Publications by authors named "Francoise Morel"

42 Publications

Transmembrane Nox4 topology revealed by topological determination by Ubiquitin Fusion Assay, a novel method to uncover membrane protein topology.

Biochem Biophys Res Commun 2020 01 25;521(2):383-388. Epub 2019 Oct 25.

Univ.Grenoble Alpes, CNRS, CHU Grenoble Alpes, Grenoble INP(1), TIMC-IMAG, GREPI, 38000, Grenoble Cedex, France. Electronic address:

The NADPH oxidase Nox4 is a multi-pass membrane protein responsible for the generation of reactive oxygen species that are implicated in cellular signaling but may also cause pathological situations when dysregulated. Although topological organization of integral membrane protein dictates its function, only limited experimental data describing Nox4's topology are available. To provide deeper insight on Nox4 structural organization, we developed a novel method to determinate membrane protein topology in their cellular environment, named Topological Determination by Ubiquitin Fusion Assay (ToDUFA). It is based on the proteolytic capacity of the deubiquitinase enzymes to process ubiquitin fusion proteins. This straightforward method, validated on two well-known protein's topologies (IL1RI and Nox2), allowed us to discriminate rapidly the topological orientation of protein's domains facing either the nucleocytosolic or the exterior/luminal compartments. Using this method, we were able for the first time to determine experimentally the topology of Nox4 which consists of 6 transmembrane domains with its N- and C-terminus moieties facing the cytosol. While the first, third and fifth loops of Nox4 protein are extracellular; the second and fourth loops are located in the cytosolic side. This approach can be easily extended to characterize the topology of all others members of the NADPH oxidase family or any multi-pass membrane proteins. Considering the importance of protein topology knowledge in cell biology research and pharmacological development, we believe that this novel method will represent a widely useful technique to easily uncover complex membrane protein's topology.
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http://dx.doi.org/10.1016/j.bbrc.2019.10.098DOI Listing
January 2020

Interaction between p22 and Nox4 in the endoplasmic reticulum suggests a unique mechanism of NADPH oxidase complex formation.

Free Radic Biol Med 2018 02 24;116:41-49. Epub 2017 Dec 24.

Department of Physiology, Faculty of Medicine, Semmelweis University, Budapest, Hungary; "Momentum" Peroxidase Enzyme Research Group of the Semmelweis University and the Hungarian Academy of Sciences, Budapest, Hungary. Electronic address:

The p22 protein is an essential component of the phagocytic- and inner ear NADPH oxidases but its relationship to other Nox proteins is less clear. We have studied the role of p22 in the TGF-β1-stimulated HO production of primary human and murine fibroblasts. TGF-β1 induced HO release of the examined cells, and the response was dependent on the expression of both Nox4 and p22. Interestingly, the p22 protein was present in the absence of any detectable Nox/Duox expression, and the p22 level was unaffected by TGF-β1. On the other hand, Nox4 expression was dependent on the presence of p22, establishing an asymmetrical relationship between the two proteins. Nox4 and p22 proteins localized to the endoplasmic reticulum and their distribution was unaffected by TGF-β1. We used a chemically induced protein dimerization method to study the orientation of p22 and Nox4 in the endoplasmic reticulum membrane. This technique is based on the rapamycin-mediated heterodimerization of the mammalian FRB domain with the FK506 binding protein. The results of these experiments suggest that the enzyme complex produces HO into the lumen of the endoplasmic reticulum, indicating that Nox4 contributes to the development of the oxidative milieu within this organelle.
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http://dx.doi.org/10.1016/j.freeradbiomed.2017.12.031DOI Listing
February 2018

Unexpected function of the phagocyte NADPH oxidase in supporting hyperglycolysis in stimulated neutrophils: key role of 6-phosphofructo-2-kinase.

FASEB J 2017 02 31;31(2):663-673. Epub 2016 Oct 31.

Groupe de Recherche et D'etude du Processus Inflammatoire (GREPI), EA 7408, Université Grenoble Alpes, Saint-Martin-d'Hères, France;

The phagocyte NADPH oxidase 2 (Nox2) is an enzymatic complex that is involved in innate immunity, notably via its capacity to produce toxic reactive oxygen species. Recently, a proteomic analysis of the constitutively active Nox2 complex, isolated from neutrophil fractions, highlighted the presence of 6-phosphofructo-2-kinase (PFK-2). The purpose of this work was to study the relationship between PFK-2 and NADPH oxidase in neutrophils. Data have underlined a specific association of the active phosphorylated form of PFK-2 with Nox2 complex in stimulated neutrophils. In its active form, PFK-2 catalyzes the production of fructose-2,6-bisphosphate, which is the main allosteric activator of phosphofructo-1-kinase, the limiting enzyme in glycolysis. Pharmacologic inhibition of PFK-2 phosphorylation and cell depletion in PFK-2 by a small interfering RNA strategy led to a decrease in the glycolysis rate and a reduction in NADPH oxidase activity in stimulated cells. Surprisingly, alteration of Nox2 activity impacted the glycolysis rate, which indicated that Nox2 in neutrophils was not only required for reactive oxygen species production but was also involved in supporting the energetic metabolism increase that was induced by inflammatory conditions. PFK-2 seems to be a strategic element that links NADPH oxidase activation and glycolysis modulation, and, as such, is proposed as a potential therapeutic target in inflammatory diseases.-Baillet, A., Hograindleur, M.-A., El Benna, J., Grichine, A., Berthier, S., Morel, F., Paclet, M.-H. Unexpected function of the phagocyte NADPH oxidase in supporting hyperglycolysis in stimulated neutrophils: key role of 6-phosphofructo-2-kinase.
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http://dx.doi.org/10.1096/fj.201600720RDOI Listing
February 2017

[NADPH oxidase Nox4, a putative therapeutic target in osteoarthritis].

Bull Acad Natl Med 2015 Apr-May;199(4-5):673-86; discussion 686-7

The NADPH oxidases, Nox, are transmembrane hemoproteins, whose exclusive function is to reduce molecular oxygen to produce superoxide anion O2°- and consequently highly reactive oxidant and toxic oxygen species, ROS. Among the 7 NADPH oxidases expressed in humans, Nox4 is the sole Nox isoform present in human primary chondrocytes. Nox4 was suggested as one of the main actors involved in cartilage degradation in osteoarthritis. The stimulation of chondrocytes, the only cell present in cartilage, by IL-1β results in the activation of Nox4. This leads to an increase of ROS production which in turn could regulate signaling pathways sensitive to oxidative stress such as gene-encoding matrix metalloproteases MMP1, MMP13 and Adamalysin ADAMTS4. A deep understanding of Nox4 structure/function and mechanisms of regulation could lead both to the identification of new therapeutic targets and to the development of innovative strategies for appropriate osteoarthritis treatment.
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September 2016

[NADPH oxidases, Nox: new isoenzymes family].

Med Sci (Paris) 2015 Jan 6;31(1):43-52. Epub 2015 Feb 6.

GREPI (groupe de recherche et d'étude du processus inflammatoire), université Joseph Fourier, Grenoble, France EFS Rhône-Alpes, 29, avenue des Maquis du Grésivaudan, 38700 La Tronche, France.

NADPH oxidases, Nox, are a family of isoenzymes, composed of seven members, whose sole function is to produce reactive oxygen species (ROS). Although Nox catalyze the same enzymatic reaction, they acquired from a common ancestor during evolution, specificities related to their tissue expression, subcellular localization, activation mechanisms and regulation. Their functions could vary depending on the pathophysiological state of the tissues. Indeed, ROS are not only bactericidal weapons in phagocytes but also essential cellular signaling molecules and their overproduction is involved in chronic diseases and diseases of aging. The understanding of the mechanisms involved in the function of Nox and the emergence of Nox inhibitors, require a thorough knowledge of their nature and structure. The objectives of this review are to highlight, in a structure/function approach, the main similar and differentiated properties shared by the human Nox isoenzymes.
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http://dx.doi.org/10.1051/medsci/20153101012DOI Listing
January 2015

Prospective evaluation of FibroTest®, FibroMeter®, and HepaScore® for staging liver fibrosis in chronic hepatitis B: comparison with hepatitis C.

J Hepatol 2014 Jul 11;61(1):28-34. Epub 2014 Mar 11.

Clinique Universitaire d'Hépato-Gastroentérologie, Pôle DIGIDUNE, CHU de Grenoble, France; Unité INSERM/Université Grenoble Alpes U823, IAPC Institut Albert Bonniot, Grenoble, France.

Background & Aims: Fibrosis blood tests have been validated in chronic hepatitis C. Their diagnostic accuracy is less documented in hepatitis B. The aim of this study was to describe the diagnostic performance of FibroTest®, FibroMeter®, and HepaScore® for liver fibrosis in hepatitis B compared to hepatitis C.

Methods: 510 patients mono-infected with hepatitis B or C and matched on fibrosis stage were included. Blood tests were performed the day of the liver biopsy. Histological lesions were staged according to METAVIR.

Results: Fibrosis stages were distributed as followed: F0 n=76, F1 n=192, F2 n=132, F3 n=54, F4 n=56. Overall diagnostic performance of blood tests were similar between hepatitis B and C with AUROC ranging from 0.75 to 0.84 for significant fibrosis, 0.82 to 0.85 for extensive fibrosis and 0.84 to 0.87 for cirrhosis. Optimal cut-offs were consistently lower in hepatitis B compared to hepatitis C, especially for the diagnosis of extensive fibrosis and cirrhosis, with decreased sensitivity and negative predictive values. More hepatitis B than C patients with F ⩾3 were underestimated: FibroTest®: 47% vs. 26%, FibroMeter®: 24% vs. 6%, HepaScore®: 41% vs. 24%, p<0.01. Multivariate analysis showed that hepatitis B (0R 3.4, 95% CI 1.2-19.2, p<0.02) and low γGT (OR 7.3, 95% CI 2.0-27.0, p<0.003) were associated with fibrosis underestimation.

Conclusion: Overall the diagnostic performance of blood tests is similar in hepatitis B and C. The risk of underestimating significant fibrosis and cirrhosis is however greater in hepatitis B and cannot be entirely corrected by the use of more stringent cut-offs.
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http://dx.doi.org/10.1016/j.jhep.2014.02.029DOI Listing
July 2014

Heme oxygenase-1 regulates matrix metalloproteinase MMP-1 secretion and chondrocyte cell death via Nox4 NADPH oxidase activity in chondrocytes.

PLoS One 2013 20;8(6):e66478. Epub 2013 Jun 20.

Université Joseph Fourier, AGIM GREPI FRE CNRS 3405, Grenoble, France.

Interleukin-1β (IL-1β) activates the production of reactive oxygen species (ROS) and secretion of MMPs as well as chondrocyte apoptosis. Those events lead to matrix breakdown and are key features of osteoarthritis (OA). We confirmed that in human C-20/A4 chondrocytes the NADPH oxidase Nox4 is the main source of ROS upon IL-1β stimulation. Since heme molecules are essential for the NADPH oxidase maturation and activity, we therefore investigated the consequences of the modulation of Heme oxygenase-1 (HO-1), the limiting enzyme in heme catabolism, on the IL-1β signaling pathway and more specifically on Nox4 activity. Induction of HO-1 expression decreased dramatically Nox4 activity in C-20/A4 and HEK293 T-REx™ Nox4 cell lines. Unexpectedly, this decrease was not accompanied by any change in the expression, the subcellular localization or the maturation of Nox4. In fact, the inhibition of the heme synthesis by succinylacetone rather than heme catabolism by HO-1, led to a confinement of the Nox4/p22(phox) heterodimer in the endoplasmic reticulum with an absence of redox differential spectrum highlighting an incomplete maturation. Therefore, the downregulation of Nox4 activity by HO-1 induction appeared to be mediated by carbon monoxide (CO) generated from the heme degradation process. Interestingly, either HO-1 or CO caused a significant decrease in the expression of MMP-1 and DNA fragmentation of chondrocytes stimulated by IL-1β. These results all together suggest that a modulation of Nox4 activity via heme oxygenase-1 may represent a promising therapeutic tool in osteoarthritis.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0066478PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3688771PMC
February 2014

Quinone compounds regulate the level of ROS production by the NADPH oxidase Nox4.

Biochem Pharmacol 2013 Jun 9;85(11):1644-54. Epub 2013 Apr 9.

Université Joseph Fourier, GREPI AGIM FRE 3405, CNRS, EPHE, Grenoble, France.

NADPH oxidase Nox4 is expressed in a wide range of tissues and plays a role in cellular signaling by providing reactive oxygen species (ROS) as intracellular messengers. Nox4 oxidase activity is thought to be constitutive and regulated at the transcriptional level; however, we challenge this point of view and suggest that specific quinone derivatives could modulate this activity. In fact, we demonstrated a significant stimulation of Nox4 activity by 4 quinone derivatives (AA-861, tBuBHQ, tBuBQ, and duroquinone) observed in 3 different cellular models, HEK293E, T-REx™, and chondrocyte cell lines. Our results indicate that the effect is specific toward Nox4 versus Nox2. Furthermore, we showed that NAD(P)H:quinone oxidoreductase (NQO1) may participate in this stimulation. Interestingly, Nox4 activity is also stimulated by reducing agents that possibly act by reducing the disulfide bridge (Cys226, Cys270) located in the extracellular E-loop of Nox4. Such model of Nox4 activity regulation could provide new insight into the understanding of the molecular mechanism of the electron transfer through the enzyme, i.e., its potential redox regulation, and could also define new therapeutic targets in diseases in which quinones and Nox4 are implicated.
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http://dx.doi.org/10.1016/j.bcp.2013.03.023DOI Listing
June 2013

Molecular interface of S100A8 with cytochrome b558 and NADPH oxidase activation.

PLoS One 2012 10;7(7):e40277. Epub 2012 Jul 10.

Groupe de Recherche et d'Etude du Processus Inflammatoire, Laboratoire Aging Imaging Modeling, Formation de Recherche en Évolution Centre National de la Recherche Scientifique CNRS 3405, Université Joseph Fourier UJF, Grenoble, France.

S100A8 and S100A9 are two calcium binding Myeloid Related Proteins, and important mediators of inflammatory diseases. They were recently introduced as partners for phagocyte NADPH oxidase regulation. However, the precise mechanism of their interaction remains elusive. We had for aim (i) to evaluate the impact of S100 proteins on NADPH oxidase activity; (ii) to characterize molecular interaction of either S100A8, S100A9, or S100A8/S100A9 heterocomplex with cytochrome b(558); and (iii) to determine the S100A8 consensus site involved in cytochrome b(558)/S100 interface. Recombinant full length or S100A9-A8 truncated chimera proteins and ExoS-S100 fusion proteins were expressed in E. coli and in P. aeruginosa respectively. Our results showed that S100A8 is the functional partner for NADPH oxidase activation contrary to S100A9, however, the loading with calcium and a combination with phosphorylated S100A9 are essential in vivo. Endogenous S100A9 and S100A8 colocalize in differentiated and PMA stimulated PLB985 cells, with Nox2/gp91(phox) and p22(phox). Recombinant S100A8, loaded with calcium and fused with the first 129 or 54 N-terminal amino acid residues of the P. aeruginosa ExoS toxin, induced a similar oxidase activation in vitro, to the one observed with S100A8 in the presence of S100A9 in vivo. This suggests that S100A8 is the essential component of the S100A9/S100A8 heterocomplex for oxidase activation. In this context, recombinant full-length rS100A9-A8 and rS100A9-A8 truncated 90 chimera proteins as opposed to rS100A9-A8 truncated 86 and rS100A9-A8 truncated 57 chimeras, activate the NADPH oxidase function of purified cytochrome b(558) suggesting that the C-terminal region of S100A8 is directly involved in the molecular interface with the hemoprotein. The data point to four strategic (87)HEES(90) amino acid residues of the S100A8 C-terminal sequence that are involved directly in the molecular interaction with cytochrome b(558) and then in the phagocyte NADPH oxidase activation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0040277PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3393751PMC
March 2013

Recombinant Nox4 cytosolic domain produced by a cell or cell-free base systems exhibits constitutive diaphorase activity.

Biochem Biophys Res Commun 2012 Mar 3;419(3):453-8. Epub 2012 Feb 3.

GREPI AGIM FRE CNRS-UJF, Université Joseph Fourier, Grenoble, France.

The membrane protein NADPH (nicotinamide adenine dinucleotide phosphate) oxidase Nox4 constitutively generates reactive oxygen species differing from other NADPH oxidases activity, particularly in Nox2 which needs a stimulus to be active. Although the precise mechanism of production of reactive oxygen species by Nox2 is well characterized, the electronic transfer throughout Nox4 remains unclear. Our study aims to investigate the initial electronic transfer step (diaphorase activity) of the cytosolic tail of Nox4. For this purpose, we developed two different approaches to produce soluble and active truncated Nox4 proteins. We synthesized soluble recombinant proteins either by in vitro translation or by bacteria induction. While proteins obtained by bacteria induction demonstrate an activity of 4.4 ± 1.7 nmol/min/nmol when measured against iodonitro tetrazolium chloride and 20.5 ± 2.8 nmol/min/nmol with cytochrome c, the soluble proteins produced by cell-free expression system exhibit a diaphorase activity with a turn-over of 26 ± 2.6 nmol/min/nmol when measured against iodonitro tetrazolium chloride and 48 ± 20.2 nmol/min/nmol with cytochrome c. Furthermore, the activity of the soluble proteins is constitutive and does not need any stimulus. We also show that the cytosolic tail of the isoform Nox4B lacking the first NADPH binding site is unable to demonstrate any diaphorase activity pointing out the importance of this domain.
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http://dx.doi.org/10.1016/j.bbrc.2012.01.136DOI Listing
March 2012

The NADPH oxidase cytosolic component p67phox is constitutively phosphorylated in human neutrophils: Regulation by a protein tyrosine kinase, MEK1/2 and phosphatases 1/2A.

Biochem Pharmacol 2011 Nov 20;82(9):1145-52. Epub 2011 Jul 20.

INSERM, U773, Centre de Recherche Biomédicale Bichat Beaujon CRB3, Paris F-75018, France.

Neutrophils play a key role in host defense and inflammation through the production of superoxide anion and other reactive oxygen species (ROS) by the enzyme complex NADPH oxidase. The cytosolic NADPH oxidase component, p67phox, has been shown to be phosphorylated in human neutrophils but the pathways involved in this process are largely unknown. In this study, we show that p67phox is constitutively phosphorylated in resting human neutrophils and that neutrophil stimulation with PMA further enhanced this phosphorylation. Inhibition of the constitutively active serine/threonine phosphatases type 1 and type 2A (PP1/2A) by calyculin A resulted in the enhancement of p67phox phosphorylation. Constitutive and calyculin A-induced phosphorylation of p67phox was completely inhibited by the protein tyrosine kinase inhibitor genistein and partially inhibited by the MEK1/2 inhibitor PD98059, but was unaffected by GF109203X, wortmannin and SB203580, inhibitors of PKC, PI3K and p38MAP kinase, respectively. Two-dimensional phosphopeptide mapping revealed that constitutive and calyculin A-induced p67phox phosphorylation occurred on the same major sites. Interestingly, calyculin A enhanced formyl-Met-Leu-Phe (fMLP)-induced superoxide production, while genistein inhibited this process. Taken together, these results suggest that (i) p67phox undergoes a continual cycle of phosphorylation/dephosphorylation in resting cells; (ii) p67phox phosphorylation is controlled by MEK1/2 and an upstream tyrosine kinase; (iii) PP1/2A directly or indirectly antagonize this process. Thus, these pathways could play a role in regulating ROS production by human neutrophils at inflammatory sites.
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http://dx.doi.org/10.1016/j.bcp.2011.07.070DOI Listing
November 2011

Coupling of 6-phosphogluconate dehydrogenase with NADPH oxidase in neutrophils: Nox2 activity regulation by NADPH availability.

FASEB J 2011 Jul 28;25(7):2333-43. Epub 2011 Mar 28.

Groupe de Recherche et d'Etude du Processus Inflammatoire (GREPI), Centre National de la Recherche Scientifique (CNRS), Université Joseph Fourier (UJF), Grenoble, France.

It is well known that activation of the phagocyte NADPH oxidase requires the association of cytosolic proteins (p67-phox, p47-phox, p40-phox, and Rac) with the membrane cytochrome b(558), leading to its conformation change. Recently, the phagocyte NADPH oxidase complex was isolated in a constitutively active form. In this complex, 6-phosphogluconate dehydrogenase (6PGDH), an enzyme involved in the production of intracellular NADPH, was identified. This protein was absent from the oxidase complex isolated from B lymphocytes, suggesting a specific interaction with the neutrophil NADPH oxidase. To clarify the implication of 6PGDH in the NADPH oxidase activity, a siRNA approach was conducted in neutrophil-like PLB985 cells. NADPH oxidase activity of siRNA-transfected cells was shown to be decreased. Similar results were obtained in vitro, after reconstitution of oxidase activity with subcellular fractions isolated from siRNA-transfected cells. Interestingly, the Michaelis constant (K(m)) of Nox2 for NADPH increases in 6PGDH-depleted cells. Moreover, 6PGDH coimmunoprecipitated with oxidase cytosolic factors from cytosol of stimulated cells. Data suggested that the affinity of Nox2 for NADPH is increased in the presence of 6PGDH on cell stimulation. The present work proposes a new way of NADPH oxidase activity regulation by modulating Nox2 affinity for NADPH.
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http://dx.doi.org/10.1096/fj.10-173807DOI Listing
July 2011

The E-loop is involved in hydrogen peroxide formation by the NADPH oxidase Nox4.

J Biol Chem 2011 Apr 22;286(15):13304-13. Epub 2011 Feb 22.

Institut für Kardiovaskuläre Physiologie, Goethe-Universität, 60596 Frankfurt am Main, Germany.

In contrast to the NADPH oxidases Nox1 and Nox2, which generate superoxide (O(2)(·-)), Nox4 produces hydrogen peroxide (H(2)O(2)). We constructed chimeric proteins and mutants to address the protein region that specifies which reactive oxygen species is produced. Reactive oxygen species were measured with luminol/horseradish peroxidase and Amplex Red for H(2)O(2) versus L-012 and cytochrome c for O(2)(·-). The third extracytosolic loop (E-loop) of Nox4 is 28 amino acids longer than that of Nox1 or Nox2. Deletion of E-loop amino acids only present in Nox4 or exchange of the two cysteines in these stretches switched Nox4 from H(2)O(2) to O(2)(·-) generation while preserving expression and intracellular localization. In the presence of an NO donor, the O(2)()-producing Nox4 mutants, but not wild-type Nox4, generated peroxynitrite, excluding artifacts of the detection system as the apparent origin of O(2)(·-). In Cos7 cells, in which Nox4 partially localizes to the plasma membrane, an antibody directed against the E-loop decreased H(2)O(2) but increased O(2)(·-) formation by Nox4 without affecting Nox1-dependent O(2)(·-) formation. The E-loop of Nox4 but not Nox1 and Nox2 contains a highly conserved histidine that could serve as a source for protons to accelerate spontaneous dismutation of superoxide to form H(2)O(2). Mutation of this but not of four other conserved histidines also switched Nox4 from H(2)O(2) to O(2)(·-) formation. Thus, H(2)O(2) formation is an intrinsic property of Nox4 that involves its E-loop. The structure of the E-loop may hinder O(2)(·-) egress and/or provide a source for protons, allowing dismutation to form H(2)O(2).
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http://dx.doi.org/10.1074/jbc.M110.192138DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3075677PMC
April 2011

New insight into the Nox4 subcellular localization in HEK293 cells: first monoclonal antibodies against Nox4.

Biochimie 2011 Mar 12;93(3):457-68. Epub 2010 Nov 12.

GREPI Timc-Imag UMR-CNRS 5525, Enzymologie/CHU Albert Michallon, Grenoble, France.

Nox4, a member of Nox family of NADPH oxidase expressed in nonphagocytic cells, is a major source of reactive oxygen species in many cell types. But understanding of the role of Nox4 in the production of ROS and of regulation mechanism of oxidase activity is largely unknown. This study reports for the first time the generation and characterization of 5 mAbs against a recombinant Nox4 protein (AA: 206-578). Among 5 novel mAbs, 3 mAbs (8E9, 5F9, 6B11) specifically recognized Nox4 protein in HEK293 transfected cells or human kidney cortex by western blot analysis; mAb 8E9 reacted with intact tet-induced T-REx™ Nox4 cells in FACS studies. The other 2 mAbs 10B4 and 7C9 were shown to have a very weak reactivity after purification. Immunofluorescence confocal microscopy showed that Nox4 localized not only in the perinuclear and endoplasmic reticulum regions but also at the plasma membrane of the cells which was further confirmed by TIRF-microscopy. Epitope determination showed that mAb 8E9 recognizes a region on the last extracellular loop of Nox4, while mAbs 6B11 and 5F9 are directed to its cytosolic tail. Contrary to mAb 6B11, mAb 5F9 failed to detect Nox4 at the plasma membrane. Cell-free oxidase assays demonstrated a moderate but significant inhibition of constitutive Nox4 activity by mAbs 5F9 and 6B11. In conclusion, 5 mAbs raised against Nox4 were generated for the first time. 3 of them will provide powerful tools for a structure/function relationship of Nox4 and for physiopathological investigations in humans.
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http://dx.doi.org/10.1016/j.biochi.2010.11.001DOI Listing
March 2011

Synovial fluid proteomic fingerprint: S100A8, S100A9 and S100A12 proteins discriminate rheumatoid arthritis from other inflammatory joint diseases.

Rheumatology (Oxford) 2010 Apr 25;49(4):671-82. Epub 2010 Jan 25.

GREPI CNRS UMR 5525, Grenoble University, Grenoble, France.

Objective: We investigated SF and serum proteomic fingerprints of patients suffering from RA, OA and other miscellaneous inflammatory arthritides (MIAs) in order to identify RA-specific biomarkers.

Methods: SF profiles of 65 patients and serum profiles of 31 patients were studied by surface-enhanced laser desorption and ionization-time-of-flight-mass spectrometry technology. The most discriminating RA biomarkers were identified by matrix-assisted laser desorption ionization-time of flight and their overexpression was confirmed by western blotting and ELISA.

Results: Three biomarkers of 10 839, 10 445 and 13 338 Da, characterized as S100A8, S100A12 and S100A9 proteins, were the most up-regulated proteins in RA SF. Their expression was about 10-fold higher in RA SF vs OA SF. S100A8 exhibited a sensitivity of 82% and a specificity of 69% in discriminating RA from other MIAs, whereas S100A12 displayed a sensitivity of 79% and a specificity of 64%. Three peptides of 3351, 3423 and 3465 Da, corresponding to the alpha-defensins-1, -2 and -3, were also shown to differentiate RA from other MIAs with weaker sensitivity and specificity. Levels of S100A12, S100A8 and S100A9 were statistically correlated with the neutrophil count in MIA SF but not in the SF of RA patients. S100A8, S100A9, S100A12 and alpha-defensin expression in serum was not different in the three populations.

Conclusion: The most enhanced proteins in RA SF, the S100A8, S100A9 and S00A12 proteins, distinguished RA from MIA with high accuracy. Possible implication of resident cells in this increase may play a role in RA physiopathology.
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http://dx.doi.org/10.1093/rheumatology/kep452DOI Listing
April 2010

New p22-phox monoclonal antibodies: identification of a conformational probe for cytochrome b 558.

J Innate Immun 2009 29;1(6):556-69. Epub 2009 Jul 29.

GREPI, TIMC-Imag UMR 5525 CNRS/Université Joseph Fourier, Laboratoire d'Enzymologie, CHU Grenoble, Grenoble, France.

The phagocyte NADPH oxidase, belonging to the NADPH oxidase family (Nox), is dedicated to the production of bactericidal reactive oxygen species. The enzyme catalytic center is the cytochrome b(558), formed by 2 subunits, Nox2 (gp91-phox) and p22-phox. Cytochrome b(558) activation results from a conformational change induced by cytosolic regulatory proteins (p67-phox, p47-phox, p40-phox and Rac). The catalytic subunit is Nox2, while p22-phox is essential for both Nox2 maturation and the membrane anchorage of regulatory proteins. Moreover, it has been shown to be necessary for novel Nox activity. In order to characterize both p22-phox topology and cytochrome b(558) conformational change, 6 monoclonal antibodies were produced against purified cytochrome b(558). Phage display epitope mapping combined with a truncation analysis of recombinant p22-phox allowed the identification of epitope regions. Some of these antibodies almost completely inhibited in vitro reconstituted NADPH oxidase activity. Data analysis identified antibodies that recognized epitopes involved in either Nox2 maturation or Nox2 activation. Moreover, flow cytometry analysis and confocal microscopy performed on stimulated neutrophils showed that the monoclonal antibody 12E6 bound preferentially active cytochrome b(558). These monoclonal antibodies provided novel and unique probes to investigate maturation, activation and activity, not only of Nox2 but also of novel Nox.
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http://dx.doi.org/10.1159/000231977DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2919509PMC
May 2010

Regulation of the phagocyte NADPH oxidase activity: phosphorylation of gp91phox/NOX2 by protein kinase C enhances its diaphorase activity and binding to Rac2, p67phox, and p47phox.

FASEB J 2009 Apr 21;23(4):1011-22. Epub 2008 Nov 21.

INSERM U773, Centre de Recherche Biomédicale Bichat Beaujon CRB3, Université Paris 7, Paris, France.

Neutrophils generate microbicidal oxidants through activation of a multicomponent enzyme called NADPH oxidase. During activation, the cytosolic NADPH oxidase components (p47(phox), p67(phox), p40(phox), and Rac2) translocate to the membranes, where they associate with flavocytochrome b(558), which is composed of gp91(phox)/NOX2 and p22(phox), to form the active system. During neutrophil stimulation, p47(phox), p67(phox), p40(phox), and p22(phox) are phosphorylated; however, the phosphorylation of gp91(phox)/NOX2 and its potential role have not been defined. In this study, we show that gp91(phox) is phosphorylated in stimulated neutrophils. The gp91(phox) phosphoprotein is absent in neutrophils from chronic granulomatous disease patients deficient in gp91(phox), which confirms that this phosphoprotein is gp91(phox). The protein kinase C inhibitor GF109203X inhibited phorbol 12-myristate 13-acetate-induced phosphorylation of gp91(phox), and protein kinase C (PKC) phosphorylated the recombinant gp91(phox)- cytosolic carboxy-terminal flavoprotein domain. Two-dimensional tryptic peptide mapping analysis showed that PKC phosphorylated the gp91(phox)-cytosolic tail on the same peptides that were phosphorylated on gp91(phox) in intact cells. In addition, PKC phosphorylation increased diaphorase activity of the gp91(phox) flavoprotein cytosolic domain and its binding to Rac2, p67(phox), and p47(phox). These results demonstrate that gp91(phox) is phosphorylated in human neutrophils by PKC to enhance its catalytic activity and assembly of the complex. Phosphorylation of gp91(phox)/NOX2 is a novel mechanism of NADPH oxidase regulation.
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http://dx.doi.org/10.1096/fj.08-114553DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2660639PMC
April 2009

[Molecular aspects of chronic granulomatous disease. "the NADPH oxidase complex"].

Authors:
Françoise Morel

Bull Acad Natl Med 2007 Feb;191(2):377-90; discussion 390-2

GREPI TIMC-Imag UMR CNRS 5525 Laboratoire d'Enzymologie / DBPC, CHU - BP 217-38043 Grenoble.

Chronic granulomatous disease (CGD) is a hereditary illness generally occurring in childhood, in the form of recurrent severe infections. The main pathogens are staphylococci and aspergilli. It results from a failure of professional phagocytes, and particularly neutrophils, to produce superoxide ions O2- and their derivatives, which protect cells from bacterial, invasion through an oxidative and toxic defence mechanism. At an infection site. contact between the neutrophils and microorganisms or an inflammatory mediator triggers a respiratory burst, which results in the activation of the NADPH oxidase enzyme complex. NADPH depletes surrounding oxygen to yield O2-. In its active form. NADPH oxidase is an assembly of two components, namely the membrane cytochrome b558 (consisting o two subunits, gp91-phox and p22-phox) and soluble protein factors present in the resting neutrophil cytoplasm. Transfer of these cytosolic factors and their anchorage to cytochrome b558 determines the activity of NADPH oxidase. The respiratory burst lasts no more than a few minutes, but the precise mechanisms underlying its termination are not well known. In chronic granulomatous disease, neutrophils have lost their bactericidal capacity The most frequent form is hereditary and X-linked; in this case, the affected gene is CYBB, which encodes gp91-phox, the catalytic subunit of cytochrome b558. In autosomal and recessive forms of CGD the mutations affect the genes encoding p22-phox, p67-phox or p47-phox. We have unraveled the assembly mechanisms of the NADPH oxidase complex and have demonstrated that the cytosolic factor p67-phox is the determining element: it triggers both the assembly and the activation of NI4DPH oxidase. Binding of p67-phox to cytochrome b558 induces a gradual conformational change of cytochrome b558, which then becomes capable of transferring electrons produced in the cytoplasm from NADPH to oxygen, reducing the latter to O2-. The isolation of NADPH oxidase in its active and assembled form has allowed us to identify the activation partners of the oxidase complex. We also demonstrated that calcium-binding myeloid-related proteins (MRP). that are abundant in neutrophil cytoplasm, play a fundamental role in this activation. CGD patient management is essentially based on long-term high-dose prophylactic antibiotic administration. Gene therapy is promising but some distance away from practical application. We are currently investigating a new therapeutic concept that consists of transferring cytochrome b558 protein directly into deficient cells (initially the PLB 985 X cell line), encapsulated in proteoliposomes, which are hydrophobic.
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February 2007

Liposome-mediated cellular delivery of active gp91(phox).

PLoS One 2007 Sep 12;2(9):e856. Epub 2007 Sep 12.

HumProTher, UMR-CNRS 5525, Université Joseph Fourier, Centre Hospitalier Universitaire, Laboratoire d'Enzymologie/DBPC/BP 217, Centre Hospitalier Universitaire de Grenoble, Grenoble, France.

Background: Gp91(phox) is a transmembrane protein and the catalytic core of the NADPH oxidase complex of neutrophils. Lack of this protein causes chronic granulomatous disease (CGD), a rare genetic disorder characterized by severe and recurrent infections due to the incapacity of phagocytes to kill microorganisms.

Methodology: Here we optimize a prokaryotic cell-free expression system to produce integral mammalian membrane proteins.

Conclusions: Using this system, we over-express truncated forms of the gp91(phox) protein under soluble form in the presence of detergents or lipids resulting in active proteins with a "native-like" conformation. All the proteins exhibit diaphorase activity in the presence of cytosolic factors (p67(phox), p47(phox), p40(phox) and Rac) and arachidonic acid. We also produce proteoliposomes containing gp91(phox) protein and demonstrate that these proteins exhibit activities similar to their cellular counterpart. The proteoliposomes induce rapid cellular delivery and relocation of recombinant gp91(phox) proteins to the plasma membrane. Our data support the concept of cell-free expression technology for producing recombinant proteoliposomes and their use for functional and structural studies or protein therapy by complementing deficient cells in gp91(phox) protein.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0000856PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1955831PMC
September 2007

Concerted activities of nitric oxide synthases and NADPH oxidases in PLB-985 cells.

Biochem Biophys Res Commun 2007 Sep 20;361(2):493-8. Epub 2007 Jul 20.

Laboratoire PASTEUR, Ecole Normale Supérieure, CNRS UMR 8640, Département de Chimie, 24 rue Lhomond, 75005 Paris, France.

Oxidative stress is a metabolic situation used by immune cells to provide protection against infection. Under activation by threatening elements, phagocytes produce chemically toxic molecules, namely the reactive oxygen species (ROS) and reactive nitrogen species (RNS). This mechanism involves two types of enzymes: NAPDH oxidases (NOX) and NO synthases (NOS), which activities are versatile and not fully understood yet. In this regard, we studied in real-time the release of bursts of ROS and RNS by single PLB-985 cells, originating from a myeloid cell line prone to differentiate into neutrophil or monocyte-like phagocytes. A selective electrochemical detection of each ROS or RNS was conducted at platinized carbon fiber microelectrodes positioned at micrometric distances from single cells. Our results show (1) the existence of a NO synthase activity in PLB-985 cells and (2) the ability of NO synthases to provide a NOX activity in cells where NADPH oxidase (NOX2) is knocked out.
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http://dx.doi.org/10.1016/j.bbrc.2007.07.041DOI Listing
September 2007

New insights into the membrane topology of the phagocyte NADPH oxidase: characterization of an anti-gp91-phox conformational monoclonal antibody.

Biochimie 2007 Sep 20;89(9):1145-58. Epub 2007 Feb 20.

GREPI EA 2938, Lab. Enzymologie/DBPC, CHU Grenoble, BP 217, 38043 Grenoble Cedex 9, France.

Cytochrome b(558) is the catalytic core of the phagocyte NADPH oxidase that mediates the production of bactericidal reactive oxygen species. Cytochrome b(558) is formed by two subunits gp91-phox and p22-phox (1/1), non-covalently associated. Its activation depends on the interaction with cytosolic regulatory proteins (p67-phox, p47-phox, p40-phox and Rac) leading to an electron transfer from NADPH to molecular oxygen and to the release of superoxide anions. Several studies have suggested that the activation process was linked to a change in cytochrome b(558) conformation. Recently, we confirmed this hypothesis by isolating cytochrome b(558) in a constitutively active form. To characterize active and inactive cytochrome b(558) conformations, we produced four novel monoclonal antibodies (7A2, 13B6, 15B12 and 8G11) raised against a mixture of cytochrome b(558) purified from both resting and stimulated neutrophils. The four antibodies labeled gp91-phox and bound to both native and denatured cytochrome b(558). Interestingly, they were specific of extracellular domains of the protein. Phage display mapping combined to the study of recombinant gp91-phox truncated forms allowed the identification of epitope regions. These antibodies were then employed to investigate the NADPH oxidase activation process. In particular, they were shown to inhibit almost completely the NADPH oxidase activity reconstituted in vitro with membrane and cytosol. Moreover, flow cytometry analysis and confocal microscopy performed on stimulated neutrophils pointed out the capacity of the monoclonal antibody 13B6 to bind preferentially to the active form of cytochrome b(558). All these data suggested that the four novel antibodies are potentially powerful tools to detect the expression of cytochrome b(558) in intact cells and to analyze its membrane topology. Moreover, the antibody 13B6 may be conformationally sensitive and used as a probe for identifying the active NADPH oxidase complex in vivo.
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http://dx.doi.org/10.1016/j.biochi.2007.01.010DOI Listing
September 2007

Prospective comparison of six non-invasive scores for the diagnosis of liver fibrosis in chronic hepatitis C.

J Hepatol 2007 May 26;46(5):775-82. Epub 2007 Jan 26.

Département d'Hépato-gastroentérologie, CHU de Grenoble, BP 217, 38043 Grenoble cedex 9, France.

Background/aims: Non-invasive markers of liver fibrosis have recently been developed as an alternative to liver biopsy. The aim of this study was to compare the diagnostic performance of 6 scores (MP3, Fibrotest, Fibrometer, Hepascore, Forns' score and APRI).

Methods: We studied 180 chronic hepatitis C patients. Liver fibrosis was staged according to the METAVIR scoring system.

Results: Overall diagnostic performance of scores determined by AUROCs ranged from 0.86 for Fibrometer to 0.78 for Forns' score (NS) for discriminating F0F1 versus F2F3F4. For discriminating F0F1F2 versus F3F4, AUROCs ranged from 0.91 for Fibrometer to 0.78 for Forns' score (p<0.02). Significant or extensive fibrosis was predicted in 10-86% of patients with positive predictive value (PPV) ranging from 55% to 94%. Using logistic regression, statistical independence was demonstrated for MP3, Fibrotest and APRI. Diagnostic performance of paired-combination scores was then evaluated. The best combinations could select one-third of patients for whom either absence of significant fibrosis or presence of extensive fibrosis could be predicted with more than 90% of certainty.

Conclusions: Current non-invasive scores give reliable information on liver fibrosis in one-third of chronic hepatitis C patients, especially when used in combination.
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http://dx.doi.org/10.1016/j.jhep.2006.12.013DOI Listing
May 2007

Regulation of phagocyte NADPH oxidase activity: identification of two cytochrome b558 activation states.

FASEB J 2007 Apr 16;21(4):1244-55. Epub 2007 Jan 16.

GREPI EA 2938, Laboratory Enzymologie/DBPC, CHU Grenoble BP 217, 38043, Grenoble Cedex 9, France.

Activation of the phagocyte NADPH oxidase (phox) requires the association of cytosolic proteins (p67-phox, p47-phox, p40-phox, and Rac1/2) with the membrane cytochrome b558, leading to a hemoprotein conformation change. To clarify this mechanism, the phagocyte NADPH oxidase complex was isolated through cytochrome b558 purification after three chromatographic steps. The purified neutrophil complex was constitutively active in the absence of an amphiphile agent with a maximum turnover (125 mol O2(-) x s(-1) x mol heme b(-1)), indicating that cytochrome b558 has been activated by cytosolic proteins and is in an "open conformation," able to transfer a maximum rate of electrons. In contrast, the phox complex prepared with B lymphocyte cytosol shows a lower constitutive turnover (approximately 50 mol O2(-) x s(-1) x mol heme b(-1)). Analysis of phox complex components by Western blot and mass spectrometry showed the presence of cytosolic factors (especially p67-phox) and structural proteins (moesin, ezrin). To investigate the difference in activity of phox complexes, we evaluated the effect of MRP8 and MRP14, specifically expressed in neutrophils, on the activity of the B lymphocyte complex. MRPs induce the switch between the partially and the fully "open" cytochrome b558 conformation. Moreover, their effect was independent of p67-phox. Data point out two potential cytochrome b558 activation states.
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http://dx.doi.org/10.1096/fj.06-6852comDOI Listing
April 2007

Leu505 of Nox2 is crucial for optimal p67phox-dependent activation of the flavocytochrome b558 during phagocytic NADPH oxidase assembly.

J Leukoc Biol 2007 Jan 23;81(1):238-49. Epub 2006 Oct 23.

Groupe de Recherche et d'Etude du Processus Inflammatoire, Université Joseph Fourier, Laboratoire d'Enzymologie, Centre Hospitalier Universitaire, Grenoble CHU 38043, Cedex 9, France.

The role of Leu505 of Nox2 on the NADPH oxidase activation process was investigated. An X-CGD PLB-985 cell line expressing the Leu505Arg Nox2 mutant was obtained, exactly mimicking the phenotype of a previously published X91+-CGD case. In a reconstituted cell-free system (CFS), NADPH oxidase and iodonitrotetrazolium (INT) reductase activities were partially maintained concomitantly with a partial cytosolic factors translocation to the plasma membrane. This suggests that assembly and electron transfer from NADPH occurred partially in the Leu505Arg Nox2 mutant. Moreover, in a simplified CFS using purified mutant cytochrome b558 and recombinant p67phox, p47phox, and Rac1proteins, we found that the Km for NADPH and for NADH was about three times higher than those of purified WT cytochrome b558, indicating that the Leu505Arg mutation induces a slight decrease of the affinity for NADPH and NADH. In addition, oxidase activity can be extended by increasing the amount of p67phox in the simplified CFS assay. However, the maximal reconstituted oxidase activity using WT purified cytochrome b558 could not be reached using mutant cytochrome b558. In a three-dimensional model of the C-terminal tail of Nox2, Leu505 appears to have a strategic position just at the entry of the NADPH binding site and at the end of the alpha-helical loop (residues 484-504), a potential cytosolic factor binding region. The Leu505Arg mutation seems to affect the oxidase complex activation process through alteration of cytosolic factors binding and more particularly the p67phox interaction with cytochrome b558, thus affecting NADPH access to its binding site.
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http://dx.doi.org/10.1189/jlb.0905541DOI Listing
January 2007

NAD(P)H oxidase activity of Nox4 in chondrocytes is both inducible and involved in collagenase expression.

Antioxid Redox Signal 2006 Sep-Oct;8(9-10):1485-96

GREPI EA 2938 UJF, Lab Enzymology/DBPC, Universitary Hospital A. Michallon, Grenoble, France [corrected]

Reactive oxygen species (ROS) are regulators of redox-sensitive cell signaling pathways. In osteoarthritis, human interleukin-1beta is implicated in cartilage destruction through an ROS-dependent matrix metalloproteinase production. To determine the molecular source of ROS production in the human IL-1beta (hIL-1beta)-sensitive chondrocyte immortalized cell line C-20/A4, transfected cells were constructed that overexpress NAD(P)H oxidases. First, RT-PCR analysis showed that the C-20/A4 cell line expressed Nox2, Nox4, p22( phox ), and p67( phox ), but not p47( phox ). It was found that ROS production by C-20/A4 chondrocytes does not depend on PMA and ionomycin activation. This indicates that Nox2 was not involved in the production of ROS. In C- 20/A4 cells that overexpress Nox4, hIL-1beta stimulated ROS production three times more than the normal production of C-20/A4 cells. Moreover, there was a fourfold increase in the production of collagenase (MMP-1) by chondrocytes that overexpress Nox4. Interestingly, MMP-1 production in cells that overexpress Nox2 was not sensitive to hIL-1beta. These data suggest that under hIL-1beta stimulation, C-20/A4 chondrocytes produce MMP-1 through a Nox4-mediated, ROS-dependent pathway.
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http://dx.doi.org/10.1089/ars.2006.8.1485DOI Listing
January 2007

Increased clozapine plasma concentrations and side effects induced by smoking cessation in 2 CYP1A2 genotyped patients.

Ther Drug Monit 2005 Aug;27(4):539-43

Hôpitaux Universitaires de Genève, Département de Psychiatrie, Service de Psychiatrie Adulte, Boulevard St Georges 16-18, 1205 Genève, Switzerland.

Clozapine, an atypical antipsychotic, depends mainly on cytochrome P4501A2 (CYP1A2) for its metabolic clearance. CYP1A2 is inducible by smoking, and lower plasma concentrations of clozapine are measured in smokers than in nonsmokers. Case reports have been published on the effects of discontinuing smoking in patients receiving clozapine, which might lead to elevated plasma concentrations and severe side effects. We present 2 cases on the consequences of smoking cessation in patients receiving this drug. In the first patient, smoking cessation resulted, within 2 weeks, in severe sedation and fatigue, with an approximately 3-fold increase of plasma clozapine concentrations. In the second patient, a very high plasma concentration of clozapine (3004 ng/mL) was measured 6 days following a 16-day stay in a general hospital, during which smoking was prohibited. In the latter patient, the replacement of omeprazole, a strong CYP1A2 inducer, by pantoprazole, a weaker CYP1A2 inducer, could have contributed, in addition to smoking cessation, to the observed strong increase of plasma clozapine concentrations. Genotyping of the 2 patients revealed that they were carriers of the AA genotype for the -164C>A polymorphism (CYP1A2*1F) in intron 1 of CYP1A2 gene, which has previously been shown to confer a high inducibility of CYP1A2 by smoking. Thus, at the initiation of clozapine treatment, smoking patients should be informed that, if they decide to stop smoking, they are encouraged to do so but must inform their prescriber beforehand. Also, because of the increased use of no-smoking policies in many hospitals, studies examining the consequences of such policies on the pharmacokinetics/pharmacodynamics of drugs metabolized by CYP1A2, taking into account different CYP1A2 genotypes, are needed.
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http://dx.doi.org/10.1097/01.ftd.0000164609.14808.93DOI Listing
August 2005

NADPH oxidase homologs are required for normal cell differentiation and morphogenesis in Dictyostelium discoideum.

Biochim Biophys Acta 2005 Jun 8;1744(2):199-212. Epub 2005 Mar 8.

Laboratoire de Biochimie et Biophysique des Systèmes Intégrés (UMR5092 CNRS), Département de Réponse et Dynamique Cellulaires, CEA-Grenoble, France.

Membrane-associated NADPH oxidase complexes catalyse the production of the superoxide anion radical from oxygen and NADPH. In mammalian systems, NADPH oxidases form a family of at least seven isoforms that participate in host defence and signalling pathways. We report here the cloning and the characterisation of slime mould Dictyostelium discoideum homologs of the mammalian heme-containing subunit of flavocytochrome b (gp91(phox)) (NoxA, NoxB and NoxC), of the small subunit of flavocytochrome b (p22(phox)) and of the cytosolic factor p67(phox). Null-mutants of either noxA, noxB, noxC or p22(phox) show aberrant starvation-induced development and are unable to produce spores. The overexpression of NoxA(myc2) in noxA null strain restores spore formation. Remarkably, the gene alg-2B, coding for one of the two penta EF-hand proteins in Dictyostelium, acts as a suppressor in noxA, noxB, and p22(phox) null-mutant strains. Knockout of alg-2B allows noxA, noxB or p22(phox) null-mutants to return to normal development. However, the knockout of gene encoding NoxC, which contains two penta EF-hands, is not rescued by the invalidation of alg-2B. These data are consistent with a hypothesis connecting superoxide and calcium signalling during Dictyostelium development.
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http://dx.doi.org/10.1016/j.bbamcr.2005.02.004DOI Listing
June 2005

Crucial role of two potential cytosolic regions of Nox2, 191TSSTKTIRRS200 and 484DESQANHFAVHHDEEKD500, on NADPH oxidase activation.

J Biol Chem 2005 Apr 31;280(15):14962-73. Epub 2005 Jan 31.

Groupe de Recherche et d'Etude du Processus Inflammatoire EA 2938 Université Joseph Fourier, Laboratoire Enzymologie, Centre Hospitalier Universitaire, 38043 Grenoble cedex 9, France.

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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http://dx.doi.org/10.1074/jbc.M500226200DOI Listing
April 2005