Methods for Analyzing the Impacts of Natural Uranium on <em>In Vitro</em> 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 ...

Biosciences and Biotechnologies Institute (BIAM)

About 20% of uranyl ions in serum are associated with the protein pool. A few of them such as transferrin have been characterized, but most still have to be identified to obtain a better explanation of the biochemical toxicology and kinetics of uranium. We designed an in vitro sensitive procedure involving a combination of bidimensional chromatography with time-resolved fluorescence, coupled with proteomic analysis, to identify uranium-binding proteins in human serum fractions. Ten novel targets were identified and validated using purified proteins and inductively coupled plasma mass spectrometry. Of these, ceruloplasmin, hemopexin, and two complement proteins displayed the capacity to bind uranium with stoichiometry greater than 1 mole of uranium per mole of protein. Not all of these targets are metalloproteins, suggesting that uranyl ions can use a wide variety of binding sites and coordination strategies. These data provide additional insights into a better understanding of uranium chemical toxicity.

Screening of human serum proteins for uranium binding.

It has been established that transferrin binds a variety of metals. These include toxic uranyl ions which form rather stable uranyl-transferrin derivatives. We determined the extent to which the iron binding sites might accommodate the peculiar topographic profile of the uranyl ion and the consequences of its binding on protein conformation. Indeed, metal intake via endocytosis of the transferrin/transferrin receptor depends on the adequate coordination of the metal in its site, which controls protein conformation and receptor binding. Using UV-vis and Fourier transform infrared difference spectroscopy coupled to a microdialysis system, we showed that at both metal binding sites two tyrosines are uranyl ligands, while histidine does not participate with its coordination sphere. Analysis by circular dichroism and differential scanning calorimetry (DSC) showed major differences between structural changes associated with interactions of iron or uranyl with apotransferrin. Uranyl coordination reduces the level of protein stabilization compared to iron, but this may be simply related to partial lobe closure. The lack of interaction between uranyl-TF and its receptor was shown by flow cytometry using Alexa 488-labeled holotransferrin. We propose a structural model summarizing our conclusion that the uranyl-TF complex adopts an open conformation that is not appropriate for optimal binding to the transferrin receptor.

Structural consequences of binding of UO2(2+) to apotransferrin: can this protein account for entry of uranium into human cells?

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.

Bone as target organ for metals: the case of f-elements.

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.

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

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.

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

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.

Claude Vidaud

CEA,
Direction de la Recherche Fondamentale (DRF),
CEA, Direction de la Recherche Fondamentale (DRF)

Biosciences and Biotechnologies Institute (BIAM)

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

Claude Vidaud

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}CEA,Direction de la Recherche Fondamentale (DRF),CEA, Direction de la Recherche Fondamentale (DRF)

Biosciences and Biotechnologies Institute (BIAM)

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

CEA,
Direction de la Recherche Fondamentale (DRF),
CEA, Direction de la Recherche Fondamentale (DRF)