Françoise Morel

The E-loop Is Involved in Hydrogen Peroxide Formation by the NADPH Oxidase Nox4

In contrast to the NADPH oxidases Nox1 and Nox2, which generate superoxide (O2̇̄), Nox4 produces hydrogen peroxide (H2O2). 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 H2O2 versus L-012 and cytochrome c for O2̇̄. 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 H2O2 to O2̇̄ generation while preserving expression and intracellular localization. In the presence of an NO donor, the O2̇̄-producing Nox4 mutants, but not wild-type Nox4, generated peroxynitrite, excluding artifacts of the detection system as the apparent origin of O2̇̄. In Cos7 cells, in which Nox4 partially localizes to the plasma membrane, an antibody directed against the E-loop decreased H2O2 but increased O2̇̄ formation by Nox4 without affecting Nox1-dependent O2̇̄ 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 H2O2. Mutation of this but not of four other conserved histidines also switched Nox4 from H2O2 to O2̇̄ formation. Thus, H2O2 formation is an intrinsic property of Nox4 that involves its E-loop. The structure of the E-loop may hinder O2̇̄ egress and/or provide a source for protons, allowing dismutation to form H2O2.

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

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.

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

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.

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

BACKGROUND & AIMSnFibrosis 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.nnnMETHODSn510 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.nnnRESULTSnFibrosis 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.nnnCONCLUSIONnOverall 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.

Intervertebral disk degeneration and herniation: the role of metalloproteinases and cytokines.

This article reviews the role of metabolic factors, including metalloproteinases and cytokines, in the occurrence of degenerative disk disease and disk herniation. Given that mechanical factors alone cannot cause disk degeneration, studies must explore metabolic, genetic, nutritional, and age-related factors. Zinc metalloproteinases exert particularly important effects, not only directly, but also indirectly through promotion of neovascularization. The production of these enzymes is dependent on a number of cytokines and on the cell changes they induce. This complex effect acts both on disk matrix degeneration and on the pain generated by contact between the protruding disk and the nerve roots. However, it can have a favorable effect by promoting resorption of the herniated disk. Available data on the role for mechanical factors on the disk chondrocyte metabolism and on metalloproteinase production show that mechanical and metabolic factors interact closely to produce disk disorders.

Molecular Interface of S100A8 with Cytochrome b558 and NADPH Oxidase Activation

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/gp91phox and p22phox. 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 87HEES90 amino acid residues of the S100A8 C-terminal sequence that are involved directly in the molecular interaction with cytochrome b558 and then in the phagocyte NADPH oxidase activation.

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

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 b558, 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‐trans‐fected cells. Interestingly, the Michaelis constant (Km) 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.—Baillet, A., Xu, R., Grichine, A., Berthier, S., Morel, F., Paclet, M.‐H. Coupling of 6‐phosphogluconate dehydrogenase with NADPH oxidase in neutrophils: Nox2 activity regulation by NADPH availability. FASEB J. 25, 2333–2343 (2011). www.fasebj.org

Differences in MMPs and TIMP-1 expression between intervertebral disc and disc herniation.

Joint Bone Spine - In Press.Proof corrected by the author Available online since lundi 8 octobre 2012