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1.
Angew Chem Int Ed Engl ; 61(26): e202203198, 2022 06 27.
Artigo em Inglês | MEDLINE | ID: mdl-35466512

RESUMO

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.


Assuntos
Corantes Fluorescentes , Urânio , Fluorescência , Peptídeos/química , Urânio/química
2.
J Biol Chem ; 293(20): 7689-7702, 2018 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-29626095

RESUMO

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.


Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Redes e Vias Metabólicas , Estresse Oxidativo , Oxigênio/metabolismo , Regulação Bacteriana da Expressão Gênica , Oxirredução
3.
Chemistry ; 25(36): 8570-8578, 2019 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-30908736

RESUMO

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.


Assuntos
Osteopontina/química , Compostos de Urânio/química , Sítios de Ligação , Cálcio/química , Cálcio/metabolismo , Dicroísmo Circular , Osteopontina/metabolismo , Peptídeos Cíclicos/síntese química , Peptídeos Cíclicos/química , Peptídeos Cíclicos/metabolismo , Espectrometria de Massas por Ionização por Electrospray , Compostos de Urânio/metabolismo
4.
Chemistry ; 25(53): 12332-12341, 2019 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-31206850

RESUMO

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.


Assuntos
Gema de Ovo/metabolismo , Fosvitina/metabolismo , Cálcio/metabolismo , Humanos , Ferro/metabolismo , Magnésio/metabolismo , Minerais , Fósforo/química , Fosvitina/química , Espectroscopia por Absorção de Raios X
5.
Analyst ; 144(20): 5928-5933, 2019 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-31490474

RESUMO

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.


Assuntos
Cobre/química , Isótopos/química , Neurônios/química , Neurônios/efeitos dos fármacos , Proteínas/química , Urânio/farmacologia , Radioisótopos de Cobre , Humanos , Espectrometria de Massas/métodos , Metabolômica/métodos , Urânio/química
6.
Arch Toxicol ; 93(8): 2141-2154, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31222525

RESUMO

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.


Assuntos
Neurônios Dopaminérgicos/efeitos dos fármacos , Urânio/toxicidade , Células Cultivadas , Neurônios Dopaminérgicos/metabolismo , Gluconeogênese , Glicólise , Humanos , Complexo de Endopeptidases do Proteassoma/fisiologia , Ligação Proteica , Proteômica , Urânio/metabolismo
7.
Proc Natl Acad Sci U S A ; 113(49): 14007-14012, 2016 12 06.
Artigo em Inglês | MEDLINE | ID: mdl-27872304

RESUMO

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.


Assuntos
Fracionamento Químico/métodos , Urânio/análise , Técnicas de Cultura de Células , Linhagem Celular/metabolismo , Humanos , Isótopos , Neurônios/metabolismo , Urânio/metabolismo
8.
Arch Toxicol ; 91(4): 1903-1914, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-27585666

RESUMO

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.


Assuntos
Autofagia/efeitos dos fármacos , Calcificação Fisiológica/efeitos dos fármacos , Osteoblastos/efeitos dos fármacos , Urânio/toxicidade , Animais , Linhagem Celular , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Osteoblastos/metabolismo , Osteoblastos/patologia , Osteossarcoma/metabolismo , Ratos , Termodinâmica , Urânio/administração & dosagem
9.
Extremophiles ; 20(3): 301-10, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-27039108

RESUMO

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.


Assuntos
Adaptação Fisiológica , Metabolismo Energético , Genoma Bacteriano , Photobacterium/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Transporte de Elétrons , Glucose/metabolismo , Pressão Hidrostática , Isoenzimas/genética , Isoenzimas/metabolismo , Maltose/metabolismo , Metilaminas/metabolismo , Oxirredutases N-Desmetilantes/genética , Oxirredutases N-Desmetilantes/metabolismo , Photobacterium/metabolismo , Água do Mar/microbiologia
10.
Inorg Chem ; 55(1): 29-36, 2016 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-26684435

RESUMO

In case of a nuclear event, contamination (broad or limited) of the population or of specific workers might occur. In such a senario, the fate of actinide contaminants may be of first concern, in particular with regard to human target organs like the skeleton. To improve our understanding of the toxicological processes that might take place, a mechanistic approach is necessary. For instance, ∼50% of Pu(IV) is known from biokinetic data to accumulate in bone, but the underlining mechanisms are almost unknown. In this context, and to obtain a better description of the toxicological mechanisms associated with actinides(IV), we have undertaken the investigation, on a molecular scale, of the interaction of thorium(IV) with osteopontin (OPN) a hyperphosphorylated protein involved in bone turnover. Thorium is taken here as a simple model for actinide(IV) chemistry. In addition, we have selected a phosphorylated hexapeptide (His-pSer-Asp-Glu-pSer-Asp-Glu-Val) that is representative of the peptidic sequence involved in the bone interaction. For both the protein and the biomimetic peptide, we have determined the local environment of Th(IV) within the bioactinidic complex, combining isothermal titration calorimetry, attenuated total reflectance Fourier transform infrared spectroscopy, theoretical calculations with density functional theory, and extended X-ray absorption fine structure spectroscopy at the Th LIII edge. The results demonstrate a predominance of interaction of metal with the phosphate groups and confirmed the previous physiological studies that have highlighted a high affinity of Th(IV) for the bone matrix. Data are further compared with those of the uranyl case, representing the actinyl(V) and actinyl(VI) species. Last, our approach shows the importance of developing simplified systems [Th(IV)-peptide] that can serve as models for more biologically relevant systems.


Assuntos
Elementos da Série Actinoide/metabolismo , Osso e Ossos/metabolismo , Osteopontina/fisiologia , Tório/química , Humanos , Oligopeptídeos/fisiologia , Osteopontina/química , Espectroscopia de Infravermelho com Transformada de Fourier
11.
Electrophoresis ; 36(11-12): 1374-82, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25630637

RESUMO

Identification of uranyl transport proteins is key to develop efficient detoxification approaches. Therefore, analytical approaches have to be developed to cope with the complexity of biological media and allow the analysis of metal speciation. CE-ICP/MS was used to combine the less-intrusive character and high separation efficiency of CE with the sensitive detection of ICP/MS. The method was based on the incubation of samples with uranyl prior to the separation. Electrophoretic buffers were compared to select a 10 mM Tris to 15 mM NaCl buffer, which enabled analyses at pH 7.4 and limited dissociation. This method was applied to the analysis of a serum. Two main fractions were observed. By comparison with synthetic mixtures of proteins, the first one was attributed to fetuin and in a lesser extent to HSA, and the second one to uranyl unbound to proteins. The analysis showed that fetuin was likely to be the main target of uranyl. CE-ICP/MS was also used to investigate the behavior of the fetuin-uranyl complex, in the presence of carbonate, an abundant complexing agent of uranyl in blood. This method enabled association constants determination, suggesting the occurrence of both FETUA(UO2(2+)) and FETUA(UO2(2+))(CO3(2-)) complexes, depending on the carbonate concentration.


Assuntos
Proteínas de Transporte/metabolismo , Eletroforese Capilar/métodos , Espectrometria de Massas/métodos , Urânio/metabolismo , Soluções Tampão , Ligação Proteica
12.
J Biol Inorg Chem ; 20(3): 497-507, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25534663

RESUMO

Bone is the main target organ for the storage of several toxic metals, including uranium. But the mode of action of uranium on bones remains poorly understood. To better assess the impact of uranium on bone cells, synthetic biomimetic apatites encompassing a controlled amount of uranium were prepared and analyzed. This study revealed the physicochemical impact of uranium on apatite mineralization: the presence of the metal induces a loss of crystallinity and a lower mineralization rate. The prepared samples were then used as substrates for bone cell culture. Osteoblasts were not sensitive to the presence of uranium in the support, whereas previous results showed a deleterious effect of uranium introduced into a cell culture solution. This work should therefore have some original prospects within the context of toxicological studies concerning the effect of metallic cations on bone cell systems.


Assuntos
Apatitas/química , Materiais Biomiméticos/química , Urânio/química , Animais , Proliferação de Células , Sobrevivência Celular , Células , Camundongos , Microscopia Eletrônica de Varredura , Osteoblastos/citologia
13.
Chem Res Toxicol ; 28(12): 2304-12, 2015 Dec 21.
Artigo em Inglês | MEDLINE | ID: mdl-26566067

RESUMO

The redox state of disulfide bonds is implicated in many redox control systems, such as the cysteine-cystine couple. Among proteins, ubiquitous cysteine-rich metallothioneins possess thiolate metal binding groups susceptible to metal exchange in detoxification processes. CeO2 NPs are commonly used in various industrial applications due to their redox properties. These redox properties that enable dual oxidation states (Ce(IV)/Ce(III)) to exist at their surface may act as oxidants for biomolecules. The interaction among metallothioneins, cysteine, and CeO2 NPs was investigated through various biophysical approaches to shed light on the potential effects of the Ce(4+)/Ce(3+) redox system on the thiol groups of these biomolecules. The possible reaction mechanisms include the formation of a disulfide bridge/Ce(III) complex resulting from the interaction between Ce(IV) and the thiol groups, leading to metal unloading from the MTs, depending on their metal content and cluster type. The formation of stable Ce(3+) disulfide complexes has been demonstrated via their fluorescence properties. This work provides the first evidence of thiol concentration-dependent catalytic oxidation mechanisms between pristine CeO2 NPs and thiol-containing biomolecules.


Assuntos
Cério/química , Cisteína/química , Dissulfetos/química , Nanopartículas Metálicas/química , Compostos de Sulfidrila/química , Animais , Dicroísmo Circular , Concentração de Íons de Hidrogênio , Fígado/patologia , Oxirredução , Coelhos
14.
Inorg Chem ; 54(24): 11688-96, 2015 Dec 21.
Artigo em Inglês | MEDLINE | ID: mdl-26632864

RESUMO

Silver(I) is an unphysiological ion that, as the physiological copper(I) ion, shows high binding affinity for thiolate ligands; its toxicity has been proposed to be due to its capability to replace Cu(I) in the thiolate binding sites of proteins involved in copper homeostasis. Nevertheless, the nature of the Ag(I)-thiolate complexes formed within cells is poorly understood, and the details of Ag(I) coordination in such complexes in physiologically relevant conditions are mostly unknown. By making use of X-ray absorption spectroscopy (XAS), we characterized the Ag(I) binding sites in proteins related to copper homeostasis, such as the chaperone Atox1 and metallothioneins (MTs), as well as in bioinspired thiolate Cu(I) chelators mimicking these proteins, in solution and at physiological pH. Different Ag(I) coordination environments were revealed: the Ag-S bond length was found to correlate to the Ag(I) coordination number, with characteristic values of 2.40 and 2.49 Å in AgS2 and AgS3 sites, respectively, comparable to the values reported for crystalline Ag(I)-thiolate compounds. The bioinspired Cu(I) chelator L(1) is proven to promote the unusual trigonal AgS3 coordination and, therefore, can serve as a reference compound for this environment. In the Cu(I)-chaperone Atox1, Ag(I) binds in digonal coordination to the two Cys residues of the Cu(I) binding loop, with the AgS2 characteristic bond length of 2.40 ± 0.01 Å. In the multinuclear Ag(I) clusters of rabbit and yeast metallothionein, the average Ag-S bond lengths are 2.48 ± 0.01 Å and 2.47 ± 0.01 Å, respectively, both indicative of the predominance of trigonal AgS3 sites. This work lends insight into the coordination chemistry of silver in its most probable intracellular targets and might help in elucidating the mechanistic aspects of Ag(I) toxicity.


Assuntos
Cobre/química , Prata/química , Espectroscopia por Absorção de Raios X/métodos , Sítios de Ligação
15.
Anal Bioanal Chem ; 407(22): 6619-25, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26084548

RESUMO

After internal contamination, uranium rapidly distributes in the body; up to 20 % of the initial dose is retained in the skeleton, where it remains for years. Several studies suggest that uranium has a deleterious effect on the bone cell system, but little is known regarding the mechanisms leading to accumulation of uranium in bone tissue. We have performed synchrotron radiation-based micro-X-ray fluorescence (SR µ-XRF) studies to assess the initial distribution of uranium within cortical and trabecular bones in contaminated rats' femurs at the micrometer scale. This sensitive technique with high spatial resolution is the only method available that can be successfully applied, given the small amount of uranium in bone tissue. Uranium was found preferentially located in calcifying zones in exposed rats and rapidly accumulates in the endosteal and periosteal area of femoral metaphyses, in calcifying cartilage and in recently formed bone tissue along trabecular bone. Furthermore, specific localized areas with high accumulation of uranium were observed in regions identified as micro-vessels and on bone trabeculae. These observations are of high importance in the study of the accumulation of uranium in bone tissue, as the generally proposed passive chemical sorption on the surface of the inorganic part (apatite) of bone tissue cannot account for these results. Our study opens original perspectives in the field of exogenous metal bio-mineralization.


Assuntos
Fêmur/metabolismo , Exposição à Radiação/análise , Espectrometria por Raios X/métodos , Urânio/farmacocinética , Absorção de Radiação/fisiologia , Animais , Fêmur/química , Fêmur/citologia , Técnicas In Vitro , Masculino , Ratos , Ratos Sprague-Dawley , Distribuição Tecidual , Urânio/análise
16.
Chemistry ; 19(34): 11261-9, 2013 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-23824755

RESUMO

Herein, we describe the structural investigation of one possible uranyl binding site inside a nonstructured protein. This approach couples spectroscopy, thermodynamics, and theoretical calculations (DFT) and studies the interaction of uranyl ions with a phosphopeptide, thus mimicking a possible osteopontin (OPN) hydroxyapatite growth-inhibition site. Although thermodynamical aspects were investigated by using time-resolved laser fluorescence spectroscopy (TRLFS) and isothermal titration calorimetry (ITC), structural characterization was performed by extended X-ray absorption fine structure (EXAFS) at the U LIII -edge combined with attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy. From the vibrational and fluorescence spectra, several structural models of a UO2 (2+) /peptide complex were developed and subsequently refined by using theoretical calculations to fit the experimental EXAFS obtained. The structural effect of the pH value was also considered under acidic to moderately acidic conditions (pH 1.5-5.5). Most importantly, the uranyl/peptide coordination environment was similar to that of the native protein.


Assuntos
Osteopontina/química , Urânio/química , Durapatita/química , Íons/química , Modelos Moleculares , Osteopontina/metabolismo , Fosfopeptídeos/química , Fosforilação , Ligação Proteica , Espectroscopia de Infravermelho com Transformada de Fourier , Termodinâmica , Urânio/metabolismo
17.
Chem Res Toxicol ; 26(5): 645-53, 2013 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-23527557

RESUMO

Uranium is a natural actinide present as uranyl U(VI) species in aqueous environments. Its toxicity is considered to be chemical rather than radiotoxicological. Whatever the route of entry, uranyl reaches the blood, is partly eliminated via the kidneys, and accumulated in the bones. In serum, its speciation mainly involves carbonate and proteins. Direct identification of labile uranyl-protein complexes is extremely difficult because of the complexity of this matrix. Thus, until now the biodistribution of the metal in serum has not been described, and therefore, little is known about the metal transport mechanisms leading to bone accumulation. A rapid screening method based on a surface plasmon resonance (SPR) technique was used to determine the apparent affinities for U(VI) of the major serum proteins. A first biodistribution of uranyl was obtained by ranking the proteins according to the criteria of both their serum concentrations and affinities for this metal. Despite its moderate concentration in serum, fetuin-A (FETUA) was shown to exhibit an apparent affinity within the 30 nM range and to carry more than 80% of the metal. This protein involved in bone mineralization aroused interest in characterizing the U(VI) and FETUA interaction. Using complementary chromatographic and spectroscopic approaches, we demonstrated that the protein can bind 3 U(VI) at different binding sites exhibiting Kd from ∼30 nM to 10 µM. Some structural modifications and functional properties of FETUA upon uranyl complexation were also controlled. To our knowledge, this article presents the first identification of a uranyl carrier involved in bone metabolism along with the characterization of its metal binding sites.


Assuntos
Urânio/sangue , Urânio/metabolismo , alfa-2-Glicoproteína-HS/metabolismo , Animais , Sítios de Ligação , Osso e Ossos/metabolismo , Bovinos , Ressonância de Plasmônio de Superfície , Urânio/química , alfa-2-Glicoproteína-HS/química
18.
J Hazard Mater ; 446: 130668, 2023 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-36608581

RESUMO

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.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Urânio , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Compostos Férricos/metabolismo , Membrana Celular/metabolismo , Cátions/química , Cátions/metabolismo , Urânio/química , Proteínas de Ligação ao Cálcio/metabolismo
19.
Chem Res Toxicol ; 25(6): 1161-75, 2012 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-22458510

RESUMO

The skeleton is a target organ for most metals. This leads to their bioaccumulation, either as storage of useful oligoelements or as a protection against damage by toxic elements. The different events leading to their accumulation in this organ, under constant remodeling, are not fully understood, nor the full subsequent impact on bone metabolism. This lack of knowledge is particularly true for lanthanides and actinides, whose use has been increasing over recent decades. These metals, known as f-elements, present chemical similarities and differences. After a comparison of the biologically relevant physicochemical properties of lanthanides and actinides, and a brief reminder of the main events of bone metabolism, this review considers the results published over the past decade regarding the interaction between bones and f-elements. Emphasis will be given to the molecular events, which constitute the basis of the most recent toxicological studies in this domain but still need further investigation. Ionic exchanges with the inorganic matrix, interactions with bone proteins, and cellular mechanism disturbances are mainly considered in this review.


Assuntos
Elementos da Série Actinoide/toxicidade , Osso e Ossos/efeitos dos fármacos , Elementos da Série dos Lantanídeos/toxicidade , Elementos da Série Actinoide/química , Elementos da Série Actinoide/farmacocinética , Animais , Osso e Ossos/química , Osso e Ossos/metabolismo , Humanos , Elementos da Série dos Lantanídeos/química , Elementos da Série dos Lantanídeos/farmacocinética
20.
Neurotoxicology ; 82: 35-44, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33166614

RESUMO

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.


Assuntos
Neurônios Dopaminérgicos/metabolismo , Urânio/metabolismo , Linhagem Celular , Neurônios Dopaminérgicos/química , Humanos , Compostos Organometálicos/metabolismo , Espectrometria por Raios X , Síncrotrons , Urânio/análise
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