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

The p22(phox) 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(phox) in the TGF-beta1-stimulated H2O2 production of primary human and murine fibroblasts. TGF-beta1 induced H2O2 release of the examined cells, and the response was dependent on the expression of both Nox4 and p22(phox). Interestingly, the p22(phox) protein was present in the absence of any detectable Nox/Duox expression, and the p22(phox) level was unaffected by TGF-beta1. On the other hand, Nox4 expression was dependent on the presence of p22(phox), establishing an asymmetrical relationship between the two proteins. Nox4 and p22(phox) proteins localized to the endoplasmic reticulum and their distribution was unaffected by TGF-beta1. We used a chemically induced protein dimerization method to study the orientation of p22(phox) 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 H2O2 into the lumen of the endoplasmic reticulum, indicating that Nox4 contributes to the development of the oxidative milieu within this organelle

When an Intramolecular Disulfide Bridge Governs the Interaction of DUOX2 with Its Partner DUOXA2

Aims: The dual oxidase 2 (DUOX2) protein belongs to the NADPH oxidase (NOX) family. As H2O2 generator, it plays a key role in both thyroid hormone biosynthesis and innate immunity. DUOX2 forms with its maturation factor, DUOX activator 2 (DUOXA2), a stable complex at the cell surface that is crucial for the H2O2-generating activity, but the nature of their interaction is unknown. The contribution of some cysteine residues located in the N-terminal ectodomain of DUOX2 in a surface protein?protein interaction is suggested. We have investigated the involvement of different cysteine residues in the formation of covalent bonds that could be of critical importance for the function of the complex. Results: We report the identification and the characterization of an intramolecular disulfide bond between cys-124 of the N-terminal ectodomain and cys-1162 of an extracellular loop of DUOX2, which has important functional implications in both export and activity of DUOX2. This intramolecular bridge provides structural support for the formation of interdisulfide bridges between the N-terminal domain of DUOX2 and the two extracellular loops of its partner, DUOXA2. Innovation: Both stability and function of the maturation factor, DUOXA2, are dependent on the oxidative folding of DUOX2, indicating that DUOX2 displays a chaperone-like function with respect to its partner. Conclusions: The oxidative folding of DUOX2 that takes place in the endoplasmic reticulum (ER) appears to be a key event in the trafficking of the DUOX2/DUOXA2 complex as it promotes an appropriate conformation of the N-terminal region, which is propitious to subsequent covalent interactions with the maturation factor, DUOXA2. Antioxid. Redox Signal. 23, 724?733.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/140308/1/ars.2015.6265.pd

Étude des différentes isoformes de NADPH :O2 oxydoréductases humaines

La famille enzymatique Nox comprend sept NAPH:O2 oxydoréductases membranaires dont la fonction est de catalyser la formation d'espèces réactives de l'oxygène (ROS) telles que l'anion superoxyde et le peroxyde d'hydrogène. La réaction est initiée par l'oxydation de NADPH cytosolique ; les électrons ainsi libérés traversent la bicouche lipidique pour finalement réduire le dioxygène extérieur. Le premier homologue caractérisé, noté Nox2 ou gp91phox, produit d'immenses quantités d'O20- indispensables à l'activité microbicide des cellules phagocytaires. En revanche, les autres isoformes synthétisent de faibles quantités de ROS impliquées dans des processus physiologiques non toxiques. La Nox3 dans l'oreille interne, est responsable de l'équilibre physique chez le rongeur. La Duox2 thyroïdienne associée à la thyroperoxydase, joue un rôle clef dans la synthèse des hormones thyroïdiennes. Cependant, les fonctions des homologues Nox1, Nox4, Nox5 et Duox1 ne sont pas clairement circonscrites.CHATENAY M.-PARIS 11-BU Pharma. (920192101) / SudocSudocFranceF

Cloning of S-Adenosyl-l-Methionine:C-24-Δ-Sterol-Methyltransferase (ERG6) from Leishmania donovani and Characterization of mRNAs in Wild-Type and Amphotericin B-Resistant Promastigotes

The 24-alkylated sterols have been shown previously to be absent in membranes of amphotericin B (AmB)-resistant Leishmania donovani promastigotes, suggesting that the S- adenosyl-l-methionine:C-24-Δ-sterol-methyltransferase (SCMT or ERG6) was not functional or not expressed in AmB-resistant (AmB-R) parasites. From an L. donovani wild-type clone, we cloned two cDNAs with an identical open reading frame encoding a putative SCMT, the enzyme responsible for a first sterol methylation at the C-24 position. The two cDNAs differed by their 3′-untranslated region (3′-UTR) and 5′-UTR sequences. One transcript (A) had a normal structure with a spliced leader and was highly expressed in normal cells but absent in AmB-R cells. The other (B), which did not possess the spliced leader sequence, was weakly expressed in normal cells but strongly expressed in AmB-R cells. As a functional test, ERG6 null mutant Saccharomyces cerevisiae yeasts were transformed using the pYES2.1 TOPO TA expression vector containing the candidate SCMT1/ERG6 coding sequence cloned from L. donovani. The transformed yeasts exhibited C-24 alkylated sterol expression, mainly ergosterol, within their membranes, proving that the isolated cDNA encodes on a SCMT responsible for sterol methylation. In AmB-R L. donovani promastigotes, the absence of the normal transcript (A) and the expression of an abnormal species (B) devoid of a spliced leader could explain the absence of sterol methylation in these cells. Further studies using a homologous system will allow us to draw conclusions about the relationship between SCMT expression and AmB resistance in Leishmania

Duox maturation factors form cell surface complexes with Duox affecting the specificity of reactive oxygen species generation

Dual oxidases (Duox1 and Duox2) are plasma membrane-targeted hydrogen peroxide generators that support extracellular hemoperoxidases. Duox activator 2 (Duoxa2), initially described as an endoplasmic reticulum resident protein, functions as a maturation factor needed to deliver active Duox2 to the cell surface. However, less is known about the Duox1/Duoxa1 homologues. We identified four alternatively spliced Duoxa1 variants and explored their roles in Duox subcellular targeting and reconstitution. Duox1 and Duox2 are functionally rescued by Duoxa2 or the Duoxa1 variants that contain the third coding exon. All active maturation factors are cotransported to the cell surface when coexpressed with either Duox1 or Duox2, consistent with detection of endogenous Duoxa1 on apical plasma membranes of the airway epithelium. In contrast, the Duoxa proteins are retained in the endoplasmic reticulum when expressed without Duox. Duox1/Duoxa1α and Duox2/Duoxa2 pairs produce the highest levels of hydrogen peroxide, as they undergo Golgi-based carbohydrate modifications and form stable cell surface complexes. Cross-functioning pairs that do not form stable complexes produce less hydrogen peroxide and leak superoxide. These findings suggest Duox activators not only promote Duox maturation, but they function as part of the hydrogen peroxide-generating enzyme.—Morand, S., Ueyama, T., Tsujibe, S., Saito, N., Korzeniowska, A., Leto, T. L. Duox maturation factors form cell surface complexes with Duox affecting the specificity of reactive oxygen species generation

Targeting and Regulation of Reactive Oxygen Species Generation by Nox Family NADPH Oxidases

Nox family NADPH oxidases serve a variety of functions requiring reactive oxygen species (ROS) generation, including antimicrobial defense, biosynthetic processes, oxygen sensing, and redox-based cellular signaling. We explored targeting, assembly, and activation of several Nox family oxidases, since ROS production appears to be regulated both spatially and temporally. Nox1 and Nox3 are similar to the phagocytic (Nox2-based) oxidase, functioning as multicomponent superoxide-generating enzymes. Factors regulating their activities include cytosolic activator and organizer proteins and GTP-Rac. Their regulation varies, with the following rank order: Nox2 > Nox1 > Nox3. Determinants of subcellular targeting include: (a) formation of Nox-p22phox heterodimeric complexes allowing plasma membrane translocation, (b) phospholipids-binding specificities of PX domain-containing organizer proteins (p47phox or Nox organizer 1 (Noxo1 and p40phox), and (c) variably splicing of Noxo1 PX domains directing them to nuclear or plasma membranes. Dual oxidases (Duox1 and Duox2) are targeted by different mechanisms. Plasma membrane targeting results in H2O2 release, not superoxide, to support extracellular peroxidases. Human Duox1 and Duox2 have no demonstrable peroxidase activity, despite their extensive homology with heme peroxidases. The dual oxidases were reconstituted by Duox activator 2 (Duoxa2) or two Duoxa1 variants, which dictate maturation, subcellular localization, and the type of ROS generated by forming stable complexes with Duox. Antioxid Redox Signal. 11, 2607–2619

The nonphagocytic NADPH oxidase Duox1 mediates a positive feedback loop during T cell receptor signaling.

Production of reactive oxygen species, often by NADPH (reduced form of nicotinamide adenine dinucleotide phosphate) oxidases, plays a role in the signaling responses of cells to many receptor stimuli. Here, we describe the function of the calcium-dependent, nonphagocytic NADPH oxidase Duox1 in primary human CD4(+) T cells and cultured T cell lines. Duox1 bound to inositol 1,4,5-trisphosphate receptor 1 and was required for early T cell receptor (TCR)-stimulated production of hydrogen peroxide (H(2)O(2)) through a pathway that was dependent on TCR-proximal kinases. Transient or stable knockdown of Duox1 inhibited TCR signaling, especially phosphorylation of tyrosine-319 of zeta chain-associated protein kinase of 70 kilodaltons (ZAP-70), store-operated entry of calcium ions (Ca(2+)), and activation of extracellular signal-regulated kinase. The production of cytokines was also inhibited by knockdown of Duox1. Duox1-mediated inactivation of Src homology 2 domain-containing protein tyrosine phosphatase 2 promoted the phosphorylation of ZAP-70 and its association with the Src family tyrosine kinase Lck and the CD3zeta chain of the TCR complex. Thus, we suggest that activation of Duox1, downstream of proximal TCR signals, generates H(2)O(2) that acts in a positive feedback loop to enhance and sustain further TCR signaling.Journal ArticleResearch Support, N.I.H. ExtramuralResearch Support, N.I.H. IntramuralResearch Support, Non-U.S. Gov'tSCOPUS: ar.jinfo:eu-repo/semantics/publishe

Hepatitis E Virus Infection in Sheltered Homeless Persons, France

EA MERS CT3 Enjeu 3International audienc

Prevalence and impact of GBV-C, SEN-V and HBV occult infections in HIV–HCV co-infected patients on HCV therapy

International audienceBackground/AimsIt has been suggested that, in HIV–HCV co-infected patients, co-infections with other viruses may affect the response to HCV therapy. We aimed to assess the prevalence of GBV-C, SEN-V and occult HBV infections, their impact on HCV and HIV infections and on the response to HCV therapy in HIV–HCV co-infected patients. Methods Three-hundred and sixty eight patients were tested before starting interferon–ribavirin for the presence of occult hepatitis B DNA, GBV-C RNA and SEN-V DNA by using real time PCR. Clinical, immunological, virological, histological characteristics and response to HCV therapy were compared according to the presence or not of each viral co-infection. Results HBV DNA, GBV-C RNA and SEN-V DNA were found in 5 (1.4%, CI95%: 0.2–2.4%), 104 (29.9%, CI95%: 25.1–34.7%) and 209 patients (57.9%, CI95%: 52.8–63.0%), respectively. GBV-C positive patients had significantly higher CD4 count at baseline, during and after HCV therapy, even after stratification on antiretroviral treatment. No other significant difference was observed according to the presence or not of GBV-C or SEN-V co-infection, in particular regarding virological responses to HCV combination therapy. Conclusions There is no reason to withhold HCV therapy in HIV infected patients who have access to HAART, because of occult HBV, GBV-C or SEN-V co-infections.</p