Severe quantitative loss of protein is often observed in high-resolution two-dimensional electrophoresis of membrane proteins, while the resolution is usually not affected. To improve the solubility ...of proteins in this technique, we tested denaturing cocktails containing various detergents and chaotropes. Best results were obtained with a denaturing solution containing urea, thiourea, and zwitterionic detergents, synthesized for this purpose. Among the dozen detergents synthesized and tested, amidosulfobetaines with an alkyl tail containing 14-16 carbons proved most efficient, solubilizing previously undetected membrane proteins.
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•Wilson’s disease requires novel therapies to improve patients’ quality of life.•A new lipophilic pro-chelator derived from an efficient Cu(I) chelator is obtained.•The pro-chelator ...protects hepatocytes against cellular copper stress.•The pro-chelator can be efficiently loaded into lipid nanovectors.•The nanolipid formulation is a promising therapeutic strategy for Wilson’s disease.
Copper homeostasis is finely regulated in human to avoid any detrimental impact of free intracellular copper ions. Upon copper accumulation, biliary excretion is triggered in liver thanks to trafficking of the ATP7B copper transporter to bile canaliculi. However, in Wilson’s disease this protein is mutated leading to copper accumulation. Current therapy uses Cu chelators acting extracellularly and requiring a life-long treatment with side effects. Herein, a new Cu(I) pro-chelator was encapsulated in long-term stable nanostructured lipid carriers. Cellular assays revealed that the pro-chelator protects hepatocytes against Cu-induced cell death. Besides, the cellular stresses induced by moderate copper concentrations, including protein unfolding, are counteracted by the pro-chelator. These data showed the pro-chelator efficiency to deliver intracellularly an active chelator that copes with copper stress and surpasses current and under development chelators. Although its biological activity is more mitigated, the pro-chelator nanolipid formulation led to promising results. This innovative approach is of outmost importance in the quest of better treatments for Wilson’s disease.
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•The increasing use of Ag nanoparticles is raising safety concerns for Human.•AgNPs are subject to various transformations before and after absorption in mammals.•A gap exists between ...in vitro and in vivo studies leading to discrepancies.•Cutting-edge imaging methods and speciation analysis provide clues for in vivo study.
Silver nanoparticles (AgNPs) are exponentially used in various consumer products including medical devices. This production leads to an increasing human exposure to silver in different forms. Indeed, AgNPs are subject to various transformations in aqueous aerobic conditions that trigger the production of Ag(I) species. The main environmental transformation produces the non-toxic species silver sulfide. Transformations occurring in mammals are more diverse and mainly depend on the interaction of AgNPs with thiol, chloride and proteins. Any of these species have a different impact on AgNPs and induces AgNP dissolution into Ag(I) species, aggregation and/or stabilization. The transformations occurring also depend on the exposure route. The main one is dietary but medical exposure is also growing with the massive use of nanosilver as biocide in medical devices. For the former, AgNP modifications and Ag distribution has been extensively studied using in vitro and in vivo models, while data related to medical use of nanosilver are scarce. However, most of the in vitro and in vivo data often remain inconsistent. In this review, we describe both in vitro, in cellulo and in vivo data about AgNP transformations, silver speciation and biodistribution. We try to reconcile all these data and describe the latest methods for the future studies of AgNP fate in mammals.
To identify proteins involved in cellular adaptive responses to zinc, a comparative proteome analysis between a previously developed high zinc- and cadmium-resistant human epithelial cell line (high ...zinc-resistant HeLa cells, HZR) and the parental HeLa cells has been carried out. Differentially produced proteins included cochaperones, proteins associated with oxido-reductase activities, and ubiquitin. Biochemical pathways to which these proteins belong were probed for their involvement in the resistance of both cell lines against cadmium toxicity. Among ER stressors, thapsigargin sensitized HZR cells, but not HeLa cells, to cadmium toxicity more acutely than tunicamycin, implying that these cells heavily relied on proper intracellular calcium distribution. The similar sensitivity of both HeLa and HZR cells to inhibitors of the proteasome, such as MG-132 or lactacystin, excluded improved proteasome activity as a mechanism associated with zinc adaptation of HZR cells. The enzyme 4-hydroxyphenylpyruvate dioxygenase (HPPD) was overproduced in HZR cells as compared to HeLa cells. It transforms HPP to homogentisate in the second step of tyrosine catabolism. Inhibition of HPPD decreased the resistance of HZR cells against cadmium, but not that of HeLa cells, suggesting that adaptation to zinc overload and increased HPP removal are linked in HZR cells.
From Secretome Analysis to Immunology Villiers, Christian; Chevallet, Mireille; Diemer, Hélène ...
Molecular & cellular proteomics,
June 2009, 20090601, 2009-06-00, Letnik:
8, Številka:
6
Journal Article
Recenzirano
Odprti dostop
Dendritic cells are known to be activated by a wide range of microbial products, leading to cytokine production and increased levels of membrane markers such as major histocompatibility complex class ...II molecules. Such activated dendritic cells possess the capacity to activate naïve T cells. In the present study we demonstrated that immature dendritic cells secrete both the YM1 lectin and lipocalin-2. By testing the ligands of these two proteins, chitosan and siderophores, respectively, we also demonstrated that chitosan, a degradation product of various fungal and protozoal cell walls, induces an activation of dendritic cells at the membrane level, as shown by the up-regulation of membrane proteins such as class II molecules, CD80 and CD86 via a TLR4-dependent mechanism, but is not able to induce cytokine production. This led to the production of activated dendritic cells unable to stimulate T cells. However, costimulation with other microbial products overcame this partial activation and restored the capacity of these activated dendritic cells to stimulate T cells. In addition, successive stimulation with chitosan and then by lipopolysaccharide induced a dose-dependent change in the cytokinic IL-12/IL-10 balance produced by the dendritic cells.
Copper oxide nanoparticles (CuO-NP) were studied for their toxicity and mechanism of action on hepatocytes (HepG2), in relation to Cu homeostasis disruption. Indeed, hepatocytes, in the liver, are ...responsible for the whole body Cu balance and should be a major line of defence in the case of exposure to CuO-NP. We investigated the early responses to sub-toxic doses of CuO-NP and compared them to equivalent doses of Cu added as salt to see if there is a specific nano-effect related to Cu homeostasis in hepatocytes. The expression of the genes encoding the Cu-ATPase ATP7B, metallothionein 1X, heme oxygenase 1, heat shock protein 70, superoxide dismutase 1, glutamate cysteine ligase modifier subunit, metal responsive element-binding transcription factor 1 and zinc transporter 1 was analyzed by qRT-PCR. These genes are known to be involved in response to Cu, Zn and/or oxidative stresses. Except for MTF1, ATP7B and SOD1, we clearly observed an up regulation of these genes expression in CuO-NP treated cells, as compared to CuCl2. In addition, ATP7B trafficking from the Golgi network to the bile canaliculus membrane was observed in WIF-B9 cells, showing a need for Cu detoxification. This shows an increase in the intracellular Cu concentration, probably due to Cu release from endosomal CuO-NP solubilisation. Our data show that CuO-NP enter hepatic cells, most probably by endocytosis, bypassing the cellular defence mechanism against Cu, thus acting as a Trojan horse. Altogether, this study suggests that sub-toxic CuO-NP treatments induce successively a Cu overload, a Cu-Zn exchange on metallothioneins and MTF1 regulation on both Cu and Zn homeostasis.
The use of nanomaterials is constantly increasing in electronics, cosmetics, food additives, and is emerging in advanced biomedical applications such as theranostics, bio-imaging and therapeutics. ...However their safety raises concerns and requires appropriate methods to analyze their fate in vivo.
In this review, we describe the current knowledge about the toxicity of labile metal (ZnO, CuO and Ag) nanoparticles (NPs) both at the organism and cellular levels, and describe the pathways that are triggered to maintain cellular homeostasis. We also describe advanced elemental imaging approaches to analyze intracellular NP fate. Finally, we open the discussion by presenting recent developments in terms of synthesis and applications of Ag and CuO NPs.
Labile metal nanoparticles (MeNPs) release metal ions that trigger a cellular response involving biomolecules binding to the ions followed by regulation of the redox balance. In addition, specific mechanisms are set up by the cell in response to physiological ions such as Cu(I) and Zn(II). Among all types of NPs, labile MeNPs induce the strongest inflammatory responses which are most probably due to the combined effects of the NPs and of its released ions. Interestingly, recent developments in imaging technologies enable the intracellular visualization of both the NPs and their ions and promise new insights into nanoparticle fate and toxicity.
The exponential use of nanotechnologies associated with the difficulties of assessing their impact on health and the environment has prompted scientists to develop novel methodologies to characterize these nanoobjects in a biological context.
•Labile metal nanoparticles release ions in biological media.•Labile metal NPs induce inflammation and metal homeostasis disruption.•CuO- and ZnO-NP exposure trigger physiological detoxifying mechanisms.•Synchrotron-based methods enable to follow the distribution of both ions and NPs.•Safer-by-design labile metal NPs are in progress for nanomedicine applications.
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Metals are essential for life and their concentration and distribution in organisms are tightly regulated. Indeed, in their free form, most transition metal ions are toxic. Therefore, ...an excess of physiologic metal ions or the uptake of non-physiologic metal ions can be highly detrimental to the organism. It is thus fundamental to understand metal distribution under physiological, pathological or environmental conditions, for instance in metal-related pathologies or upon environmental exposure to metals. Elemental imaging techniques can serve this purpose, by allowing the visualization and the quantification of metal species in tissues down to the level of cell organelles. Synchrotron radiation-based X-ray fluorescence (SR-XRF) microscopy is one of the most sensitive techniques to date, and great progress was made to reach nanoscale spatial resolution. Here we propose a correlative method to couple SR-XRF to electron microscopy (EM), with the possibility to quantify selected elemental contents in a specific organelle of interest with 50 × 50 nm2 raster scan resolution. We performed EM and SR-XRF on the same section of hepatocytes exposed to silver nanoparticles, in order to identify mitochondria through EM and visualize Ag co-localized with these organelles through SR-XRF. We demonstrate the accumulation of silver in mitochondria, which can reach a 10-fold higher silver concentration compared to the surrounding cytosol. The sample preparation and experimental setup can be adapted to other scientific questions, making the correlative use of SR-XRF and EM suitable to address a large panel of biological questions related to metal homeostasis.
Titanium dioxide nanoparticles (TiO
-NPs) are increasingly used in consumer products for their particular properties. Even though TiO
is considered chemically stable and insoluble, studying their ...behavior in biological environments is of great importance to figure their potential dissolution and transformation. The interaction between TiO
-NPs with different sizes and crystallographic forms (anatase and rutile) and the strong chelating enterobactin (
) siderophore was investigated to look at a possible dissolution. For the first time, direct evidence of anatase TiO
-NP surface dissolution or solubilization (i.e., the removal of Ti atoms located at the surface) in a biological medium by this siderophore was shown and the progressive formation of a hexacoordinated titanium-enterobactin (Ti-
) complex observed. This complex was characterized by UV-visible and Fourier transform infrared (FTIR) spectroscopy (both supported by Density Functional Theory calculations) as well as electrospray ionization mass spectrometry (ESI-MS) and X-ray photoelectron spectroscopy (XPS). A maximum of ca. 6.3% of Ti surface atoms were found to be solubilized after 24 h of incubation, releasing Ti-
complexes in the micromolar range that could then be taken up by bacteria in an iron-depleted medium. From a health and environmental point of view, the effects associated to the solubilization of the E171 TiO
food additive in the presence of enterobactin and the entrance of the Ti-enterobactin complex in bacteria were questioned.
Titanium dioxide nanoparticles (TiOsub.2-NPs) are increasingly used in consumer products for their particular properties. Even though TiOsub.2 is considered chemically stable and insoluble, studying ...their behavior in biological environments is of great importance to figure their potential dissolution and transformation. The interaction between TiOsub.2-NPs with different sizes and crystallographic forms (anatase and rutile) and the strong chelating enterobactin (ent) siderophore was investigated to look at a possible dissolution. For the first time, direct evidence of anatase TiOsub.2-NP surface dissolution or solubilization (i.e., the removal of Ti atoms located at the surface) in a biological medium by this siderophore was shown and the progressive formation of a hexacoordinated titanium–enterobactin (Ti–ent) complex observed. This complex was characterized by UV–visible and Fourier transform infrared (FTIR) spectroscopy (both supported by Density Functional Theory calculations) as well as electrospray ionization mass spectrometry (ESI-MS) and X-ray photoelectron spectroscopy (XPS). A maximum of ca. 6.3% of Ti surface atoms were found to be solubilized after 24 h of incubation, releasing Ti–ent complexes in the micromolar range that could then be taken up by bacteria in an iron-depleted medium. From a health and environmental point of view, the effects associated to the solubilization of the E171 TiOsub.2 food additive in the presence of enterobactin and the entrance of the Ti–enterobactin complex in bacteria were questioned.
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