The plasma membrane-associated cation-binding protein PCaP1 of Arabidopsis thaliana is a uranyl-binding protein.

Uranium (U) is a naturally-occurring radionuclide that is toxic to living organisms. Given that proteins are primary targets of U(VI), their identification is an essential step towards understanding the mechanisms of radionuclide toxicity, and possibly detoxification. Here, we implemented a chromatographic strategy including immobilized metal affinity chromatography to trap protein targets of uranyl in Arabidopsis thaliana. This procedure allowed the identification of 38 uranyl-binding proteins (UraBPs) from root and shoot extracts. Among them, UraBP25, previously identified as plasma membrane-associated cation-binding protein 1 (PCaP1), was further characterized as a protein interacting in vitro with U(VI) and other metals using spectroscopic and structural approaches, and in planta through analyses of the fate of U(VI) in Arabidopsis lines with altered PCaP1 gene expression. Our results showed that recombinant PCaP1 binds U(VI) in vitro with affinity in the nM range, as well as Cu(II) and Fe(III) in high proportions, and that Ca(II) competes with U(VI) for binding. U(VI) induces PCaP1 oligomerization through binding at the monomer interface, at both the N-terminal structured domain and the C-terminal flexible region. Finally, U(VI) translocation in Arabidopsis shoots was affected in pcap1 null-mutant, suggesting a role for this protein in ion trafficking in planta.

A Simple Fluorescence Affinity Assay to Decipher Uranyl-Binding to Native Proteins.

Determining the affinity of proteins for uranyl is key to understand the toxicity of this cation and to further develop decorporation strategies. However, usual techniques to achieve that goal often require specific equipment and expertise. Here, we propose a simple, efficient, fluorescence-based method to assess the affinity of proteins and peptides for uranyl, at equilibrium and in buffered solution. We first designed and characterized an original uranyl-binding fluorescent probe. We then built a reference scale for uranyl affinity in solution, relying on signal quenching of our fluorescent probe in presence of high-affinity uranyl-binding peptides. We finally validated our approach by re-evaluating the uranyl-binding affinity of four native proteins. We envision that this tool will facilitate the reliable and reproducible assessment of affinities of peptides and proteins for uranyl.

Cytoplasmic aggregation of uranium in human dopaminergic cells after continuous exposure to soluble uranyl at non-cytotoxic concentrations.

Uranium exposure can lead to neurobehavioral alterations in particular of the monoaminergic system, even at non-cytotoxic concentrations. However, the mechanisms of uranium neurotoxicity after non-cytotoxic exposure are still poorly understood. In particular, imaging uranium in neurons at low intracellular concentration is still very challenging. We investigated uranium intracellular localization by means of synchrotron X-ray fluorescence imaging with high spatial resolution (< 300 nm) and high analytical sensitivity (< 1 µg.g-1 per 300 nm pixel). Neuron-like SH-SY5Y human cells differentiated into a dopaminergic phenotype were continuously exposed, for seven days, to a non-cytotoxic concentration (10 µM) of soluble natural uranyl. Cytoplasmic submicron uranium aggregates were observed accounting on average for 62 % of the intracellular uranium content. In some aggregates, uranium and iron were co-localized suggesting common metabolic pathways between uranium and iron storage. Uranium aggregates contained no calcium or phosphorous indicating that detoxification mechanisms in neuron-like cells are different from those described in bone or kidney cells. Uranium intracellular distribution was compared to fluorescently labeled organelles (lysosomes, early and late endosomes) and to fetuin-A, a high affinity uranium-binding protein. A strict correlation could not be evidenced between uranium and the labeled organelles, or with vesicles containing fetuin-A. Our results indicate a new mechanism of uranium cytoplasmic aggregation after non-cytotoxic uranyl exposure that could be involved in neuronal defense through uranium sequestration into less reactive species. The remaining soluble fraction of uranium would be responsible for protein binding and for the resulting neurotoxic effects.

Fetuin exhibits a strong affinity for plutonium and may facilitate its accumulation in the skeleton.

After entering the blood, plutonium accumulates mainly in the liver and the bones. The mechanisms leading to its accumulation in bone are, however, completely unknown. We already know that another uptake pathway not involving the transferrin-mediated pathways is suspected to intervene in the case of the liver. Fetuin, a protein playing an important role in bone metabolism, is proposed as a potential transporter of Pu from serum to bone. For the first time, the binding constants of these two proteins (transferrin and fetuin) with tetravalent plutonium at physiological pH (pH 7.0) were determined by using capillary electrophoresis (CE) coupled with inductively coupled plasma mass spectrometry (ICP-MS). Their very close values (log10 KPuTf = 26.44 ± 0.28 and log10 KPuFet = 26.20 ± 0.24, respectively) suggest that transferrin and fetuin could compete to chelate plutonium, either in the blood or directly at bone surfaces in the case of Pu deposits. We performed competition reaction studies demonstrating that the relative distribution of Pu-protein complexes is fully explained by thermodynamics. Furthermore, considering the average concentrations of transferrin and fetuin in the blood, our calculation is consistent with the bio-distribution of Pu observed in humans.

Isotopic variations of copper at the protein fraction level in neuronal human cells exposed in vitro to uranium.

The study of isotopic variations of endogenous and toxic metals in fluids and tissues is a recent research topic with an outstanding potential in biomedical and toxicological investigations. Most of the analyses have been performed so far in bulk samples, which can make the interpretation of results entangled, since different sources of stress or the alteration of different metabolic processes can lead to similar variations in the isotopic compositions of the elements in bulk samples. The downscaling of the isotopic analysis of elements at the sub-cellular level, is considered as a more promising alternative. Here we present for the first time the accurate determination of Cu isotopic ratios in four main protein fractions from lysates of neuron-like human cells exposed in vitro to 10 µM of natural uranium for seven days. These protein fractions were isolated by Size Exclusion Chromatography and analysed by Multi-Collector Inductively Coupled Plasma Mass Spectrometry to determine the Cu isotopic variations in each protein fraction with regard to the original cell lysate. Values obtained, expressed as δ65Cu, were -0.03 ± 0.14 ‰ (Uc, k = 2), -0.55 ± 0.20 ‰ (Uc, k = 2), -0.32 ± 0.21 ‰ (Uc, k = 2) and +0.84 ± 0.21 ‰ (Uc, k = 2) for the four fractions, satisfying the mass balance. The results obtained in this preliminary study pave the way for dedicated analytical developments to identify new specific disease biomarkers, to gain insight into stress-induced altered metabolic processes, as well as to decipher metabolic pathways of toxic elements.

How Do Actinyls Interact with Hyperphosphorylated Yolk Protein Phosvitin?

The development of the nuclear industry has raised multiple questions about its impact on the biotope and humans. Proteins are key biomolecules in cell machinery and essential in deciphering toxicological processes. Phosvitin was chosen as a relevant model for phosphorylated proteins because of its important role as an iron, calcium, and magnesium storage protein in egg yolk. A multitechnique spectroscopic investigation was performed to reveal the coordination geometry of two oxocations of the actinide family (actinyl UVI , NpV ) in speciation with phosvitin. IR spectroscopy revealed phosphoryl groups as the main functional groups interacting with UVI . This was confirmed through laser luminescence spectroscopy (U) and UV/Vis absorption spectroscopy (Np). For UVI , X-ray absorption spectroscopy at the LIII edge revealed a small contribution of bidentate binding present, along with predominantly monodentate binding of phosphoryl groups; for NpV , uniquely bidentate binding was revealed. As a perspective to this work, X-ray absorption spectroscopy speciation of UVI and NpV in the extracted yolk of living eggs of the dogfish Scyliorhinus canicula was determined; this corroborated the binding of phosphorous together with a reduction of the actinyl moiety. Such data are essential to pinpoint the mechanisms of heavy metals (actinyls) accumulation and toxicity in oviparous organisms, and therefore, contribute to a shift from descriptive approaches to predictive toxicology.

Deciphering the uranium target proteins in human dopaminergic SH-SY5Y cells.

Uranium (U) is the heaviest naturally occurring element ubiquitously present in the Earth's crust. Human exposure to low levels of U is, therefore, unavoidable. Recently, several studies have clearly pointed out that the brain is a sensitive target for U, but the mechanisms leading to the observed neurological alterations are not fully known. To deepen our knowledge of the biochemical disturbances resulting from U(VI) toxicity in neuronal cells, two complementary strategies were set up to identify the proteins that selectively bind U(VI) in human dopaminergic SH-SY5Y cells. The first strategy relies on the selective capture of proteins capable of binding U(VI), using immobilized metal affinity chromatography, and starting from lysates of cells grown in a U(VI)-free medium. The second strategy is based on the separation of U-enriched protein fractions by size-exclusion chromatography, starting from lysates of U(VI)-exposed cells. High-resolution mass spectrometry helped us to highlight 269 common proteins identified as the urano-proteome. They were further analyzed to characterize their cellular localization and biological functions. Four canonical pathways, related to the protein ubiquitination system, gluconeogenesis, glycolysis, and the actin cytoskeleton proteins, were particularly emphasized due to their high content of U(VI)-bound proteins. A semi-quantification was performed to concentrate on the ten most abundant proteins, whose physico-chemical characteristics were studied in particular depth. The selective interaction of U(VI) with these proteins is an initial element of proof of the possible metabolic effects of U(VI) on neuronal cells at the molecular level.

Phosphate-Rich Biomimetic Peptides Shed Light on High-Affinity Hyperphosphorylated Uranyl Binding Sites in Phosphoproteins.

Some phosphoproteins such as osteopontin (OPN) have been identified as high-affinity uranyl targets. However, the binding sites required for interaction with uranyl and therefore involved in its toxicity have not been identified in the whole protein. The biomimetic approach proposed here aimed to decipher the nature of these sites and should help to understand the role of the multiple phosphorylations in UO2 2+ binding. Two hyperphosphorylated cyclic peptides, pS168 and pS1368 containing up to four phosphoserine (pSer) residues over the ten amino acids present in the sequences, were synthesized with all reactions performed in the solid phase, including post-phosphorylation. These ß-sheet-structured peptides present four coordinating residues from four amino acid side chains pointing to the metal ion, either three pSer and one glutamate in pS168 or four pSer in pS1368 . Significantly, increasing the number of pSer residues up to four in the cyclodecapeptide scaffolds produced molecules with an affinity constant for UO2 2+ that is as large as that reported for osteopontin at physiological pH. The phosphate-rich pS1368 can thus be considered a relevant model of UO2 2+ coordination in this intrinsically disordered protein, which wraps around the metal ion to gather four phosphate groups in the UO2 2+ coordination sphere. These model hyperphosphorylated peptides are highly selective for UO2 2+ with respect to endogenous Ca2+ , which makes them good starting structures for selective UO2 2+ complexation.

Is hydroxypyridonate 3,4,3-LI(1,2-HOPO) a good competitor of fetuin for uranyl metabolism?

Uranium is widespread in the environment, resulting both from natural occurrences and anthropogenic activities. Its toxicity is mainly chemical rather than radiological. In the blood it is transported as uranyl UO22+ cation and forms complexes with small ligands like carbonates and with some proteins. From there it reaches the skeleton, its main target organ for accumulation. Fetuin is a serum protein involved in biomineralization processes, and it was demonstrated to be the main UO22+-binder in vitro. Fetuin's life cycle ends in bone. It is thus suspected to be a key protagonist of U accumulation in this organ. Up to now, there has been no effective treatment for the removal of U from the body and studies devoted to the interactions involving chelating agents with both UO22+ and its protein targets are lacking. The present work aims at studying the potential role of 3,4,3-LI(1,2-HOPO) as a promising chelating agent in competition with fetuin. The apparent affinity constant of 3,4,3-LI(1,2-HOPO) was first determined, giving evidence for its very high affinity similar to that of fetuin. Chromatography experiments, aimed at identifying the complexes formed and quantifying their UO22+ content, and spectroscopic structural investigations (XAS) were carried out, demonstrating that 3,4,3-LI(1,2-HOPO) inhibits/limits the formation of fetuin-uranyl complexes under stoichiometric conditions. But surprisingly, possible ternary complexes stable enough to remain present after the chromatographic process were identified under sub-stoichiometric conditions of HOPO versus fetuin. These results contribute to the understanding of the mechanisms accounting for U residual accumulation despite chelation therapy after internal contamination.

Impact of uranium uptake on isotopic fractionation and endogenous element homeostasis in human neuron-like cells.

The impact of natural uranium (U) on differentiated human neuron-like cells exposed to 1, 10, 125, and 250 µM of U for seven days was assessed. In particular, the effect of the U uptake on the homeostatic modulation of several endogenous elements (Mg, P, Mn, Fe, Zn, and Cu), the U isotopic fractionation upon its incorporation by the cells and the evolution of the intracellular Cu and Zn isotopic signatures were studied. The intracellular accumulation of U was accompanied by a preferential uptake of 235U for cells exposed to 1 and 10 µM of U, whereas no significant isotopic fractionation was observed between the extra- and the intracellular media for higher exposure U concentrations. The U uptake was also found to modulate the homeostasis of Cu, Fe, and Mn for cells exposed to 125 and 250 µM of U, but the intracellular Cu isotopic signature was not modified. The intracellular Zn isotopic signature was not modified either. The activation of the non-specific U uptake pathway might be related to this homeostatic modulation. All together, these results show that isotopic and quantitative analyses of toxic and endogenous elements are powerful tools to help deciphering the toxicity mechanisms of heavy metals.

Uranium exposure of human dopaminergic cells results in low cytotoxicity, accumulation within sub-cytoplasmic regions, and down regulation of MAO-B.

Natural uranium is an ubiquitous element present in the environment and human exposure to low levels of uranium is unavoidable. Although the main target of acute uranium toxicity is the kidney, some concerns have been recently raised about neurological effects of chronic exposure to low levels of uranium. Only very few studies have addressed the molecular mechanisms of uranium neurotoxicity, indicating that the cholinergic and dopaminergic systems could be altered. The main objective of this study was to investigate the mechanisms of natural uranium toxicity, after 7-day continuous exposure, on terminally differentiated human SH-SY5Y cells exhibiting a dopaminergic phenotype. Cell viability was first assessed showing that uranium cytotoxicity only occurred at high exposure concentrations (> 125 µM), far from the expected values for uranium in the blood even after occupational exposure. SH-SY5Y differentiated cells were then continuously exposed to 1, 10, 125 or 250 µM of natural uranium for 7 days and uranium quantitative subcellular distribution was investigated by means of micro-PIXE (Particle Induced X-ray Emission). The subcellular element imaging revealed that uranium was located in defined perinuclear regions of the cytoplasm, suggesting its accumulation in organelles. Uranium was not detected in the nucleus of the differentiated cells. Quantitative analysis evidenced a very low intracellular uranium content at non-cytotoxic levels of exposure (1 and 10 µM). At higher levels of exposure (125 and 250 µM), when cytotoxic effects begin, a larger and disproportional intracellular accumulation of uranium was observed. Finally the expression of dopamine-related genes was quantified using real time qRT-PCR. The expression of monoamine oxidase B (MAO-B) gene was statistically significantly decreased after exposure to uranium while other dopamine-related genes were not modified. The down regulation of MAO-B was confirmed at the protein level. This original result suggests that the inhibition of dopamine catabolism, but also of other MAO-B substrates, could constitute selective effects of uranium neurotoxicity.

The ErpA/NfuA complex builds an oxidation-resistant Fe-S cluster delivery pathway.

Fe-S cluster-containing proteins occur in most organisms, wherein they assist in myriad processes from metabolism to DNA repair via gene expression and bioenergetic processes. Here, we used both in vitro and in vivo methods to investigate the capacity of the four Fe-S carriers, NfuA, SufA, ErpA, and IscA, to fulfill their targeting role under oxidative stress. Likewise, Fe-S clusters exhibited varying half-lives, depending on the carriers they were bound to; an NfuA-bound Fe-S cluster was more stable (t½ = 100 min) than those bound to SufA (t½ = 55 min), ErpA (t½ = 54 min), or IscA (t½ = 45 min). Surprisingly, the presence of NfuA further enhanced stability of the ErpA-bound cluster to t½ = 90 min. Using genetic and plasmon surface resonance analyses, we showed that NfuA and ErpA interacted directly with client proteins, whereas IscA or SufA did not. Moreover, NfuA and ErpA interacted with one another. Given all of these observations, we propose an architecture of the Fe-S delivery network in which ErpA is the last factor that delivers cluster directly to most if not all client proteins. NfuA is proposed to assist ErpA under severely unfavorable conditions. A comparison with the strategy employed in yeast and eukaryotes is discussed.

Methods for Analyzing the Impacts of Natural Uranium on In Vitro Osteoclastogenesis.

Uranium has been shown to interfere with bone physiology and it is well established that this metal accumulates in bone. However, little is known about the effect of natural uranium on the behavior of bone cells. In particular, the impact of uranium on osteoclasts, the cells responsible for the resorption of the bone matrix, is not documented. To investigate this issue, we have established a new protocol using uranyl acetate as a source of natural uranium and the murine RAW 264.7 cell line as a model of osteoclast precursors. Herein, we detailed all the assays required to test uranium cytotoxicity on osteoclast precursors and to evaluate its impact on the osteoclastogenesis and on the resorbing function of mature osteoclasts. The conditions we have developed, in particular for the preparation of uranyl-containing culture media and for the seeding of RAW 264.7 cells allow to obtain reliable and highly reproductive results. Moreover, we have optimized the use of software tools to facilitate the analysis of various parameters such as the size of osteoclasts or the percentage of resorbed matrix.

A new procedure for high precision isotope ratio determinations of U, Cu and Zn at nanogram levels in cultured human cells: What are the limiting factors?

The monitoring of isotopic fractionations in in vitro cultured human cell samples is a very promising and under-exploited tool to help identify the metabolic processes leading to disease-induced isotopic fractionations or decipher metabolic pathways of toxic metals in these samples. One of the limitations is that the analytes are often present at small amounts, ranging from tens to hundreds of ng, thus making challenging low-uncertainty isotope ratio determinations. Here we present a new procedure for U, Cu and Zn purification and isotope ratio determinations in cultured human neuron-like cells exposed to natural U. A thorough study of the influence of the limiting factors impacting the uncertainty of δ238U, δ66Zn and δ65Cu is also carried out. These factors include the signal intensity, which determines the within-day measurement reproducibility, the procedural blank correction and the matrix effects, which determine the accuracy of the mass bias correction models. Given the small Cu and U amounts in the cell samples, 15-30 and 20ng respectively, a highly efficient sample introduction system was employed in order to improve the analyte transport to the plasma and, hence, the signal intensity. With this device, the procedural blanks became the main uncertainty source of δ238U and δ65Cu values, accounting over 65% of the overall uncertainty. The matrix effects gave rise to inaccuracies in the mass bias correction models for samples finally dissolved in the minimal volumes required for the analysis, 100-150µL, leading to biases for U and Cu. We will show how these biases can be cancelled out by dissolving the samples in volumes of at least 300µL for Cu and 450µL for U. Using our procedure, expanded uncertainties (k = 2) of around 0.35‰ for δ238U and 0.15‰ for δ66Zn and δ65Cu could be obtained. The analytical approach presented in this work is also applicable to other biological microsamples and can be extended to other elements and applications.

Reactivity of U-associated osteopontin with lactoferrin: a one-to-many complex.

Uranium is the heaviest natural element, mainly found in aqueous medium as the hexavalent uranyl ion (UO22+). Bones are the main organs in which uranium accumulates, depending on as yet unknown molecular and cellular mechanisms. Recently, it has been revealed that osteopontin (OPN), a protein involved in bio-mineralization processes, and its main naturally occurring cleaved form (fOPN), have nanomolar affinities for UO22+. The binding of UO22+ is due to both the phosphorylation sites and acidic residues of these proteins and is accompanied by a slight gain in secondary structure. OPN is an Intrinsically Disordered Protein (IDP), a family of proteins which play a crucial role in several interaction networks, where phosphorylations are thought to be key elements. OPN has been shown to bind lactoferrin (LF) and the two proteins have antagonist functions in the modulation of the bio-mineralization process. However, to date, there has been no evidence that UO22+ and LF compete in their binding to OPN or not. Based on a series of convergent experimental data, this study first addressed in detail the LF/fOPN interaction and proposed a LF:fOPN 4/1 maximal stoichiometry. Moreover the phosphorylations were demonstrated to be necessary for the stability of such complexes. The interaction of preformed UO22+/fOPN complexes with LF was also investigated and the occurrence of several entities involving the three partners was demonstrated. These complexes did not reveal any significant conformational changes compared to those obtained in the absence of UO22+. The results have shown not only that LF and UO22+ do not compete, but also that these complexes are likely to be more stable than LF/fOPN complexes, as indicated by their melting temperature (Tm) values. The potential impact of those uranyl-stabilized ternary complexes on some biological pathways now remains to be assessed. Nonetheless, this work has contributed to shedding light on the formation of stable ternary complexes involving a large structured protein, an IDP and an exogenous metal.

Interaction of silver nanoparticles with metallothionein and ceruloplasmin: impact on metal substitution by Ag(i), corona formation and enzymatic activity.

The release of Ag(i) from silver nanoparticles (AgNPs) unintentionally spread in the environment is suspected to impair some key biological functions. In comparison with AgNO3, in-depth investigations were carried out into the interactions between citrate-coated AgNPs (20 nm) and two metalloproteins, intracellular metallothionein 1 (MT1) and plasmatic ceruloplasmin (Cp), both involved in metal homeostasis. These were chosen for their physiological relevance and the diversity of their various native metals bound because of thiol groups and/or their structural differences. Transmission electron microscopy (TEM), and dynamic light scattering (DLS), UV-vis and circular dichroism (CD) spectroscopies were used to study the effects of such intricate interactions on AgNP dissolution and proteins in terms of metal exchanges and structural modifications. The isolation of the different populations formed together with on-line quantifications of their metal content were performed by asymmetrical flow field-flow fractionation (AF4) linked to inductively coupled plasma mass spectrometry (ICP-MS). For the 2 proteins, Ag(i) dissolved from the AgNPs, substituted for the native metal, to different extents and with different types of dynamics for the corona formed: the MT1 rapidly surrounded the AgNPs with the transient reticulate corona thus promoting their dissolution associated with the metal substitution, whereas the Cp established a more stable layer around the AgNPs, with a limited substitution of Cu and a decrease in its ferroxidase activity. The accessibility and lability of the metal binding sites inside these proteins and their relative affinities for Ag(i) are discussed, taking into account the structural characteristics of the proteins.

Effect of natural uranium on the UMR-106 osteoblastic cell line: impairment of the autophagic process as an underlying mechanism of uranium toxicity.

Natural uranium (U), which is present in our environment, exerts a chemical toxicity, particularly in bone where it accumulates. Generally, U is found at oxidation state +VI in its oxocationic form [Formula: see text] in aqueous media. Although U(VI) has been reported to induce cell death in osteoblasts, the cells in charge of bone formation, the molecular mechanism for U(VI) effects in these cells remains poorly understood. The objective of our study was to explore U(VI) effect at doses ranging from 5 to 600 µM, on mineralization and autophagy induction in the UMR-106 model osteoblastic cell line and to determine U(VI) speciation after cellular uptake. Our results indicate that U(VI) affects mineralization function, even at subtoxic concentrations (<100 µM). The combination of thermodynamic modeling of U with EXAFS data in the culture medium and in the cells clearly indicates the biotransformation of U(VI) carbonate species into a meta-autunite phase upon uptake by osteoblasts. We next assessed U(VI) effect at 100 and 300 µM on autophagy, a survival process triggered by various stresses such as metal exposure. We observed that U(VI) was able to rapidly activate autophagy but an inhibition of the autophagic flux was observed after 24 h. Thus, our results indicate that U(VI) perturbs osteoblastic functions by reducing mineralization capacity. Our study identifies for the first time U(VI) in the form of meta-autunite in mammalian cells. In addition, U(VI)-mediated inhibition of the autophagic flux may be one of the underlying mechanisms leading to the decreased mineralization and the toxicity observed in osteoblasts.

Evidence of isotopic fractionation of natural uranium in cultured human cells.

The study of the isotopic fractionation of endogen elements and toxic heavy metals in living organisms for biomedical applications, and for metabolic and toxicological studies, is a cutting-edge research topic. This paper shows that human neuroblastoma cells incorporated small amounts of uranium (U) after exposure to 10 µM natural U, with preferential uptake of the 235U isotope with regard to 238U. Efforts were made to develop and then validate a procedure for highly accurate n(238U)/n(235U) determinations in microsamples of cells. We found that intracellular U is enriched in 235U by 0.38 ± 0.13‰ (2σ, n = 7) relative to the exposure solutions. These in vitro experiments provide clues for the identification of biological processes responsible for uranium isotopic fractionation and link them to potential U incorporation pathways into neuronal cells. Suggested incorporation processes are a kinetically controlled process, such as facilitated transmembrane diffusion, and the uptake through a high-affinity uranium transport protein involving the modification of the uranyl (UO22+) coordination sphere. These findings open perspectives on the use of isotopic fractionation of metals in cellular models, offering a probe to track uptake/transport pathways and to help decipher associated cellular metabolic processes.

Investigation of uranium interactions with calcium phosphate-binding proteins using ICP/MS and CE-ICP/MS.

During long-term exposure, uranium accumulates in bone. Since uranium in U(vi) complexes shares similar coordination properties to calcium, this toxicant is assumed to be exchanged with calcium ions at the surfaces of bone mineral crystals. Recently, two proteins involved in bone turnover, fetuin A and osteopontin, were shown to exhibit a high affinity for U(vi). A common biochemical feature of both fetuin A and osteopontin is their inhibiting role in calcium phosphate precipitation. Therefore it is conceivable that complexation of U(vi) with these proteins may alter their interaction with calcium and/or calcium phosphate. Quantitative analyses of calcium, phosphorus and uranium performed using inductively coupled plasma/mass spectrometry (ICP/MS) demonstrated the inhibition of the precipitation of calcium phosphate by fetuin A and osteopontin for 2 h. In addition, the presence of U(vi) did not seem to alter the duration of this process. However, dynamic light scattering studies revealed that the size of the colloidal particles formed with osteopontin was altered by the presence of U(vi) in a concentration-dependent manner. Finally, using hyphenated capillary electrophoresis-ICP/MS (CE-ICP/MS), we showed that in these systems, at a low concentration of U(vi) (protein : U(vi) 8 : 1), U(vi) might remain in solution by forming a complex with proteins and not by sequestration of a precipitate of either autunite or uranyl orthophosphate.

Genomic and physiological analysis reveals versatile metabolic capacity of deep-sea Photobacterium phosphoreum ANT-2200.

Bacteria of the genus Photobacterium thrive worldwide in oceans and show substantial eco-physiological diversity including free-living, symbiotic and piezophilic life styles. Genomic characteristics underlying this variability across species are poorly understood. Here we carried out genomic and physiological analysis of Photobacterium phosphoreum strain ANT-2200, the first deep-sea luminous bacterium of which the genome has been sequenced. Using optical mapping we updated the genomic data and reassembled it into two chromosomes and a large plasmid. Genomic analysis revealed a versatile energy metabolic potential and physiological analysis confirmed its growth capacity by deriving energy from fermentation of glucose or maltose, by respiration with formate as electron donor and trimethlyamine N-oxide (TMAO), nitrate or fumarate as electron acceptors, or by chemo-organo-heterotrophic growth in rich media. Despite that it was isolated at a site with saturated dissolved oxygen, the ANT-2200 strain possesses four gene clusters coding for typical anaerobic enzymes, the TMAO reductases. Elevated hydrostatic pressure enhances the TMAO reductase activity, mainly due to the increase of isoenzyme TorA1. The high copy number of the TMAO reductase isoenzymes and pressure-enhanced activity might imply a strategy developed by bacteria to adapt to deep-sea habitats where the instant TMAO availability may increase with depth.