Metallomics

The microenvironment (TME) undergoes significant modification in tumor progression. TME comprises locally secreted molecules, cells, and metal ions. Metals interact with the cell surface or are internalized, modulating downstream pathways related to adhesion, migration, and proliferation. Our group showed that tumor-bearing mice present altered Mn distribution. Mn-exposed tumor cells showed in vitro higher proliferation and migration, associated with changes in glycocalyx organization. In this work, we modulated, in vitro, tumor cells metallome, and evaluated metals internalization, cellular distribution and its early effect on migration. Cell viability and survival were evaluated using the MTT and clonogenic assays. Metal retention and distribution were assessed with inductively coupled plasma optical emission spectroscopy (ICP-OES) and high-resolution X-ray fluorescence (XRF). Cell migration was evaluated with wound healing assay. Our results indicate that an early interaction between Mn and the cell surface, probably the negatively-charged glycocalyx, induces morphological changes that lead to increased invasiveness. Future investigations will help to better understand the mechanisms of Mn retention and internalization during tumor progression.

COQ7 is a mitochondrial hydroxylase that catalyzes the penultimate step of the biosynthesis of coenzyme Q (CoQ; ubiquinone). CoQ is an obligate component of the mitochondrial electron transport chain and an antioxidant. CoQ deficiencies due to mutations in CoQ biosynthetic enzymes are severe genetic disorders often manifesting as mitochondrial disease syndrome. COQ7 is part of the relatively rare class of di-iron carboxylate enzymes, which carry out a wide range of reactions. In a previous study we described how COQ7 activity is inhibited in mammalian cells after treatment with iron chelating agents. Here, we report that manganese exposure of mouse cells leads to decreased COQ7 activity and resulting CoQ deficiency, which might participate in manganese toxicity. We find that the presence of cobalt can interfere with the inhibition of COQ7 by manganese. We present evidence that both manganese inhibition and cobalt interference are the result of metal exchange at the di-iron active site of COQ7. We present findings that suggest that 1) cobalt has greater affinity for the active site of COQ7 than both iron and manganese and, 2) that iron replacement by cobalt at the active site preserves catalytic activity.

Cadmium, being a highly toxic metal, perturbs cellular homeostasis by forming stable complexes with numerous thiol-active proteins, ultimately leading to severe liver and lung damage. Despite its well-documented toxicity, the molecular mechanisms governing cadmium export remain poorly understood. Given the chemical similarity between cadmium and copper, we investigated whether the canonical copper-exporting ATPases, ATP7A and ATP7B participate in cadmium handling. Upon Cd treatment in hepatocytes, ATP7B undergoes trafficking to lysosomes via the retromer complex, as also observed in the case of elevated copper, accompanied by the upregulation of acidic lysosomal populations. In contrast, ATP7A expressed in lung adenocarcinoma cells, though exhibit vesicular redistribution upon Cd exposure, does not mediate lysosomal sequestration, suggesting distinct deployment of late secretory pathways by the two copper ATPases in response to cadmium. We have also observed that ATP7B-/- hepatocytes exhibit increased sensitivity to Cd exposure compared to wild-type cells. Whereas, overexpressing the ATP7B amino-terminal copper-binding domain in bacteria alleviates cadmium-induced stress, indicating its capacity to sequester Cd. Caenorhabditis elegans lacking copper-ATPase cua-1, displayed increased Cd sensitivity, while mutants (glo-1-/-), deficient in lysosome-related organelles (LRO), and (lmp-1-/-), deficient in lysosomal membrane glycoprotein, showed reduced resistance to cadmium toxicity. Treatment of the worm with cadmium increases the abundance of lysosomes marked by elevation in lysosomal biogenesis and functional genes, reinforcing the importance of lysosomal pathways in cadmium detoxification. To summarise, we delineated the non-canonical role of copper ATPases and lysosomes in cadmium-induced cellular toxicity.

The concentration-dependent decrease in viable cells is a well-documented phenomenon in cytotoxicity assays for most toxic substances. We report that arsenite (As3+), a widely recognized oxidative toxicant, exhibited lower cytotoxic effects at 300 {micro}M compared to 100 {micro}M As3+ in CHO-K1 and Jurkat cells. Formation of stress granules (SGs), which appear in the cytoplasm shortly after exposure to hypertonicity, heat shock, and high concentrations of As3+ is considered as a pro-survival cellular event. We hypothesized that unusual cytotoxicity profile of As3+ could be attributed to SG formation. In both CHO-K1 and Jurkat cells stably expressing GFP-tagged G3BP1, SGs were more rapidly and distinctly induced by 300 {micro}M As3+ than 100 {micro}M As3+. Other toxic metals and a metalloid such as Cd2+, Cu2+, Ag+, and Se4+ did not clearly induce SG formation and instead reduced the viability in a concentration-dependent manner. Exposure to As3+ led to phosphorylation of eIF2, a key regulator of polysome stability and a hallmark of SG formation. Depletion of intracellular glutathione (GSH) increased the susceptibility of cells to As3+, highlighting its role in cellular defense mechanisms. Exposure to As3+ activated small ubiquitin-like modifier (SUMO) which is implicated in phase separation. However, neither depletion of GSH nor overexpression of SUMO contributed As3+-induced SG formation. Consistently, THP-1 and HL60 cells exposed to As3+ also exhibited non-canonical cytotoxic features, albeit at higher concentrations (1 mM). These findings underscore the need for further mechanistic investigations into As3+-induced SG formation, given that As3+ is a promising anti-cancer agent, and resistance of tumor cells to As3+ is a critical issue.

Zinc (Zn2+) is an essential metal in biology and its bioavailability is highly regulated. Many cell types exhibit fluctuations in Zn2+ that appear to play an important role in cellular function. However, the detailed molecular mechanisms by which Zn2+ dynamics influence cell physiology remain enigmatic. Here, we use a combination of fluorescent biosensors and cell perturbations to define how changes in intracellular Zn2+ impact kinase signaling pathways. By simultaneously monitoring Zn2+ dynamics and kinase activity in individual cells, we quantify changes in labile Zn2+ and directly correlate changes in Zn2+ with ERK and Akt activity. Under our experimental conditions, Zn2+ fluctuations are not toxic and do not activate stress-dependent kinase signaling. We demonstrate that while Zn2+ can non-specifically inhibit phosphatases leading to sustained kinase activation, ERK and Akt are predominantly activated via upstream signaling, and through a common node via Ras. We provide a framework for quantification of Zn2+ fluctuations and correlate these fluctuations with signaling events in single cells to shed light on the role that Zn2+ dynamics play in healthy cell signaling. Significance StatementWhile zinc (Zn2+) is a vital ion for cell function and human health, little is known about the role it plays in regulating cell signaling. Here, we use fluorescent tools to study the interaction between Zn2+ and cell signaling pathways that play a role in cell growth and proliferation. Importantly, we use small, non-toxic Zn2+concentrations to ensure that our Zn2+ changes are closer to what cells would experience in the body and not stress-inducing. We also demonstrate that these signaling changes are driven by Ras activation, which contradicts one of the major hypotheses in the field. Our sensors shed light on how cells respond to a very important micronutrient in real time.

The complex interplay between metal abundance, transport mechanisms, cell distribution, and tumor progression-related biological pathways (e.g., metabolism, collagen remodeling) remains poorly understood. Traditionally, genes and metals have been studied in isolation, limiting insights into their interactions. Recent advances in spatial transcriptomics and elemental profiling now enable comprehensive exploration of tissue-wide metal-gene interactions, though integration remains challenging. In this proof-of-concept study, we investigated metal-dependent signaling within the tumor microenvironment of a unique colorectal cancer (CRC) tumor. We implemented a spatial multimodal workflow which integrated elemental imaging, gene expression, cellular composition, and histopathological features to uncover metals-related pathways through spatially resolved differential expression analysis. Preliminary findings revealed significant associations, for instance: elevated iron correlated with mesenchymal phenotypes located at the tumors proliferative front, reflecting epithelial-to-mesenchymal transition pathways, and extracellular matrix remodeling. High concentrations of copper were predominantly localized in regions of active tumor growth and associated with the upregulation of immune response genes. This proof-of-concept workflow demonstrates the feasibility of integrating elemental imaging with spatial transcriptomics to identify metals-based gene correlates. Future application of this workflow to larger patient cohorts will pave the way for expansive comparisons across the metallome and transcriptome, ultimately identifying novel targets for tumor progression biomarkers and therapeutic interventions.

Cisplatin (CDDP), a widely used chemotherapy drug is imported in mammalian cells by copper transporter CTR1. We observed that CDDP cytotoxicity is significantly reduced in CTR1 deficient yeast and in copper loaded mammalian cells indicating its shared uptake pathway with copper. CTR1 resides on the plasma membrane undergoes endocytosis during CDDP import as in copper, involving its amino-terminal His-Met-motifs and the conserved 150MXXM154 pore motif. Unlike Copper, CDDP directs endocytosed CTR1 toward lysosomes rather than being sorted through VPS35-positive endosome. CDDP enhances lysosomal acidity and cathepsin activity. Furthermore, CDDP selectively downregulate cuproproteins, CCS and SOD1, disrupting copper homeostasis. This study highlights the unique aspects of copper versus CDDP uptake by CTR1 and its differential downstream ramification on lysosomes and cuproproteins.

We recently produced three web-based calculators that predict in vivo metal occupancies of proteins, based on the metal affinities of a protein of interest along with estimates of the availabilities of the labile buffered pools of metals inside a cell. Metal availabilities were calculated from the calibrated responses of DNA-binding, metal-sensing, transcriptional regulators. The availability of intracellular Fe(II) was estimated to be similar in E. coli grown under anaerobic conditions compared to cells grown aerobically in LB medium. The purpose of this article is to archive the background data that underpins the release of a new calculator for hyperaerated cells grown in flasks with baffles, with relatively low culture volumes plus high shaking speeds to give elevated oxygenation. The intracellular availability of Fe(II) calculated from the responses of the intracellular Fe(II) sensor Fur was estimated to be significantly lower in these hyperaerated cells than either of the previous values determined for anaerobic or aerobic cultures. The total number of atoms of Mn(II) per cell increased in hyperaerated cells albeit with only modest change in intracellular Mn(II) availability as estimated from the responses of the Mn(II) sensor MntR. Accurate determination of intracellular Ni(II) availability will require further calibration of the magnitude of the responses of the Ni(II) sensor NikR in hyperaerated cells to take account of the state of Fnr. The hyperaerated metalation calculator is made available online and as a spreadsheet, for use by others.

Iron overload, notably caused by hereditary hemochromatosis, is an excess storage of iron in various organs which cause tissue damage and may promote tumorigenesis. To manage that disorder, free iron depletion can be induced by iron chelators like deferoxamine which are gaining interest also in the cancer field since iron stock could be a potent target for managing tumorigenesis. Curcumin, a well-known active substance extracted from the turmeric rhizome, has shown to be destabilizing endoplasmic reticulum and secondarily lysozomes, increasing mitophagy/autophagy and subsequent apoptosis. Recent findings show that cells treated with curcumin exhibit also a decrease in ferritin, which is consistent with its chemical structure and iron chelating activity. Here we investigated how curcumin would play on the intracellular effects of iron overload via Fe-Nitriloacetic acid or Ferric ammonium citrate loading in Huh-7 cells and explore consequences in terms of antioxidant activity, autophagy, or apoptotic signal transduction. With T51B and RL-34 epithelial cells experiments, we brought evidence that curcumin-iron complexation abolishes both curcumin-induced autophagy and apoptosis together with the tumorigenic action of iron overload.

Non-thermal atmospheric pressure plasmas have an antiseptic activity beneficial in different medical applications. In a genome-wide screening, hydrogen peroxide and superoxide were identified as key species contributing to the antibacterial effects of plasma while [FeS] cluster proteins emerged as potential cellular targets. We investigated the impact of plasma treatment on [FeS] cluster homeostasis in Escherichia coli treated for 1 min with the effluent of a microscale atmospheric pressure plasma jet ({micro}APPJ). Mutants defective in [FeS] cluster synthesis and maintenance lacking the SufBC2D scaffold protein complex or desulfurase IscS were hypersensitive to plasma treatment. Monitoring the activity of [FeS] cluster proteins of the tricarboxylic acid cycle (aconitase, fumarase, succinate dehydrogenase) and malate dehydrogenase (no [FeS] clusters), we identified cysteine, iron, superoxide dismutase, and catalase as determinants of plasma sensitivity. Survival rates, enzyme activity, and restoration of enzyme activity after plasma treatment were superior in mutants with elevated cysteine levels and in the wildtype under iron replete conditions. Mutants with elevated hydrogen peroxide and superoxide detoxification capacity over-expressing sodA and katE showed full protection from plasma-induced enzyme inactivation and survival rates increased from 34% (controls) to 87%. Our study indicates that metabolic and genetic adaptation of bacteria may result in plasma tolerance and resistance, respectively. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/631878v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@119626eorg.highwire.dtl.DTLVardef@18d2973org.highwire.dtl.DTLVardef@9c5568org.highwire.dtl.DTLVardef@1ab4e58_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstractC_FLOATNO C_FIG HighlightsO_LIEnzymes with [FeS] clusters are rapidly inactivated by plasma C_LIO_LIClusters damaged by plasma are repaired in vivo using iron and cysteine C_LIO_LIOver-expression of sodA and katE completely prevents disruption of [FeS] clusters by plasma C_LIO_LIPlasma resistance is increased threefold by SodA and KatE over-production C_LIO_LIPre-adaptation of E. coli to O2- increases plasma tolerance C_LI

Many proteins bind transition metal ions as cofactors to carry out their biological functions. Despite binding affinities for divalent transition metal ions being predominantly dictated by the Irving-Williams series for wild-type proteins, in vivo metal ion binding specificity is ensured by intracellular mechanisms that regulate free metal ion concentrations. However, a growing area of biotechnology research considers the use of metal-binding proteins in vitro to purify specific metal ions from wastewater, where specificity is dictated by the proteins metal binding affinities. A goal of metalloprotein engineering is to modulate these affinities to improve a proteins specificity towards a particular metal; however, the quantitative relationship between the affinities and the equilibrium metal-bound protein fractions depends on the underlying binding kinetics. Here we demonstrate a high-throughput intrinsic tryptophan fluorescence quenching method to validate kinetic models in multi-metal solutions for CcNikZ-II, a nickel-binding protein from Clostridium carboxidivorans. Using our validated models, we quantify the relationship between binding affinity and specificity in different classes of metal-binding models for CcNikZ-II. We further demonstrate that principles for improving specificity through changes in binding affinity are qualitatively different depending on the competing metals, highlighting the power of mechanistic models to guide metalloprotein engineering targets.

With the rise of resistance to last resort antibiotics, metal-based antimicrobials have re-emerged as an alternative to prevent and manage infections. The group 11 metals (copper, silver, gold), historically known for their usage in coins and similar chemical properties, have demonstrated promising bactericidal activity. Despite their efficiency, we do not have a complete understanding for how bacteria are eradicated by metal ions and how they respond to metal-induced stress. One understudied aspect of these metal-bacteria interactions are prolonged exposure models, as other studies tend to focus on the acute toxic response of antimicrobial metals. We used RNA-seq profiling to understand the Escherichia coli physiological response to sublethal inhibitory antimicrobial coinage metal stress after 10 hours of incubation. Gene expression patterns of the adaptive and intrinsic response elicited by each metal were identified, including increased essential metal uptake (Ag, Cu, Au), cysteine biosynthesis (Cu, Au), change of the metal ion oxidation state (Cu, Au), efflux of metal stressor (Cu), protein translation and ribosome biogenesis (Au), and cell envelope stress response (Ag). In this paper, we highlight the remarkable differences and similarities in the transcriptomic response profile of E. coli to these antimicrobial metal elements. IMPORTANCEDogma has existed that all antimicrobial metals kill bacteria the same way, leading to the assumption that bacteria respond the same way to metal toxicity. Nowadays, we have a better understanding why some metal elements are more toxic than others, but questions remain in relation to how bacteria adapt to survive and thrive when challenged by different metal-based antimicrobials. Our study advances the field by characterizing the type of bacterial response(s) to acclimate and grow during a prolonged exposure of silver, copper and gold - metallic elements that are known for their antimicrobial activity. Taking advantage of well-characterized Escherichia coli, we propose a model that summarizes our findings after comparing the shared and unique responses to each of these metals. This information enhances our understanding of bacterial tolerance to metal-based antimicrobials, which can lead to improved drug development strategies as society continues to search for alternatives against antibiotic-resistant pathogens.

Here we provide the background data supporting an additional web-based calculator to predict in-cell metalation of proteins within E. coli BW25113 grown in M9 media. Intracellular metal availabilities have been estimated from the calibrated responses of DNA-binding, metal-sensing, transcriptional regulators. We have previously tested and validated metalation calculator predictions. They confirm that when proteins are expressed heterologously in E. coli in LB medium, they can become mismatched to metal availability and be mismetalated. Mg2+GTP-CobW from Rhodobacter is predicted to be correctly metalated with cobalt in E. coli grown in M9 media, although mismetalated with zinc in LB-grown heterologous cells. Similarly, MncA from Synechocystis PCC 6803 is predicted to be correctly metalated with manganese in E. coli grown in M9 media, although mismetalated with iron in LB-grown heterologous cells. The M9 metalation calculator is available online and as a spreadsheet for use in optimising metalation in engineering biology. An MncA-refined metalation calculator for idealised cells, reflecting the midpoint of intracellular metal availabilities, is also included and available online.

Colorectal cancer (CRC) is the second leading cause of cancer-related mortality in the United States. CRC tumors exhibit aberrant iron accumulation, which supports tumor cell proliferation through multiple metabolic pathways. However, the oncogenic benefits of elevated iron must be counterbalanced by its potential to catalyze oxidative damage via reactive oxygen species generated from labile, redox-active iron. Ferroptosis is a regulated, non-apoptotic form of cell death characterized by iron-dependent lipid peroxidation. This process is tightly controlled by the selenoenzyme glutathione peroxidase 4 (GPX4), which reduces lipid peroxides and can be pharmacologically inhibited by agents such as RSL3 and JKE1674. A key source of redox-active iron is the labile iron pool (LIP), yet its role in regulating ferroptosis remains incompletely defined. To examine this, we supplemented CRC cells with exogenous iron following pharmacologic induction of ferroptosis. Iron supplementation significantly reduced cell viability, suggesting that expansion of the LIP potentiates ferroptotic cell death. However, whether ferroptosis is accompanied by dynamic changes in the LIP, and if such changes are mechanistically required for its potentiation, were unknown. To further characterize this response, we profiled the expression of iron regulatory genes under ferroptotic conditions and observed no change in transcriptional response in iron homeostasis genes. Using a reactivity-based probe of labile iron, we found that the LIP did not measurably increase during ferroptosis induction with GPX4 inhibition or inhibition of the SLC7A11 cysteine/glutamate antiporter. These findings suggest that the LIP does not expand upon pharmacological initiated ferroptosis, despite the potentiating effect of exogenous iron supplementation.

Copper(I) is essential for all life forms. Though Cu(II) is most abundant state in environment, its reduction to Cu(I) is prerequisite for bio-utilization, by a mechanism that is uncharacterized. We show that in human Copper Transporter-1, two amino-terminal methionine-histidine clusters and neighbouring aspartates distinctly binds Cu(II) and Cu(I) preceding its import. The endocytosis of hCTR1 from basolateral membrane of polarized epithelia to Common-Recycling-Endosomes is dependent on copper reduction and Cu(I) coordination by methionines. The transient binding of both Cu(II) and Cu(I) during the reduction process facilitated by aspartates acts as another crucial determinant of hCTR1 endocytosis. Mutating 7Met-Gly-Met9 and Asp13 abrogates copper uptake and endocytosis that is correctable by reduced and non-reoxidizable Cu(I). Histidines clusters are crucial for hCTR1 functioning at limiting copper. Finally, we show that two N-terminal His-Met-Asp clusters exhibit functional complementarity in regulating Cu(I)-induced hCTR1 endocytosis. We propose a mechanistic model where His-Met-Asp residues of amino-terminal hCTR1 coordinates copper and maintains its reduced state crucial for uptake.

Any disturbances in cellular iron regulation can lead to dysfunctions and various diseases. However, our current understanding of iron dynamics in living cells is hampered by the lack of high-resolution iron reporter systems capable of monitoring the iron status of individual cells dynamically. To address this challenge, we have developed the Live-Cell Iron Mapping System (LIMS), which consists of two distinct genetically encoded fluorescent iron reporter systems: the rFeRepS and IronFist. The rFeRepS (ratiometric Iron Reporter Systems) code for two genetically encoded constructs that translate the iron status of individual cells into a ratiometric fluorescent signal. The ratio signal reflects the cellular iron status, based on endogenous iron-responsive proteins. A high rFeRepS ratio indicates ferritin-based cellular iron storage, while a low ratio implies transferrin receptor (TfR)-based active cellular iron uptake. The IronFist (Iron Fluorescent Indicators based on Stability and Translation) is derived from the hemerythrin-like domain (Hr) of the F-box and leucine-rich protein 5 (FXBL5), which undergoes a structural rearrangement upon binding to ferrous iron, preventing its ubiquitination and subsequent degradation. In the IronFist design, we fused either the blue or green fluorescent protein (FP) variant, mTagBFP2 or mNeonGreen, to the C-terminus of Hr and co-expressed mCherry via a ribosomal skipping sequence as a reference protein for normalization. Thus, IronFist dynamically translates fluctuations of the labile iron pool into a ratiometric fluorescent signal. We demonstrated the functionality of both iron reporter systems by treating cells with different iron formulations including iron carbohydrate nanoparticles. Our results show clear cell-to-cell variations in the response to high iron treatments.

Copper is an essential metalloelement that plays key fundamental roles in both health and pathology, and is increasingly been implicated in molecular pathogenesis of many cancer types. It has shown promise as a replacement to cisplatin in coordination complexes presently in mainstream chemotherapeutic practices. In this study, two newly synthesized water-soluble ternary copper (II) mixed ligand complexes; complex 1 - (Cu(4-mphen)(tyr)(H2O)]NO3{middle dot}2H2O)(C.1) and complex 2 - (Cu(5-mphen)(tyr)(H2O)]NO3{middle dot}2H2O (C.2) where (4-m= 4-methyl; 5-m = 5-methyl; phen-1, 10 = phenanthroline; tyr = tyrosine)), were investigated on adenocarcinomic human alveolar basal epithelial cell, A549 and non-cancerous human bronchial epithelial cell, BEAS-2B for their antiproliferative effects using the XTT assay (cytotoxicity), Comet assay (genotoxicity) and DCFH-DA assay (intracellular ROS) tests. C.1 was significantly more cytotoxic in A549 than C.2. Data from the Comet and ROS assay tests support each other. C.2 caused more copper-induced DNA damage, possibly through significant induction of ROS-mediated oxidative damage in the cancer cell, but a minimal insignificant ROS rise in normal cells. These results can only be preliminary and further studies are required to better understand the cellular effects and functional interactions of these agents, for an efficient therapeutic design and application.

ABSTRACTIron is the fundamental element for numerous physiological functions. Reduced ferrous (Fe2+) and oxidized ferric (Fe3+) are the two ionized iron states in the living organisms. In the cell membrane, divalent metal ion transporter 1 (DMT1) is responsible for cellular uptake of Fe2+, whereas transferrin receptors (TFR) carry transferrin (TF)-bound Fe3+. In this study we performed, for the first time, detailed analysis of the action of Fe ions on cytoplasmic free calcium ion concentration ([Ca2+]i) in astrocytes. Using qPCR and immunocytochemistry we identified DMT1 and TFR in astrocytes in primary cultures, in acutely isolated astrocytes and in brain tissue preparations; in situ both DMT1 and TFR are concentrated in astroglial perivascular endfeet. Administration of Fe2+ or Fe3+ in low μM concentrations evoked Ca2+ signals in astrocytes in vitro and in vivo. Iron ions triggered increase in [Ca2+]i by acting through two distinct molecular cascades. Uptake of Fe2+ by DMT1 inhibited astroglial Na+-K+-ATPase (NKA), which led to an elevation in cytoplasmic Na+ concentration (as measured by SBFI probe), thus reversing Na+/Ca2+ exchanger (NCX) thereby generating Ca2+ influx. Uptake of Fe3+ by TF-TFR stimulated phospholipase C to produce inositol 1,4,5-trisphosphate (InsP3), thus trigering InsP3 receptor-mediated Ca2+ release from the endoplasmic reticulum. Iron-induced Ca2+ signals promote astroglial release of arachidonic acid and prostaglandin E2 cytokines by activating cytosolic phospholipase A2 (cPLA2) and NF-κB signalling cascade. In summary, these findings reveal new mechanisms of iron-induced astrocytic signalling operational in conditions of iron overload, in response to which astrocytes actively accumulate excessive iron and activate neuroprotective pathways.Competing Interest StatementThe authors have declared no competing interest.View Full Text

Metals facilitate catalysis during cellular metabolism, but heterogeneity of metal content at single-cell level within and between cell populations is poorly characterized. This is important because deficiencies of biometals, for example iron, are enormously prevalent worldwide. Here we quantify metal content of single-cells using inductively-coupled plasma mass spectrometry. To develop the method, we used rhodium and iridium-intercalated Jurkat cells, obtaining >0.96% r2 cross-analytical correlation with mass cytometry. We quantified iron and calcium mass/cell for murine T-lymphocytes with 3% and 8% 2-sigma intra-precision, respectively, when assessing thousands of cells/minute. T-lymphocytes exposed to a 625-fold difference in extracellular iron concentrations maintained close iron homeostatic control, varying [~]20% in iron content. Nevertheless, this relatively small variation strongly correlated with changes in cellular activation characteristics measured by flow cytometry. We also assessed human B-cell iron content, which was [~]10-fold higher than murine T-lymphocytes. Overall, we demonstrate rapid iron quantification at single-cell level in different cell types and relate cellular iron content to cell function. TeaserPrecise and rapid iron metallomics of lymphocytes by single cell ICP-MS is a powerful approach for accessing signatures of immunological status.

Metal(loid) salts have been used to treat infectious diseases due to their exceptional biocidal properties at low concentrations. However, the mechanism of their toxicity has yet to be fully elucidated. The production of reactive oxygen species (ROS) has been linked to the toxicity of soft metal(loid)s such as Ag(I), Au(III), As(III), Cd(II), Hg(II), and Te(IV). Nevertheless, few reports have described the direct, or ROS-independent, effects of some of these soft-metal(loid)s on bacteria, including the dismantling of iron-sulphur clusters [4Fe-4S] and the accumulation of porphyrin IX. Here, we used genome-wide genetic, proteomic, and biochemical approaches under anaerobic conditions to evaluate the direct mechanisms of toxicity of these metal(loid)s in Escherichia coli. We found that certain soft-metal(loid)s promote protein aggregation in a ROS-independent manner. This aggregation occurs during translation in the presence of Ag(I), Au(III), Hg(II), or Te(IV) and post-translationally in cells exposed to Cd(II) or As(III). We determined that aggregated proteins were involved in several essential biological processes that could lead to cell death. For instance, several enzymes involved in amino acid biosynthesis were aggregated after soft-metal(loid) exposure, disrupting intracellular amino acid concentration. We also propose a possible mechanism to explain how soft-metal(loid)s act as proteotoxic agents.