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The maturation pathway for the nickel-dependent enzyme urease utilizes the protein UreE as a metallochaperone to supply Ni(II) ions. In Helicobacter pylori urease maturation also requires HypA and HypB, accessory proteins that are commonly associated with hydrogenase maturation. Herein we report on the characterization of a protein complex formed between HypA and the UreE2 dimer. Nuclear magnetic resonance (NMR) coupled with molecular modelling show that the protein complex apo, Zn-HypA•UreE2, forms between the rigorously conserved Met-His-Glu (MHE motif) Ni-binding N-terminal sequence of HypA and the two conserved His102A and His102B located at the dimer interface of UreE2. This complex forms in the absence of Ni(II) and is supported by extensive protein contacts that include the use of the C-terminal sequences of UreE2 to form additional strands of β-sheet with the Ni-binding domain of HypA. The Ni-binding properties of apo, Zn-HypA•UreE2 and the component proteins were investigated by isothermal titration calorimetry using a global fitting strategy that included all of the relevant equilibria, and show that the Ni,Zn-HypA•UreE2 complex contains a single Ni(II)-binding site with a sub-nanomolar KD. The structural features of this novel Ni(II) site were elucidated using proteins produced with specifically deuterated amino acids, protein point mutations, and the analyses of X-ray absorption spectroscopy, hyperfine shifted NMR features, as well as molecular modeling coupled with quantum-mechanical calculations. The results show that the complex contains a six-coordinate, high-spin Ni(II) site with ligands provided by both component proteins.

Acquisition and homeostasis of essential metals during host colonization by bacterial pathogens rely on metal uptake, trafficking, and storage proteins. How these factors have evolved within bacterial pathogens is poorly defined. Urease, a nickel enzyme, is essential for Helicobacter pylori to colonize the acidic stomach. Our previous data suggest that acquisition of nickel transporters and a histidine-rich protein (HRP) involved in nickel storage in H. pylori and gastric Helicobacter spp. have been essential evolutionary events for gastric colonization. Using bioinformatics, proteomics, and phylogenetics, we extended this analysis to determine how evolution has framed the repertoire of HRPs among 39 Epsilonproteobacteria; 18 gastric and 11 non-gastric enterohepatic (EH) Helicobacter spp., as well as 10 other Epsilonproteobacteria. We identified a total of 213 HRPs distributed in 22 protein families named orthologous groups (OGs) with His-rich domains, including 15 newly described OGs. Gastric Helicobacter spp. are enriched in HRPs (7.7 ± 1.9 HRPs/strain) as compared to EH Helicobacter spp. (1.9 ± 1.0 HRPs/strain) with a particular prevalence of HRPs with C-terminal histidine-rich domains in gastric species. The expression and nickel-binding capacity of several HRPs was validated in five gastric Helicobacter spp. We established the evolutionary history of new HRP families, such as the periplasmic HP0721-like proteins and the HugZ-type heme oxygenases. The expansion of histidine-rich extensions in gastric Helicobacter spp. proteins is intriguing but can tentatively be associated with the presence of the urease nickel enzyme. We conclude that this HRP expansion is associated with unique properties of organisms that rely on large intracellular nickel amounts for their survival.

International audience; Helicobacter pylori HypA (HpHypA) is a metallochaperone necessary for maturation of [Ni,Fe]-hydrogenase and urease, the enzymes required for colonization and survival of H. pylori in the gastric mucosa. HpHypA contains a structural Zn(II) site and a unique Ni(II) binding site at the N-terminus. X-ray absorption spectra suggested that the Zn(II) coordination depends on pH and on the presence of Ni(II). This study was performed to investigate the structural properties of HpHypA as a function of pH and Ni(II) binding, using NMR spectroscopy combined with DFT and molecular dynamics calculations. The solution structure of apo,Zn-HpHypA, containing Zn(II) but devoid of Ni(II), was determined using 2D, 3D and 4D NMR spectroscopy. The structure suggests that a Ni-binding and a Zn-binding domain, joined through a short linker, could undergo mutual reorientation. This flexibility has no physiological effect on acid viability or urease maturation in H. pylori. Atomistic molecular dynamics simulations suggest that Ni(II) binding is important for the conformational stability of the N-terminal helix. NMR chemical shift perturbation analysis indicates that no structural changes occur in the Zn-binding domain upon addition of Ni(II) in the pH 6.3-7.2 range. The structure of the Ni(II) binding site was probed using 1H NMR spectroscopy experiments tailored to reveal hyperfine-shifted signals around the paramagnetic metal ion. On this basis, two possible models were derived using quantum-mechanical DFT calculations. The results provide a comprehensive picture of the Ni(II) mode to HpHypA, important to rationalize, at the molecular level, the functional interactions of this chaperone with its protein partners.

The study area (Szklary Massif, SW Poland) comprises three sites of different soil provenance: (1) natural serpentine Cambisol, (2) anthroposol situated on waste dump and (3) cultivated Inceptisol developed on glacial tills next to the dump. Potentially toxic elements (PTE) have either lithogenic or anthropogenic origins in these sites. The chemical partitioning of Co, Cr, Cu, Ni, Pb and Zn among solid forms was determined by sequential extractions coupled with direct mineral identifications (SEM, electron microprobe analysis – EMPA, and XRD). Examination of solid residues after several extraction steps was conducted in order to discuss the indirect speciation obtained by the extraction method. Total concentrations of PTE having anthropogenic origin greatly exceed those of lithogenic origin. Mobility of studied PTE is variable in the different environments except for Cr which is always mostly found in residual fractions of extractions. Cu and Pb are more mobile than Cr and Co in all soils. Zn is more stable (Cu > Pb > Ni > Co > Zn >> Cr) in the serpentine soil and cultivated epipedon (Pb > Cu > Zn > Ni > Co >> Cr) than in the anthroposol (Zn > Cu ⩾ Pb > Ni > Co >> Cr). PTE of lithogenic origin are generally less mobile than those from anthropogenic origin except Ni which is more mobile in the serpentine soil. Nonetheless, mineral forms of metals better determine their mobility than metal origin. Identification by direct methods of the PTE mineral form was not possible for metals present at low concentrations (Cu, Pb). However, direct mineralogical examinations of the solid residues of several extractions steps improved the assessment of the PTE solid speciation and mobility, particularly for Cr, Ni and Zn.

International audience; Peroxiredoxin 6 (Prdx6) is a "moonlighting" protein with both GSH peroxidase and phospholipase A(2) (PLA(2)) activities. This protein is responsible for degradation of internalized dipalmitoylphosphatidylcholine, the major phospholipid component of lung surfactant. The PLA(2) activity is inhibited by surfactant protein A (SP-A). We postulate that SP-A regulates the PLA(2) activity of Prdx6 through direct protein-protein interaction. Recombinant human Prdx6 and SP-A isolated from human alveolar proteinosis fluid were studied. Measurement of kinetic constants at pH 4.0 (maximal PLA(2) activity) showed K(m)0.35 mm and V(max) 138 nmol/min/mg of protein. SP-A inhibited PLA(2) activity non-competitively with K(i) 10 mug/ml and was Ca(2+) -independent. Activity at pH 7.4 was approximately 50% less, and inhibition by SP-A was partially dependent on Ca(2+). Interaction of SP-A and Prdx6 at pH 7.4 was shown by Prdx6-mediated inhibition of SP-A binding to agarose beads, a pull-down assay using His-tagged Prdx6 and Ni(2) -chelating beads, co-immunoprecipitation from lung epithelial cells and from a binary mixture of the two proteins, binding after treatment with a trifunctional cross-linker, and size-exclusion chromatography. Analysis by static light scattering and surface plasmon resonance showed calcium-independent SP-A binding to Prdx6 at pH 4.0 and partial Ca(2+) dependence of binding at pH 7.4. These results indicate a direct interaction between SP-A and Prdx6, which provides a mechanism for regulation of the PLA(2) activity of Prdx6 by SP-A.

Bacterial peptide deformylase (PDF) belongs to a sub-family of metalloproteases that catalyse the removal of the N-terminal formyl group from newly synthesised proteins. PDF is essential in prokaryotes and conserved throughout the eubacteria. It is therefore considered an attractive target for developing new antibacterial agents. Here, we report the crystal structures of four bacterial deformylases, free or bound to the naturally occurring antibiotic actinonin, including two from the major bacterial pathogens Pseudomonas aeruginosa and Staphylococcus aureus. The overall tertiary structure is essentially conserved but shows significant differences, namely at the C terminus, which are directly related to the deformylase type (i.e. I or II) they belong to. The geometry around the catalytic metal ion exhibits a high level of similarity within the different enzymes, as does the binding mode of actinonin to the various deformylases. However, some significant structural differences are found in the vicinity of the active site, highlighting the structural and molecular requirements for the design of a deformylase inhibitor active against a broad spectrum of bacterial strains.

Vascular endothelial growth factor (VEGF) is mitogenic for endothelial cells and has been shown to induce angiogenesis and endothelial cell migration through stimulation of endothelial tyrosine-kinase receptors. Here, using confocal microscopy and the patch-clamp technique on endothelial cells, membrane permeability to calcium as well as cytoplasmic and nuclear free calcium levels have been investigated in the first stages of tyrosine-kinase receptor activation by VEGF. VEGF (0.5nM) as well as inositol trisphosphate (IP3) induced an activation of membrane calcium-permeable channels exhibiting a similar low conductance in the range of 10 pS. The VEGF-triggered activation of these calcium channels, mediated by IP3 and involving the intracellular calcium stores, results in an increase in both cytoplasmic and nuclear calcium levels in endothelial cells, potentially modulating gene expression. Finally, the effect of Ni2+, a calcium channel blocker, on endothelial cell proliferation has been studied. The results show that inhibition of extracellular calcium influx significantly inhibits VEGF-induced cell proliferation. In the process of cell stimulation by VEGF, and possibly by other growth factors, activation of calcium channels could then be a key step in calcium-regulated gene expression and cell activation. These results suggest that the use of calcium channel blockers could be a novel way of prevention or reversion of VEGF-induced tumoral angiogenesis.

International audience; The ABC-type importer NikABCDE mediates nickel acquisition in Escherichia coli. The periplasmic nickel-binding component NikA has a folding similar to that of the peptide transporter OppA and does not bind free nickel. Instead, we showed that the metal is tetra-coordinated by an organic tri-dentate molecule and His416. Conversely, it has been recently reported that NikA binds Ni-(L-His)2 and that addition of histidine increases the rate of nickel uptake in vivo. Here, we report the structure of NikA/Ni-(L-His)2 and show that histidine binding differs from peptide binding in OppA. The structure also confirms the central role of His416 in nickel binding.

International audience; Nickel is an essential metal for Helicobacter pylori, as it is the co-factor of two enzymes crucial for col-onization, urease and hydrogenase. Nickel is taken up by specific transporters and its intracellular homeostasis depends on nickel-binding proteins to avoid toxicity. Nickel trafficking is controlled by the Ni(II)-dependent transcriptional regulator NikR. In contrast to other NikR proteins, NikR from H. pylori is a pleiotropic regulator that depending on the target gene acts as an activator or a repres-sor. We systematically quantified the in vivo Ni 2+-NikR response of 11 direct NikR targets that encode functions related to nickel metabolism, four activated and seven repressed genes. Among these, four targets were characterized for the first time (hpn, hpn-like, hydA and hspA) and NikR binding to their promoter regions was demonstrated by electrophoretic mobility shift assays. We found that NikR-dependent repression was generally set up at higher nickel concentrations than activation. Kinetics of the regulation revealed a gradual and temporal NikR-mediated response to nickel where activation of nickel-protection mechanisms takes place before repression of nickel uptake. Our in vivo study demonstrates, for the first time, a chronological hierarchy in the NikR-dependent tran-scriptional response to nickel that is coherent with the control of nickel homeostasis in H. pylori.

Background Nickel, chromium VI, and cadmium have been identified as lung carcinogens in highly exposed cohorts. The purpose of this study was to examine the etiological link between lung cancer and these metals in occupations, that usually entail lower levels of exposure than those seen in historical cohorts. Methods Two population-based case–control studies were conducted in Montreal, from 1979 to 1986 and from 1996 to 2001, comprising 1,598 cases and 1,965 controls. A detailed job history was obtained to evaluate lifetime occupational exposure to many agents, including nickel, chromium VI, and cadmium compounds. Results Lung cancer odds ratios were increased only among former or non-smokers: 2.5 (95% CI: 1.3–4.7) for nickel exposure, 2.4 (95% CI: 1.2–4.8) for chromium VI, and 4.7 (95% CI: 1.5–14.3) for cadmium. The metals did not increase risk among smokers. Conclusions While excess risks due to these metal compounds were barely discernable among smokers, carcinogenic effects were seen among non-smokers. Am. J. Ind. Med. 53:476–485, 2010. © 2010 Wiley-Liss, Inc.

Based on a pluridisciplinary research programme on New Caledonia's lagoon (2004-2008), this paper addresses economic, ecological and political issues in order to implement integrated coastal zone management (ICZM) in this French Pacific territory. The nickel mining industry constitutes the core of the re-balancing economic and social strategy between the Northern and Southern provinces. But major impacts on the coastal environment of metal-processing plants, harbours, and decades of mine exploitation have released a controversy. A short diachronic analysis suggests that such environmental concerns prompted the emergence of collective actions to among civil society, customary and institutional stakeholders. The inscription of New Caledonia lagoon and reef areas in the UNESCO World Heritage List in 2008 would be both an outcome and a catalyst of this on-going process. Looking beyond the reefs towards the mainland and watersheds for the construction of local socio-ecological systems, we assume that the current stakes could result in the initiation of ICZM in New Caledonia.

International audience; NikR is a transcriptional metalloregulator central in the mandatory response to acidity of Helicobacter pylori that controls the expression of numerous genes by binding to specific promoter regions. NikR/DNA interactions were proposed to rely on protein activation by Ni(II) binding to high-affinity (HA) and possibly secondary external (X) sites. We describe a biochemical characterization of HpNikR mutants that shows that the HA sites are essential but not sufficient for DNA binding, while the secondary external (X) sites and residues from the HpNikR dimer-dimer interface are important for DNA binding. We show that a second metal is necessary for HpNikR/DNA binding, but only to some promoters. Small-angle X-ray scattering shows that HpNikR adopts a defined conformation in solution, resembling the cis-conformation and suggests that nickel does not trigger large conformational changes in HpNikR. The crystal structures of selected mutants identify the effects of each mutation on HpNikR structure. This study unravels key structural features from which we derive a model for HpNikR activation where: (i) HA sites and an hydrogen bond network are required for DNA binding and (ii) metallation of a unique secondary external site (X) modulates HpNikR DNA binding to low-affinity promoters by disruption of a salt bridge.

International audience; Solar ultraviolet (UV) radiation has hazardous effects on human health that are, in part, associated with its immunosuppressive effects via the induction of interleukin (IL)-10 production. Although IL-10 is produced by both T helper type 2 (Th2) cells and T-regulatory type 1 (Tr1) cells, the relative contribution of either subset in UV radiation-induced immunosuppression has not been established. Here, we show that T cells isolated from non-treated allergic contact dermatitis (ACD) reactions, 48 h following nickel challenge and propagated for 7-10 days in the presence of IL-2, were mainly CD4(+) and produced IL-10, but little interferon-gamma. A single sub-erythematous solar-simulated radiation (SSR) prior to antigen challenge exposure resulted in a clinical attenuation of the intensity of ACD reactions which was associated with a significant increase in both the magnitude of IL-10 production by skin-infiltrating T cells and the frequency of IL-10-producing Tr1 cells. Skin-infiltrating T cells in SSR-exposed, as well as non-exposed, ACD reactions showed a perturbed T-cell receptor (TCR)-Vbeta repertoire, without overexpression of a particular TCR-Vbeta gene product, indicating the presence of high frequencies of nickel non-specific T cells in ACD reactions. These results show that a single sub-erythematous SSR induces immunosuppression via the cutaneous infiltration of IL-10-producing Tr1, and to a lesser extent, Th2 cells.

International audience; Soluble nickel compounds are carcinogenic to humans although the mechanism by which they cause cancer remains unclear. One major consequence of exposure to nickel is the stabilization of hypoxia inducible factor-1alpha (HIF-1alpha), a protein known to be overexpressed in a variety of cancers. In this study, we report a persistent stabilization of HIF-1alpha by nickel chloride up to 72 h after the removal of nickel from the culture media. In addition, we show that the HIF-prolyl hydroxylases (PHD's) are inhibited when cells are exposed to nickel and that they remain repressed for up to 72 h after nickel is removed. We then show that nickel can inhibit purified HIF-PHD's 2 in vitro, through direct interference with the enzyme. Through theoretical calculations, we also demonstrate that nickel may be able to replace the iron in the active site of this enzyme, providing a plausible mechanism for the persistent inhibition of HIF-PHD's by nickel. The data presented suggest that nickel can interfere with HIF-PHD directly and does not inhibit the enzyme by simply depleting cellular factors, such as iron or ascorbic acid. Understanding the mechanisms by which nickel can inhibit HIF-PHD's and stabilize HIF-1alpha may be important in the treatment of cancer and ischemic diseases.

International audience; NikR proteins are bacterial metallo-regulatory transcription factors that control the expression of the nickel uptake system and/or nickel containing enzymes such as urease, and are involved in the acid stress response. Here, a comparative study is reported on NikR from Helicobacter pylori (HpNikR) and Escherichia coli (EcNikR), as well as the Q2E mutant of EcNikR. Most attention was focused on the Ni(II) binding properties of these proteins, as a function of pH. The influence of the pH on the Ni(II) binding and aggregation properties was studied using gel filtration analysis and UV-visible absorption spectroscopy in the presence of an increasing concentration of nickel. Q2E and wt EcNikR are identical in Ni(II) binding but the Q2E mutant is impaired to some extent in DNA-binding. For EcNikR it is shown that between pH 6 and 8, addition of Ni(II) above 1 equiv. induces mass aggregation and precipitation, concomitant with binding of Ni(II) up to a maximum of 5-8 Ni(II) ions per monomer. The Ni(II) site with highest affinity is the well-described square planar site with three histidines and one cysteine ligands. Aggregation is complete in the presence of less than 1 extra equiv. of Ni(II) and aggregation is fully reversible and precipitates are rapidly solubilized by addition of EDTA. The sensitivity of EcNikR to aggregation decreases with decreasing pH, concurrent with histidines being the main ligands of the site responsible for aggregation. HpNikR does not display aggregation except at alkaline pH, where 3 Ni(II) equiv. are needed. The participation of a cluster consisting of surface-exposed histidines present in EcNikR but not in HpNikR, is proposed to be involved in aggregation. Our results on HpNikR are compatible with the crystallographic data and with the ability of this protein to bind more than one nickel.

International audience; Soluble nickel compounds are likely human carcinogens. The mechanism by which soluble nickel may contribute to carcinogenesis is unclear, though several hypotheses have been proposed. Here we verify the ability of nickel to enter the cell via the divalent metal ion transporter 1 (DMT1) and disturb cellular iron homeostasis. Nickel may interfere with iron at both an extracellular level, by preventing iron from being transported into the cell, and at an intracellular level, by competing for iron sites on enzymes like the prolyl hydroxylases that modify hypoxia inducible factor-1alpha (HIF-1alpha). Nickel was able to decrease the binding of the Von Hippel-Lindau (VHL) protein to HIF-1alpha, indicating a decrease in prolyl hydroxylase activity. The ability of nickel to affect various iron dependent processes may be an important step in nickel dependent carcinogenesis. In addition, understanding the mechanisms by which nickel activates the HIF-1alpha pathway may lead to new molecular targets in fighting cancer.

International audience; Transcription factors of the T-domain family regulate many developmental processes. We have isolated from the sea urchin a new member of the Tbx2 subfamily: coquillette. Coquillette has a late zygotic expression whose localization is dynamic: at the blastula stage it is restricted to the aboral side of most of the presumptive ectoderm and endoderm territories and from gastrulation on, to the aboral-most primary mesenchyme cells. Perturbation of coquillette function delays gastrulation and strongly disorganizes the skeleton of the larva. Coquillette is sensitive to alteration of the oral-aboral (OA) axis and we identify goosecoid, which controls oral and aboral fates in the ectoderm, as a probable upstream regulator. Coquillette appears to be an integral part of the patterning system along the OA axis.

International audience; The specificity of recombinant (2-->3)-alpha-sialyltransferase (ST3Gal-III), expressed in baculovirus-infected insect cells, has been determined with various oligosaccharide acceptors and sugar-nucleotide donors using a fluorescence based assay. Recombinant ST3Gal-III tagged with a polyhistidine tail was immobilized on Ni(2+)-NTA-Agarose as an active enzyme for use in the synthesis of three sialylated oligosaccharides: (i) the divalent molecule [alpha-Neu5Ac-(2-->3)-D-Galp-(1-->4)-beta-D-GlcpNAc-O-CH(2)](2)-C-(CH(2)OBn)(2) (12); (ii) the dansylated derivative, alpha-Neu5Ac-(2-->3)-D-Galp-(1-->3)-beta-D-GlcpNAc-O-(CH(2))(6)-NH-dansyl and; (iii) the tetrasacharide alpha-Neu5Ac-(2-->3)-beta-D-Galp-(1-->4)-beta-D-GlcpNAc-(1-->2)-alpha-D-Manp-O-CH(3). Compound 12 was itself prepared from the divalent N-acetyllactosamine molecule built on pentaerythritol by a chemo-enzymatic route.

International audience; Juvenile Helix aspersa snails exposed in field microcosms were used to assess the transfer of Cd, Cu, Ni, Pb and Zn from forest soils amended with liquid and composted sewage sludge. Zn concentrations and contents were significantly higher in snails exposed to liquid and composted sludge after 5 and 7 weeks of exposure, when compared with control. Trends were less clear for the other metals. Present results show that Zn, among the cocktail of metallic trace elements (MTE) coming from sewage sludge disposal, represents the principal concern for food chain transfer and secondary poisoning risks. The microcosm design used in this experiment was well suited for relatively long-term (about 2 months) active biomonitoring with H. aspersa snails. The snails quickly indicated the variations of MTE concentrations in their immediate environment. Therefore, the present study provides a simple but efficient field tool to evaluate MTE bioavailability and transfer.

International audience; To gain more insight about Escherichia coli tmRNA structure, NiCR, a square planar macrocyclic nickel (II) complex, was used to probe guanine N7 exposure. On the basis of this additional structural information, a refined secondary structure of the molecule is proposed. In addition to its known specificity for guanine N7, we show here that the chemical probe can also cleave at specific uridine residues. In contrast to the alkaline-labile modification of guanine, the reactivity of NiCR at these uridine residues results in direct strand scission. To better characterize the uridine cleavage sites and assess the importance of the RNA structure for the reaction to occur, smaller RNA molecules derived from one pseudoknot (PK4) of E. coli tmRNA containing two uridine cleavage sites were engineered and probed. It is shown that this pseudoknot can fold by itself in solution and that the expected uridine residues are also cleaved by the nickel complex, suggesting that only a local sequence and/or structural context is required for cleavage. In E. coli tmRNA, the five uridine cleavage sites are located in double-stranded regions. These sites contain a G-U wobble base-pair and a downstream uridine which is cleaved. Using smaller RNAs derived from one stem of PK4, systematic changes in the proposed recognition motif indicate that the G-U pair is required for cleavage. Furthermore, there is no cleavage if the G-U pair is reversed. If the recognition motif is moved within the stem, the cleavage site moves accordingly. Additionally, if the recognition motif is changed such that the G-U pair is flanked by two uridine residues, the reactivity occurs only at the 3' uridine. Radical quenching studies have indicated that sulfate radical, as in the case of guanine oxidation, is involved in uridine oxidation. Although additional studies are required to better characterize the reaction, this paper reports a novel specificity for a chemical probe which may be useful for investigating structural motifs involving G-U pairs in folded RNAs.

International audience; The dissociation constant of Ni(II) for Escherichia coli NikR was determined using three independent techniques, including binding kinetics, and shown to be in the sub-micromolar range.