Essential Transition Metal Research Articles

2D-DIGE as a proteomic biomarker discovery tool in environmental studies with Procambarus clarkii.

Metal content in digestive gland and gills of P. clarkii crayfish reflects their contents in sediments at sites of Doñana National Park and its surroundings. Accumulation of essential and toxic transition metals is paralleled by clear signs of oxidative stress to lipids and proteins and by significant deregulation of many proteins involved in protein folding, mitochondrial respiratory imbalance and inflammatory response. These results indicate that P. clarkii is an excellent bioindicator to be used in aquatic ecosystems quality monitoring. Additionally, results evidence that the anthropogenic activities carried out around Doñana National Park represent an extremely serious threat to this unique Biosphere Reserve and pose a risk to the environment and their inhabitants health. The identified deregulated proteins provide information about the metabolic pathways and/or physiological processes affected by pollutant-elicited oxidative stress, may also be useful as biomarkers of environmental pollution and have the added value of providing information about the molecular responses of this environmentally and economically interesting animal.

Copper chaperone Atox1 plays role in breast cancer cell migration

Copper (Cu) is an essential transition metal ion required as cofactor in many key enzymes. After cell uptake of Cu, the metal is transported by the cytoplasmic Cu chaperone Atox1 to P1B-type ATPases in the Golgi network for incorporation into Cu-dependent enzymes in the secretory path. Cu is vital for many steps of cancer progression and Atox1 was recently suggested to have additional functionality as a nuclear transcription factor. We here investigated the expression level, cellular localization and role in cell migration of Atox1 in an aggressive breast cancer cell line upon combining immunostaining, microscopy and a wound healing assay. We made the unexpected discovery that Atox1 accumulates at lamellipodia borders of migrating cancer cells and Atox1 silencing resulted in migration defects as evidenced from reduced wound closure. Therefore, we have discovered an unknown role of the Cu chaperone Atox1 in breast cancer cell migration.

Identification of Two Conserved Residues Involved in Copper Release from Chloroplast PIB-1-ATPases

Copper is an essential transition metal for living organisms. In the plant model Arabidopsis thaliana, half of the copper content is localized in the chloroplast, and as a cofactor of plastocyanin, copper is essential for photosynthesis. Within the chloroplast, copper delivery to plastocyanin involves two transporters of the PIB-1-ATPases subfamily: HMA6 at the chloroplast envelope and HMA8 in the thylakoid membranes. Both proteins are high affinity copper transporters but share distinct enzymatic properties. In the present work, the comparison of 140 sequences of PIB-1-ATPases revealed a conserved region unusually rich in histidine and cysteine residues in the TMA-L1 region of eukaryotic chloroplast copper ATPases. To evaluate the role of these residues, we mutated them in HMA6 and HMA8. Mutants of interest were selected from phenotypic tests in yeast and produced in Lactococcus lactis for further biochemical characterizations using phosphorylation assays from ATP and Pi Combining functional and structural data, we highlight the importance of the cysteine and the first histidine of the CX3HX2H motif in the process of copper release from HMA6 and HMA8 and propose a copper pathway through the membrane domain of these transporters. Finally, our work suggests a more general role of the histidine residue in the transport of copper by PIB-1-ATPases.

Essential Transition Metal Ion Complexation as a Strategy to Improve the Antimicrobial Activity of Organic Drugs

Essential Transition Metal Ion Complexation as a Strategy to Improve the Antimicrobial Activity of Organic Drugs

Conserved methionine dictates substrate preference in Nramp-family divalent metal transporters

Natural resistance-associated macrophage protein (Nramp) family transporters catalyze uptake of essential divalent transition metals like iron and manganese. To discriminate against abundant competitors, the Nramp metal-binding site should favor softer transition metals, which interact either covalently or ionically with coordinating molecules, over hard calcium and magnesium, which interact mainly ionically. The metal-binding site contains an unusual, but conserved, methionine, and its sulfur coordinates transition metal substrates, suggesting a vital role in their transport. Using a bacterial Nramp model system, we show that, surprisingly, this conserved methionine is dispensable for transport of the physiological manganese substrate and similar divalents iron and cobalt, with several small amino acid replacements still enabling robust uptake. Moreover, the methionine sulfur's presence makes the toxic metal cadmium a preferred substrate. However, a methionine-to-alanine substitution enables transport of calcium and magnesium. Thus, the putative evolutionary pressure to maintain the Nramp metal-binding methionine likely exists because it-more effectively than any other amino acid-increases selectivity for low-abundance transition metal transport in the presence of high-abundance divalents like calcium and magnesium.

Lipocalin 2 alleviates iron toxicity by facilitating hypoferremia of inflammation and limiting catalytic iron generation.

Iron is an essential transition metal ion for virtually all aerobic organisms, yet its dysregulation (iron overload or anemia) is a harbinger of many pathologic conditions. Hence, iron homeostasis is tightly regulated to prevent the generation of catalytic iron (CI) which can damage cellular biomolecules. In this study, we investigated the role of iron-binding/trafficking innate immune protein, lipocalin 2 (Lcn2, aka siderocalin) on iron and CI homeostasis using Lcn2 knockout (KO) mice and their WT littermates. Administration of iron either systemically or via dietary intake strikingly upregulated Lcn2 in the serum, urine, feces, and liver of WT mice. However, similarly-treated Lcn2KO mice displayed elevated CI, augmented lipid peroxidation and other indices of organ damage markers, implicating that Lcn2 responses may be protective against iron-induced toxicity. Herein, we also show a negative association between serum Lcn2 and CI in the murine model of dextran sodium sulfate (DSS)-induced colitis. The inability of DSS-treated Lcn2KO mice to elicit hypoferremic response to acute colitis, implicates the involvement of Lcn2 in iron homeostasis during inflammation. Using bone marrow chimeras, we further show that Lcn2 derived from both immune and non-immune cells participates in CI regulation. Remarkably, exogenous rec-Lcn2 supplementation suppressed CI levels in Lcn2KO serum and urine. Collectively, our results suggest that Lcn2 may facilitate hypoferremia, suppress CI generation and prevent iron-mediated adverse effects.

The biological chemistry of the transition metal "transportome" of Cupriavidus metallidurans.

This review tries to illuminate how the bacterium Cupriavidus metallidurans CH34 is able to allocate essential transition metal cations to their target proteins although these metals have similar charge-to-surface ratios and chemical features, exert toxic effects, compete with each other, and occur in the bacterial environment over a huge range of concentrations and speciations. Central to this ability is the "transportome", the totality of all interacting metal import and export systems, which, as an emergent feature, transforms the environmental metal content and speciation into the cellular metal mélange. In a kinetic flow equilibrium resulting from controlled uptake and efflux reactions, the periplasmic and cytoplasmic metal content is adjusted in a way that minimizes toxic effects. A central core function of the transportome is to shape the metal ion composition using high-rate and low-specificity reactions to avoid time and/or energy-requiring metal discrimination reactions. This core is augmented by metal-specific channels that may even deliver metals all the way from outside of the cell to the cytoplasm. This review begins with a description of the basic chemical features of transition metal cations and the biochemical consequences of these attributes, and which transition metals are available to C. metallidurans. It then illustrates how the environment influences the metal content and speciation, and how the transportome adjusts this metal content. It concludes with an outlook on the fate of metals in the cytoplasm. By generalization, insights coming from C. metallidurans shed light on multiple transition metal homoeostatic mechanisms in all kinds of bacteria including pathogenic species, where the "battle" for metals is an important part of the host-pathogen interaction.

Organic and inorganic mercurials have distinct effects on cellular thiols, metal homeostasis, and Fe-binding proteins in Escherichia coli.

The protean chemical properties of the toxic metal mercury (Hg) have made it attractive in diverse applications since antiquity. However, growing public concern has led to an international agreement to decrease its impact on health and the environment. During a recent proteomics study of acute Hg exposure in E. coli, we also examined the effects of inorganic and organic Hg compounds on thiol and metal homeostases. On brief exposure, lower concentrations of divalent inorganic mercury Hg(II) blocked bulk cellular thiols and protein-associated thiols more completely than higher concentrations of monovalent organomercurials, phenylmercuric acetate (PMA) and merthiolate (MT). Cells bound Hg(II) and PMA in excess of their available thiol ligands; X-ray absorption spectroscopy indicated nitrogens as likely additional ligands. The mercurials released protein-bound iron (Fe) more effectively than common organic oxidants and all disturbed the Na(+)/K(+) electrolyte balance, but none provoked efflux of six essential transition metals including Fe. PMA and MT made stable cysteine monothiol adducts in many Fe-binding proteins, but stable Hg(II) adducts were only seen in CysXxx(n)Cys peptides. We conclude that on acute exposure: (a) the distinct effects of mercurials on thiol and Fe homeostases reflected their different uptake and valences; (b) their similar effects on essential metal and electrolyte homeostases reflected the energy dependence of these processes; and (c) peptide phenylmercury-adducts were more stable or detectable in mass spectrometry than Hg(II)-adducts. These first in vivo observations in a well-defined model organism reveal differences upon acute exposure to inorganic and organic mercurials that may underlie their distinct toxicology.

Identification of mutations allowing Natural Resistance Associated Macrophage Proteins (NRAMP) to discriminate against cadmium.

Each essential transition metal plays a specific role in metabolic processes and has to be selectively transported. Living organisms need to discriminate between essential and non-essential metals such as cadmium (Cd(2+) ), which is highly toxic. However, transporters of the natural resistance-associated macrophage protein (NRAMP) family, which are involved in metal uptake and homeostasis, generally display poor selectivity towards divalent metal cations. In the present study we used a unique combination of yeast-based selection, electrophysiology on Xenopus oocytes and plant phenotyping to identify and characterize mutations that allow plant and mammalian NRAMP transporters to discriminate between their metal substrates. We took advantage of the increased Cd(2+) sensitivity of yeast expressing AtNRAMP4 to select mutations that decrease Cd(2+) sensitivity while maintaining the ability of AtNRAMP4 to transport Fe(2+) in a population of randomly mutagenized AtNRAMP4 cDNAs. The selection identified mutations in three residues. Among the selected mutations, several affect Zn(2+) transport, whereas only one, E401K, impairs Mn(2+) transport by AtNRAMP4. Introduction of the mutation F413I, located in a highly conserved domain, into the mammalian DMT1 transporter indicated that the importance of this residue in metal selectivity is conserved among NRAMP transporters from plant and animal kingdoms. Analyses of overexpressing plants showed that AtNRAMP4 affects the accumulation of metals in roots. Interestingly, the mutations selectively modify Cd(2+) and Zn(2+) accumulation without affecting Fe transport mediated by NRAMP4 in planta. This knowledge may be applicable for limiting Cd(2+) transport by other NRAMP transporters from animals or plants.

Unresolved questions in human copper pump mechanisms.

Copper (Cu) is an essential transition metal providing activity to key enzymes in the human body. To regulate the levels and avoid toxicity, cells have developed elaborate systems for loading these enzymes with Cu. Most Cu-dependent enzymes obtain the metal from the membrane-bound Cu pumps ATP7A/B in the Golgi network. ATP7A/B receives Cu from the cytoplasmic Cu chaperone Atox1 that acts as the cytoplasmic shuttle between the cell membrane Cu importer, Ctr1 and ATP7A/B. Biological, genetic and structural efforts have provided a tremendous amount of information for how the proteins in this pathway work. Nonetheless, basic mechanistic-biophysical questions (such as how and where ATP7A/B receives Cu, how ATP7A/B conformational changes and domain-domain interactions facilitate Cu movement through the membrane, and, finally, how target polypeptides are loaded with Cu in the Golgi) remain elusive. In this perspective, unresolved inquiries regarding ATP7A/B mechanism will be highlighted. The answers are important from a fundamental view, since mechanistic aspects may be common to other metal transport systems, and for medical purposes, since many diseases appear related to Cu transport dysregulation.

Phosphonate-functionalized three-dimensional gold nanocomposite as a sensitive interface for facile electrochemical stripping detection of trace copper(II) ions

In biological systems, specific status of the essential transition metal ions in body fluids is of clinical significance for sustaining normal, healthy individual. In this work, we demonstrate an efficient and controllable phosphonate-functionalized interface, self-assembled on the surface of three-dimensional nanoporous gold electrode, that responds to trace copper(II) ions in aqueous solution with appropriate sensitivity and stability, when combined with facile differential pulse anodic stripping voltammetry (DPASV), thus offering the opportunity to probe physiological and pathological condition of cellular metals with spatial fidelity. The structural morphology and electrochemical properties of the as-prepared phosphonate-functionalized three-dimensional (3D) gold electrodes were characterized by scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), dynamic contact angle analysis and cyclic voltammetry. The results indicated that, by use of cost-effective materials of short-chain MPPA (3-mercaptopropylphosphonic acid), the derived less densely packed self-assembled monolayers (SAMs), with evenly-spread terminal –PO3H2 functionality at intermediate surface coverage unlike its long-chain counterparts, exhibited great performance for trace analysis of heavy metal ions of copper(II) in aqueous environment. Incorporated with 3D gold substrate, possessing highly interconnected hierarchical porous structure beneficial for mass transport, a linear dynamic range from 0.05 to 350μM to the detection of copper ions, was obtained under the optimized conditions with a relatively low detection limits. The proposed sensing interface using phosphonic moieties as functional group holds promise to generally estimate metal accumulation, trafficking, and function or toxicity in living systems.

Host-imposed manganese starvation of invading pathogens: two routes to the same destination.

During infection invading pathogens must acquire all essential nutrients, including first row transition metals, from the host. To combat invaders, the host exploits this fact and restricts the availability of these nutrients using a defense mechanism known as nutritional immunity. While iron sequestration is the most well-known aspect of this defense, recent work has revealed that the host restricts the availability of other essential elements, notably manganese (Mn), during infection. Furthermore, these studies have revealed that the host utilizes multiple strategies that extend beyond metal sequestration to prevent bacteria from obtaining these metals. This review will discuss the mechanisms by which bacteria attempt to obtain the essential first row transition metal ion Mn during infection, and the approaches utilized by the host to prevent this occurrence. In addition, this review will discuss the impact of host-imposed Mn starvation on invading bacteria.

Optimization of triacetylfusarinine C and ferricrocin productions in Aspergillus fumigatus.

Iron is an essential element for all microorganisms. Bacteria and fungi produce versatile siderophores for binding and storing this essential transition metal when its availability is limited in the environment. The aim of the study was to optimize the fermentation medium of Aspergillus fumigatus for siderophore production. Triacetyl-fusarinine C and ferricrocin yields were dependent on glucose and glycine supplementations as well as the initial pH of the culture media. The optimal fermentation medium for triacetylfusarinine C production contained 8% glucose, 0.4% glycine and the initial pH was set to 5.9. Meanwhile, maximal ferricrocin yields were recorded in the presence of 10% glucose, 0.5% glycine and at an initial pH of 7.4. Under optimized fermentation conditions, the yields for triacetylfusarinine C and ferricrocin increased up to 2.9 g/l culture medium and 18.9 mg/g mycelium, respectively.

Toxicity of Copper on Isolated Liver Mitochondria: Impairment at Complexes I, II, and IV Leads to Increased ROS Production

Oxidative damage has been implicated in disorders associated with abnormal copper metabolism and also Cu(2+) overloading states. Besides, mitochondria are one of the most important targets for Cu(2+), an essential redox transition metal, induced hepatotoxicity. In this study, we aimed to investigate the mitochondrial toxicity mechanisms on isolated rat liver mitochondria. Rat liver mitochondria in both in vivo and in vitro experiments were obtained by differential ultracentrifugation and the isolated liver mitochondria were then incubated with different concentrations of Cu(2+). Our results showed that Cu(2+) induced a concentration and time-dependent rise in mitochondrial ROS formation, lipid peroxidation, and mitochondrial membrane potential collapse before mitochondrial swelling ensued. Increased disturbance in oxidative phosphorylation was also shown by decreased ATP concentration and decreased ATP/ADP ratio in Cu(2+)-treated isolated mitochondria. In addition, collapse of mitochondrial membrane potential (MMP), mitochondrial swelling, and release of cytochrome c following of Cu(2+) treatment were well inhibited by pretreatment of mitochondria with CsA and BHT. Our results showed that Cu(2+) could interact with respiratory complexes (I, II, and IV). This suggests that Cu(2+)-induced liver toxicity is the result of metal's disruptive effect on liver hepatocyte mitochondrial respiratory chain that is the obvious cause of Cu(2+)-induced ROS formation, lipid peroxidation, mitochondrial membrane potential decline, and cytochrome c expulsion which start cell death signaling.

Conformational switching in a copper‐sensing operon repressor (CsoR) (LB246)

Copper is an essential transition metal for living organisms but it is detrimental in excess. The metalloregulatory protein copper‐sensing operon repressor (CsoR) in bacteria has evolved to prevent cytoplasmic copper toxicity. Cu(I)‐binding to tetrameric CsoRs mediates transcriptional derepression of copper resistance genes but the mechanism is unknown. A phylogenetic analysis of 227 DUF156 protein members including biochemically or structurally characterized CsoR/RcnR repressors reveals that Geobacillus thermodenitrificans (Gt) CsoR characterized here is representative of CsoRs from pathogenic bacilli Listeria monocytogenes and Bacillus anthracis. The 2.56 Å structure of Cu(I)‐bound Gt CsoR reveals that Cu(I) binding induces a kink in the α2‐helix between two conserved copper‐ligating residues and folds an N‐terminal tail (residues 12‐19) over the Cu(I) binding site. NMR studies of Gt CsoR reveal that this tail is flexible in the apo‐state with these dynamics quenched upon Cu(I) binding. Small angle X‐ray scattering (SAXS) experiments on an N‐terminally truncated Gt CsoR (∆2‐10) reveal that the Cu(I)‐bound tetramer is hydrodynamically more compact than is the apo‐state. A mutational analysis of residues critical to N‐terminal tail folding reveals that these residues function to stabilize the apoprotein‐DNA complex and/or control the extent of allosteric negative regulation of DNA binding by Cu(I), but to varying degrees. The mechanism of Cu(I)‐mediated allosteric switching in CsoRs is discussed.Grant Funding Source: Supported by NIH grant GM042569

Experimental phasing using zinc and sulfur anomalous signals measured at the zinc absorption peak

Iron is an essential transition metal required for bacterial growth and survival. Excess free iron can lead to the generation of reactive oxygen species that can cause severe damage to cellular functions. Cells have developed iron-sensing regulators to maintain iron homeostasis at the transcription level. The ferric uptake regulator (Fur) is an iron-responsive regulator that controls the expression of genes involved in iron homeostasis, bacterial virulence, stress resistance, and redox metabolism. Here, we report the expression, purification, crystallization, and phasing of the apo-form of Bacillus subtilis Fur (BsFur) in the absence of regulatory metal ions. Crystals were obtained by microbatch crystallization method at 295 K and diffraction data at a resolution of 2.6 Å was collected at the zinc peak wavelength (λ=1.2823 Å). Experimental phasing identified the positions of one zinc atom and four sulfur atoms of cysteine residues coordinating the zinc atom, indicating that the data contained a meaningful anomalous scattering originating from the ordered zinc-coordinating sulfur atoms, in spite of the small anomalous signals of sulfur atoms at the examined wavelength.

In vivo zinc toxicity phenotypes provide a sensitized background that suggests zinc transport activities for most of the Drosophila Zip and ZnT genes

Members of the ZIP (SLC39A) and ZnT (SLC30A) families of transmembrane domain proteins are predicted to transport the essential transition metal zinc across membranes, regulating cellular zinc content and distribution via uptake and efflux at the outer plasma and organellar membranes. Twenty-four ZIP and ZnT proteins are encoded in mammalian genomes, raising questions of whether all actually transport zinc, whether several function together in the same tissues/cell types, and how the activity of these transporters is coordinated. To address these questions, we have taken advantage of the ability to manipulate several genes simultaneously in targeted cell types in Drosophila. Previously we reported zinc toxicity phenotypes caused by combining overexpression of a zinc uptake gene, dZip42C.1, with suppression of a zinc efflux gene, dZnT63C. Here we show that these phenotypes can be used as a sensitized in vivo system to detect subtle alterations in zinc transport activity that would be buffered in healthy cells. Using two adult tissues, the fly eye and midline (thorax/abdomen), we find that when overexpressed, most of the 17 Drosophila Zip and ZnT genes modify the zinc toxicity phenotypes in a manner consistent with their predicted zinc transport activity. In most cases, we can reconcile that activity with the cellular localization of an enhanced green fluorescent protein tagged version of the protein. Additionally, targeted suppression of each gene by RNA interference reveals several of the fly Zip and ZnT genes are required in the eye, indicating that numerous independent zinc transport genes are acting together in a single tissue.

Manganese acquisition and homeostasis at the host-pathogen interface.

Pathogenic bacteria acquire transition metals for cell viability and persistence of infection in competition with host nutritional defenses. The human host employs a variety of mechanisms to stress the invading pathogen with both cytotoxic metal ions and oxidative and nitrosative insults while withholding essential transition metals from the bacterium. For example, the S100 family protein calprotectin (CP) found in neutrophils is a calcium-activated chelator of extracellular Mn and Zn and is found in tissue abscesses at sites of infection by Staphylococcus aureus. In an adaptive response, bacteria have evolved systems to acquire the metals in the face of this competition while effluxing excess or toxic metals to maintain a bioavailability of transition metals that is consistent with a particular inorganic “fingerprint” under the prevailing conditions. This review highlights recent biological, chemical and structural studies focused on manganese (Mn) acquisition and homeostasis and connects this process to oxidative stress resistance and iron (Fe) availability that operates at the human host-pathogen interface.

Mutants impaired in vacuolar metal mobilization identify chloroplasts as a target for cadmium hypersensitivity in Arabidopsis thaliana

Cadmium (Cd) is highly toxic to plants causing growth reduction and chlorosis. It binds thiols and competes with essential transition metals. It affects major biochemical processes such as photosynthesis and the redox balance, but the connection between cadmium effects at the biochemical level and its deleterious effect on growth has seldom been established. In this study, two Cd hypersensitive mutants, cad1-3 impaired in phytochelatin synthase (PCS1), and nramp3nramp4 impaired in release of vacuolar metal stores, have been compared. The analysis combines genetics with measurements of photosynthetic and antioxidant functions. Loss of AtNRAMP3 and AtNRAMP4 function or of PCS1 function leads to comparable Cd sensitivity. Root Cd hypersensitivities conferred by cad1-3 and nramp3nramp4 are cumulative. The two mutants contrast in their tolerance to oxidative stress. In nramp3nramp4, the photosynthetic apparatus is severely affected by Cd, whereas it is much less affected in cad1-3. In agreement with chloroplast being a prime target for Cd toxicity in nramp3nramp4, the Cd hypersensitivity of this mutant is alleviated in the dark. The Cd hypersensitivity of nramp3nramp4 mutant highlights the critical role of vacuolar metal stores to supply essential metals to plastids and maintain photosynthetic function under Cd and oxidative stresses.

Effect of Dose-Response of Zinc and Manganese on Siderophores Production

Problem statement: This study was conducted to find and determine whether the siderophores of the four environmental Pseudomonas spp. isolates possess a sequestering activity towards essential transition metals (Zn and Mn) other than iron. Approach: Four fluorescent Pseudomonads isolated from various environments, were characterized analytically (Isoelectric focusing), biologically (pyoverdine-mediated uptake) and genetically (16S rDNA sequencing). By means of spectrophotometric measurements, it was possible to establish and compare the levels of pyoverdine production, in two different nutrient-poor media. Results: The strains were assigned, by sequencing, to P. fluorescens, P. aeruginosa, P. putida and P. mosselii isolated, respectively from soil, compost, sea water and waste water treatment plant. These bacterial strains were recognized as producing diver’s yellow-green siderophores types, when grown under conditions of iron starvation. The highest metabolite concentration was obtained with PsC132 and PsTp171 strains isolated respectively from compost and waste water treatment plant, in CAA medium. Strains grown in CAA medium exhibit a higher PVD level compared to SM medium. Mn (II) was found to promote pyoverdine biosynthesis, but rather, Zn (II) had no significant effect on siderophore production when compared to control medium. For both strains PsS29 and PsC132, the increase of iron concentration quenched siderophore production especially above 20 μM. Pyoverdine level declined with the high concentration of zinc but increased with Manganese concentration ranging up to 70 μM (in case of PsC132) and 300 μM (in case of PsS29). Conclusion/Recommendations: The ability of fluorescent Pseudomonas, isolated from wastewater treatment plant and from compost, to sequester zinc, point to a unique advantage of these species for divers bioremediation applications.