Incorporation of uranium into a biomimetic apatite: physicochemical and biological aspects.

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.

Characterization of UO2(2+) binding to osteopontin, a highly phosphorylated protein: insights into potential mechanisms of uranyl accumulation in bones.

Bones are one of the few organs in which uranyl (UO2(2+)) accumulates. This large dioxo-cation displays affinity for carboxylates, phenolates and phosphorylated functional groups in proteins. The noncollagenous protein osteopontin (OPN) plays an important role in bone homeostasis. It is mainly found in the extracellular matrix of mineralized tissues but also in body fluids such as milk, blood and urine. Furthermore, OPN is an intrinsically disordered protein, which, like other proteins of the SIBLING family, contains a polyaspartic acid sequence and numerous patterns of alternating acidic and phosphorylated residues. All these properties led to the hypothesis that this protein could be prone to UO2(2+) binding. In this work, a simple purification procedure enabling highly purified bovine (bOPN) and human OPN (hOPN) to be obtained was developed. Various biophysical approaches were set up to study the impact of phosphorylations on the affinity of OPN for UO2(2+) as well as the formation of stable complexes originating from structural changes induced by the binding of this metal cation. The results obtained suggest a new mechanism of the interaction of UO2(2+) with bone metabolism and a new role for OPN as a metal transporter.

Alternate dipping preparation of biomimetic apatite layers in the presence of carbonate ions.

The classical simulated body fluids method cannot be employed to prepare biomimetic apatites encompassing metallic ions that lead to very stable phosphates. This is the case for heavy metals such as uranium, whose presence in bone mineral after contamination deserves toxicological study. We have demonstrated that existing methods, based on alternate dipping into calcium and phosphate ions solutions, can be adapted to achieve this aim. We have also especially studied the impact of the presence of carbonate ions in the medium as these are necessary to avoid hydrolysis of the contaminating metallic cations. Both the apatite-collagen complex method and a standard chemical (STD) method employing only mineral solutions lead to biomimetic apatites when calcium and carbonate ions are introduced simultaneously. The obtained materials were fully characterized and we established that the STD method tolerates the presence of carbonate ions much better, and this leads to homogeneous samples. Emphasis was set on the repeatability of the method to ensure the relevancy of further work performed on series of samples. Finally, osteoblasts cultured on these samples also proved a similar yield and standard-deviation in their adenosine triphosphate content when compared to commercially available substrates designed to study of such cell cultures.

Revision of the biodistribution of uranyl in serum: is fetuin-A the major protein target?

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.

Characterisation of the purified human sodium/iodide symporter reveals that the protein is mainly present in a dimeric form and permits the detailed study of a native C-terminal fragment.

The sodium/iodide symporter is an intrinsic membrane protein that actively transports iodide into thyroid follicular cells. It is a key element in thyroid hormone biosynthesis and in the radiotherapy of thyroid tumours and their metastases. Sodium/iodide symporter is a very hydrophobic protein that belongs to the family of sodium/solute symporters. As for many other membrane proteins, particularly mammalian ones, little is known about its biochemistry and structure. It is predicted to contain 13 transmembrane helices, with an N-terminus oriented extracellularly. The C-terminal, cytosolic domain contains approximately one hundred amino acid residues and bears most of the transporter's putative regulatory sites (phosphorylation, sumoylation, di-acide, di-leucine or PDZ-binding motifs). In this study, we report the establishment of eukaryotic cell lines stably expressing various human sodium/iodide symporter recombinant proteins, and the development of a purification protocol which allowed us to purify milligram quantities of the human transporter. The quaternary structure of membrane transporters is considered to be essential for their function and regulation. Here, the oligomeric state of human sodium/iodide symporter was analysed for the first time using purified protein, by size exclusion chromatography and light scattering spectroscopy, revealing that the protein exists mainly as a dimer which is stabilised by a disulfide bridge. In addition, the existence of a sodium/iodide symporter C-terminal fragment interacting with the protein was also highlighted. We have shown that this fragment exists in various species and cell types, and demonstrated that it contains the amino-acids [512-643] from the human sodium/iodide symporter protein and, therefore, the last predicted transmembrane helix. Expression of either the [1-512] truncated domain or the [512-643] domain alone, as well as co-expression of the two fragments, was performed, and revealed that co-expression of [1-512] with [512-643] allowed the reconstitution of a functional protein. These findings constitute an important step towards an understanding of some of the post-translational mechanisms that finely tune iodide accumulation through human sodium/iodide symporter regulation.

Surface plasmon resonance for rapid screening of uranyl affine proteins.

A sensitive immunoassay based on SPR analysis was developed to measure uranyl cation (UO(2)(2+)) affinity for any protein in a free state under physiological conditions. The technique involves immobilization of a specific monoclonal antibody (mAb) raised against UO(2)(2+) and 1,10-phenanthroline-2,9-dicarboxylic acid (DCP) used as a probe of UO(2)(2+) captured by the mAb. Calibration curves were established for accurate determination of UO(2)(2+) concentrations with a detection limit of 7 nM. The remaining free UO(2)(2+) could be accurately quantified from the different protein-metal equilibrium and a dose-response curve established for K(D) determination. This generic method was applied not only to proteins such as transferrin and albumin but also to small phosphonated ligands. Its robustness allows the fast UO(2)(2+) K(D) determination of any kind of macromolecules and small ligands using very few amount of compounds, thus opening new prospects in the field of uranium toxicity.

High-affinity uranyl-specific antibodies suitable for cellular imaging.

Monoclonal antibodies (mAbs) have proved to be valuable models for the study of protein-metal interactions, and previous reports have described very specific antibodies to chelated metal ions, including uranyl. We raised specific mAbs against UO2(2+)-DCP-BSA (DCP, 1,10-phenanthroline-2,9-dicarboxylic acid) to generate new sets of antibodies that might cross-react with various complexed forms of uranyl in different environments for further application in the field of toxicology. Using counter-screening with UO2(2+)-DCP-casein, we selected two highly specific mAbs against uranyl-DCP ( K D 10-100 pM): U04S and U08S. Competitive assays in the presence of different metal ions (UO2(2+), Fe (3+), Zn2+, Cu2+, and Ca2+) showed that uranyl in solution can act as a good competitor, suggesting some antibody ability to cross-react with chelating groups other than DCP in the UO2(2+) equatorial coordination plane. Interestingly, one of the antibodies could be used for revealing uranyl cations in cell samples. Fluorescence activated cell sorting analyses after immunolabeling revealed the interaction of uranyl with human kidney cells HK2. The intracellular accumulation of uranyl could be directly visualized by metal-immunostaining using fluorescent-labeled mAb. Our results suggest that U04S mAb epitopes mostly include the uranyl fraction and its paratopes can accommodate a wide variety of chelating groups.

Copper-mediated homo-dimerisation for the HAH1 metallochaperone.

The HAH1 metallochaperone is a key protein implicated in copper homeostasis in human cells. Using as solid-phase based assay completed with Biacore studies, we provided evidence that HAH1 forms homo-dimers in the presence of copper. Biacore analysis allowed us to determine the kinetic parameters of this interaction, characterised by an apparent affinity constant of 6muM. Moreover, we demonstrated that copper-loaded HAH1 interacts independently with each of the six individual metal-binding domains of the copper-translocating Menkes ATPase. Finally, the homo-dimerisation of the metallochaperone was confirmed in living cells by using fluorescence resonance energy transfer. Results have been discussed in the context of intracellular copper control.