest the involvement of glycoprotein synthesis, protein kinase activation, and calcium ions in the stimulated expression of Mo3e by activated human mononuclear phagocytes. Anti-Mo3e antibody blocks the human monocyte response to migration inhibitory factor (MIF), which indicates an association between the expression of Mo3e antigen and responsiveness to MIF.     

NADPH-oxidase activation and cognition in Alzheimer disease progression

Superoxide production via NADPH-oxidase (NOX) has been shown to play a role in a variety of neurological disorders, including Alzheimer disease (AD). To improve our understanding of the NOX system and cognitive impairment, we studied the various protein components of the phagocytic isoform (gp91^phox, or NOX2) in the frontal and temporal cortex of age- and postmortem-matched samples. Individuals underwent antemortem cognitive testing and postmortem histopathologic assessment to determine disease progression and assignment to one of the following groups: no cognitive impairment (NCI), preclinical AD, mild cognitive impairment (MCI), early AD, and mild-to-moderate AD. Biochemical methods were used to determine overall NOX activity as well as levels of the various subunits (gp91^phox, p67^phox, p47^phox, p40^phox, and p22^phox). Overall enzyme activity was significantly elevated in the MCI cohort in both cortical regions compared to the NCI cohort. This activity level remained elevated in the AD groups. Only the NOX cytosolic subunit proteins (p67^phox, p47^phox, and p40^phox ) were significantly elevated with disease progression; the membrane-bound subunits (gp91^phox and p22^phox) remained stable. In addition, there was a robust correlation between NOX activity and the individual's cognitive status such that as the enzyme activity increased, cognitive performance decreased. Collectively, these data show that upregulated NADPH-oxidase in frontal and temporal cortex suggests that increases in NOX-associated redox pathways might participate in early pathogenesis and contribute to AD progression.     

The phagocyte 47-kilodalton cytosolic oxidase protein is an early reactant in activation of the respiratory burst

Activation of the phagocyte NADPH oxidase requires participation of membrane-bound cytochrome b558 and cytosol proteins of 47 kDa (p47) and 67 kDa (p67). We examined the sequence of participation of p47 and p67 in activation of the oxidase using an arachidonate-activated cell-free superoxidase (O2-) generating assay requiring phagocyte membrane and cytosol. Neutrophil cytosol from patients with certain forms of autosomal recessive chronic granulomatous disease (CGD) lack either p47 or p67. Initial incubation of membrane and arachidonate with CGD cytosol deficient in either p47 or p67 fails to generate superoxide in the cell-free assay until addition of complementary cytosol. CGD cytosol was incubated with arachidonate and membrane for 5-15 min and the lag time of O2- generation was measured after addition of complementary CGD cytosol. The lag time is shortened when p47, but not p67, is present in the initial incubation. We have previously shown that the peptide, RGVHFIF, corresponding to a cytoplasmic carboxyl-terminal domain of the large subunit of cytochrome b558, inhibits activation of NADPH oxidase in the cell-free assay, but does not affect the enzyme activity of fully assembled oxidase. Experiments with sequential addition of complementary CGD cytosols were performed as above, except that RGVHFIF was added after the initial incubation. The peptide failed to inhibit when added after initial incubation if p47 was present during that incubation. In contrast, the peptide markedly inhibited oxidase activity if p47 was absent during the initial incubation. These results suggest that p47, but not p67, is a participant with membrane and/or other cytosol components in early arachidonate-dependent reactions. In the absence of p67, these reactions culminate in the irreversible formation of a metastable activation intermediate that is insensitive to inhibition by RGVHFIF. After addition of p67, this activation intermediate subsequently reacts to form the active NADPH oxidase.     

The cytosolic subunit p67 of the NADPH-oxidase complex does not bind NADPH

The NADPH-oxidase of phagocytic cells is a multicomponent enzyme that generates superoxide. It comprises a membrane flavocytochrome b₅₅₈ and four cytosolic proteins; p67^phox, p47^phox, p40^phox and Rac. The NADPH-binding site of this complex was shown to be located on the flavocytochrome b₅₅₈. However, a number of studies have suggested the presence of another site on the p67^phox subunit which is the key activating component. Using several approaches like tryptophan quenching fluorescence measurement, inhibition by 2′,3′-dialdehyde NADPH, and free/bound NADPH concentration measurements, we demonstrate that no NADPH binds on p67^phox, thus definitively solving the controversy on the number and location of the NADPH-binding sites on this complex.     

Retinoic acid-induced differentiation-specific, C-kinase-dependent phosphorylation of cytosolic 44 and 32 kDa proteins in HL-60 cells

The effect of various differentiation-inducers on the activity of Ca2+, phospholipid-dependent protein kinase (C-kinase) activity and endogenous protein phosphorylation by the kinase were examined in the extracts of HL-60 cells. Although all of the inducers, retinoic acid, dibutyryl cAMP, nicotinamide, dimethylsulfoxide, and 3-aminobenzamide increased the cytosolic C-kinase activity accompanied with the differentiation into mature myelocytes, only retinoic acid markedly enhanced Ca2+, phospholipid-dependent phosphorylation of 44 and 32 kDa proteins in the cytosol. These results suggest that the differentiation pathway induced by retinoic acid is different from the pathways induced by other inducers.     

Purification and characterization of a differentiation-specific sialoglycoprotein of lactating-guinea-pig mammary tissue.

A large acidic glycoprotein, PAS-I, was purified from the fat-globule membrane of guinea-pig milk. Threonine and serine accounted for over 30 mol% of the amino acids, and galactose, N-acetylgalactosamine, N-acetylglucosamine, mannose and sialic acid were the principal sugars detected. On a molar basis, sialic acid accounted for over 60% of the total sugar. Removal of sialic acid by treatment with neuraminidase revealed the presence of binding sites for peanut (Arachis hypogaea) agglutinin, a lectin specific for the sugar sequence beta-D-Gal-(beta 1----3)-D-GalNac (the T antigen). The distribution of PAS-I-related epitopes, defined by five monoclonal antibodies, was determined in the mammary gland and in other guinea-pig tissues. PAS-I was maximally expressed on the apical surfaces of secretory cells in lactating mammary tissue and was either absent, or present in much lower amounts, in the glands of virgin or pregnant animals. PAS-I epitopes were not detected in liver, heart, spleen, pancreas, ovary, uterus, lung or intestine, either by immunofluorescence microscopy or by immunoblotting techniques. Several of the PAS-I-specific antibodies bound to mucins of high Mr in human fat-globule membrane, and similarities and differences between PAS-I and the human mucins are discussed. PAS-I and epitopes of this glycoprotein will be useful as indicators of differentiation in mammary cells and of markers of the apical surface of these cells during lactation.     

NSP-interacting GTPase: A cytosolic protein as cofactor for nuclear shuttle proteins

Despite the significant progress in the identification of essential components of the nuclear transport machinery, some events of this process are still unclear. Particularly, functional information about the release of nuclear-exported macromolecules at the cytoplasmic side of the nuclear pore complex and their subsequent trans-cytoplasmic movement is lacking. Recently, we identified a cytoplasmic GTPase, designated NIG (NSP-interacting GTPase), which may play a relevant role in these processes. NIG interacts in vivo with the geminivirus NSP and promotes the translocation of the viral protein from the nucleus to the cytoplasm where it is redirected to the cell surface to interact with the viral movement protein, MP. Here we position the NIG function into the mechanistic model for the intracellular trafficking of viral DNA and discuss the putative role of NIG in general cellular nucleocytoplasmic transport of nucleic acid-protein complexes.Key words: geminivirus, NIG, NSP, nucleocytoplasmic trafficking, transport activity     

Identification of distinct activation pathways of the human neutrophil NADPH-oxidase

The stimulation of the human neutrophil NADPH-oxidase is initiated by a variety of agonists, which appear to utilize more than one activation pathway. We have discerned that opsonized zymosan (OZ) stimulates O2- release by a mechanism distinct from that of phorbol myristate acetate (PMA). PMA differs from OZ stimulation in its susceptibility to H-7 (a protein kinase inhibitor) inhibition of O2- release and the lack of PMA-initiated release of radiolabeled arachidonic acid ([3H]AA) from prelabeled cells. That AA release was linked to O2- generation in OZ-stimulated cells was suggested by the finding that mepacrine, a phospholipase inhibitor, exhibits parallel dose response inhibition for both O2- generation and [3H]AA release, whereas mepacrine did not significantly inhibit the O2- generation induced by PMA. The specific involvement of phospholipase A2 (PLA2) in the release of AA was indicated by the lack of release of [3H]oleate, which is not released by PLA2 in intact cells; [3H]AA released from phosphatidylinositol and phosphatidylcholine and not accompanied by the formation of [3H]-arachidonyl phosphatidic acid, thus eliminating the involvement of phospholipase C; and the inhibition of [3H]AA release by p-bromophenacyl bromide, a specific PLA2 inhibitor. The reduction of O2- formation by inhibitors of AA metabolism (BW755C, acetylsalicylic acid, and indomethacin) further supports a linkage between AA release and O2- generation. That [3H]AA release, like O2- generation, in OZ-stimulated cells was calcium dependent further differentiates OZ from calcium-independent PMA activation. These studies in toto suggest that OZ stimulation of the NADPH-oxidase differs from PMA, in that the particulate stimulus is PLA2 mediated and independent of protein kinase C.     

Cell-free expression and assembly of ATP synthase

Cell-free (CF) expression technologies have emerged as promising methods for the production of individual membrane proteins of different types and origin. However, many membrane proteins need to be integrated in complex assemblies by interaction with soluble and membrane-integrated subunits in order to adopt stable and functionally folded structures. The production of complete molecular machines by CF expression as advancement of the production of only individual subunits would open a variety of new possibilities to study their assembly mechanisms, function, or composition. We demonstrate the successful CF formation of large molecular complexes consisting of both membrane-integrated and soluble subunits by expression of the atp operon from Caldalkalibacillus thermarum strain TA2.A1 using Escherichia coli extracts. The operon comprises nine open reading frames, and the 542-kDa F₁F_o-ATP synthase complex is composed of 9 soluble and 16 membrane-embedded proteins in the stoichiometry α₃β₃γδ?ab₂c₁₃. Complete assembly into the functional complex was accomplished in all three typically used CF expression modes by (i) solubilizing initial precipitates, (ii) cotranslational insertion into detergent micelles or (iii) cotranslational insertion into preformed liposomes. The presence of all eight subunits, as well as specific enzyme activity and inhibition of the complex, was confirmed by biochemical analyses, freeze-fracture electron microscopy, and immunogold labeling. Further, single-particle analysis demonstrates that the structure and subunit organization of the CF and the reference in vivo expressed ATP synthase complexes are identical. This work establishes the production of highly complex molecular machines in defined environments either as proteomicelles or as proteoliposomes as a new application of CF expression systems.     

Phosphoinositide 3-kinase activity is required for retinoic acid-induced expression and activation of the tissue transglutaminase

Tissue transglutaminase (TGase) is a dual function enzyme that couples an ability to bind GTP with transamidation activity. Retinoic acid (RA) consistently induces TGase expression and activation, and it was recently shown that increased TGase expression protected cells from apoptosis. To better understand how RA regulates TGase, we considered whether RA employed pro-survival signaling pathways to mediate TGase expression and activation. It was found that RA stimulation of NIH3T3 cells activated ERK and phosphoinositide 3-kinase (PI3K); however, only PI3K activation was necessary for RA-induced TGase expression. The overexpression of a constitutively active form of PI3K did not induce TGase expression, indicating that PI3K signaling was necessary but not sufficient for TGase expression. The exposure of cells expressing exogenous TGase to the PI3K inhibitor, LY294002, reduced the ability of TGase to be photoaffinity-labeled with [alpha-(32)P]GTP, providing evidence that PI3K regulates the GTP binding activity of TGase as well as its expression. Moreover, cell viability assays showed that incubation of RA-treated cells with LY294002 together with the TGase inhibitor, monodansylcadaverine (MDC), converted RA from a differentiation factor to an apoptotic stimulus. These findings demonstrate that PI3K activity is required for the RA-stimulated expression and GTP binding activity of TGase, thereby linking the up-regulation of TGase with a well established cell survival factor.     

Effect of detonation nanodiamonds on phagocyte activity

Detonation ND (nanodiamond) holds much promise for biological studies and medical applications. Properties like size of particles, inclination for modification of their surface and unambiguous biocompatibility are crucial. Of prime importance is interaction between ND and immune cells, which supervise foreign intrusion into an organism and eliminate it. Neutrophils are more reactive in inflammatory response implementing cytotoxical arsenal including ROS (reactive oxygen species). The aim of the work was to estimate the ability of two ND samples (produced by Diamond Center and PlasmaChem) to keep the vitality of neutrophils from the inflammatory site. The ability of cells to generate ROS in the presence of ND particles is considered as indicating their biocompatibility. IR spectra and size of particles in the samples were characterized. Acid modification of ND was carried out to get the luminescent form. In the biological aspect, ND demonstrated up or down action, depending on the concentration, time and conditions of activation of cells. Weak action of ND in whole blood was obtained possibly owing to the ND adsorbed plasma proteins, which mask active functional groups to interact with the cell membrane. ND did not influence the viability of isolated inflammatory neutrophils in low and moderate concentrations and suppressed it in high concentrations (≥1 g/l). Addition of ND to the cell suspension initiated concentration-dependent reaction to produce ROS similar to respiratory burst. ND up-regulated response to bacterial formylpeptide, but up- and down-modified (low or high concentrations, accordingly) response to such bacterial agents as OZ (opsonized zymosan), which neutrophils swallow up by oxygen-dependent phagocytosis. Localization of the particles on the cell surface as into the cells was identified by monitoring the intrinsic fluorescence of oxidized ND. The various mechanisms that could account for penetration of ND particles into the cell are discussed. Common conclusion concerns compatibility of ND with living neutrophils from inflammatory site and their normal functioning for infection safeguard.     

NADPH-oxidase activity and lipid peroxidation in neutrophils from rats fed fat-rich diets

In order to investigate the effect of fat-rich diets on neutrophil functions, 21 day-aged rats were fed for 6 weeks with a control diet consisting of a regular laboratory rodent chow (4 per cent final fat content), a control diet supplied with soybean oil (15 per cent final fat content), or a control diet supplied with coconut oil (15 per cent final fat content). Glycogen-elicited peritoneal neutrophils from rats fed soybean and coconut oil-enriched diets presented a reduction in spontaneous and PMA-stimulated H2O2 generation relative to neutrophils from rats fed the control diet. The activity of superoxide dismutase, glutathione peroxidase and catalase did not change in animals fed fat-rich diets. In addition, the capacity to generate O2-, spontaneously or in response to PMA, did not change in neutrophils from animals fed fat-rich diets. Values attained matched those observed in animals fed the control diet, regardless of the method used to measure O2-, the superoxide dismutase-inhibitable reduction of cytochrome c or the lucigenin-dependent chemiluminescence. However, the initial rate of O2- generation both in resting neutrophils and in PMA-stimulated cells was significantly reduced when animals were fed with coconut or soybean oil-enriched diets due, at least in part, to a reduction in the activity of glucose-6-phosphate dehydrogenase. The concentration of thiobarbituric acid reactive substances, an index of lipid peroxidation, was increased in animals fed both fat-rich diets. This was accompanied by an increase in arachidonic acid content in these cells. Results presented suggest that lipid peroxidation in neutrophils from animals fed fat-rich diets may be associated with a consumption of H2O2 yielding more reactive oxygen-derived species such as the hydroxyl radical.