Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:1.6.3.1 (NADPH oxidase)
 11,281 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Superoxide production by phagocytic white blood cells requires the assembly of an NADPH oxidase from membrane and cytosolic proteins. Recombinant cytosolic proteins p47phox and p67phox and neutrophil membranes were used to purify a third cytosolic component that is necessary and sufficient for cell-free reconstitution of NADPH oxidase. The component was isolated as a complex of rho-GDP dissociation inhibitor (rho-GDI) and two members of the rho subfamily of ras-related guanine nucleotide binding proteins, rac2 and CDC42Hs. Oxidase reconstitution with these pure cytosolic proteins was unaffected by GTP gamma S but was inhibited by GDP beta S, suggesting that the active complex contained endogenous bound GTP. Direct binding of rho-GDI to the GTP gamma S-bound forms of these G-proteins was demonstrated by gel filtration following exchange with radiolabeled guanine nucleotide. rho-GDI was shown to be nonessential for cell-free oxidase reconstitution in experiments that compared the activities of pure recombinant forms of these G-proteins. Recombinant rac augmented superoxide production, while recombinant CDC42Hs, which shares 70% amino acid sequence identity with rac, did not. Three highly conserved regions of rac1 and rac2 were noted as markedly divergent in CDC42Hs. It is proposed that one or more of these regions of rac may be involved in the specific interaction of rac with the other NADPH oxidase protein(s).
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PMID:Regulation of the human neutrophil NADPH oxidase by rho-related G-proteins. 850 89

Angiotensin II is a multifunctional hormone that affects both contraction and growth of vascular smooth muscle cells through a complex series of intracellular signaling events initiated by the interaction of angiotensin II with the AT1 receptor. The cellular response to angiotensin II is multiphasic, involving stimulation within seconds of phospholipase C and Ca2+ mobilization; activation within minutes of phospholipase D, A2, protein kinase C, and MAP kinase; and stimulation after a period of hours of gene transcription and NADH/NADPH oxidase activity. Angiotensin II also activates numerous intracellular tyrosine kinases. In this respect, it shares some aspects of signaling with growth factor and cytokine receptors, including activation of phospholipase C-gamma, src, and ras; association of shc with grb2; and stimulation of the Jak/STAT pathway. The cellular events responsible for this unique series of events may involve receptor movement and the creation of a signaling domain. Elucidation of these pathways is important to our understanding of AT1 receptor function as a final effector of the renin-angiotensin system.
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PMID:Angiotensin II signaling in vascular smooth muscle. New concepts. 903 29

Previously, our laboratory reported that lactosylceramide (LacCer) stimulated human aortic smooth muscle cell proliferation via specific activation of p44 mitogen-activated protein kinase (MAPK) in the p21(ras)/Raf-1/MEK2 pathway and induced expression of the transcription factor c-fos downstream to the p44 MAPK signaling cascade (Bhunia A. K., Han, H., Snowden, A., and Chatterjee S. (1996) J. Biol. Chem. 271, 10660-10666). In the present study, we explored the role of free oxygen radicals in LacCer-mediated induction of cell proliferation. Superoxide levels were measured by the lucigenin chemiluminescence method, MAPK activity was measured by immunocomplex kinase assays, and Western blot analysis and c-fos expression were measured by Northern blot assay. We found that LacCer (10 microM) stimulates endogenous superoxide production (7-fold compared with control) in human aortic smooth muscle cells specifically by activating membrane-associated NADPH oxidase, but not NADH or xanthine oxidase. This process was inhibited by an inhibitor of NADPH oxidase, diphenylene iodonium (DPI), and by antioxidants, N-acetyl-L-cysteine (NAC) or pyrrolidine dithiocarbamate. NAC and DPI both abrogated individual steps in the signaling pathway leading to cell proliferation. For example, the p21(ras).GTP loading, p44 MAPK activity, and induction of transcription factor c-fos all were inhibited by NAC and DPI as well as an antioxidant pyrrolidine dithiocarbamate or reduced glutathione (GSH). In contrast, depletion of GSH by L-buthionine (S, R)-sulfoximine up-regulated the above described signaling cascade. In sum, LacCer, by virtue of activating NADPH oxidase, produces superoxide (a redox stress signaling molecule), which mediates cell proliferation via activation of the kinase cascade. Our findings may explain the potential role of LacCer in the pathogenesis of atherosclerosis involving the proliferation of aortic smooth muscle cells.
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PMID:Redox-regulated signaling by lactosylceramide in the proliferation of human aortic smooth muscle cells. 918 53

Sphingolipids and their metabolic products are now known to have second-messenger functions in a variety of cellular signaling pathways. Lactosylceramide (LacCer), a glycosphingolipid (GSL) present in vascular cells such as endothelial cells, smooth muscle cells, macrophages, neutrophils, platelets, and monocytes, contributes to atherosclerosis. Large amounts of LacCer accumulate in fatty streaks, intimal plaque, and calcified intimal plaque, along with oxidized low density lipoproteins (Ox-LDLs), growth factors, and proinflammatory cytokines. A possible role for LacCer in vascular cell biology was suggested when this GSL was found to stimulate the proliferation in vitro of aortic smooth muscle cells (ASMCs). A further link of LacCer in atherosclerosis was uncovered by the finding that Ox-LDLs stimulated specifically the biosynthesis of LacCer. Ox-LDL-stimulated endogenous synthesis of LacCer by activation of UDP-Gal:GlcCer,beta1-4galtransferase (GalT-2) is an early step in this signaling pathway. In turn, LacCer serves as a lipid second messenger that orchestrates a signal transduction pathway, ultimately leading to cell proliferation. This signaling pathway includes LacCer-mediated activation of NADPH oxidase that produces superoxide. Such superoxide molecules stimulate the GTP loading of p21(ras). Subsequently, the kinase cascade (Raf-1, Mek2, and p44MAPK [mitogen-activated protein kinase]) is activated. The phosphorylated form of p44MAPK translocates from the cytoplasm to the nucleus and engages in c-fos expression, proliferating cell nuclear antigen (PCNA) such as cyclin activation, and cell proliferation takes place. Interestingly, D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP), an inhibitor of GalT-2, can abrogate the Ox-LDL-mediated activation of GalT-2, the signal kinase cascade noted above, as well as cell proliferation. Additional studies have revealed that LacCer mediates the tumor necrosis factor-alpha (TNF-alpha)-induced nuclear factor-kappaB expression and intercellular adhesion molecule (ICAM-1) expression in vascular endothelial cells via the redox-dependent transcriptional pathway. LacCer also stimulates the expression of CD11/CD8, or Mac-1, on the surface of human neutrophils. Collectively, this phenomenon may contribute to the adhesion of neutrophils or monocytes to the endothelial cell surface and thus initiate the process of atherosclerosis. In addition, the LacCer-mediated proliferation of ASMCs may contribute to the progression of atherosclerosis. On the other hand, programmed cell death (apoptosis) by proinflammatory cytokines such as TNF-alpha, interleukin-1, and high concentrations of Ox-LDL occur via activation of a cell membrane-associated neutral sphingomyelinase (N-SMase). N-SMase hydrolyzes sphingomyelin into ceramide and phosphocholine. In turn, ceramide or a homologue serves as an important stress-signaling molecule. Interestingly, an antibody against N-SMase can abrogate Ox-LDL- and TNF-alpha-induced apoptosis and therefore may be useful for in vivo studies of apoptosis in experimental animals. Because plaque stability is an integral aspect of atherosclerosis management, activation of N-SMase and subsequent apoptosis may be vital events in the onset of plaque rupture, stroke, or heart failure. Interestingly, in human liver cells, N-SMase action mediates the TNF-alpha-induced maturation of the sterol regulatory-element binding protein. Moreover, a cell-permeable ceramide can reconstitute the phenomenon above in a sterol-independent fashion. Such findings may provide new avenues for therapy for patients with atherosclerosis. The findings described here indicate an important role for sphingolipids in vascular biology and provide an exciting opportunity for further research in vascular disease and atherosclerosis.
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PMID:Sphingolipids in atherosclerosis and vascular biology. 976 22

The oncogenic ras protein controls signal-transduction pathways that are critical for cell proliferation and tumorigenesis. Here, we demonstrate that v-Ha-ras-transduced human keratinocyte HaCaT cells produced significantly larger amounts of superoxide than did control cell lines. The superoxide generation was mediated by the transduced ras protein, because superoxide generation was modified by an inhibitor, lovastatin, that inhibits ras farnesylation during ras protein maturation. Superoxide generation was also inhibited by diphenylene iodonium, an inhibitor of flavoproteins, including NADPH oxidase, but not by rotenone, an inhibitor of the respiratory chain of the mitochondria. Those observations suggested that a phagocytic-like NADPH oxidase exists in keratinocytes that could be activated by the dominant activated v-Ha-ras and produce superoxide. Overexpression of manganese-containing superoxide dismutase and copper and zinc-containing superoxide dismutase cDNA via adenovirus infection also attenuated superoxide generation. Previous work has demonstrated that extracellular superoxide dismutase (SOD) can lower superoxide generation; this is the first report that intracellular SOD could also modify the amount of superoxide production from the cells. This report implies that superoxide radical may act as a second messenger molecule of oncogenic ras.
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PMID:Superoxide generation in v-Ha-ras-transduced human keratinocyte HaCaT cells. 1055 93

Synapsins are synaptic vesicle-associated phosphoproteins involved in synapse formation and regulation of neurotransmitter release. Recently, synapsin I has been found to bind the Src homology 3 (SH3) domains of Grb2 and c-Src. In this work we have analyzed the interactions between synapsins and an array of SH3 domains belonging to proteins involved in signal transduction, cytoskeleton assembly, or endocytosis. The binding of synapsin I was specific for a subset of SH3 domains. The highest binding was observed with SH3 domains of c-Src, phospholipase C-gamma, p85 subunit of phosphatidylinositol 3-kinase, full-length and NH(2)-terminal Grb2, whereas binding was moderate with the SH3 domains of amphiphysins I/II, Crk, alpha-spectrin, and NADPH oxidase factor p47(phox) and negligible with the SH3 domains of p21(ras) GTPase-activating protein and COOH-terminal Grb2. Distinct sites in the proline-rich COOH-terminal region of synapsin I were found to be involved in binding to the various SH3 domains. Synapsin II also interacted with SH3 domains with a partly distinct binding pattern. Phosphorylation of synapsin I in the COOH-terminal region by Ca(2+)/calmodulin-dependent protein kinase II or mitogen-activated protein kinase modulated the binding to the SH3 domains of amphiphysins I/II, Crk, and alpha-spectrin without affecting the high affinity interactions. The SH3-mediated interaction of synapsin I with amphiphysins affected the ability of synapsin I to interact with actin and synaptic vesicles, and pools of synapsin I and amphiphysin I were shown to associate in isolated nerve terminals. The ability to bind multiple SH3 domains further implicates the synapsins in signal transduction and protein-protein interactions at the nerve terminal level.
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PMID:Specificity of the binding of synapsin I to Src homology 3 domains. 1089 72

It has been shown that oxidative stress occurs in chronic hepatitis C. Release of reactive oxygen species (ROS) from sequestered phagocytes and activated resident macrophages represents the predominant component of oxidative stress in the liver. However, little is known about the ability of the monocyte to produce ROS in response to protein of hepatitis C virus. In this study, we investigated the ROS production in human monocytes stimulated by several viral proteins of hepatitis C virus. Human monocytes from healthy blood donors were incubated with recombinant viral protein: Core, NS3, NS4, and NS5. ROS production was measured by chemiluminescence. Only NS3 triggered ROS production in human monocytes. Generated ROS were mainly the anion superoxide. NS3 also induced a rapid and transient increase in intracellular calcium concentration measured by a video digital microscopy technique. By using different metabolic inhibitors, we showed that ROS production requires calcium influx, tyrosine kinases, and the stress-activated protein kinase, p38. The study of p47(PHOX) phosphorylation and translocation showed that NADPH oxidase was activated and involved in ROS production induced by NS3. In a second experiment, NS3 inhibited the oxidative burst induced by phorbol 12-myristate 13-acetate. These results indicate that NS3 activates NADPH oxidase and modulates ROS production, which may be involved in the natural history of hepatitis C infection.
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PMID:Nonstructural 3 protein of hepatitis C virus triggers an oxidative burst in human monocytes via activation of NADPH oxidase. 1130 37

Accumulating evidence has suggested that cellular production of superoxide acts as an intracellular messenger to regulate gene expression and modulate cellular activities. In this report, we set out to investigate the role of active H-ras-mediated superoxide production on tumor cell malignancy in a SV-40 transformed human lung WI-38 VA-13 cell line. Stable transfection and expression of constitutively active mutant V12-H-ras (V12-H-ras) dramatically increased intracellular production of superoxide. The expression of V12-H-ras significantly enhanced cell proliferation, migration and resistance to TNF-alpha treatment compared to that of parental and vector control cells, while expression of wild type H-ras (WT-H-ras) only had modest effects. Upon scavenging by superoxide dismutase and other molecules that decrease the intracellular level of active H-ras mediated superoxide production, cell proliferation, migration and resistance to TNF-alpha were significantly reduced. Furthermore, we demonstrated that the activation of membrane NADPH oxidase activity by expression of active H-ras contributed to the intracellular superoxide production. The causal relationship between membrane superoxide production and increased cell proliferation, migration, and resistance to TNF-alpha by the expression of active H-ras, has provided direct evidence to demonstrate that superoxide acts as an intracellular messenger to cascade ras oncogenic signal relay and to modulate tumor malignant activity.
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PMID:Elevated superoxide production by active H-ras enhances human lung WI-38VA-13 cell proliferation, migration and resistance to TNF-alpha. 1131 92

Nitric oxide (NO) mediates apoptosis induction in fibroblasts with constitutive src or induced ras oncogene expression, whereas nontransformed parental cells and revertants are not affected. This direct link between the transformed phenotype and sensitivity to NO-mediated apoptosis induction seems to be based on the recently described extracellular superoxide anion generation by transformed cells, as NO-mediated apoptosis induction in transformed cells is inhibited by extracellular superoxide dismutase (SOD), by SOD mimetics and by apocynin, an inhibitor of NADPH oxidase. Furthermore, nonresponsive nontransformed cells can be rendered sensitive for NO-mediated apoptosis induction when they are supplemented with xanthine oxidase/xanthine as an extracellular source for superoxide anions. As superoxide anions and NO readily interact in a diffusion-controlled reaction to generate peroxynitrite, peroxynitrite seems to be the responsible apoptosis inducer in NO-mediated apoptosis induction. In line with this conclusion, NO-mediated apoptosis induction in superoxide anion-generating transformed cells is inhibited by the peroxynitrite scavengers ebselen and FeTPPS. Moreover, direct application of peroxynitrite induces apoptosis both in transformed and nontransformed cells, indicating that peroxynitrite is no selective apoptosis inducer per se, but that selective apoptosis induction in transformed cells by NO is achieved through selective peroxynitrite generation. The interaction of NO with target cell derived superoxide anions represents a novel concept for selective apoptosis induction in transformed cells. This mechanism may be the basis for selective apoptosis induction by natural antitumor systems (like macrophages, natural killer cells, granulocytes) that utilize NO for antitumor action. Apoptosis induction mediated by NO involves mitochondrial depolarization and is blocked by Bcl-2 overexpression.
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PMID:Nitric oxide mediates apoptosis induction selectively in transformed fibroblasts compared to nontransformed fibroblasts. 1208 14

Neutrophil priming by agents such as TNF-alpha and GM-CSF causes a dramatic increase in the response of these cells to secretagogue agonists and affects the capacity of neutrophils to induce tissue injury. In view of the central role of phosphatidylinositol 3-kinase (PI3-kinase) in regulating NADPH oxidase activity we examined the influence of priming agents on agonist-stimulated phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) accumulation in human neutrophils. Pretreatment of neutrophils with TNF-alpha or GM-CSF, while not influencing fMLP-stimulated PtdIns(3,4,5)P3 accumulation at 5 s, caused a major increase in PtdIns(3,4,5)P3 at later times (10-60 s), which paralleled the augmented superoxide anion (O2-) response. The intimate relationship between PtdIns(3,4,5)P3 accumulation and O2- release was confirmed using platelet-activating factor, which caused full but transient priming of both responses. Likewise, LY294002, a PI3-kinase inhibitor, and genistein, a tyrosine kinase inhibitor, caused parallel inhibition of O2- generation and PtdIns(3,4,5)P3 accumulation; in contrast, radicicol, which inhibits receptor-mediated activation of p85 PI3-kinase, had no effect on either response. Despite major increases in PI3-kinase activity observed in p85 and anti-phosphotyrosine immunoprecipitates in growth factor-stimulated smooth muscle cells, no such increase was observed in primed/stimulated neutrophils. In contrast, both fMLP and TNF-alpha alone caused a 3-fold increase in PI3-kinase activity in p110gamma PI3-kinase immunoprecipitates. p21(ras) activation (an upstream regulator of PI3-kinase) was unaffected by priming. These data demonstrate that timing and magnitude of PtdIns(3,4,5)P3 accumulation in neutrophils correlate closely with O2- generation, that PI3-kinase-gamma is responsible for the enhanced PtdIns(3,4,5)P3 production seen in primed cells, and that factors other than activation of p21(ras) underlie this response.
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PMID:Regulation of phosphatidylinositol 3-kinase activity and phosphatidylinositol 3,4,5-trisphosphate accumulation by neutrophil priming agents. 1221 55


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