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PubMed1996

Targeting the plant alternative oxidase protein to Schizosaccharomyces pombe mitochondria confers cyanide-insensitive respiration.

The Sauromatum guttatum alternative oxidase has been expressed in Schizosaccharomyces pombe under the control of the thiamine-repressible nmt1 promoter. Alternative oxidase protein and activity were detected both in spheroplasts and isolated mitochondria, indicating that the enzyme is expressed in a functional form and confers cyanide-resistant respiration to S. pombe, which is sensitive to inhibition by octyl-gallate. Protein import studies revealed that the precursor form of the alternative oxidase protein is efficiently imported into isolated mitochondria and processed to its mature form comparable to that observed with potato mitochondria. Western blot analysis and respiratory studies revealed that the alternative oxidase protein is expressed in the inner mitochondrial membrane in its reduced (active) form. Treatment of mitochondria with diamide and dithiothreitol resulted in interconversion of the reduced and oxidized species and modulation of respiratory activity. The addition of pyruvate did not effect either the respiratory rate or expression of the reduced species of the protein. To our knowledge this is the first time that the alternative oxidase has been effectively targeted to and integrated into the inner mitochondrial membrane of S. pombe, and we conclude that the expression of a single polypeptide is sufficient for alternative oxidase activity.

Antimycin A↗
PubMed1996

Rac "insert region" is a novel effector region that is implicated in the activation of NADPH oxidase, but not PAK65.

The small GTPase Rac assembles with the cytosolic p47(phox) and p67(phox) and the membrane-associated flavocytochrome b558 to form the multicomponent respiratory burst oxidase. Mutation of amino acids in a region of Rac (residues 26-45), homologous to an effector region in Ras, was previously shown to interfere with Rac binding to the oxidase. Herein we have elucidated an additional region in Rac involved in regulating oxidase activity. Rho family small GTPases contain a 12-amino acid "insert" region (residues 124-135) that is not present in Ras. Point mutations in and deletion of this region were constructed and used for in vitro studies of the activation of PAK65 and NADPH oxidase. Apparent binding constants (based on EC50 values) of the mutant Rac proteins for the oxidase are at least 13-25-fold higher than for wild-type Rac. Mutations in the insert region versus the 26-45 effector region resulted in distinct kinetic consequences, pointing to different roles for these two protein regions: mutations in the insert region but not the 26-45 effector region resulted in an increase in the EC50 for p67(phox). Although mutations in the 26-45 amino acid effector region showed markedly diminished activation of both PAK and the NADPH oxidase, insert region mutations did not affect activation of PAK. We propose that the combinatorial use of the 26-45 effector region and the insert region provides the Rho family GTPases with versatility in their specificity for several downstream targets.

Amino Acid Sequence↗
PubMed1997

Effect of metal on 2,4,5-trihydroxyphenylalanine (topa) quinone biogenesis in the Hansenula polymorpha copper amine oxidase.

Previous studies of wild-type and mutant forms of a recombinant copper amine oxidase from Hansenula polymorpha, expressed in Saccharomyces cerevisiae, have indicated a self-processing mechanism for 2,4,5-trihydroxyphenylalanine (topa) quinone biogenesis involving the active site copper (Cai, D., and Klinman, J. P. (1994) J. Biol. Chem. 269, 32039-32042). In contrast to prokaryotic copper amine oxidases, however, it has not been possible to initiate topa quinone formation by the addition of exogenous copper to precursor H. polymorpha amine oxidase lacking copper. Metal analysis of copper-depleted wild-type enzyme reveals 0.2-0.3 mol copper, together with 0.6 mol zinc. Despite changes in the zinc and copper levels in growth media, the level of zinc in purified enzyme remains fairly constant. Further, we have been unable to displace protein-bound zinc by exogenously added copper. The H. polymorpha amine oxidase gene was subsequently expressed in Escherichia coli and found to be almost completely free of copper and zinc. In vitro reconstitution of this apoprotein confirms that zinc binds to H. polymorpha amine oxidase and prevents reconstitution with copper. By contrast, addition of copper first to apoprotein leads to formation of topa quinone and stable activity in the presence of added zinc. These findings indicate efficient binding of either zinc or copper to a site that undergoes little or no exchange. The data confirm that topa quinone biogenesis in the H. polymorpha system is catalyzed by copper and occurs in the absence of added factors. We conclude that the mechanisms of cofactor biogenesis in pro- and eukaryotic systems are likely to be similar or identical. The results described herein imply different pathways for the in vivo assembly of heterologously expressed amine oxidases in S. cerevisiae and E. coli.

Amine Oxidase (Copper-Containing)↗
PubMed1997

Characterization of the native lysine tyrosylquinone cofactor in lysyl oxidase by Raman spectroscopy.

Lysine tyrosylquinone (LTQ) recently has been identified as the active site cofactor in lysyl oxidase by isolation and characterization of a derivatized active site peptide. Reported in this study is the first characterization of the underivatized cofactor in native lysyl oxidase by resonance Raman (RR) spectrometry. The spectrum is characterized by a unique set of vibrational modes in the 1200 to 1700 cm-1 region. We show that the RR spectrum of lysyl oxidase closely matches that of a synthetic LTQ model compound, 4-n-butylamino-5-ethyl-1,2-benzoquinone, in aqueous solutions but differs significantly from those of other topa quinone-containing amine oxidases under similar conditions. Furthermore, we have observed the same 18O shift of the C=O stretch in both the lysyl oxidase enzyme and the LTQ cofactor model compound. The RR spectra of different model compounds and their D shifts give additional evidence for the protonation state of LTQ cofactor in the enzyme. The overall similarity of these spectra and their shifts shows that the lysyl oxidase cofactor and the model LTQ compound have the same structure and properties. These data provide strong and independent support for the new cofactor structure, unambiguously ruling out the possibility that the structure originally reported had been derived from a spurious side reaction during the derivatization of the protein and isolation of the active site peptide.

Animals↗
PubMed1997

Irreversible inhibition of lysyl oxidase by homocysteine thiolactone and its selenium and oxygen analogues. Implications for homocystinuria.

Homocysteine thiolactone, selenohomocysteine lactone, and homoserine lactone were found to be competitive, irreversible inhibitors of lysyl oxidase, with KI values of 21 +/- 3 microM, 8.3 +/- 2.2 microM, and 420 +/- 56 microM, respectively. The first order rate constants for inactivation (k2) of the enzyme varied over a much smaller range, ranging from 0.12 to 0.18 to 0.28 min-1 for the Se-, thio-, and O-lactones, respectively. Mutually exclusive labeling of the enzyme by [1-14C]beta-aminopropionitrile, [U-14C]phenylhydrazine, or [35S]homocysteine thiolactone was observed. These labeling results, together with the closely similar perturbations of the near UV-visible spectra of lysyl oxidase and of a model of its lysine tyrosylquinone cofactor by the thiolactone, indicate that the lactones likely derivatize and reduce the active site carbonyl cofactor. Substitution with deuterium at the alpha-carbon of the thiolactone caused a deuterium kinetic isotope effect on k2 of 3.2 +/- 0.2, consistent with the involvement of rate-limiting alpha-proton abstraction during lactone-induced inactivation of the enzyme. The activities of plasma amine oxidase and diamine oxidase were only minimally reduced at concentrations of the sulfur or selenium lactones that fully inhibited lysyl oxidase. Thus, these lactones constitute a new category of mechanism-based inactivators selective for lysyl oxidase. Further, these results may relate to the development of connective tissue defects seen in homocystinuria.

Animals↗
PubMed1997

Nitric oxide inactivates NADPH oxidase in pig neutrophils by inhibiting its assembling process.

The effects of nitric oxide (NO) on superoxide (O-2) generation of the NADPH oxidase in pig neutrophils were studied. NO dose-dependently suppressed O-2 generation of both neutrophil NADPH oxidase and reconstituted NADPH oxidase. Effects of NO on NADPH-binding site and the redox centers including FAD and low spin heme in cytochrome b558 and the electron transfer rates from NADPH to heme via FAD were examined under anaerobic conditions. Both reaction rates and the Km value for NADPH were unchanged by NO. Visible and EPR spectra of cytochrome b558 showed that the structure of heme was unchanged by NO, indicating that NO does not affect the redox centers of the oxidase. In reconstituted NADPH oxidase system, NO did not inhibit O-2 generation of the oxidase when added after activation. The addition of NO to the membrane component or the cytosol component inhibited the activity by 24.0 +/- 5.3 or 37.4 +/- 7.1%, respectively. The addition of NO during the activation process or to the cytosol component simultaneously with myristate inhibited the activity by 74.0 +/- 5.2 or 70.0 +/- 8.3%, respectively, suggesting that cytosol protein(s) treated with myristate becomes susceptible to NO. Peroxynitrite did not interfere with O-2 generation.

Animals↗
PubMed1998

Essential requirement of cytosolic phospholipase A2 for activation of the phagocyte NADPH oxidase.

Arachidonic acid (AA) can trigger activation of the phagocyte NADPH oxidase in a cell-free assay. However, a role for AA in activation of the oxidase in intact cells has not been established, nor has the AA generating enzyme critical to this process been identified. The human myeloid cell line PLB-985 was transfected to express p85 cytosolic phospholipase A2 (cPLA2) antisense mRNA and stable clones were selected that lack detectable cPLA2. cPLA2-deficient PLB-985 cells differentiate similarly to control PLB-985 cells in response to retinoic acid or 1,25-dihydroxyvitamin D3, indicating that cPLA2 is not involved in the differentiation process. Neither cPLA2 nor stimulated [3H]AA release were detectable in differentiated cPLA2-deficient PLB-985 cells, demonstrating that cPLA2 is the major type of PLA2 activated in phagocytic-like cells. Despite the normal synthesis of NADPH oxidase subunits during differentiation of cPLA2-deficient PLB-985 cells, these cells fail to activate NADPH oxidase in response to a variety of soluble and particulate stimuli, but the addition of exogenous AA fully restores oxidase activity. This establishes an essential requirement of cPLA2-generated AA for activation of phagocyte NADPH oxidase.

Biological Transport↗
PubMed1998

Mapping of functional domains in p47(phox) involved in the activation of NADPH oxidase by "peptide walking".

The superoxide generating NADPH oxidase of phagocytes consists, in resting cells, of a membrane-associated electron transporting flavocytochrome (cytochrome b559) and four cytosolic proteins as follows: p47(phox), p67(phox), p40(phox), and the small GTPase, Rac(1 or 2). Activation of the oxidase is consequent to the assembly of a membrane-localized multimolecular complex consisting of cytochrome b559 and the cytosolic components. We used "peptide walking" (Joseph, G., and Pick, E. (1995) J. Biol. Chem. 270, 29079-29082) for mapping domains in the amino acid sequence of p47(phox) participating in the molecular events leading to the activation of NADPH oxidase. Ninety-five overlapping pentadecapeptides, with a four-residue offset between neighboring peptides, spanning the complete p47(phox) sequence, were tested for the ability to inhibit NADPH oxidase activation in a cell-free system. This consisted of solubilized macrophage membranes, recombinant p47(phox), p67(phox), and Rac1, and lithium dodecyl sulfate, as the activator. Eight functional domains were identified and labeled a-h. These were (N- and C-terminal residue numbers are given for each domain) as follows: a (21-35); b (105-119); c (149-159); d (193-207); e (253-267); f (305-319); g (325-339), and h (373-387). Four of these domains (c, d, e, and g) correspond to or form parts of regions shown before to participate in NADPH oxidase assembly. Thus, domain c corresponds to a region on the N-terminal boundary of the first src homology 3 (SH3) domain, whereas domains d and e represent more precisely defined sites within the full-length first and second SH3 domains, respectively. Domain g overlaps an extensively investigated arginine-rich region. Domains a and b, in the N-terminal half of p47(phox), and domains f and h, in the C-terminal half, represent newly identified entities, for which there is no earlier experimental evidence of involvement in NADPH oxidase activation. "Peptide walking" was also applied to the identification of domains in p47(phox) mediating binding to p67(phox). This was done by quantifying, by enzyme-linked immunosorbent assay, the binding of p67(phox), in solution, to a series of 95 overlapping biotinylated p47(phox) peptides, attached to streptavidin-coated 96-well plates. A single proline-rich domain (residues 357-371) was found to bind p67(phox) in the absence and presence of lithium dodecyl sulfate.

Amino Acid Sequence↗
PubMed1998

A highly conserved glutamate residue (Glu-270) is essential for plant alternative oxidase activity.

We have previously demonstrated that expression of a Sauromatum guttatum alternative oxidase in Schizosaccharomyces pombe confers cyanide-resistant respiratory activity on these cells (Albury, M. S., Dudley, P., Watts, F. Z., and Moore, A. L. (1996) J. Biol. Chem. 271, 17062-17066). Using this functional expression system we have investigated the active site of the plant alternative oxidase, which has been postulated to comprise a non-heme binuclear iron center. Mutation of a conserved glutamate (Glu-270), previously postulated to be a bridging ligand within the active site, to asparagine abolishes catalytic activity because mitochondria containing the E270N mutant protein do not exhibit antimycin A-resistant respiration. Western blot analysis, using antibodies specific for the alternative oxidase, revealed that the E270N mutant protein was targeted to and processed by S. pombe mitochondria in a manner similar to that of the wild-type protein. It is possible that lack of antimycin A-insensitive respiration observed in mitochondria containing the E270N mutant protein is due to incorrect insertion of the mutant alternative oxidase into the inner mitochondrial membrane. However, Western blot analysis of subfractionated mitochondria shows that both wild-type and E270N alternative oxidase are specifically located in the inner mitochondrial membrane, suggesting that misfolding or lack of insertion is unlikely. These results provide the first experimental evidence to support the structural model in which the active site of the alternative oxidase contains a coupled binuclear iron center.

Amino Acid Substitution↗
PubMed1999

Functional expression of the plant alternative oxidase affects growth of the yeast Schizosaccharomyces pombe.

We have investigated the extent to which functional expression of the plant alternative oxidase (from Sauromatum guttatum) in Schizosaccharomyces pombe affects yeast growth. When cells are cultured on glycerol, the maximum specific growth rate is decreased from 0.13 to 0.11 h-1 while growth yield is lowered by 20% (from 1. 14 x 10(8) to 9.12 x 10(7) cells ml-1). Kinetic studies suggest that the effect on growth is mitochondrial in origin. In isolated mitochondria we found that the alternative oxidase actively competes with the cytochrome pathway for reducing equivalents and contributes up to 24% to the overall respiratory activity. Metabolic control analysis reveals that the alternative oxidase exerts a considerable degree of control (22%) on total electron flux. Furthermore, the negative control exerted by the alternative oxidase on the flux ratio of electrons through the cytochrome and alternative pathways is comparable with the positive control exerted on this flux-ratio by the cytochrome pathway. To our knowledge, this is the first paper to report a phenotypic effect because of plant alternative oxidase expression. We suggest that the effect on growth is the result of high engagement of the non-protonmotive alternative oxidase in yeast respiration that, consequently, lowers the efficiency of energy conservation and hence growth.

Gene Expression Regulation, Enzymologic↗
PubMed1999

A noninvasive fluorimetric procedure for measurement of membrane potential. Quantification of the NADPH oxidase-induced depolarization in activated neutrophils.

The electrogenic activity of the NADPH oxidase is associated with depolarization of the plasma membrane in activated neutrophils. The magnitude and consequences of this depolarization, however, remain unknown. Neutrophils are not amenable to electrophysiological determinations of membrane potential by current clamp. Instead, the occurrence of depolarization has been inferred from the use of potential-sensitive fluorescent dyes. However, such dyes partition into intracellular organelles and may yield erroneous results, particularly because the NADPH oxidase resides largely in secretory granules, where it has been claimed to become activated. We confirmed the intracellular generation of oxidase products using dihydrorhodamine, which is converted to the fluorescent rhodamine 123 when oxidized. Rhodamine 123 accumulated inside endomembrane organelles in both neutrophils and in differentiated HL60 cells, where it co-localized with the primary granule marker CD63. To estimate the surface membrane potential without interference from organelles, we devised a method based on the voltage-driven uptake of Mn(2+) across the plasmalemma. The uptake of Mn(2+) through calcium release-activated channels was measured as the rate of Indo-1 fluorescence quenching in thapsigargin-treated cells. The rate of Mn(2+) influx was found to vary when the membrane potential was manipulated using conductive ionophores and also when the NADPH oxidase was activated. A calibration curve in the positive potential range was constructed using the Na(+) ionophore SQI-Pr. Using this calibration, the membrane potential of phorbol ester-activated neutrophils was found to reach +58 +/- 6 mV, a sustained depolarization of over 100 mV compared with the resting potential. The depolarization was greatly diminished when the NADPH oxidase was inhibited with diphenylene iodonium. Together, these results indicate that the NADPH oxidase can generate a large depolarization of the plasmalemma, which should suffice to activate a variety of voltage-gated channels, including the outwardly rectifying H(+) conductance.

Membrane Potentials↗
PubMed2000

Arachidonic acid and phosphorylation synergistically induce a conformational change of p47phox to activate the phagocyte NADPH oxidase.

The superoxide-producing phagocyte NADPH oxidase can be activated by arachidonic acid (AA) or by phosphorylation of p47(phox) under cell-free conditions. The molecular mechanism underlying the activation, however, has remained largely unknown. Here we demonstrate that AA, at high concentrations (50-100 micrometer), induces direct interaction between the oxidase factors p47(phox) and p22(phox) in parallel with superoxide production. The interaction, being required for the oxidase activation, is mediated via the Src homology 3 (SH3) domains of p47(phox) (p47-(SH3)(2)), which are intramolecularly masked in a resting state. We also show that AA disrupts complexation of p47-(SH3)(2) with its intramolecular target fragment (amino acids 286-340) without affecting association of p47-(SH3)(2) with p22(phox), indicating that the disruption plays a crucial role in the induced interaction with p22(phox). Phosphorylation of p47(phox) by protein kinase C partially replaces the effects of AA; treatment of the SH3 target fragment with PKC in vitro results in a completely impaired interaction with p47-(SH3)(2), and the same treatment of the full-length p47(phox) leads to both interaction with p22(phox) and oxidase activation without AA, but to a lesser extent. Furthermore, phosphorylated p47(phox) effectively binds to p22(phox) and activates the oxidase in the presence of AA at low concentrations (1-5 micrometer), where an unphosphorylated protein only slightly supports superoxide production. Thus AA, at high concentrations, fully induces the interaction of p47(phox) with p22(phox) by itself, whereas, at low concentrations, AA synergizes with phosphorylation of p47(phox) to facilitate the interaction, thereby activating the NADPH oxidase.

Arachidonic Acid↗
PubMed2000

Targeting of Rac1 to the phagocyte membrane is sufficient for the induction of NADPH oxidase assembly.

The superoxide (O(2))-generating NADPH oxidase complex of phagocytes consists of a membrane-associated flavocytochrome (cytochrome b(559)) and four cytosolic proteins, p47(phox), p67(phox), p40(phox), and the small GTPase Rac (Rac1 or -2). NADPH oxidase activation (O(2) production) is elicited as the consequence of assembly of some or all cytosolic components with cytochrome b(559). This process can be reproduced in an in vitro system consisting of phagocyte membranes, p47(phox), p67(phox), and Rac, activated by an anionic amphiphile. We now show that post-translationally processed (prenylated) Rac1 initiates NADPH oxidase assembly, expressed in O(2) production, in a cell-free system containing phagocyte membrane vesicles and p67(phox), in the absence of an activating amphiphile and of p47(phox). Prenylated Cdc42Hs, a GTPase closely related to Rac, is inactive under the same conditions. Results obtained with phagocyte membrane vesicles can be reproduced fully by replacing these with partially purified cytochrome b(559), incorporated in phosphatidylcholine vesicles. Prenylated, but not nonprenylated, Rac1 binds spontaneously to phagocyte membrane vesicles and also to artificial, protein-free, phosphatidylcholine vesicles, a process counteracted by GDP dissociation inhibitor for Rho. Binding of prenylated Rac1 to membrane vesicles is accompanied by the recruitment of p67(phox) to the same location and the formation of an assembled NADPH oxidase complex, producing O(2) upon the addition of NADPH. Amphiphile and p47(phox)-independent NADPH oxidase activation by prenylated Rac1 is inhibited by Rho GDP dissociation inhibitor and by phosphatidylcholine vesicles, both competing with membrane for prenylated Rac1. We conclude that, in vitro, targeting of Rac to the phagocyte membrane is sufficient for the induction of NADPH oxidase assembly, suggesting that the principal or, possibly, the only role of Rac is to recruit cytosolic p67(phox) to the membrane environment, to be followed by the interaction of p67(phox) with cytochrome b(559).

Amino Acid Sequence↗
PubMed2001

Role of neutrophil NADPH oxidase in the mechanism of tumor necrosis factor-alpha -induced NF-kappa B activation and intercellular adhesion molecule-1 expression in endothelial cells.

In this study, we explored a novel function of polymorphonuclear neutrophils (PMN) NAD(P)H oxidase in the mechanism of tumor necrosis factor-alpha (TNFalpha)-induced NF-kappaB activation and intercellular adhesion molecule-1 (ICAM-1) expression in endothelial cells. Studies were made in mice lacking the p47(phox) subunit of NAD(P)H oxidase as well as in cultured mouse lung vascular endothelial cells (MLVEC) from these mice. In response to TNFalpha challenge, NF-kappaB activation and ICAM-1 expression were significantly attenuated in lungs of p47(phox)(-/-) mice as compared with wild-type (WT) mice. The attenuated NF-kappaB activation in p47(phox)(-/-) mice was secondary to inhibition of NIK activity and subsequent IkappaBalpha degradation. Induction of neutropenia using anti-PMN serum prevented the initial TNFalpha-induced NF-kappaB activation and ICAM-1 expression in WT mice, indicating the involvement of PMN NAD(P)H oxidase in signaling these responses. Moreover, the responses were restored upon repletion with PMN obtained from WT mice but not with PMN from p47(phox)(-/-) mice. These findings were recapitulated in MLVEC co-cultured with PMN, suggesting that NF-kappaB activation and resultant ICAM-1 expression in endothelial cells occurred secondary to oxidants generated by the PMN NAD(P)H oxidase complex. The functional relevance of the PMN NAD(P)H oxidase in mediating TNFalpha-induced ICAM-1-dependent endothelial adhesivity was evident by markedly reduced adhesion of p47(phox)(-/-) PMN in co-culture experiments. Thus, oxidant signaling by the PMN NAD(P)H oxidase complex is an important determinant of TNFalpha-induced NF-kappaB activation and ICAM-1 expression in endothelial cells.

Animals↗
PubMed2002

Rac activation induces NADPH oxidase activity in transgenic COSphox cells, and the level of superoxide production is exchange factor-dependent.

Transient expression of constitutively active Rac1 derivatives, (G12V) or (Q61L), was sufficient to induce phagocyte NADPH oxidase activity in a COS-7 cell model in which human cDNAs for essential oxidase components, gp91(phox), p22(phox), p47(phox), and p67(phox), were expressed as stable transgenes. Expression of constitutively active Rac1 in "COS(phox)" cells induced translocation of p47(phox) and p67(phox) to the membrane. Furthermore, translocation of p47(phox) was induced in the absence of p67(phox) expression, even though Rac does not directly bind p47(phox). Rac effector domain point substitutions (A27K, G30S, D38A, Y40C), which can selectively eliminate interaction with different effector proteins, impaired Rac1V12-induced superoxide production. Activation of endogenous Rac1 by expression of constitutively active Rac-guanine nucleotide exchange factor (GEF) derivatives was sufficient to induce high level NADPH oxidase activity in COS(phox) cells. The constitutively active form of the hematopoietic-specific GEF, Vav1, was the most effective at activating superoxide production, despite detection of higher levels of Rac1-GTP upon expression of constitutively active Vav2 or Tiam1 derivatives. These data suggest that Rac can play a dual role in NADPH oxidase activation, both by directly participating in the oxidase complex and by activating signaling events leading to oxidase assembly, and that Vav1 may be the physiologically relevant GEF responsible for activating this Rac-regulated complex.

Animals↗
PubMed2002

The HIV-1 Nef protein and phagocyte NADPH oxidase activation.

Nef, a multifunctional HIV protein, activates the Vav/Rac/p21-activated kinase (PAK) signaling pathway. Given the potential role of this pathway in the activation of the phagocyte NADPH oxidase, we have investigated the effect of the HIV-1 Nef protein on the phagocyte respiratory burst. Microglia (cell line and primary culture) were transduced with lentiviral expression vectors. Expression of Nef did not activate the NADPH oxidase by itself but led to a massive enhancement of the responses to a variety of stimuli (Ca(2+) ionophore, formyl peptide, endotoxin). These effects were not caused by up-regulation of phagocyte NADPH oxidase subunits. Nef mutants lacking motifs involved in the interaction with Vav and PAK failed to reproduce the effects of wild type Nef, suggesting a role for the Vav/Rac/PAK signaling pathway. The following results suggest a key role for Rac in the priming effect of Nef. (i) Inactivation of Rac by Clostridium difficile toxin B abolished the Nef effect. (ii) The fraction of activated Rac1 was increased in Nef-transduced cells, and (iii) the dominant positive Rac1(V12) mutant mimicked the effect of Nef. These results are to our knowledge the first analysis of the effect of Rac activation on the NADPH oxidase in intact phagocytes. Rac activation is not sufficient to stimulate the phagocyte NADPH oxidase; however, it markedly enhances the NADPH oxidase response to other stimuli.

Amino Acid Motifs↗
PubMed2002

Cytosolic phospholipase A2 (cPLA2) regulation of human monocyte NADPH oxidase activity. cPLA2 affects translocation but not phosphorylation of p67(phox) and p47(phox).

The NADPH oxidase of human monocytes is activated upon exposure to opsonized zymosan and a variety of other stimuli to catalyze the formation of superoxide anion. Assembly of the NADPH oxidase complex is believed to be a highly regulated process, and molecular mechanisms responsible for this regulation have yet to be fully elucidated. We have previously reported that cytosolic phospholipase A(2) (cPLA(2)) expression and activity are essential for superoxide anion production in activated human monocytes. In this study, we investigated the mechanisms involved in cPLA(2) regulation of NADPH oxidase activation by evaluating the effects of cPLA(2) on translocation and phosphorylation of p67(phox) and p47(phox). We report that translocation and phosphorylation of p67(phox), as well as p47(phox), occur upon activation of human monocytes and that decreased cPLA(2) protein expression, mediated by antisense oligodeoxyribonucleotides (AS-ODN) specific for cPLA(2) mRNA, blocked the stimulation-induced translocation of p47(phox) and p67(phox) from the cytosol to the membrane fraction. Inhibition of translocation of both p47(phox) and p67(phox) by cPLA(2) AS-ODN was above 85%. Arachidonic acid (AA), a product of cPLA(2) enzymatic activity, completely restored translocation of both of these oxidase components in the AS-ODN-treated, cPLA(2)-deficient human monocytes. These results represent the first report that cPLA(2) activity or AA is required for p67(phox) and p47(phox) translocation in human monocytes. Although cPLA(2) was required for translocation of p47(phox) and p67(phox), it did not influence phosphorylation of these components. These results suggest that one mechanism of cPLA(2) regulation of NADPH oxidase activity is to control the arachidonate-sensitive assembly of the complete oxidase complex through modulating the translocation of both p47(phox) and p67(phox). These studies provide insight into the mechanisms by which activation signals are transduced to allow the induction of superoxide anion production in human monocytes.

Cells, Cultured↗
PubMed2002

EPR studies of the mitochondrial alternative oxidase. Evidence for a diiron carboxylate center.

The alternative oxidase (AOX) is a ubiquinol oxidase found in the mitochondrial respiratory chain of plants as well as some fungi and protists. It has been predicted to contain a coupled diiron center on the basis of a conserved sequence motif consisting of the proposed iron ligands, four glutamate and two histidine residues. However, this prediction has not been experimentally verified. Here we report the high level expression of the Arabidopsis thaliana alternative oxidase AOX1a as a maltose-binding protein fusion in Escherichia coli. Reduction and reoxidation of a sample of isolated E. coli membranes containing the alternative oxidase generated an EPR signal characteristic of a mixed-valent Fe(II)/Fe(III) binuclear iron center. The high anisotropy of the signal, the low value of the g-average tensor, and a small exchange coupling (-J) suggest that the iron center is hydroxo-bridged. A reduced membrane preparation yielded a parallel mode EPR signal with a g-value of about 15. In AOX containing a mutation of a putative glutamate ligand of the diiron center (E222A or E273A) the EPR signals are absent. These data provide evidence for an antiferromagnetic-coupled binuclear iron center, and together with the conserved sequence motif, identify the alternative oxidase as belonging to the growing family of diiron carboxylate proteins. The alternative oxidase is the first integral membrane protein in this family, and adds a new catalytic activity (ubiquinol oxidation) to this group of enzymatically diverse proteins.

Amino Acid Motifs↗