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1.
Role of the small GTPase Rac in p22phox-dependent NADPH oxidases 总被引:2,自引:0,他引:2
The superoxide-producing phagocyte NADPH oxidase gp91(phox)/Nox2 and the non-phagocytic oxidases Nox1 and Nox3 each form a complex in the membrane with p22(phox), which provides both stabilization and a docking site for organizer proteins. The p22(phox)-complexed Nox2 and Nox1 are dormant on their own, and their activation requires soluble supportive proteins such as a Nox organizer (p47(phox) or Noxo1) and a Nox activator (p67(phox) or Noxa1). The small GTPase Rac directly binds to the activators, and thus plays an essential role in the Nox2-based oxidase containing p47(phox) and p67(phox) or a positive role in Nox1 activity supported by Noxo1 and Noxa1. Although Nox3 complexed with p22(phox) constitutively produce superoxide, the production can be enhanced by supportive proteins. Here we compare the roles of Rac in these p22(phox)-dependent oxidases using the organizer and activator in different combinations. Expression of constitutively active Rac1(Q61L) is essential for activation of the Nox2- or Nox1-based oxidase containing the organizer p47(phox) and either p67(phox) or Noxa1. When these oxidases use Noxo1 as an organizer instead of p47(phox), they produce a small but significant amount of superoxide without expression of Rac1(Q61L), although the production is enhanced by Rac1(Q61L). Thus p47(phox) is likely related to strict dependence on Rac. The Nox3-based oxidase has a similar tendency in the change of the dependence: Rac plays a positive role in Nox3 activation in the presence of p47(phox) and either p67(phox) or Noxa1, whereas Rac fails to upregulate Nox3 activity when p47(phox) is replaced with Noxo1. We also demonstrate that, in the Nox3-based oxidase containing solely p67(phox) as supportive protein, expression of Rac1(Q61L) enhances not only superoxide production but also membrane translocation of p67(phox). Since the enhancements are not observed with a mutant p67(phox) defective in binding to Rac, this GTPase appear to directly recruit p67(phox) to the membrane. 相似文献
2.
Kawano M Miyamoto K Kaito Y Sumimoto H Tamura M 《Archives of biochemistry and biophysics》2012,519(1):1-7
Noxa1 was discovered as an activating factor for Nox1, an O(2)(-)-generating enzyme. Subsequent studies have shown that Noxa1 is colocalized with Nox2 in several cell types, including vascular cells. Nox2 activation by Noxa1 has been examined in reconstituted model cells. However, little is known about the kinetic properties of Noxa1 in Nox2 activation. In the present study, we used purified cyt.b(558) (Nox2 plus p22(phox)), Rac(Q61L), and Noxo1 to examine the ability of Noxa1 to activate Nox2. In the pure reconstitution system, Noxa1 activated Nox2 with lower efficiency than p67(phox), a canonical activator of Nox2. The EC(50) value of Noxa1 was considerably higher than that of p67(phox). The V(max) value with Noxa1 and Noxo1 was one-third of that with p67(phox) and p47(phox). The EC(50) value of Noxo1 or Rac(Q61L) was also higher when Noxa1 was used. The affinity of FAD for the oxidase and the stability of the active complex were remarkably low when Noxa1 and Noxo1 were used compared with p67(phox) and p47(phox). The stability was not improved by fusion of Noxa1 with Rac(Q61L). These findings show that Noxa1 has quite different kinetic properties from p67(phox) and suggest that Noxa1 may function as a moderate activator of Nox2. 相似文献
3.
Several Nox family NADPH oxidases function as multicomponent enzyme systems. We explored determinants of assembly of the multicomponent oxidases Nox1 and Nox3 and examined the involvement of Rac1 in their regulation. Both enzymes are supported by p47phox and p67phox or homologous regulators called Noxo1 and Noxa1, although Nox3 is less dependent on these cofactors for activity. Plasma membrane targeting of Noxa1 depends on Noxo1, through tail-to-tail interactions between these proteins. Noxa1 can support Nox1 without Noxo1, when targeted to the plasma membrane by fusing membrane-binding sequences from Rac1 (amino acids 183 to 192) to the C terminus of Noxa1. However, membrane targeting of Noxa1 is not sufficient for activation of Nox1. Both the Noxo1-independent and -dependent Nox1 systems involve Rac1, since they are affected by Rac1 mutants or Noxa1 mutants defective in Rac binding or short interfering RNA-mediated Rac1 silencing. Nox1 or Nox3 expression promotes p22phox transport to the plasma membrane, and both oxidases are inhibited by mutations in the p22phox binding sites (SH3 domains) of the Nox organizers (p47phox or Noxo1). Regulation of Nox3 by Rac1 was also evident from the effects of mutant Rac1 or mutant Nox3 activators (p67phox or Noxa1) or Rac1 silencing. In the absence of Nox organizers, the Nox activators (p67phox or Noxa1) colocalize with Rac1 within ruffling membranes, independently of their ability to bind Rac1. Thus, Rac1 regulates both oxidases through the Nox activators, although it does not appear to direct the subcellular localization of these activators. 相似文献
4.
Ueno N Takeya R Miyano K Kikuchi H Sumimoto H 《The Journal of biological chemistry》2005,280(24):23328-23339
Nox3, a member of the superoxide-producing NADPH oxidase (Nox) family, participates in otoconia formation in mouse inner ears, which is required for perception of balance and gravity. The activity of other Nox enzymes such as gp91(phox)/Nox2 and Nox1 is known to absolutely require both an organizer protein (p47(phox) or Noxo1) andanactivatorprotein (p67(phox) or Noxa1); for the p47(phox)-dependent activation of these oxidases, treatment of cells with stimulants such as phorbol 12-myristate 13-acetate is also indispensable. Here we show that ectopic expression of Nox3 in various types of cells leads to phorbol 12-myristate 13-acetate-independent constitutive production of a substantial amount of superoxide under the conditions where gp91(phox) and Nox1 fail to generate superoxide, i.e. in the absence of the oxidase organizers and activators. Nox3 likely forms a functional complex with p22(phox); Nox3 physically interacts with and stabilizes p22(phox), and the Nox3-dependent superoxide production is totally dependent on p22(phox). The organizers p47(phox) and Noxo1 are capable of enhancing the superoxide production by Nox3 in the absence of the activators, and the enhancement requires the interaction of the organizers with p22(phox), further indicating a link between Nox3 and p22(phox). The p47(phox)-enhanced Nox3 activity is further facilitated by p67(phox) or Noxa1, whereas the activators cancel the Noxo1-induced enhancement. On the other hand, the small GTPase Rac, essential for the gp91(phox) activity, is likely dispensable to the Nox3 system. Thus Nox3 functions together with p22(phox) as an enzyme constitutively producing superoxide, which can be distinctly regulated by combinatorial use of the organizers and activators. 相似文献
5.
Direct interaction of the novel Nox proteins with p22phox is required for the formation of a functionally active NADPH oxidase 总被引:9,自引:0,他引:9
Ambasta RK Kumar P Griendling KK Schmidt HH Busse R Brandes RP 《The Journal of biological chemistry》2004,279(44):45935-45941
Nox1 and Nox4, homologues of the leukocyte NADPH oxidase subunit Nox2 (gp91phox) mediate superoxide anion formation in various cell types. However, their interactions with other components of the NADPH oxidase are poorly defined. We determined whether a direct interaction of Nox1 and Nox4 with the p22phox subunit of the NADPH oxidase occurs. Using confocal microscopy, co-localization of p22phox with Nox1, Nox2, and Nox4 was observed in transiently transfected vascular smooth muscle cells (VSMC) and HEK293 cells. Plasmids coding for fluorescent fusion proteins of p22phox and the Nox proteins with cyan- and yellow-fluorescent protein (cfp and yfp, respectively) were constructed and expressed in VSMC and HEK293 cells. The cfp-tagged p22phox expression level increased upon cotransfection with Nox1 or Nox4. Protein-protein interaction between the fluorescent fusion proteins of p22phox and the Nox partners was observed using the fluorescence resonance energy transfer technique. Immunoprecipitation of native Nox1 from human VSMC revealed co-precipitation of p22phox. Immunoprecipitation from transfected HEK293 cells revealed co-precipitation of native p22phox with yfp-tagged Nox1, Nox2, and Nox4. Following mutation of a histidine (corresponding to the position 115 in human Nox2) to leucine, this interaction was abolished. Transfection of rat p22phox (but not Noxo1 and Noxa1) increased the radical generation in cells expressing Nox4. We provide evidence that p22phox directly interacts with Nox1 and Nox4, to form an superoxide-generating NADPH oxidase and demonstrate that mutation of the potential heme binding site in the Nox proteins disrupts the complex formation of Nox1 and Nox4 with p22phox. 相似文献
6.
Yamamoto A Kami K Takeya R Sumimoto H 《Biochemical and biophysical research communications》2007,352(2):560-565
NADPH oxidase organizer 1 (Noxo1), harboring a PX domain, two SH3 domains, and a proline-rich region (PRR), participates in activation of superoxide-producing Nox-family NADPH oxidases. Here, we show that Noxo1 supports superoxide production in a cell-free system for gp91(phox)/Nox2 activation by arachidonic acid. This lipid enhances an SH3-mediated binding of Noxo1 to p22(phox), a protein complexed with Nox oxidases; the binding is known to be required for Nox activation. We also demonstrate that the bis-SH3 domain directly interacts with the Noxo1 PRR. The interaction appears to prevent the bis-SH3 domain and PRR from binding to their target proteins; disruption of the intramolecular interaction facilitates Noxo1 binding to p22(phox) and also allows the PRR to associate with the Nox activator Noxa1, which association is crucial for Nox activation as well. These findings suggest that Nox activation involves a conformational change leading to disruption of the bis-SH3-PRR interaction in Noxo1. 相似文献
7.
Ambasta RK Schreiber JG Janiszewski M Busse R Brandes RP 《Free radical biology & medicine》2006,41(2):193-201
NADPH oxidase is the most important source of oxygen-derived radicals (ROS) in the vascular wall. In vascular smooth muscle cells (VSMC), NADPH oxidase is characterized by the expression of the membrane subunit Nox1, which is activated by cytoplasmic proteins binding to its activation domain. We set out to identify the cytoplasmic protein involved in NADPH oxidase activation in mouse VSMC. Western blot analysis revealed that human endothelial cells and leukocytes but not VSMC from the aorta of the rat and the mouse express the classic NADPH oxidase activator p67phox. In mouse VSMC, however, the p67phox homologue Noxa1 was detected. Using antibodies generated against mouse Noxa1, the protein was observed in the cytosolic fraction of mouse VSMC with a molecular weight of about 51 kDa. Immunohistochemistry revealed that Noxa1 is expressed in the smooth muscle layer but not in endothelium or the adventitia of the mouse carotid artery. Fluorescent fusion proteins of Noxa1 were observed to be expressed in the cytoplasm of VSMC and coexpression of the NADPH oxidase organizer Noxo1 targeted the complex to membrane. An antisense plasmid of Noxa1 attenuated the endogenous Noxa1 protein expression in VSMC. This plasmid attenuated the ROS formation in mouse VSMC as detected using L012 chemiluminescence and prevented the agonist-induced ROS production in response to basic fibroblast growth factor and epidermal growth factor. In conclusion, these data indicate that Noxa1 replaces p67phox in VSMC and plays a central role in the activation of the NADPH oxidase in the vascular wall. 相似文献
8.
Expression and function of Noxo1gamma, an alternative splicing form of the NADPH oxidase organizer 1
Activation of the superoxide-producing NADPH oxidase Nox1 requires both the organizer protein Noxo1 and the activator protein Noxa1. Here we describe an alternative splicing form of Noxo1, Noxo1gamma, which is expressed in the testis and fetal brain. The Noxo1gamma protein contains an additional five amino acids in the N-terminal PX domain, a phosphoinositide-binding module; the domain plays an essential role in supporting superoxide production by NADPH oxidase (Nox) family oxidases including Nox1, gp91(phox)/Nox2, and Nox3, as shown in this study. The PX domain isolated from Noxo1gamma shows a lower affinity for phosphoinositides than that from the classical splicing form Noxo1beta. Consistent with this, in resting cells, Noxo1gamma is poorly localized to the membrane, and thus less effective in activating Nox1 than Noxo1beta, which is constitutively present at the membrane. On the other hand, cell stimulation with phorbol 12-myristate 13-acetate (PMA), an activator of Nox1-3, facilitates membrane translocation of Noxo1gamma; as a result, Noxo1gamma is equivalent to Noxo1beta in Nox1 activation in PMA-stimulated cells. The effect of the five-amino-acid insertion in the Noxo1 PX domain appears to depend on the type of Nox; in activation of gp91(phox)/Nox2, Noxo1gamma is less active than Noxo1beta even in the presence of PMA, whereas Noxo1gamma and Noxo1beta support the superoxide-producing activity of Nox3 to the same extent in a manner independent of cell stimulation. 相似文献
9.
Activation of the non-phagocytic superoxide-producing NADPH oxidase Nox1, complexed with p22(phox) at the membrane, requires its regulatory soluble proteins Noxo1 and Noxa1. However, the role of the small GTPase Rac remained to be clarified. Here we show that Rac directly participates in Nox1 activation via interacting with Noxa1. Electropermeabilized HeLa cells, ectopically expressing Nox1, Noxo1, and Noxa1, produce superoxide in a GTP-dependent manner, which is abrogated by expression of a mutant Noxa1(R103E), defective in Rac binding. Superoxide production in Nox1-expressing HeLa and Caco-2 cells is decreased by depletion or sequestration of Rac; on the other hand, it is enhanced by expression of the constitutively active Rac1(Q61L), but not by that of a mutant Rac1 with the A27K substitution, deficient in binding to Noxa1. We also demonstrate that Nox1 activation requires membrane recruitment of Noxa1, which is normally mediated via Noxa1 binding to Noxo1, a protein tethered to the Nox1 partner p22(phox): the Noxa1-Noxo1 and Noxo1-p22(phox) interactions are both essential for Nox1 activity. Rac likely facilitates the membrane localization of Noxa1: although Noxa1(W436R), defective in Noxo1 binding, neither associates with the membrane nor activates Nox1, the effects of the W436R substitution are restored by expression of Rac1(Q61L). The Rac-Noxa1 interaction also serves at a step different from the Noxa1 localization, because the binding-defective Noxa1(R103E), albeit targeted to the membrane, does not support superoxide production by Nox1. Furthermore, a mutant Noxa1 carrying the substitution of Ala for Val-205 in the activation domain, which is expected to undergo a conformational change upon Rac binding, fully localizes to the membrane but fails to activate Nox1. 相似文献
10.
Inoue Y Ogasawara M Moroi T Satake M Azumi K Moritomo T Nakanishi T 《Immunogenetics》2005,57(7):520-534
To illuminate the origins of NADPH oxidase (Nox), we identified cDNA clones encoding Nox2, Nox4, p22 phagocyte oxidase (phox),
p47phox, and p67phox in a chordate phylogenetically distant to the vertebrates, the sea squirt Ciona intestinalis. We also examined the spatiotemporal expression of these genes in embryos and juveniles. The sequences of the Nox2, Nox4,
p22phox, p47phox, and p67phox cDNAs contained open reading frames encoding 581, 811, 175, 461, and 515 amino acids, respectively.
The level of identities between the deduced Nox2, Nox4, p22phox, p47phox, and p67phox amino acid sequences and their corresponding
human components were 54.0, 31.0, 44.4, 36.0, and 26.2%, respectively. Despite these low identities, the functional domains
of the C. intestinalis and human NADPH oxidase and Nox4 are highly conserved. The genomic organizations of the components of the NADPH oxidase gene
except for p67phox (a single exon gene) and the Nox4 gene in C. intestinalis are highly similar to those of the corresponding human NADPH oxidase genes. Further, the analyzed part of the C. intestinalis genome and EST database do not seem to present p40phox and Nox5. The Nox2, p22phox, p47phox, and p67phox genes were specifically
expressed in the blood cells of juveniles. The Nox4 gene was expressed in blood cells and endostyle of juveniles. These results
suggest that C. intestinalis NADPH oxidase components possess potential functional activities similar to those of human, but the manner in which cytosolic
phox proteins in C. intestinalis interact is different from that in human. 相似文献
11.
Takeya R Ueno N Kami K Taura M Kohjima M Izaki T Nunoi H Sumimoto H 《The Journal of biological chemistry》2003,278(27):25234-25246
The catalytic core of a superoxide-producing NADPH oxidase (Nox) in phagocytes is gp91phox/Nox2, a membrane-integrated protein that forms a heterodimer with p22phox to constitute flavocytochrome b558. The cytochrome becomes activated by interacting with the adaptor proteins p47phox and p67phox as well as the small GTPase Rac. Here we describe the cloning of human cDNAs for novel proteins homologous to p47phox and p67phox, designated p41nox and p51nox, respectively; the former is encoded by NOXO1 (Nox organizer 1), and the latter is encoded by NOXA1 (Nox activator 1). The novel homologue p41nox interacts with p22phox via the two tandem SH3 domains, as does p47phox. The protein p51nox as well as p67phox can form a complex with p47phox and with p41nox via the C-terminal SH3 domain and binds to GTP-bound Rac via the N-terminal domain containing four tetratricopeptide repeat motifs. These bindings seem to play important roles, since p47phox and p67phox activate the phagocyte oxidase via the same interactions. Indeed, p41nox and p51nox are capable of replacing the corresponding classical homologue in activation of gp91phox. Nox1, a homologue of gp91phox, also can be activated in cells, when it is coexpressed with p41nox and p51nox, with p41nox and p67phox, or with p47phox and p51nox; in the former two cases, Nox1 is partially activated without any stimulants added, suggesting that p41nox is normally in an active state. Thus, the novel homologues p41nox and p51nox likely function together or in combination with a classical one, thereby activating the two Nox family oxidases. 相似文献
12.
Yanzhang Li 《Biochemical and biophysical research communications》2010,397(1):5-126
Glycated albumin, an early-glycation Amadori-modified protein, stimulates transforming growth factor-β (TGF-β) expression and increases the production of the extracellular matrix proteins in mesangial cells, contributing to the pathogenesis of diabetic nephropathy. Glycated albumin has been shown to increase NADPH oxidase-dependent superoxide formation in mesangial cells. However, the mechanisms are not well understood. Therefore, in the present studies, we determined the mechanisms by which glycated albumin activates NADPH oxidase in primary rat mesangial cells and its contribution to glycated albumin-induced TGF-β expression and extracellular matrix protein production. Our data showed that glycated albumin treatment stimulated NADPH oxidase activity and increased the formation of superoxide formation in rat mesangial cells. Moreover, glycated albumin treatment stimulated the expression and phosphorylation of p47phox, one of the cytosolic regulatory subunits of the NADPH oxidase. However, the levels of other NADPH oxidase subunits including Nox1, Nox2, Nox4, p22phox, and p67phox were not altered by glycated albumin. Moreover, siRNA-mediated knockdown of p47phox inhibited glycated albumin-induced NADPH oxidase activity and superoxide formation. Glycated albumin-induced TGF-β expression and extracellular matrix production (fibronectin) was also inhibited by p47phox knock down. Taken together, these data suggest that up-regulation of p47phox is involved in glycated albumin-mediated activation of NADPH oxidase, leading to glycated albumin-induced expression of TGF-β and extracellular matrix proteins in mesangial cells and contributing to the development of diabetic nephropathy. 相似文献
13.
Masahito Kawano Rey IshiiYuki Yoshioka Takehito FukudaMinoru Tamura 《Archives of biochemistry and biophysics》2013
Noxa1 activates Nox2 together with Noxo1 and Rac in a pure reconstitution system, but the resulting activity is considerably lower than that induced by p67phox and p47phox. In this study, we found that C-terminal-truncated forms of Noxa1 exhibited higher activities than full-length Noxa1. Of the truncations examined, Noxa1(1-225) showed the highest ability for activation. Kinetic studies revealed that Noxa1(1-225) had a threefold higher Vmax value than full-length Noxa1 with a similar EC50 value. The affinities of Noxo1 and RacQ61L were not much altered by the truncation. Conversely, the affinity of FAD for the Nox2 complex was enhanced after the truncation. In the absence of Noxo1, Noxa1(1-225) showed much higher activity with a lower EC50 than full-length Noxa1. Noxa1(1-225) showed comparable activity to that of p67phox with either Noxo1 or p47phox, although the stability was lower than that with p67phox and p47phox. These findings indicate that the role of the C-terminal half of Noxa1 is autoinhibition. The data suggest a two-step autoinhibition mechanism, comprising self-masking to interrupt the binding to the oxidase, and holding of the activation domain in a suboptimal position to the oxidase. This study reveals that when both types of inhibition are released, Noxa1 achieves high-level superoxide production. 相似文献
14.
Nox organizer 1 (Noxo1), a p47(phox) homolog, is produced as four isoforms with unique N-terminal PX domains derived by alternative mRNA splicing. We compared the subcellular distribution of these isoforms or their isolated PX domains produced as GFP fusion proteins, as well as their ability to support Nox1 activity in several transfected models. Noxo1alpha, beta, gamma, and delta show different subcellular localization patterns, determined by their PX domains. In HEK293 cells, Noxo1beta exhibits prominent plasma membrane binding, Noxo1gamma shows plasma membrane and nuclear associations, and Noxo1alpha and delta localize primarily on intracellular vesicles or cytoplasmic aggregates, but not the plasma membrane. Nox1 activity correlates with Noxo1 plasma membrane binding in HEK293 cells, since Noxo1beta supports the highest activity and Noxo1gamma and Noxo1alpha support moderate or low activities, respectively. In COS-7 cells, where Noxo1alpha localizes on the plasma membrane, the activities supported by the three isoforms (alpha, beta, and gamma) do not differ significantly. The PX domains of beta and gamma bind the same phospholipids, including phosphatidic acid. These results indicate that the variant PX domains are unique determinants of Noxo1 localization and Nox1 function. Finally, the overexpressed Noxo1 isoforms do not affect p22(phox) localization, although Nox1 is needed to transport p22(phox) to the plasma membrane. 相似文献
15.
Jiang F Lim HK Morris MJ Prior L Velkoska E Wu X Dusting GJ 《Redox report : communications in free radical research》2011,16(6):223-229
Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is upregulated in a variety of tissues in obesity. It is still unclear as to whether NADPH oxidase upregulation in a specific tissue is part of a systemic response. Here we analyzed the expression pattern of NADPH oxidase in vascular, adipose, and kidney tissues in a rat model of diet-induced obesity. After weaning, rats were fed either a normal or high-fat diet for 12 weeks. The high-fat diet resulted in 20% increased body weight. In the aorta, Nox4 expression was increased by three-fold in obese rats. Upregulations of p22phox and p47phox in adipose, and Nox4, p22phox, and p47phox in kidney were observed in obesity. Marked increases in plasma leptin and insulin were observed, with more modest changes in adiponectin in obese rats. The average systolic blood pressure in the obese group was 11 mmHg higher than that of lean rats (P < 0.005). There was a significant correlation between blood pressure and aortic Nox4 expression (P < 0.01). In cultured vascular smooth muscle cells, adiponectin reduced the expression of Nox4 in a protein kinase A-dependent manner. Our results suggest that upregulation of NADPH oxidase in multiple tissues during obesity appears to be a systemic response. At least in vitro, adiponectin may have a protective antioxidant role by suppressing vascular NADPH oxidase expression. The association between NADPH oxidase Nox4 expression in the vasculature and the elevated blood pressure in obesity requires further investigation. 相似文献
16.
E Rebelato TR Mares-Guia MF Graciano L Labriola LR Britto HM Garay-Malpartida R Curi MC Sogayar AR Carpinelli 《Life sciences》2012,91(7-8):244-249
AIMS: NADPH oxidase (NOX) is a known source of superoxide anions in phagocytic and non-phagocytic cells. In this study, the presence of this enzyme in human pancreatic islets and the importance of NADPH oxidase in human β-cell function were investigated. MAIN METHODS AND KEY FINDINGS: In isolated human pancreatic islets, the expression of NADPH oxidase components was evidenced by real-time PCR (p22(PHOX), p47(PHOX) and p67(PHOX)), Western blotting (p47(PHOX) and p67(PHOX)) and immunohistochemistry (p47(PHOX), p67(PHOX) and gp91(PHOX)). Immunohistochemistry experiments showed co-localization of p47(PHOX), p67(PHOX) and gp91(PHOX) (isoform 2 of NADPH oxidase-NOX2) with insulin secreting cells. Inhibition of NADPH oxidase activity impaired glucose metabolism and glucose-stimulated insulin secretion. SIGNIFICANCE: These findings demonstrate the presence of the main intrinsic components of NADPH oxidase comprising the NOX2 isoform in human pancreatic islets, whose activity also contributes to human β-cell function. 相似文献
17.
18.
《Redox report : communications in free radical research》2013,18(6):223-229
AbstractNicotinamide adenine dinucleotide phosphate (NADPH) oxidase is upregulated in a variety of tissues in obesity. It is still unclear as to whether NADPH oxidase upregulation in a specific tissue is part of a systemic response. Here we analyzed the expression pattern of NADPH oxidase in vascular, adipose, and kidney tissues in a rat model of diet-induced obesity. After weaning, rats were fed either a normal or high-fat diet for 12 weeks. The high-fat diet resulted in 20% increased body weight. In the aorta, Nox4 expression was increased by three-fold in obese rats. Upregulations of p22phox and p47phox in adipose, and Nox4, p22phox, and p47phox in kidney were observed in obesity. Marked increases in plasma leptin and insulin were observed, with more modest changes in adiponectin in obese rats. The average systolic blood pressure in the obese group was 11 mmHg higher than that of lean rats (P < 0.005). There was a significant correlation between blood pressure and aortic Nox4 expression (P < 0.01). In cultured vascular smooth muscle cells, adiponectin reduced the expression of Nox4 in a protein kinase A-dependent manner. Our results suggest that upregulation of NADPH oxidase in multiple tissues during obesity appears to be a systemic response. At least in vitro, adiponectin may have a protective antioxidant role by suppressing vascular NADPH oxidase expression. The association between NADPH oxidase Nox4 expression in the vasculature and the elevated blood pressure in obesity requires further investigation. 相似文献
19.
Li XJ Grunwald D Mathieu J Morel F Stasia MJ 《The Journal of biological chemistry》2005,280(15):14962-14973
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. 相似文献
20.
Guichard C Pedruzzi E Fay M Ben Mkaddem S Coant N Daniel F Ogier-Denis E 《Médecine sciences : M/S》2006,22(11):953-959
Reactive oxygen species (ROS) generated by the NADPH oxidases are conventionally thought to be cytotoxic and mutagenic and at high levels induce an oxidative stress response. The phagocyte NADPH oxidase catalyzes the NADPH-dependent reduction of molecular oxygen to generate superoxide O2-., which can dismute to generate ROS species. Together, these ROS participate in host defence by killing or damaging invading microbes. Flavocytochrome b558 is the catalytic core of the phagocyte NADPH oxidase and consists of a large glycoprotein gp91phox or Nox-2 and a small protein p22phox. The other components of the NADPH oxidase are cytosolic proteins, namely p67phox, p47phox, p40phox and Rac. A defect in any of the genes encoding gp91phox, p22phox, p67phox or p47phox results in chronic granulomatous disease, a genetic disorder characterized by severe and recurrent infections. Evidence is rapidly accumulating that low level of ROS were produced by NADPH oxidase homologs in non-phagocytic cells. To date, six human homologs (Nox-1, Nox-3, Nox-4, Nox-5, Duox-1 and Duox-2) have been recently identified in a variety of non-phagocytic cells. The identification of Nox-1 was quickly followed by the cloning of Nox-3, Nox-4, and Nox-5. In parallel, two very large members of the Nox family were discovered, namely Duox-1 and Duox-2, initially also referred to as thyroid oxidases. The physiological functions of Nox-dependent ROS generation are in progress and still require detailed characterization. Activation mechanisms and tissue distribution of the different members of the Nox family are very different, suggesting distinct physiological functions. Nox family enzymes are likely to be involved in a variety of physiological events including cell proliferation, host defence, differentiation, apoptosis, senescence and activation of growth-related signaling pathways. An increase and a decrease in the function of Nox enzymes can contribute to a wide range of pathological processes. 相似文献