Regulation Of Metal Homeostasis Research Articles

Association of Metal Homeostasis and (p)ppGpp Regulation in the Pathophysiology of Enterococcus faecalis.

In Enterococcus faecalis, the regulatory nucleotides pppGpp and ppGpp, collectively, (p)ppGpp, are required for growth in blood, survival within macrophages, and virulence. However, a clear understanding of how (p)ppGpp promotes virulence in E. faecalis and other bacterial pathogens is still lacking. In the host, the essential transition metals iron (Fe) and manganese (Mn) are not readily available to invading pathogens because of a host-driven process called nutritional immunity. Considering its central role in adaptation to nutritional stresses, we hypothesized that (p)ppGpp mediates E. faecalis virulence through regulation of metal homeostasis. Indeed, supplementation of serum with either Fe or Mn restored growth and survival of the Δrel ΔrelQ [(p)ppGpp0] strain to wild-type levels. Using a chemically defined medium, we found that (p)ppGpp accumulates in response to either Fe depletion or Mn depletion and that the (p)ppGpp0 strain has a strong growth requirement for Mn that is alleviated by Fe supplementation. Although inactivation of the nutrient-sensing regulator codY restored some phenotypes of the (p)ppGpp0 strain, transcriptional analysis showed that the (p)ppGpp/CodY network does not promote transcription of known metal transporters. Interestingly, physiologic and enzymatic investigations suggest that the (p)ppGpp0 strain requires higher levels of Mn in order to cope with high levels of endogenously produced reactive oxygen species (ROS). Because (p)ppGpp mediates antibiotic persistence and virulence in several bacteria, our findings have broad implications and provide new leads for the development of novel therapeutic and preventive strategies against E. faecalis and beyond.

Adverse effects of parental zinc deficiency on metal homeostasis and embryonic development in a zebrafish model

The high prevalence of zinc deficiency is a global public health concern, and suboptimal maternal zinc consumption has been associated with adverse effects ranging from impaired glucose tolerance to low birthweights. The mechanisms that contribute to altered development and poor health in zinc deficient offspring are not completely understood. To address this gap, we utilized the Danio rerio model and investigated the impact of dietary zinc deficiency on adults and their developing progeny. Zinc deficient adult fish were significantly smaller in size, and had decreases in learning and fitness. We hypothesized that parental zinc deficiency would have an impact on their offspring's mineral homeostasis and embryonic development. Results from mineral analysis showed that parental zinc deficiency caused their progeny to be zinc deficient. Furthermore, parental dietary zinc deficiency had adverse consequences for their offspring including a significant increase in mortality and decreased physical activity. Zinc deficient embryos had altered expression of genes that regulate metal homeostasis including several zinc transporters (ZnT8, ZnT9) and the metal-regulatory transcription factor 1 (MTF-1). Zinc deficiency was also associated with decreased expression of genes related to diabetes and pancreatic development in the embryo (Insa, Pax4, Pdx1). Decreased expression of DNA methyltransferases (Dnmt4, Dnmt6) was also found in zinc deficient offspring, which suggests that zinc deficiency in parents may cause altered epigenetic profiles for their progeny. These data should inform future studies regarding zinc deficiency and pregnancy and suggest that supplementation of zinc deficient mothers prior to pregnancy may be beneficial.

Imaging metals in Caenorhabditis elegans.

Systemic trafficking and storage of essential metal ions play fundamental roles in living organisms by serving as essential cofactors in various cellular processes. Thereby metal quantification and localization are critical steps in understanding metal homeostasis, and how their dyshomeostasis might contribute to disease etiology and the ensuing pathologies. Furthermore, the amount and distribution of metals in organisms can provide insight into their underlying mechanisms of toxicity and toxicokinetics. While in vivo studies on metal imaging in mammalian experimental animals are complex, time- and resource-consuming, the nematode Caenorhabditis elegans (C. elegans) provides a suitable comparative and complementary model system. Expressing homologous genes to those inherent to mammals, including those that regulate metal homeostasis and transport, C. elegans has become a powerful tool to study metal homeostasis and toxicity. A number of recent technical advances have been made in the development and application of analytical methods to visualize metal ions in C. elegans. Here, we briefly summarize key findings and challenges of the three main techniques and their application to the nematode, namely sensing fluorophores, microbeam synchrotron radiation X-ray fluorescence as well as laser ablation (LA) coupled to inductively coupled plasma-mass spectrometry (ICP-MS).

AdcAII of Streptococcus pneumoniae Affects Pneumococcal Invasiveness.

Across bacterial species, metal binding proteins can serve functions in pathogenesis in addition to regulating metal homeostasis. We have compared and contrasted the activities of zinc (Zn2+)-binding lipoproteins AdcA and AdcAII in the Streptococcus pneumoniae TIGR4 background. Exposure to Zn2+-limiting conditions resulted in delayed growth in a strain lacking AdcAII (ΔAdcAII) when compared to wild type bacteria or a mutant lacking AdcA (ΔAdcA). AdcAII failed to interact with the extracellular matrix protein laminin despite homology to laminin-binding proteins of related streptococci. Deletion of AdcA or AdcAII led to significantly increased invasion of A549 human lung epithelial cells and a trend toward increased invasion in vivo. Loss of AdcAII, but not AdcA, was shown to negatively impact early colonization of the nasopharynx. Our findings suggest that expression of AdcAII affects invasiveness of S. pneumoniae in response to available Zn2+ concentrations.

Regulation of Copper and Iron Homeostasis by Metal Chelators: A Possible Chemotherapy for Alzheimer’s Disease

With the increase of life expectancy of humans in more than two-thirds of the countries in the World, aging diseases are becoming the frontline health problems. Alzheimer's disease (AD) is now one of the major challenges in drug discovery, since, with the exception of memantine in 2003, all clinical trials with drug candidates failed over the past decade. If we consider that the loss of neurons is due to a high level of oxidative stress produced by nonregulated redox active metal ions like copper linked to amyloids of different sizes, regulation of metal homeostasis is a key target. The difficulty for large copper-carrier proteins to directly extract copper ions from metalated amyloids might be considered as being at the origin of the rupture of the copper homeostasis regulation in AD brains. So, there is an urgent need for new specific metal chelators that should be able to regulate the homeostasis of metal ions, specially copper and iron, in AD brains. As a consequence of that concept, chelators promoting metal excretion from brain are not desired. One should favor ligands able to extract copper ions from sinks (amyloids being the major one) and to transfer these redox-active metal ions to copper-carrier proteins or copper-containing enzymes. Obviously, the affinity of these chelators for the metal ion should not be a sufficient criterion, but the metal specificity and the ability of the chelators to release the metal under specific biological conditions should be considered. Such an approach is still largely unexplored. The requirements for the chelators are very high (ability to cross the brain-blood barrier, lack of toxicity, etc.), few chemical series were proposed, and, among them, biochemical or biological data are scarce. As a matter of fact, the bioinorganic pharmacology of AD represents less than 1% of all articles dedicated to AD drug research. The major part of these articles deals with an old and rather toxic drug, clioquinol and related analogs, that do not efficiently extract copper from soluble amyloids. We have designed and developed new tetradendate ligands such as 21 and PA1637 based on bis(8-aminoquinolines) that are specific for copper chelation and are able to extract copper(II) from amyloids and then can release copper ion upon reduction with a biological reducing agent. These studies contribute to the understanding of the physicochemical properties of the tetradentate copper ligands compared with bidentate ligands like clioquinol. One of these copper ligands, PA1637, after selection with a nontransgenic mouse model that is able to efficiently monitor the loss of episodic memory, is currently under preclinical development.

How do heavy metal ATPases contribute to hyperaccumulation?

AbstractPlants contain metal chelators and transporters that regulate metal homeostasis. The HMA3 and HMA4 genes encode proteins belonging to the PIB‐type. Recently, some studies on the characterization of the HMA2 gene from several plants for potential application in phytoremediation have been reported. This ATPase family of integral membrane transporter proteins that transport transition metals are probably involved in mediating metal‐resistant and metal‐hyperaccumulating traits. While HMA3 may contribute to metal detoxification by sequestering Cd into the vacuole, HMA4 acts as a physiological master switch during the process of metal hyperaccumulation, and HMA2 and HMA4 play roles in root to shoot metal translocation. The focus of this review is the discussion on the different functions of HMA3, HMA4, and HMA2 in various plant species. It is hypothesized that a refined distinction and description of the roles of several types of these metal transporters in plants will be essential for the development of schemes to genetically modify plants that accumulate specific metals, with subsequent use in either phytoremediation or in improving human nutrition. Enhanced expression of HMA3 and HMA4 is a prerequisite for hyperaccumulation and hyperresistance in hyperaccumulators. Through genetic engineering, these genes are able to generate nontoxic, metal‐free crops to produce safe food.

Trafficking of the Menkes copper transporter ATP7A is regulated by clathrin-, AP-2–, AP-1–, and Rab22-dependent steps

The transporter ATP7A mediates systemic copper absorption and provides cuproenzymes in the trans-Golgi network (TGN) with copper. To regulate metal homeostasis, ATP7A constitutively cycles between the TGN and plasma membrane (PM). ATP7A trafficking to the PM is elevated in response to increased copper load and is reversed when copper concentrations are lowered. Molecular mechanisms underlying this trafficking are poorly understood. We assess the role of clathrin, adaptor complexes, lipid rafts, and Rab22a in an attempt to decipher the regulatory proteins involved in ATP7A cycling. While RNA interference (RNAi)-mediated depletion of caveolin 1/2 or flotillin had no effect on ATP7A localization, clathrin heavy chain depletion or expression of AP180 dominant-negative mutant not only disrupted clathrin-regulated pathways, but also blocked PM-to-TGN internalization of ATP7A. Depletion of the μ subunits of either adaptor protein-2 (AP-2) or AP-1 using RNAi further provides evidence that both clathrin adaptors are important for trafficking of ATP7A from the PM to the TGN. Expression of the GTP-locked Rab22aQ64L mutant caused fragmentation of TGN membrane domains enriched for ATP7A. These appear to be a subdomain of the mammalian TGN, showing only partial overlap with the TGN marker golgin-97. Of importance, ATP7A remained in the Rab22aQ64L-generated structures after copper treatment and washout, suggesting that forward trafficking out of this compartment was blocked. This study provides evidence that multiple membrane-associated factors, including clathrin, AP-2, AP-1, and Rab22, are regulators of ATP7A trafficking.

Iron, copper, zinc, and manganese transport and regulation in pathogenic Enterobacteria: correlations between strains, site of infection and the relative importance of the different metal transport systems for virulence

For all microorganisms, acquisition of metal ions is essential for survival in the environment or in their infected host. Metal ions are required in many biological processes as components of metalloproteins and serve as cofactors or structural elements for enzymes. However, it is critical for bacteria to ensure that metal uptake and availability is in accordance with physiological needs, as an imbalance in bacterial metal homeostasis is deleterious. Indeed, host defense strategies against infection either consist of metal starvation by sequestration or toxicity by the highly concentrated release of metals. To overcome these host strategies, bacteria employ a variety of metal uptake and export systems and finely regulate metal homeostasis by numerous transcriptional regulators, allowing them to adapt to changing environmental conditions. As a consequence, iron, zinc, manganese, and copper uptake systems significantly contribute to the virulence of many pathogenic bacteria. However, during the course of our experiments on the role of iron and manganese transporters in extraintestinal Escherichia coli (ExPEC) virulence, we observed that depending on the strain tested, the importance of tested systems in virulence may be different. This could be due to the different set of systems present in these strains, but literature also suggests that as each pathogen must adapt to the particular microenvironment of its site of infection, the role of each acquisition system in virulence can differ from a particular strain to another. In this review, we present the systems involved in metal transport by Enterobacteria and the main regulators responsible for their controlled expression. We also discuss the relative role of these systems depending on the pathogen and the tissues they infect.

Compensatory Mutations in Predicted Metal Transporters Modulate Auxin Conjugate Responsiveness in Arabidopsis

Levels of the phytohormone indole-3-acetic acid (IAA) can be altered by the formation and hydrolysis of IAA conjugates. The isolation and characterization of Arabidopsis thaliana mutants with reduced IAA-conjugate sensitivity and wild-type IAA responses is advancing the understanding of auxin homeostasis by uncovering the factors needed for conjugate metabolism. For example, the discovery that the IAA-Ala-resistant mutant iar1 is defective in a protein in the ZIP family of metal transporters uncovered a link between metal homeostasis and IAA-conjugate sensitivity. To uncover additional factors impacting auxin conjugate metabolism, we conducted a genetic modifier screen and isolated extragenic mutations that restored IAA-amino acid conjugate sensitivity to the iar1 mutant. One of these suppressor mutants is defective in a putative cation diffusion facilitator, MTP5 (At3g12100; formerly known as MTPc2). Loss of MTP5 function restored IAA conjugate sensitivity to iar1 but not to mutants defective in IAA-amino acid conjugate amidohydrolases. Our results are consistent with a model in which MTP5 and IAR1 transport metals in an antagonistic fashion to regulate metal homeostasis within the subcellular compartment in which the IAA-conjugate amidohydrolases reside, and support previous suggestions that the ion composition in this compartment influences hydrolase activity.

Handling of Iron Oxide and Silver Nanoparticles by Astrocytes

Metal-containing nanoparticles (NPs) are currently used for various biomedical applications. Since such NPs are able to enter the brain, the cells of this organ have to deal with NPs and with NP-derived metal ions. In brain, astrocytes are considered to play a key function in regulating metal homeostasis and in protecting other brain cells against metal toxicity. Thus, among the different types of brain cells, especially astrocytes are of interest regarding the uptake and the handling of metal-containing NPs. This article summarizes the current knowledge on the consequences of an exposure of astrocytes to NPs. Special focus will be given to magnetic iron oxide nanoparticles (IONPs) and silver nanoparticles (AgNPs), since the biocompatibility of these NPs has been studied for astrocytes in detail. Cultured astrocytes efficiently accumulate IONPs and AgNPs in a time-, concentration- and temperature-dependent manner by endocytotic processes. Astrocytes are neither acutely damaged by the exposure to high concentrations of NPs nor by the prolonged intracellular presence of large amounts of accumulated NPs. Although metal ions are liberated from accumulated NPs, NP-derived iron and silver ions are not exported from astrocytes but are rather stored in proteins such as ferritin and metallothioneins which are synthesized in NP-treated astrocytes. The efficient accumulation of large amounts of metal-containing NPs and the upregulation of proteins that safely store NP-derived metal ions suggest that astrocytes protect the brain against the potential toxicity of metal-containing NPs.

BjHO-1 is involved in the detoxification of heavy metal in India mustard (Brassica juncea)

Heme oxygenase-1 (HO-1) is a stress-responsive gene coding for an enzyme catalyzing the catabolism of heme to yield biliverdin IXα, carbon monoxide (CO) and iron. However, its biological role in regulating metal homeostasis, particularly the tolerance to toxic heavy metals is poorly understood. In this study, a novel gene encoding a Brassica juncea heme oxygenase-1 (designated as BjHO-1) was cloned and functionally identified. Spatial expression of BjHO-1 showed that it was differentially expressed in cotyledon, hypocotyl, leaf and root. BjHO-1 was found to be induced significantly by heavy metal Hg. To understand whether BjHO-1 is able to regulate plant tolerance to Hg, we constructed transgenic B. juncea plants overexpressing HO-1, and showed that 35S::BjHO1 plants confer the plant resistance to Hg toxicity by improving plant dry mass, reducing Hg accumulation, and attenuating Hg-induced oxidative stress. We further cloned a 1,099 bp promoter sequence upstream of BjHO-1 using genome walking approach. Multiple stress-responsive elements were detected in the BjHO-1 promoter regions. The promoter can be activated by Zn, Cd, Hg and Pb exposure. Our results indicate that up-regulation of BjHO-1 is beneficial for limiting the uptake or accumulation of heavy metals into plants. This work also provides a new example for molecular breeding designed for plants that do not accumulate or minimizing accumulation of toxic trace metals growing on heavy metal-contaminated soils.

The Metal Chelating and Chaperoning Effects of Clioquinol: Insights from Yeast Studies

Clioquinol (CQ), a once popular antibiotic, was used to inhibit the growth of microorganisms. Recently, CQ and its analog PBT2 have shown encouraging effects in the animal and clinical trials for Alzheimer's disease (AD). However, the mechanism by which this class of molecules works remains controversial. In this work, we used the yeast Saccharomyces cerevisiae as a model to study how CQ affects molecular and cellular functions and particularly, copper, iron, and zinc homeostasis. We observed a CQ-induced inhibition of yeast growth, which could be slightly relieved by supplementation of copper or iron. Microarray results indicated that yeast cells treated with CQ sense a general deficiency in metals, despite elevated total cellular contents of copper and iron. Consistent with this, reduced activities of some metal-sensitive enzymes were observed. Intriguingly, CQ can increase the SOD1 activity, likely through Ccs1's accessibility to CQ-bound copper ions. Further studies revealed that CQ sequestrates copper and iron at the cellular membrane, likely the plasma membrane, resulting overall metal accumulation but cytosolic metal depletion. CQ's effects on metal-sensitive metalloenzymes were also verified in mammalian cell line SH-SY5Y. Together, our results revealed that CQ can regulate metal homeostasis by binding metal ions, resulting the cell sensing a state of deficiency of bioavailable metal ions while simultaneously increasing available metals to SOD1 (via Ccs1) and possibly some other metalloproteins that can access CQ-bound metals. We hope this regulation of metal homeostasis may be helpful in explaining the therapeutic effects of CQ used in disease treatment.

AdcAII, A New Pneumococcal Zn-Binding Protein Homologous with ABC Transporters: Biochemical and Structural Analysis

Regulation of metal homeostasis is vital for pathogenic bacteria facing drastic metal concentration changes in various locations within the host during invasion. Metal-binding receptors (MBRs), one of the extracellular components of ATP-binding cassette transporters, have been shown to be essential in this process. Streptococcus pneumoniae expresses two characterized MBRs: PsaA and AdcA, two extracellular lipoproteins encoded by the psaABCD and adcRCBA operons, respectively. The Mn- and Zn-uptake functions of PsaA and AdcA, respectively, have been well established. Here we describe AdcAII as a third putative S. pneumoniae MBR. The analysis of a phylogenetic tree built from the sequence alignment of 68 proteins reveals a subgroup of members displaying an unusual genetic operon organisation. The adcAII gene belongs to a 6670-nucleotide-long transcript spanning the spr0903 to spr0907 loci encoding for the CcdA, thioredoxine, YfnA, AdcAII and PhtD proteins. Two adjacent repeats of imperfect AdcR-binding consensus sequence were identified upstream of the adcAII gene, suggesting a transcriptional co-regulation of adcAII and phtD genes. Biophysical and structural studies of recombinant AdcAII were performed to identify the metal specificity of the protein. Using electrospray mass spectrometry in native conditions, we found that Zn was bound to recombinant AdcAII. Screening of the effect of 10 cationic ions on the thermal stability of AdcAII revealed that Zn had the most pronounced stabilizing effect. The crystal structure of AdcAII has been solved to 2.4 Å resolution. One Zn ion is bound to each AdcAII molecule in a symmetrical active site composed of three His and one Glu. The structure almost perfectly superimposed on the known MBR structures. The presence of a flexible 15-residue-long loop close to the metal-binding site is specific to those specialized in Zn transport. Taken together, these functional and structural data provide new perspectives related to the physiological role of AdcAII in pneumococcus Zn homeostasis.

Manipulation of intracellular magnesium levels in Saccharomyces cerevisiae with deletion of magnesium transporters

Magnesium is an important divalent ion for organisms. There have been a number of studies in vitro suggesting that magnesium affects enzyme activity. Surprisingly, there have been few studies to determine the cellular mechanism for magnesium regulation. We wished to determine if magnesium levels could be regulated in vivo. It is known that Saccharomyces cerevisiae has two magnesium transporters (ALR1 and ALR2) across the plasma membrane. We created S. cerevisiae strains with deletion of one (alr1 or alr2) or both (alr1 alr2) transporters. The deletion of ALR1 resulted in a decrease in intracellular magnesium levels. An increase from 5 to 100 mM in the exogenous magnesium level increased the intracellular levels of magnesium in the alr1 and alr1 alr2 strains, whereas the expression of magnesium transporters from S. cerevisiae or Arabidopsis thaliana led to a change of the intracellular levels of magnesium in those strains. The deletion of magnesium transporters in A. cerevisiae and overexpression of magnesium transporters from A. thaliana also affected the intracellular concentrations of a range of metal ions, which suggests that cells use non-specific transporters to help regulate metal homeostasis.

Metallothionein, zinc, and mercury levels in tissues of young rats exposed to zinc and subsequently to mercury

Several studies have described mercury toxicity and the role of metallothioneins (MT) in the detoxification and regulation of metal homeostasis. However, little data exist on this topic during the specific post-natal developmental phase in young mammals. This developmental phase is particularly important since young animals are more sensitive to toxicants than adults. The objective of this work was to investigate whether MT participates in the mechanism of protection conferred by zinc pre-treatment on the toxic effects induced by mercury in neonate rats. Pups were exposed to ZnCl 2 (5 doses of 27 mg/kg/day, s.c.) and subsequently to HgCl 2 (5 doses of 5 mg/kg/day, s.c.); metal (Zn and Hg) and MT contents were analyzed in the liver, kidney, and blood. MT was induced in the liver and kidney of pups of both Zn–sal and Zn–Hg groups, although the greatest increase was in neonates exposed to Zn only. A direct relationship exists between MT and metals for both hepatic and renal tissues, which indicates that the increase in metal levels occurs in parallel to the increase in MT content. Although the heat-treated cytosolic fraction is rich in MT and metals, higher Zn and Hg contents were detected in the insoluble fraction of all tissues. These results suggest that MT is, at least in part, responsible for preventing Hg accumulation in the liver and blood and decreasing renal toxicity.

Nicotine attenuates the β‐amyloid neurotoxicity through regulating metal homeostasis

Nicotine reduces beta-amyloidosis and has a beneficial effect against Alzheimer's disease (AD), but the underlying mechanism is not clear. The abnormal interactions of beta-amyloid (Abeta) with metal ions such as copper and zinc are implicated in the process of Abeta deposition in AD brains. In the present study, we investigated the effect of nicotine on metal homeostasis in the hippocampus and cortex of APP(V717I) (London mutant form of APP) transgenic mice. A significant reduction in the metal contents of copper and zinc in senile plaques and neuropil is observed after nicotine treatment. The densities of copper and zinc distributions in a subfield of the hippocampus CA1 region are also reduced after nicotine treatment. We further studied the mechanism of nicotine-mediated effect on metal homeostasis by using SH-SY5Y cells overexpressing the Swedish mutant form of human APP (APPsw). Nicotine treatment decreases the intracellular copper concentration and attenuates Abeta-mediated neurotoxicity facilitated by the addition of copper, and these effects are independent of the activation of nicotinic acetylcholine-receptor. These data suggest that the effect of nicotine on reducing beta-amyloidosis is partly mediated by regulating metal homeostasis.

The iron-binding protein ferritin is expressed in cells of the osteoblastic lineage in vitro and in vivo

Ferritin, a metal-binding protein responsible for maintaining the bioavailability of iron, has been demonstrated in cells of the osteoblastic lineage. Messenger RNAs encoding the light and heavy chain subunits of ferritin were detected in ROS 17 2.8 , ROS 25 1 , and UMR106 rat osteosarcoma cell lines, in fetal rat calvaria, and in primary cultures of rat calvarial osteoblast-like cells. In vivo, the expression of ferritin light-chain mRNA was observed in both active osteoblasts and in osteocytes. A 450-kD iron-binding protein was immunoprecipitated from ROS 17 2.8 cells by an antiferritin antiserum. This protein comigrated with native ferritin, and could be dissociated into subunits comigrating with ferritin light and heavy chains. Addition of extracellular Fe 59-transferrin to cultures of ROS 17 2.8 cells resulted in the sequestration of the iron in intracellular ferritin. These observations demonstrate that cells of the osteoblastic lineage possess a functional ferritin-based iron uptake and storage system capable of regulating metal homeostasis in bone.