Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:1.6.3.1 (NADPH oxidase)
11,281 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Insulin stimulation of target cells elicits a burst of H(2)O(2) that enhances tyrosine phosphorylation of the insulin receptor and its cellular substrate proteins as well as distal signaling events in the insulin action cascade. The molecular mechanism coupling the insulin receptor with the cellular oxidant-generating apparatus has not been elucidated. Using reverse transcription-PCR and Northern blot analyses, we found that Nox4, a homolog of gp91phox, the phagocytic NAD(P)H oxidase catalytic subunit, is prominently expressed in insulin-sensitive adipose cells. Adenovirus-mediated expression of Nox4 deletion constructs lacking NAD(P)H or FAD/NAD(P)H cofactor binding domains acted in a dominant-negative fashion in differentiated 3T3-L1 adipocytes and attenuated insulin-stimulated H(2)O(2) generation, insulin receptor (IR) and IRS-1 tyrosine phosphorylation, activation of downstream serine kinases, and glucose uptake. Transfection of specific small interfering RNA oligonucleotides reduced Nox4 protein abundance and also inhibited the insulin signaling cascade. Overexpression of Nox4 also significantly reversed the inhibition of insulin-stimulated IR tyrosine phosphorylation induced by coexpression of PTP1B by inhibiting PTP1B catalytic activity. These data suggest that Nox4 provides a novel link between the IR and the generation of cellular reactive oxygen species that enhance insulin signal transduction, at least in part via the oxidative inhibition of cellular protein-tyrosine phosphatases (PTPases), including PTP1B, a PTPase that has been previously implicated in the regulation of insulin action.
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PMID:The NAD(P)H oxidase homolog Nox4 modulates insulin-stimulated generation of H2O2 and plays an integral role in insulin signal transduction. 1496 67

NADPH oxidase 5 (NOX5) is a homologue of the gp91(phox) subunit of the phagocyte NADPH oxidase. NOX5 is expressed in lymphoid organs and testis and distinguished from the other NADPH oxidases by its unique N terminus, which contains three canonical EF-hands, Ca(2+)-binding domains. Upon heterologous expression, NOX5 was shown to generate superoxide in response to intracellular Ca(2+) elevations. In this study, we have analyzed the mechanism of Ca(2+) activation of NOX5. In a cell-free system, Ca(2+) elevations triggered superoxide production by NOX5 (K(m) = 1.06 microm) in an NADPH- and FAD-dependent but cytosol-independent manner. That result indicated a role for the N-terminal EF-hands in NOX5 activation. Therefore, we generated recombinant proteins of NOX5 N terminus and investigated their interactions with Ca(2+). Flow dialysis experiments showed that NOX5 N terminus contained four Ca(2+)-binding sites and allowed us to define the hitherto unidentified fourth, non-canonical EF-hand. The EF-hands of NOX5 formed two pairs: the very N-terminal pair had relatively low affinity for Ca(2+), whereas the more C-terminal pair bound Ca(2+) with high affinity. Ca(2+) binding caused a marked conformation change in the N terminus, which exposed its hydrophobic core, and became able to bind melittin, a model peptide for calmodulin targets. Using a pull-down assay, we demonstrate that the regulatory N terminus and the catalytic C terminus of NOX5 interact in a Ca(2+)-dependent way. Our results indicate that the Ca(2+)-induced conformation change of NOX5 N terminus led to enzyme activation through an intra-molecular interaction. That represents a novel mechanism of activation among NAD(P)H oxidases and Ca(2+)-activated enzymes.
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PMID:Mechanism of Ca2+ activation of the NADPH oxidase 5 (NOX5). 1498 37

The superoxide-producing phagocyte NADPH oxidase can be reconstituted in a cell-free system. The activity of NADPH oxidase is dependent on FAD, but the physiological status of FAD in the oxidase is not fully elucidated. To clarify the role of FAD in NADPH oxidase, FAD-free full-length recombinant p47(phox), p67(phox), p40(phox), and Rac were prepared, and the activity was reconstituted with these proteins and purified cytochrome b(558) (cyt b(558)) with different amounts of FAD. A remarkably high activity, over 100 micromol/s/micromol heme, was obtained in the oxidase with purified cyt b(558), ternary complex (p47-p67-p40(phox)), and Rac. From titration with FAD of the activity of NADPH oxidase reconstituted with purified FAD-devoid cyt b, the dissociation constant K(d) of FAD in cyt b(558) of reconstituted oxidase was estimated as nearly 1 nm. We also examined addition of FAD on the assembly process in reconstituted oxidase. The activity was remarkably enhanced when FAD was present during assembly process, and the efficacy of incorporating FAD into the vacant FAD site in purified cyt b(558) increased, compared when FAD was added after assembly processes. The absorption spectra of reconstituted oxidase under anaerobiosis showed that incorporation of FAD into cyt b(558) recovered electron flow from NADPH to heme. From both K(d) values of FAD and the amount of incorporated FAD in cyt b(558) of reconstituted oxidase, in combination with spectra, we propose the model in which the K(d) values of FAD in cyt b(558) is changeable after activation and FAD binding works as a switch to regulate electron transfer in NADPH oxidase.
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PMID:Binding of FAD to cytochrome b558 is facilitated during activation of the phagocyte NADPH oxidase, leading to superoxide production. 1510 59

Activation of the phagocyte NADPH oxidase involves assembly of p47(phox), p67(phox), Rac, and flavocytochrome b(558), and the activation can be triggered in a cell-free system with an anionic amphiphile. We find that the activated oxidase in a pure cell-free system was rapidly inactivated upon dilution. When the activated oxidase was treated with a chemical cross-linker, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, the half-life of the oxidase in dilution was extended from 1min to 4h at 25 degrees C. The cross-linked oxidase was resistant to inhibition by inactive flavin analogs, indicating that cross-linking prevents flavin exchange. When a fusion protein p67N-p47N plus RacQ61L was added, flavocytochrome b(558) became spontaneously active. Cross-linking of this mixture produced an oxidase that was extremely stable to dilution (t(1/2)=6.6h). Western blotting analysis showed the presence of a cross-link between p67N-p47N and RacQ61L. These results suggest that covalently linked phox components prevents FAD loss and stabilizes the longevity of the stoichiometric complex, extending the lifespan of the active oxidase.
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PMID:Inactivation of neutrophil NADPH oxidase upon dilution and its prevention by cross-link and fusion of phox proteins. 1546 35

Assembly of cytosolic factors p67(phox) and p47(phox) with cytochrome b(558) is one of the crucial keys for NADPH oxidase activation. Certain sequences of Nox2 appear to be involved in cytosolic factor interaction. The role of the D-loop (191)TSSTKTIRRS(200) and the C-terminal (484)DESQANHFAVHHDEEKD(500) of Nox2 on oxidase activity and assembly was investigated. Charged amino acids were mutated to neutral or reverse charge by directed mutagenesis to generate 21 mutants. Recombinant wild-type or mutant Nox2 were expressed in the X-CGD PLB-985 cell model. K195A/E, R198E, R199E, and RR198199QQ/AA mutations in the D-loop of Nox2 totally abolished oxidase activity. However, these D-loop mutants demonstrated normal p47(phox) translocation and iodonitrotetrazolium (INT) reductase activity, suggesting that charged amino acids of this region are essential for electron transfer from FAD to oxygen. Replacement of Nox2 D-loop with its homolog of Nox1, Nox3, or Nox4 was fully functional. In addition, fMLP (formylmethionylleucylphenylalanine)-activated R199Q-Nox2 and D-loop(Nox4)-Nox2 mutants exhibited four to eight times the NADPH oxidase activity of control cells, suggesting that these mutations lead to a more efficient oxidase activation process. In contrast, the D484T and D500A/R/G mutants of the alpha-helical loop of Nox2 exhibited no NADPH oxidase and INT reductase activities associated with a defective p47(phox) membrane translocation. This suggests that the alpha-helical loop of the C-terminal of Nox2 is probably involved in the correct assembly of the NADPH oxidase complex occurring during activation, permitting cytosolic factor translocation and electron transfer from NADPH to FAD.
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PMID:Crucial role of two potential cytosolic regions of Nox2, 191TSSTKTIRRS200 and 484DESQANHFAVHHDEEKD500, on NADPH oxidase activation. 1568 31

AMID (apoptosis-inducing factor-homologous mitochondrion-associated inducer of death; also known as PRG3 (p53-responsive gene 3)) is a human caspase-independent pro-apoptotic protein with some similarity to apoptosis-inducing factor. AMID was purified from a recombinant bacterial host, enabling biochemical analysis of the protein. AMID is a flavoprotein; possesses NAD(P)H oxidase activity; and catalyzes NAD(P)H-dependent reduction of cytochrome c and other electron acceptors, including molecular oxygen. NADPH binds approximately 10-fold tighter than NADH. AMID binds 6-hydroxy-FAD (a cofactor that accumulates only adventitiously and at low abundance in other flavoprotein enzymes) to form a stoichiometric cofactor.protein complex. AMID has a distinctive electronic spectrum due to the modified flavin. NAD(P)+ binding perturbed the spectrum, enabling determination of K(d) values for these coenzymes. 6-Hydroxy-FAD could be removed from AMID and the apoprotein reconstituted with FAD. FAD was converted to 6-hydroxy-FAD in reconstituted AMID during aerobic turnover with NADPH. AMID is a DNA-binding protein that lacks apparent DNA sequence specificity. Formation of the protein.DNA complex (i) effected a major protein conformational change and (ii) was prevented in the presence of nicotinamide coenzyme. Apo-AMID retains DNA binding activity. Our studies establish a link between coenzyme and DNA binding that likely impacts on the physiological role of AMID in cellular apoptosis.
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PMID:The human apoptosis-inducing protein AMID is an oxidoreductase with a modified flavin cofactor and DNA binding activity. 1595 87

It has been widely believed that undifferentiated human promyelocytic leukemia cells (HL-60) have no ability to generate reactive oxygen species (ROS) responding to stimuli. We report here that undifferentiated HL-60 cells possess NADPH oxidase and that generation of superoxide can be measured using a highly sensitive chemiluminescence dye, L-012. Five subunits of NADPH oxidase, namely, gp91(phox), p22(phox), p67(phox), p47(phox), and Rac 2, were detected in undifferentiated HL-60 cells by immunoblotting analysis. The contents of these NADPH oxidase components in the cells were increased with the differentiation induced by phorbol myristate acetate (PMA), except for p22(phox). Messenger RNAs of these subunits were also detected by the RT-PCR method, and their expressions increased except that of p22(phox) with the differentiation induced by PMA. Kinetic analysis using L-012 revealed that HL-60 cells generated substantial amounts of ROS by various stimulants, including formylmethionyl-leucyl-phenylalanine, PMA, myristic acid, and a Ca2+ ionophore, A23187. Both diphenyleneiodonium (an inhibitor of FAD-dependent oxidase) and apocynin (a specific inhibitor of NADPH oxidase) suppressed this stimuli-dependent ROS generation. Genistein, staurosporine, uric acid, and sodium azide inhibited the ROS generation in undifferentiated HL-60 cells in a similar way to that in undifferentiated neutrophils. These results suggested that the mechanism of ROS generation in undifferentiated HL-60 cells is the same as that in primed neutrophils.
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PMID:Mechanism and characteristics of stimuli-dependent ROS generation in undifferentiated HL-60 cells. 1611 42

It is well established that growth-factor-induced reactive oxygen species (ROS) act as second messengers in cell signaling. We have previously reported that betaPix, a guanine nucleotide exchange factor for Rac, interacts with NADPH oxidase 1 (Nox1) leading to EGF-induced ROS generation. Here, we report the identification of the domains of Nox1 and betaPix responsible for the interaction between the two proteins. GST pull-down assays show that the PH domain of betaPix binds to the FAD-binding region of Nox1. We also show that overexpression of the PH domain of betaPix results in inhibition of superoxide anion generation in response to EGF. Additionally, NADPH oxidase Organizer 1 (NoxO1) is shown to interact with the NADPH-binding region of Nox1. These results suggest that the formation of the complex consisting of Nox1, betaPix, and NoxO1 is likely to be a critical step in EGF-induced ROS generation.
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PMID:Molecular interaction of NADPH oxidase 1 with betaPix and Nox Organizer 1. 1632 88

Mitochondrial P450 type enzymes catalyze central steps in steroid biosynthesis, including cholesterol conversion to pregnenolone, 11beta and 18 hydroxylation in glucocorticoid and mineralocorticoid synthesis, C-27 hydroxylation of bile acids, and 1alpha and 24 hydroxylation of 25-OH-vitamin D. These monooxygenase reactions depend on electron transfer from NADPH via FAD adrenodoxin reductase and 2Fe-2S adrenodoxin. These systems can function as a futile NADPH oxidase, oxidizing NADPH in absence of substrate, and leak electrons via adrenodoxin and P450 to O(2), producing superoxide and other reactive oxygen species (ROS). The degree of uncoupling depends on the P450 and steroid substrate. Studies with purified proteins and overexpression in cultured cells show consistently that adrenodoxin, but not reductase, is responsible for ROS production that can lead to apoptosis. In the ovary and corpus luteum, antioxidant enzyme activities superoxide dismutase, catalase, and glutathione peroxidase parallel steroidogenesis. Antioxidant beta-carotene, alpha-tocopherol, and ascorbate can protect against oxidative damages of P450 systems. In testis Leydig cells, steroidogenesis is associated with aging of the steroidogenic capacity.
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PMID:Antioxidant protective mechanisms against reactive oxygen species (ROS) generated by mitochondrial P450 systems in steroidogenic cells. 1668 56

The FAD-dependent NAD(P)H oxidase from Lactobacillus sanfrancisensis (L.san-Nox2) catalyzes the oxidation of 2 equivalents of either NADH or NADPH and reduces 1 equivalent of O(2) to yield 2 equivalents of water. During steady-state turnover only 0.5% of the reducing equivalents are detected in solution as hydrogen peroxide, suggesting that it is not released from the enzyme after the oxidation of the first equivalent of NAD(P)H and reaction with O(2). Here we report the crystal structure of L.san-Nox2 to 1.8 A resolution. The enzyme crystallizes as a dimer with each monomer consisting of a FAD binding domain (residues 1-120), a NAD(P)H binding domain (residues 150-250), and a dimerization domain (residues 325-451). The electron density for the redox-active Cys42 residue located adjacent to the si-face FAD is consistent with oxidation to the sulfenic acid (Cys-SOH) state. The side chain of Cys42 is also observed in two conformations; in one the sulfenic acid is hydrogen bonded to His10 and in the other it hydrogen bonds with the FAD O2' atom. Surprisingly, the NAD(P)H binding domains each contain an ADP ligand as established by electron density maps and MALDI-TOF analysis of the ligands released from heat-denatured enzyme. The ADP ligand copurifies with the enzyme, and its presence does not inhibit enzyme activity. Consequently, we hypothesize that either NADPH or NADH substrates bind via a long channel that extends from the enzyme exterior and terminates at the FAD re-face. A homology model of the NADH oxidase from Lactococcus lactis (L.lac-Nox2) was also generated using the crystal structure of L.san-Nox2, which reveals several important similarities and differences between the two enzymes. HPLC analysis of ligands released from denatured L.lac-Nox2 indicates that it does not bind ADP, which correlates with the specificity of the enzyme for oxidation of NADH.
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PMID:The crystal structure of NAD(P)H oxidase from Lactobacillus sanfranciscensis: insights into the conversion of O2 into two water molecules by the flavoenzyme. 1689 66


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