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 UO22+ 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 β‐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 UO22+ that is as large as that reported for osteopontin at physiological pH. The phosphate‐rich pS1368 can thus be considered a relevant model of UO22+ coordination in this intrinsically disordered protein, which wraps around the metal ion to gather four phosphate groups in the UO22+ coordination sphere. These model hyperphosphorylated peptides are highly selective for UO22+ with respect to endogenous Ca2+, which makes them good starting structures for selective UO22+ complexation.
Uranyl complexation: Hyperphosphorylated uranyl binding sites are proposed in target phosphoproteins such as osteopontin (see graphic, OPN), an intrinsically disordered protein that can wrap around the metal ion to gather several phosphate groups in its coordination sphere. A biomimetic approach with preorganized cyclic peptides demonstrates that four phosphoserine (pSer) residues properly oriented in a β‐sheet structure lead to an affinity for uranyl similar to that of the protein.
•Natural uranium is cytotoxic towards dopaminergic cells but only at high concentrations (> 125 μM) which are not relevant for the vast majority of human exposures.•Uranium is located in defined ...cytoplasmic regions suggesting its accumulation within organelles yet to be determined.•Among the dopamine-related genes investigated, monoamine oxidase B gene expression is decreased even at 10 μM uranium exposure, far from cytotoxicity threshold.
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.
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 K d 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.
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 UO22+/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.
A methodology that couples spectroscopy, thermodynamics, and theoretical calculations explores the interaction of uranyl ions with a phosphopeptide that mimics a possible osteopontin hydroxyapatite growth‐inhibition site for osteopontin. This target is a phosphorylated intrinsically disordered protein responsible for regulating bone growth (see scheme; EXAFS=extended X‐ray absorption fine structure, ITC=isothermal titration calorimetry, TRLFS=time‐resolved laser fluorescence spectroscopy).
Since the early 40s when the first research related to the development of the atomic bomb began for the Manhattan Project, actinides (An) and their association with the use of nuclear energy for ...civil applications, such as in the generation of electricity, have been a constant source of interest and fear. In 1962, the first Society of Toxicology (SOT), led by H. Hodge, was established at the University of Rochester (USA). It was commissioned as part of the Manhattan Project to assess the impact of nuclear weapons production on workers’ health. As a result of this initiative, the retention and excretion rates of radioactive heavy metals, their physiological impact in the event of acute exposure and their main biological targets were assessed. In this context, the scientific community began to focus on the role of proteins in the transportation and
accumulation of An. The first studies focused on the identification of these proteins. Thereafter, the continuous development of physico-chemical characterization techniques has made it possible to go further and specify the modes of interaction with proteins from both a thermodynamic and structural point of view, as well as from the point of view of their biological activity. This article reviews the work performed in this area since the Manhattan Project. It is divided into three parts: first, the identification of the most affine proteins; second, the study of the affinity and structure of protein-An complexes; and third, the impact of actinide ligation on protein conformation and function.
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
{
...U
(
VI
)
O
2
2
+
}
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.
The uranyl cation (UO22+) can be suspected to interfere with the binding of essential metal cations to proteins, underlying some mechanisms of toxicity. A dedicated computational screen was used to ...identify UO22+ binding sites within a set of nonredundant protein structures. The list of potential targets was compared to data from a small molecules interaction database to pinpoint specific examples where UO22+ should be able to bind in the vicinity of an essential cation, and would be likely to affect the function of the corresponding protein. The C‐reactive protein appeared as an interesting hit since its structure involves critical calcium ions in the binding of phosphorylcholine. Biochemical experiments confirmed the predicted binding site for UO22+ and it was demonstrated by surface plasmon resonance assays that UO22+ binding to CRP prevents the calcium‐mediated binding of phosphorylcholine. Strikingly, the apparent affinity of UO22+ for native CRP was almost 100‐fold higher than that of Ca2+. This result exemplifies in the case of CRP the capability of our computational tool to predict effective binding sites for UO22+ in proteins and is a first evidence of calcium substitution by the uranyl cation in a native protein.
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.
To improve our knowledge on protein targets of uranyl ion (UO
2
2+), we set up a proteomic strategy based on immobilized metal-affinity chromatography (IMAC). The successful enrichment of UO
2
...2+-interacting proteins from human kidney-2 (HK-2) soluble cell extracts was obtained using an ion-exchange chromatography followed by a dedicated IMAC process previously described and designed for the uranyl ion. By mass spectrometry analysis we identified 64 proteins displaying varied functions. The use of a computational screening algorithm along with the particular ligand-based properties of the UO
2
2+ ion allowed the analysis and categorization of the protein collection. This profitable approach demonstrated that most of these proteins fulfill criteria which could rationalize their binding to the UO
2
2+-loaded phase. The obtained results enable us to focus on some targets for more in-depth studies and open new insights on its toxicity mechanisms at molecular level.
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.
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