Metal Metallothionein Research Articles

Metal Substitution and Solvomorphism in Alkylthiolate-Bridged Zn3 and HgZn2 Metal Clusters.

The impact of substituting Hg(II) for Zn(II) in a thiolate-bridged trinuclear cluster with parallels to a metallothionein metal cluster was investigated. A new solvomorph of [Zn(ZnL)2](ClO4)2 (1) (L = N-(2-pyridylmethyl)-N-(2-(ethylthiolato)-amine) and five solvomorphs of a new compound [Hg(ZnL)2](ClO4)2 (2) were characterized by single-crystal X-ray crystallography. The interplay of hydrogen bonding and aromatic-packing interactions in producing lamellar, 2D lamellar, and columnar arrangements of complex cations in the crystalline state is discussed. Both variable temperature proton nuclear magnetic resonance and electrospray ion-mass spectrometry (ESI-MS) suggest that the complex ions of 1 and 2 are the predominant solution species at moderate concentrations. ESI-MS was also used to monitor differences in metal ion redistribution as 1 was titrated with Hg(ClO4)2 and [HgL(ClO4)]. These studies document the facile replacement of Zn(II) by Hg(II) with the preservation of the overall structure in thiolate-rich clusters.

Complexation of the Zn, Co, Cd, and Pb ions by metallothioneins: A QM/MM simulation

Complexation of divalent metal ions (Zn, Co, Cd and Pb) with metallothioneins (MT), typical low-molecular proteins of diverse higher organisms, has been theoretically studied within the all-electron approximation B3LYP/DZP. The active site of MT (metal ions and cysteine residues) adopts the structure, in which cadmium, cobalt and zinc ions reveal a tetrahedral location of the sulfur atoms. A pyramidal structure is typical for the coordination environment of the lead complexes in the MT active site. The Gibbs energies of the active site formation were calculated accounting the influence of water medium in terms of the IEFPCM approximation. For all studied metallothioneins, the metal–active site complexes demonstrate close Gibbs formation energies per one metal ion. The influence of the amino acid environment of the MT active sites has been studied with the two-shell ONIOM model and B3LYP/DZP:UFF approximation. A simple scheme for calculation of relative metal–MT binding energies has been developed. Even under the pronounced destabilization of the lead complexes by a polypeptide shell, the total energetic effect is sufficient to make accumulation of lead ions by metallothioneins more efficient than Cd and, especially, Zn and Co. The following general trend of stability of the MT complexes has been found: Co≈Zn≪Cd≪Pb. These results compared with the experimental regularities of bioaccumulation of heavy metal salts by bacteria Bacillus are briefly discussed.

Arsenic Induction of Metallothionein and Metallothionein Induction Against Arsenic Cytotoxicity.

Human exposure to arsenic (As) can lead to oxidative stress that can become evident in organs such as the skin, liver, kidneys and lungs. Several intracellular antioxidant defense mechanisms including glutathione (GSH) and metallothionein (MT) have been shown to minimize As cytotoxicity. The current review summarizes the involvement of MT as an intracellular defense mechanism against As cytotoxicity, mostly in blood. Zinc (Zn) and selenium (Se) supplements are also proposed as a possible remediation of As cytotoxicity. In vivo and in vitro studies on As toxicity were reviewed to summarize cytotoxic mechanisms of As. Intracellular antioxidant defense mechanisms of MT are linked in relation to As cytotoxicity. Arsenic uses a different route, compared to major metal MT inducers such as Zn, to enter/exit blood cells. A number of in vivo and in vitro studies showed that upregulated MT biosynthesis in blood components are related to toxic levels of As. Despite the cysteine residues in MT that aid to bind As, MT is not the preferred binding protein for As. Nonetheless, intracellular oxidative stress due to As toxicity can be minimized, if not eliminated, by MT. Thus MT induction by essential metals such as Zn and Se supplementation could be beneficial to fight against As toxicity.

Neuroprotection and regeneration by extracellular metallothionein via lipoprotein-receptor-related proteins

Metallothionein has a well-documented protective and proregenerative effect in the mammalian brain, particularly following physical trauma and ischemia or during the onset of neurodegenerative disease. A range of mechanisms have been established for this, including metallothionein's metal binding properties and its ability to scavenge free radicals. In recent years it has become apparent that metallothionein is present in the extracellular compartment of the central nervous system and that it can interact with cell surface receptors of the lipoprotein-receptor-related protein family, including lipoprotein-receptor-related protein1 (LRP1) and megalin. These interactions activate intracellular pathways which are consistent with many of the observed effects of metallothionein in the central nervous system, including its effects on neurons, glial cells, and cells of the immune system. The evidence describing the release, receptor interactions, and subsequent physiological consequences of metallothionein is discussed in this review.

Differential ESI-MS behaviour of highly similar metallothioneins

ESI-MS can only be accepted as a quantification method when using standards with a high resemblance to the analyte(s). Unfortunately, this is usually not applicable to metallothioneins (MTs), a superfamily of singular metal-binding cysteine-rich proteins, present in all living organisms, since the absence of suitable reference material due to the high diversity among metal–MT species precludes their quantification by molecular mass spectrometry. Even thus, it is widely assumed that the intensities of the ESI-MS peaks of similar species are directly correlated with their relative concentration in the sample, and this has been extended to the determination of different MT proteins coexisting in a sample. Practically all organisms contain several MT isoforms, some of them exhibiting highly similar sequences, with conserved coordinating Cys residues. For the current analysis, we used as a model system the MT isoforms of two terrestrial snails ( Helix pomatia and Cornu aspersum). Hence, distinct samples were prepared by mixing, at different molar ratios, the recombinant HpCuMT and HpCdMT isoforms from H. pomatia, or the recombinant CaCuMT, CaCdMT and CaCdCuMT isoforms from C. aspersum, and they were analyzed by ESI-MS both at neutral pH (for Zn-loaded MT forms) and at acidic pH (for the corresponding apo-forms). The results here presented reveal that the ESI-MS peak intensity of a single MT species strongly depends on its sensitivity to be ionized, and thus, on the presence or absence of metal ions bound. Furthermore, our data demonstrate that very similar MT isoforms of the same organism with similar pI (ranging from 7.9 to 8.3) can show a clear different sensitivity to ES ionization, something that cannot be readily predicted only by consideration of their amino acid content. In conclusion, even in this optimum case, deductions about quantity features of MT samples drawn from ESI-MS measurements should be carefully considered.

Metal–metallothioneins like proteins investigation by heteroatom-tagged proteomics in two different snails as possible sentinel organisms of metal contamination in freshwater ecosystems

Metal speciation analysis in MLPs was carried out in two snails, Marisa cornuarietis and Pomacea bridgesi, in order to investigate them as possible sentinel organisms of heavy metal contamination. To carry out this study snails born in a non-contaminated environment were divided into two groups: a control group and a contaminated one with cadmium administered for 40 days. Subsequently, we investigated the speciation of the induced MLPs in exposed animals in relation to controls. In order to obtain the MLP fraction, cytosols from both snail species where subjected to size-exclusion fractionation, monitoring on-line the metal signal (Cd, Cu and Zn) by ICP-MS while protein elution was followed by on-line UV detection. MLP fraction was then separated by anion-exchange (AE)-FPLC using optimal chromatographic conditions for the separation of the different MLP isoforms in both snail species. Specific detection of separated metalloforms was carried out again by the hyphenation of the AE chromatographic system with ICP-MS. The determination of the amount of metal bound to MLPs was carried out by post-column isotope dilution analysis ICP-MS, finding that the snail M. cornuarietis accumulated higher concentrations of cadmium than P. bridgesi. Thus this first snail could therefore be a better candidate sentinel organism of pollution in natural waters. Identification and characterization of the isoforms separated in M. cornuarietis was carried out for the entire or intact isoforms by MALDI-TOF and then conventional triptic digestion was also carried out to identify the nature of the formed peptides. The presence identification of a MLP isoform of relatively low molecular weight in M. cornuarietis is reported.

Metallothionein induction related to hepatic structural perturbations and antioxidative defences in roach (Rutilus rutilus) exposed to the fungicide procymidone

A variety of stresses, hormones, glucocorticoids and cytokines are known to induce metallothioneins (MTs) in animals. The aim of this study was to investigate the effects of chemical stress induced by the dicarboximide fungicide procymidone on hepatic structure, MT content and antioxidative defences (catalase and glutathione reductase activities and glutathione content) in the common fish Rutilus rutilus. Catalase and glutathione reductase activities remained stable throughout the experiment. Four days of exposure to 0.2 or 0.4 mg l−1 of procymidone induced an obvious increase in liver MT content, perturbation of metal MT contents, and an increase in hepatic glutathione content. After 14 days' exposure, obvious and large structural alterations of the hepatic parenchyma occurred simultaneously with a decrease in MT and glutathione content. These events were interpreted as degeneration of the liver. Fish exposed for 14 days to procymidone and then placed for 14 days in clean water showed nearly complete decontamination of the liver, but MT concentrations remained high. The toxicological significance of these events is discussed.

Metallothionein isoforms separation and cadmium speciation by capillary electrophoresis with ultraviolet and quadrupole-inductively coupled plasma mass spectrometric detection

A complete characterisation of metallothioneins (MTs) in biological systems requires first an optimal separation of the different isoforms and sub-isoforms present. A method for the adequate separation of rabbit liver MT1 and MT2 mixtures by capillary zone electrophoresis (CZE) with UV detection is developed here. Different experimental conditions affecting both, peak shape and resolution achieved, are investigated in detail. Thus, the influence of the composition, ionic strength and pH of the buffer solution, and the separation voltage, have been studied. In order to improve the resolution between sub-isoforms, the electroosmotic flow has been also changed and its effect investigated by adding organic modifiers. Analytical performance characteristics are evaluated: calibration graphs were linear between 70 and 205 μg ml −1 for MT1 and between 83 and 242 μg ml −1 for MT2, while the detection limits were 4 and 3 μg ml −1, respectively. Speciation of the metal–MTs compounds has been accomplished by the coupling CZE–ICP–MS by means of different nebulisers: a conventional concentric Meinhard nebuliser, a Babington type one and a high efficiency nebuliser (HEN). Finally, real sample MTs analyses have been carried out in mussel hepatopancreas cytosols, previously exposed to a metal enriched environment.

Isoform separation of metallothioneins by capillary zone electrophoresis with Tris–tricine buffer in the presence or absence of methanol

A simple capillary electrophoretic method for analysis of metallothionein (MT) isoforms has been developed. Rabbit liver metallothionein (MT) isoforms are readily separated by capillary zone electrophoresis (CZE) with 150 mM Tris(hydroxymethyl)aminomethane (Tris)–150 mM N-Tris(hydroxymethyl)methylglycine (Tricine) buffer (pH 7.50) using an uncoated polyimide cladded fused-silica capillary column thus offering an alternative method for MT separations under original conditions. The effects of buffer concentration, buffer pH and buffer composition on the separation of MT isoforms were investigated. The effect of the addition of organic solvent, MeOH, as buffer modifier on the separation was also studied. Addition of MeOH to the buffer system enhanced the separation and additional information of the heterogeneity within each isoform group was gained. Also the effect of pH on the separation is enhanced when using organic solvent modified buffer. Enhanced information is gained by using photodiode array detection allowing recording of UV spectra of the putative isoform peaks thus offering the possibility of characterisation of the peaks obtained by comparing them to the unique characteristics of metallothionein metal–thiol bonds. The Tris–tricine method presented in this paper allows the separation of peaks that were found to need two separate runs under different conditions for separation to occur when using Tris–borate buffer. Rabbit liver MT, theoretically a mixture of MT-1 and MT-2, exhibited two major peaks, one medium sized peak and several smaller peaks. The experiments indicate that rabbit liver MT exhibited peaks which were not found in either MT-1 or MT-2 isoforms or they were found only as small residues. Both MT-1 and MT-2 samples exhibited one major peak and several smaller peaks. Residues of MT-2 were found in the MT-1 sample and vice versa.

Kallikrein gene therapy: a new strategy for hypertensive diseases

The tissue kallikrein–kinin system has been postulated to play a role in blood pressure homeostasis and the pathogenesis of clinical hypertension. To demonstrate the potential therapeutic effects of somatic gene delivery in treating hypertension, we used spontaneously hypertensive rats (SHR) as a model. The gene encoding the human tissue kallikrein was used because of its powerful hypotensive action. The human kallikrein DNA constructs were placed under the control of the metallothionein metal response element, the cytomegalovirus promoter/enhancer or the Rous sarcoma virus 3′-LTR. The human tissue kallikrein DNA constructs were incorporated into adenoviral vectors via homologous recombination. The naked plasmid DNA constructs or adenovirus containing the kallikrein gene were first introduced into kidney 293 cells and the expression of human tissue kallikrein was identified by ELISA. The kallikrein gene was delivered into SHR via intramuscular, intravenous, portal vein, intraperitoneal, and intracerebroventricular routes. A single injection of naked human kallikrein DNA constructs caused a prolonged reduction of high blood pressure for up to 8 weeks. Adenoviral-mediated gene delivery results in high efficiency of human tissue kallikrein expression. Immunoreactive human kallikrein was detected in rat serum at the highest level at 1 day post gene delivery. Portal vein delivery of a reporter gene, Ad.CMV-LacZ, results in intense staining of β-galactosidase in rat liver, suggesting that recombinant kallikrein is mainly produced in liver and secreted into the circulation. These results show that kallikrein gene delivery causes a sustained reduction of blood pressure in genetically hypertensive rats and provide important information for a potential gene therapy approach to human hypertension and related diseases.

Direct gene delivery of human tissue kallikrein reduces blood pressure in spontaneously hypertensive rats.

Hypertension is a multigene and multifactorial disorder affecting approximately 25% of the population. To demonstrate potential therapeutic effects of human tissue kallikrein in hypertension, spontaneously hypertensive rats were subjected to somatic gene therapy. Two human tissue kallikrein DNA constructs, one under the promoter control of the metallothionein metal response element and the other under the control of the Rous sarcoma virus 3'-LTR, were generated. We delivered naked DNA constructs into spontaneously hypertensive rats via intravenous injection. The expression of human tissue kallikrein in rats was identified in the heart, lung, and kidney by reverse transcription polymerase chain reaction followed by Southern blot analysis and an ELISA specific for human tissue kallikrein. A single injection of both human kallikrein plasmid DNA constructs caused a sustained reduction of blood pressure which began 1 wk after injection and continued for 6 wk. A maximal effect of blood pressure reduction of 46 mmHg in rats was observed 2-3 wk after injection with kallikrein DNA as compared to rats with vector DNA (n = 6, P < 0.05). The hypotensive effect caused by somatic gene delivery of human tissue kallikrein in hypertensive rats is reversed by subcutaneous injection of aprotinin, a potent tissue kallikrein inhibitor. No antibodies to either human tissue kallikrein or kallikrein DNA were detected in rat sera after injection of the human kallikrein gene. These results show that direct gene delivery of human tissue kallikrein causes a sustained reduction in systolic blood pressure in genetically hypertensive rats and indicate that the feasibility of kallikrein gene therapy for treating human hypertension should be studied.

Characterization of a corticotropin-releasing hormone-responsive element in the rat proopiomelanocortin gene promoter and molecular cloning of its binding protein.

A corticotropin-releasing hormone (CRH) and cAMP-responsive region (-236/-133) in the rat POMC gene promoter previously reported to confer CRH/cAMP responsiveness to heterologous reporter constructs has been characterized. DNAse footprint analysis revealed that multiple elements in this region were bound by nuclear proteins from the POMC expressing AtT20 cells. When these individual DNA elements were separately tested in heterologous reporter constructs for CRH induction, only one element, designated PCRH-RE (POMC CRH responsive element, -171/-160) was found to give strong CRH stimulation (5- to 7-fold). This element appears novel as to the possible binding factors, although it has homology to the mouse metallothionein metal regulatory element. Gel shift analyses of the PCRH-RE with AtT20 cell nuclear extracts showed marked stimulation of retarded nucleoproteins following CRH stimulation, suggesting that the possible binding factor(s) may mediate transcriptional regulation at this site. The activity of PCRH-RE binding protein was inhibited by divalent cations, with Cu2+ and Cd2+ being most effective; Zn2+ had no effect, indicating that this binding factor(s) is functionally distinct from the metallothionein metal regulatory element binding protein. A 2.6 kilobase cDNA clone encoding a protein (PCRH-REB-1) binding to this element was isolated by Southwestern screening of an AtT20 expression library with radiolabeled PCRH-RE oligonucleotides. This clone was used to isolate several other cDNA clones to determine the sequence corresponding to the entire coding region of the protein (PCRH-REB), which proved to be identical to a recently described DNA binding protein of the replication factor C complex, mRFC140/Mouse Southwestern. Primer extension and Northern blot analysis revealed that the size of the full length mRNA is about 4.9 kilobases. PCRH-REB mRNA expression is not restricted to corticotrophs but is present in a broad tissue distribution as evaluated by reverse transcription polymerase chain reaction analysis. A bacterially expressed beta-galactosidase-PCRH-REB-1 fusion protein was shown to bind PCRH-RE efficiently. Furthermore, binding of the PCRH-REB-1 fusion protein to the POMC CRH-responsive element was inhibited by divalent cations with similar sensitivities to those observed using AtT20 nuclear extracts. The predicted PCHR-REB protein sequence presents several interesting motifs: one p-Loop motif (ATP binding site), nine protein kinase A phosphorylation sites (implying a possible role in responding to the CRH-induced cAMP signal), and regions of homology to proteins involved in DNA replication and repair. PCRH-REB is, therefore, a potential transacting factor binding to a major CRH-responsive element in the POMC promoter.

Electrochemical characterization of metallothionein metal—mercaptide complexes: application of cyclic voltammetry to investigation of metalloproteins

Electrochemical studies of metalloproteins have considerable potential in the investigation of metal—complex structures. Due to a lack of information on the precise nature of the results obtained however, application of electrochemistry has been largely neglected by protein chemists. This report discusses the cyclic de voltammetry of a selection of proteins with particular reference to metallothioneins (MTs) and interpretation of waves associated with MT metal—protein complexes. Comparison of voltammograms of proteins in the presence and absence of added metal, together with scans of a selection of native MTs, has allowed for the designation of waves associated with specific metal complexes. The contrast in electrochemical activity between proteins free of thiols or disulfide bonds with those rich in these functionalities was also shown. Evidence from saturation plots, peak current versus scan rate plots and detergent competition studies with MTs, indicated that the major electrochemical process at the mercury electrode was adsorption rather than diffusion controlled. Characterization of native MT metal speciation and assessment of the fidelity of metal reconstitution of laboratory produced thionein was readily feasible using cyclic voltammetry and nanomole quantities of material.