Haem release from haemopexin by HxuA allows Haemophilus influenzae to escape host nutritional immunity

In this work we describe the identification of a copper-inducible regulon in Mycobacterium tuberculosis (Mtb). Among the regulated genes was Rv0190/MT0200, a paralogue of the copper metalloregulatory repressor CsoR. The five-locus regulon, which includes a gene that encodes the copper-protective metallothionein MymT, was highly induced in wild-type Mtb treated with copper, and highly expressed in an Rv0190/MT0200 mutant. Importantly, the Rv0190/MT0200 mutant was hyper-resistant to copper. The promoters of all five loci share a palindromic motif that was recognized by the gene product of Rv0190/MT0200. For this reason we named Rv0190/MT0200 RicR for regulated in copper repressor. Intriguingly, several of the RicR-regulated genes, including MymT, are unique to pathogenic Mycobacteria. The identification of a copper-responsive regulon specific to virulent mycobacterial species suggests copper homeostasis must be maintained during an infection. Alternatively, copper may provide a cue for the expression of genes unrelated to metal homeostasis, but nonetheless necessary for survival in a host.     

Toxicological aspects of metallothionein.

Metallothionein (MT) is expressed to a certain extent in almost all mammalian tissues. The biological significance of MT is related to its various forms MT-1, MT-2, MT-3 and MT4. For MT-1 several isoforms of the protein exist and it is likely that these isoforms are related to various functions involved in developmental processes occurring at various stages of gestation. Toxicokinetics and biochemistry of essential and toxic metals such as cadmium, zinc, mercury and copper in organs e.g. kidney, CNS, are often related to metallothionein. It is debated whether there is a relation or not for other metals e.g. selenium and bismuth. For the toxicokinetics of cadmium, MT plays an important role. By expanding techniques from experimental toxicology and biochemistry to include molecular biology methods, more specific and relevant studies can be performed of the actual role and biological function of MT. The present paper on toxicological aspects of metallothionein, presents an overview and evaluation of present knowledge concerning differences among organs and within organs of the expression of MT and how this affects tissue sensitivity to toxicity.     

Comparison of metallothionein gene expression and nonprotein thiols in ten Arabidopsis ecotypes. Correlation with copper tolerance.

Seedlings of 10 Arabidopsis ecotypes were compared with respect to copper tolerance, expression of two metallothionein genes (MT1 and MT2), and nonprotein thiol levels. MT1 was uniformly expressed in all treatments, and MT2 was copper inducible in all 10 ecotypes. MT1 and MT2 mRNA levels were compared with various growth parameters for the 10 ecotypes in the presence of 40 microM Cu2+. The best correlation (R = 0.99) was obtained between MT2 mRNA and the rate of root extension. MT2 mRNA levels also paralleled the recovery phase following inhibition by copper. Induction of MT2 mRNA was initiated at copper concentrations below the threshold for growth inhibition. In cross-induction experiments, Ag+, Cd2+, Zn2+, Ni2+, and heat shock all induced significant levels of MT2 gene expression, whereas Al3+ and salicylic acid did not. The correlation between copper tolerance and nonprotein thiol levels in the 10 ecotypes was not statistically significant. However, 2 ecotypes, Ws and Enkheim, previously shown to exhibit an acclimation response, had the highest levels of nonprotein thiols. We conclude that MT2 gene expression may be the primary determinant of ecotypic differences in the copper tolerance of nonpretreated Arabidopsis seedlings.     

Changes in zinc,copper and metallothionein contents during oocyte growth and early development of the teleost Danio rerio (zebrafish)

In the present report, we investigated zinc, copper and metallothionein (MT) contents in zebrafish oocytes and embryos. Our results demonstrate that the metal content increases during oocytes maturation. Zinc increases from 30 ng/oocyte (stage-1 oocytes) to 100 ng/oocyte (stage-3 oocytes); copper varied from 1 ng/oocyte (stage-1 oocytes) to 3.5 ng/oocyte (stage-3 oocytes). During embryogenesis, zinc and copper contents dramatically increase after fertilisation around the 512-cells stage, then slowly decrease until the mid-gastrula stage. During oocyte growth, the changes in the MT level are proportional to metal content, whereas during embryogenesis the pattern of MT accumulation does not parallel that of the two metals. Indeed, the maternal pool of MT decreases steadily during the early stages of the development until the gastrula stage. We have examined the effect of cadmium on the expression of MT during zebrafish development. After cadmium exposure, MT content increases in embryos at the blastula stage, whereas no induction occurs in embryos at the gastrula stage. However, pre-treatment of embryos at the gastrula stage with 5-aza-2'-deoxycytidine induces MT synthesis following exposure to cadmium. These observations show that changes in metal levels are not correlated to MT content in the embryo, whereas DNA methylation is one of the factors regulating MT expression.     

Reduction of copper and metallothionein in toxic milk mice by tetrathiomolybdate, but not deferiprone.

Copper is both essential for life and toxic. Aberrant regulation of copper at the level of intracellular transport has been associated with inherited diseases, including Wilson's disease (WND) in humans. WND results in accumulation of copper and the copper and zinc-binding protein metallothionein (MT) in liver and other tissues, liver degeneration, and neurological dysfunction. The toxic milk (TX) mutation in mice results in a phenotype that mimics human WND, and TX has been proposed to be a model of the disease. We characterized TX mice as a model of altered metal ion and MT levels during development, and after treatment with the metal ion chelators tetrathiomolybdate (TTM) and deferiprone (L1). We report that hepatic, renal and brain copper and MT are elevated in TX mice at 3 and 12 months of age. Zinc was significantly higher in TX mouse liver, but not brain and kidney, at both time points. Nodules appeared spontaneously in TX mouse livers at 8-12 months that maintained high copper levels, but with more normal morphology and decreased MT levels. Treatment of TX mice with TTM significantly reduced elevated hepatic copper and MT. Transient increases in blood and kidney copper accompanied TTM treatment and indicated that renal excretion was a significant route of removal. Treatment with L1, on the other hand, had no effect on liver or kidney copper and MT, but resulted in increased brain copper and MT levels. These data indicate that TTM, but not L1, may be useful in treating diseases of copper overload including WND.     

The binding of gold(I) to metallothionein

The binding of gold(I) to metallothionein, MT, has been unambiguously established by the reaction of Na₂AuTM with purified horse kidney MT. Zinc was displaced more readily than cadmium although the latter could be displaced using large Au/Cd ratios. The metal exchange reactions were complete within 2 hr of mixing. Further evidence that such reactions might be physiologically significant were obtained by studying in vitro metal displacements in the liver cytosol of in vivo metal treated rats: When Na₂AuTM was added to the cytosol of rats administered CdCl₂ in vivo, zinc, copper and cadmium were displaced in 2/1/1 ratios from the metallothionein fraction. The zinc and cadmium displacement provide direct evidence that the gold was binding to MT. Addition of Cd⁺² to liver cytosol of gold-treated rats resulted in displacement of copper and zinc, but not gold, from the MT fractions. When liver MT is prepared from rats exposed to Au or Cd, the Cd/protein ratio increased during the preparation, but the Au/protein ratio decreased. The Mt-bound metals account for 95% of the cytosolic Cd but only 15%–30% of the cytosolic gold in these studies. Thus, the nonspecific binding of gold to MT in vivo should be considered as one aspect in its equilibration among protein binding sites, which include, inter alia, metallothionein. Gold was found to coelute with zinc and cadmium in the MT fraction of rat kidney cytosol, when both Cd and Na₂AuTM were administered to the rats. The possible significance of gold binding to MT in the treatment of rheumatoid arthritis-chrysotherapy-is briefly discussed.     

A study of the role of metallothionein in the inherited copper toxicosis of dogs.

The role of metallothionein (MT) was assessed in the copper-loading disease prevalent in Bedlington terriers. Fractionation of tissue supernatants over Sephadex G-75 showed that most of the additional cytosolic copper present in liver tissue of these dogs was bound to MT, and that substantially more MT-bound copper could be solubilized by detergent plus mercaptoethanol. Zinc contents were only slightly raised, although most of the extra zinc was associated with a 4000-Mr ligand. Ion-exchange chromatography revealed two isoproteins, MT1 and MT2, in all the dog liver samples examined. In Bedlington terrier liver, copper associated with both isoproteins was increased, although the increase for MT2 was greater than for MT1. The content of MT protein was also raised, although cell-free translations and RNA blots of total liver RNA showed that this increase was not associated with a rise in MT mRNA. The significance of these results to the mechanism of copper accumulation in the Bedlington terrier disorder is discussed.     

Chemical synthesis and expression of copper metallothionein gene of Neurospora crassa

The gene coding for the Neurospora crassa copper metallothionein (MT) was synthesized and inserted in the lacZ' gene of pUC18 plasmid to give the same translational reading frame as the latter gene. The MT-beta-galactosidase fused gene was expressed in Escherichia coli to produce a fused protein in which the amino and carboxy termini of MT are linked to the beta-galactosidase through methionine residues. An MT derivative containing an extra homoserine residue at the carboxy terminus was prepared by cyanogen bromide cleavage of the fused protein followed by a reverse-phase HPLC separation. The spectral features of the MT derivative and its copper complex were similar to those of the corresponding native MTs.     

A new insight into the Ag+ and Cu+ binding sites in the metallothionein beta domain

The copper(I) and silver(I) binding properties of the beta fragment of recombinant mouse metallothionein I have been studied by electronic absorption and circular dichroism spectroscopy. When possible, the stoichiometry of the species formed was confirmed by electrospray mass spectrometry. The behaviour observed differs from that reported for the native protein. Titration of either Zn3-beta MT at pH 7 or apo-beta MT at pH 3 with Cu+ leads to the formation of species having the same stoichiometry and structure: Cu6-beta MT, Cu7-beta MT and Cu10-beta MT. In the first stage of the titration of Zn3-beta MT with Cu+ at pH 7 one additional species of formula Cu4Zn1-beta MT was detected. In contrast, the titration of Zn3-beta MT at pH 7.5 and of apo-beta MT at pH 2.5 with Ag+ proceeds through different reaction pathways, affording ZnxAg3-beta MT, Ag6-beta MT and Ag9-beta MT or Ag3-beta MT, Ag6-beta MT and Ag9-beta MT, respectively. The CD envelope corresponding to species with the same stoichiometric ratio, Ag6-beta MT and Ag9-beta MT, indicates that they have a different structure at each pH value. On the basis of the differences observed, the postulated similarity between copper and silver binding to metallothionein may be questioned.     

Metallothionein and the development of the mottled disorder in the mouse

Copper accumulates in kidney tissue of mottled (Mo) mice largely in association with a low MW cytosol protein, and the reduced copper levels in neonatal mutant liver are largely the result of a reduction in the amount of copper associated with this same protein. On the basis of ion-exchange chromatographic profile, heat stability, absence of a 280nm absorption peak, and the binding of Cd109 and Zn65 the protein mutants in the kidney is identified as metallothionein (MT). Amino acid analysis, however, failed to confirm this, and it is suggested that the high copper content of the mutant protein results in its oxidative degradation during purification, even when normal anaerobic precautions are taken. Estimates of thionein protein content of tissues from mutant and normal mice demonstrated that the levels are significantly elevated in both young and adult mutant kidney and depressed in young mutant liver, in parallel therefore with the changes in tissue copper levels. In adult mutant liver tissue, however, thionein levels are significantly raised, even though tissue copper content is normal. The synthesis and degradation of MT was examined in some detail. Incorporation of S35-cysteine in kidney MT was significantly raised in both young and adult mutant mice, while in adult tissue the rate of degradation of MT was significantly depressed. The elevated kidney MT levels arise therefore in young mutant mice from an increased rate of synthesis and in adult mice from the combined effects of increased synthesis and reduced degradation.     

Copper transporter 1, metallothionein and glutathione reductase genes are differentially expressed in tissues of sea bream (Sparus aurata) after exposure to dietary or waterborne copper

The high affinity copper transporter 1 (Ctr1), metallothionein (MT) and glutathione reductase (GR) are essential for copper uptake, sequestration and defense respectively. Following rearing on a normal commercial diet (12.6+/-0.2 mg kg(-1) Cu), sea bream were fed an experimental control diet lacking mineral mix (7.7+/-0.3 mg kg(-1) Cu), an experimental diet enhanced with Cu (135+/-4 mg kg(-1) Cu) or an experimental diet (7.7+/-0.3 mg kg(-1) Cu) whilst exposed to Cu in water (0.294+/-0.013 mg L(-1)). Fish were sampled at 0, 15 and 30 days after exposures. Fish fed the Cu-enhanced experimental diet showed lower levels of expression of Ctr1 in the intestine and liver compared to fish fed control experimental diets, whilst Ctr1 expression in the gill and kidney was unaffected by excess dietary Cu exposure. Waterborne-Cu exposure increased Ctr1 mRNA levels in the intestine and the kidney compared to experimental controls. Excess dietary Cu exposure had no effect on levels of metallothionein (MT) mRNA, and the only effect of dietary excess Cu on glutathione reductase (GR) mRNA was a decrease in the intestine. Both MT mRNA and GR were increased in the liver and gill after waterborne-Cu exposure, compared to levels in fish fed experimental control low Cu diets. Thus, Ctr1, MT and GR mRNA expression in response to excess Cu is dependent on the route of exposure. Furthermore, the tissue expression profile of sea bream Ctr1 is consistent with the known physiology of copper exposure in fish and indicates a role both in essential copper uptake and in avoidance of excess dietary and waterborne copper influx.     

5-Azacytidine increases the total cellular copper content and basal level metallothionein mRNA accumulation of human Hep G2 cells.

In this study we have demonstrated the ability of 5-azacytidine to elevate the basal level expression of the metallothionein (MT)-IF and MT-IG genes and increase the basal level expression of the MT-IIA gene in Hep G2 cells, a cell line which exhibits heavy metal inducible MT gene expression. Atomic absorption analysis of 5-azacytidine treated Hep G2 cells detected a 2-fold increase in the total cellular copper content. Pretreatment of 5-azacytidine exposed cells with hydroxyurea and cycloheximide indicated that the increase in total cellular copper content was a direct response to 5-azacytidine treatment. S1 nuclease analysis illustrated that pretreatment of Hep G2 cells with KCN, a copper specific chelator and uptake inhibitor, suppressed 5-azacytidine- and copper-inducible MT-IG gene expression. Thus, the increase in MT gene expression in response to 5-azacytidine treatment can be correlated to an increase in the total cellular copper content. Possible mechanisms on how 5-azacytidine could alter the influx/efflux of copper in Hep G2 cells are discussed.