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ARTICLE IN PRESS Journal of Trace Elements in Medicine and Biology 21 (2007) S1, 35–39 www.elsevier.de/jtemb THIRD INTERNATIONAL FESTEM SYMPOSIUM Metallothionein functions and structural characteristics Emilio Carpenè, Giulia Andreani, Gloria Isani Department of Biochemistry, University of Bologna, Via Tolara di Sopra 50, 40024 Ozzano Emilia, Bologna, Italy Received 29 June 2007; accepted 10 September 2007 Abstract Metallothioneins (MTs) are low molecular weight proteins characterized by a high cysteine content and give rise to metal-thiolate clusters. Most MTs have two metal clusters containing three and four bivalent metal ions, respectively. The MT gene family in mammals consists of four subfamilies designated MT-1 through MT-4. MT-3 is expressed predominantly in brain and MT-4 in differentiating stratified squamous epithelial cells. Many reports have addressed MT structure and function, but despite the increasing experimental data several topics remain to be clarified, and the true function of this elusive protein has yet to be disclosed. Owing to their induction by a variety of stimuli, MTs are considered valid biomarkers in medicine and environmental studies. Here, we will discuss only a few topics taken from the latest literature. Special emphasis will be placed on MT antioxidant functions, the related oxidation of cysteines, which can give rise to intra/intermolecular bridges, and the relations between MTs and diseases which could be originated by metal dysregulation. r 2007 Elsevier GmbH. All rights reserved. Keywords: Metallothionein; Structure; Function; Metals; Biomarker Introduction Metallothioneins (MTs), first isolated in the equine kidney [1], are ubiquitous low molecular weight proteins and polypeptides of extremely high metal and cysteine content which give rise to metal-thiolate clusters. Any protein or polypeptide resembling mammalian MTs can be classified as MT [2]. MTs constitute a protein superfamily of 15 families comprising many sequences inferred from both aminoacid and polynucleotide sequences obtained from all animal phyla examined to date and also from certain fungi, plants and cyanobacteria [3]. The MT gene family in mammals consists of four subfamilies designated MT-1 through MT-4. The study of MTs includes the competences in different Corresponding author. E-mail address: [email protected] (E. Carpenè). 0946-672X/$ - see front matter r 2007 Elsevier GmbH. All rights reserved. doi:10.1016/j.jtemb.2007.09.011 fields, which range from analytical chemistry and structural spectroscopy to molecular biology and medicine. There is currently no simple quantification method to detect tissue concentrations of MT whereas sophisticated 2D NMR spectroscopy shows that despite their different aminoacid sequences MTs have similar spatial structure with two metal-thiolate clusters containing three and four bivalent metal ions, respectively [4]. Increasing evidence shows that mammalian MT-1/ MT-2 isoforms are involved in zinc homeostasis and protection against heavy metal toxicity and oxidative stress. MT-3 is expressed mainly in neurons but also in glia; MT-4 is mostly present in differentiating stratified squamous epithelial cells. Many reports have described MT structure, functions [5–7] and gene expression [8], but despite the increasing data several topics await clarification and the true function of this elusive protein [9] has yet to be disclosed. ARTICLE IN PRESS 36 E. Carpenè et al. / Journal of Trace Elements in Medicine and Biology 21 (2007) S1, 35–39 Owing to their induction by a variety of stimuli, MTs are considered valid biomarkers in the medical and environmental fields. Here, we will discuss only few topics taken mostly from recent papers among the 7960 reported in Pub Med. The same site gives the following references when entering MT and the link-word in brackets: 2852 (zinc), 2781 (cadmium), 1689 (copper), 1133 (cancer), 415 (carcinoma), 55 (Alzheimer-AD), 33 (Amyotrophic Lateral Sclerosis-ALS), 15 (Parkinson), and 5 (Prion). Structure (1) Thionein: Despite the wealth of information available for the metallated mammalian MTs, the exact mechanism of the initial metal ion chelation remains unsettled as do the kinetics of removal and subsequent protein unfolding. Apo-MT has recently been reported in the cell in quantities equal to those of the metallated protein, which might indicate a potential role for MT in the absence of metals. Calculations carried out on the demetallation of CdMT1a indicate the metal free protein is structurally stable [7]. (2) Metallation: After the thionein has been synthesized at ribosome level it will be saturated with different metal ions according to the specific isoform or to the number of different concentrations of the available metals. Electrospray ionization time-of-flight mass spectrometry (ESI–TOF–MS) probing of reconstituted MT-3 demonstrates that MT-3 binds Zn and Cd ions more weakly than MT-2 but exposes higher metal-binding capacity and plasticity. The lower metal-binding affinity may be connected with its hexapeptide insert, and at the same time this acidic insert could be involved in binding additional metals [10]. (3) Dimerization: MT dimerization has been observed by several authors and is very evident in marine mussel exposed to cadmium [11,12]. However, it has only recently been demonstrated in mammals that under metal excess, the N-terminal domain is responsible for the formation of non-oxidative metal-bridged dimers, whereas under aerobic conditions, a specific intermolecular disulfide is formed between the C terminal domains. Both forms of dimers exhibit radical differences in the reactive properties of their respective cluster bound metal ions [13]. The oxidation of cysteines has been involved in the dissociative mechanism controlling free zinc fluctuations and modulation of cellular signalling pathways [14]. factor-1 (MTF-1) plays an important role in MT transcription. Several lines of evidence suggest that the highly conserved six-zinc finger DNA-binding domain of MTF1 functions as a zinc-sensing domain and the linkers between the six different fingers can actively participate in modulating MTF1 translocation to the nucleus and binding to the MT1 gene promoter [8]. Function (1) MT as scavenger of free radicals: Since the classical work of Thornalley and Vasak [15] on the scavenging activity of MT toward free hydroxyl (1OH) and superoxide (O21 ) radicals produced by the xantine/ xantine oxidase reaction much more evidence has accumulated on the antioxidant activity of MT by in vivo and in vitro experiments [16]. Several animal and cell models and free radical generating systems have been investigated. Using an epithelioma cell line from a piscine species (Cyprinus carpio) we demonstrated a protective effect of MT in radical scavenging when cells were treated with the redox cycling diquat and menadione after MT levels had been pre-induced by Cd exposure [17]. Despite the large body of literature on this topic the exact reaction involving the protein and the different radicals remains unsettled. More recent in vitro experiments with a NO donor showed that S-nitrosothiols formed in the b domain of mouse Cd7MT1 with a subsequent random formation of disulfide bonds [18]. Because zinc is preferentially bound in the b domain under natural conditions, the amount of zinc released can be fine-tuned. In turn, the released zinc can suppress the inducible NO synthase lowering NO production. (2) MT and metal detoxication and homeostasis: MT was primarily considered a protein involved in detoxification of non-essential and excess essential metals. This role is still claimed by most authors working in the MT field and often supported by data from species ranging from fungi to mammals, which could explain the wide variety of MT isoforms. Drosophila melanogaster has four MT genes, which are transcriptionally induced by heavy metals through the same metal-responsive transcription factor, MTF-1. Targeted mutagenesis demonstrated that the four MT genes exhibit distinct, yet overlapping, roles in heavy metal homeostasis and toxicity prevention [19]. A copper-specific MT isoform was shown to preferentially bind 12 copper ions in the Roman snail Elix pomatia [20]. Transcription MT and diseases The exact mechanisms controlling MT synthesis are not well understood but there is a general consensus that the metal responsive element-binding transcription There is a growing amount of exciting information on the genome changes from fertilization through the different ARTICLE IN PRESS E. Carpenè et al. / Journal of Trace Elements in Medicine and Biology 21 (2007) S1, 35–39 stages of the individual life span and the ongoing phenotype modifications caused by diseases and environmental changes. Misregulation of gene repression and activation can be related to the etiology of animal diseases in a very large number of cases, and many transcription factors belong to the zinc-finger protein family [21]. Considering that metal metabolism is dysregulated and reactive oxygen species (ROS) are produced during most pathological disorders, including neurodegenerative diseases [22] and senescence [23]; it is not surprising that MT expression varies extensively in several diseases. Neurodegenerative diseases: Cu(II)-binding amyloid-b peptides and the production of ROS have been reported to play a significant role in the progression of Alzheimer disease (AD) [22]. In vitro experiments performed with a recombinant human MT-3 expressed in Escherichia coli and reconstituted as Zn7MT-3 gave rise to a novel hypothesis in the redox silencing of copper. ESI–MS spectra suggest the formation of Cu(I)4Zn4 MT-3, the source of electrons for the reduction of Cu(II), is furnished by CysS ligands in a process concomitant with the formation of disulfide bridges and zinc release (Fig. 1). Titration of Zn7MT-3, with increasing Cu(II) concentrations, revealed the progressive disappearance of the mass peak of ZnMT and the concomitant occurrence of the Cu4Zn4-MT3 species, confirming its cooperative formation. At higher Cu(II)/protein stoichiometries, the simultaneous presence of a number of different mass peaks was detected (5 major mass peaks) [22]. Oxidation of a cytosolic factor irrespective of MT oxidation has been implicated in the nuclear trafficking of MT [24]. Comparing the MT profiles from samples of AD and control brains, it was found that without dithiothreitol (DTT) the copper and zinc levels of the MT were lower in AD. With DTT, the difference between AD and control brains was no more significant. This shows that a comparable amount of MT was present in AD and controls. However, in the case of AD, more MT were oxidized and lost bound metals. The oxidation was reversible with DTT. This comparison indicates that a significant difference between AD and control brains is not the amount of MT, but the relative part of oxidised MT. There seem to be more oxidative processes taking place in AD brains [25]. 37 Cancer: Recent studies have indicated a strong relation between the mutated zinc protein p53 and MT in tumors. MT could regulate the DNA binding of p53 through zinc transfer reaction, while the apo-MT can sequester zinc and thereby reduce the transcriptional activity of p53 [26]. In vitro a complex between MT and p53 was observed in breast cancer epithelial cells with both wild and inactive type p53. In addition, experiments based on p53 attached to glutathione-Sepharose showed that only apo-MT1 and not MT1 forms a complex with p53. This interaction may prevent binding of p53 to DNA so that p53 may not be able to act as a transcriptional factor and modulate gene transcription and apoptosis in tumor cells [27]. Myocardial hypertrophies: Pressure and volume overload produce distinct forms of cardiac hypertrophy. Gene expression profiled in rat hearts subjected to pressure overload showed that MT was one of the genes with the highest level of up-regulation. MT could be associated with caspase-3 activity suggesting that MT inhibits the apoptosis of cardiomyocytes thereby conferring a protective effect against heart failure [28]. Prion: The prion protein contains several octapeptide repeats sequences toward the N-terminus, which have binding affinity for metals such as copper, zinc and manganese. Therefore, an imbalanced metal homeostasis was claimed to generate the pathological isoform [29], even if we failed to confirm these findings. Relations between MT and the prion protein have been investigated in cattle with BSE and marked astrocytic MTi/II immunolabelling was seen in all BSE affected animals [30]. Using a cell line expressing a deoxycyclineinducible PrPC gene it was demonstrated that PrPC expression in turn induces MT expression [31]. In conclusion, these studies suggest that MT expression could be a useful biomarker of the disease phase and prognosis. MT quantification and MT as biomarker of environmental metal exposure It is generally accepted that MT is an important defense against the detoxification of non-essential metals Fig. 1. In presence of Cu(II), Zn7 MT can be partially oxidized. Cu(II) is reduced to Cu(I) by one electron which was released during the oxidation of two cysteines, in the meanwhile other two cysteines will chelate the reduced Cu(I). Overall a zinc ion and another electron are released from the oxidized MT. The fenton activity of Cu(II) will be quenched and the antioxidant activity of MT will be increased by the released zinc and electrons. ARTICLE IN PRESS 38 E. Carpenè et al. / Journal of Trace Elements in Medicine and Biology 21 (2007) S1, 35–39 like cadmium and mercury. The early induction of MT by trace metals, namely cadmium, in different species makes this protein a potential and biomarker useful to assess the ecotoxicological significance of non-essential (Cd, Hg) and essential, but potentially toxic (Cu), trace metals. The accurate measurement of MT is mandatory to assess its biomarker potential. This aspect has been revised several times [32–34]. Recent advances in speciation analysis have made a variety of promising techniques including HPLC–ICP–MS, HPLC–ESI–MS [35] and RP-HPLC coupled to fluorescence detection [36]. However, in spite of the sensitivity and accuracy of the new methods, the analysis of biological samples with these hyphenated techniques requires the presence of expensive equipment and well-trained persons, which quite often is lacking in most laboratories working in this field. Therefore, MT analysis continues to give problems and a fast and sensitive quantification method is still required. Marine molluscs can accumulate trace metals orders of magnitude higher than the concentrations present in seawaters. Therefore, molluscs have been widely used as indicators of metal pollution in marine ecosystems. Moreover selected heavy metals, such as Cd, Cu and Hg are assumed to be good inducers of MT biosynthesis. In this respect, MT is considered a valid biomarker of metal exposure in marine molluscs [33,36,37]. With regard to terrestrial animals, a significant link between MT and cadmium concentrations in kidney was recently demonstrated in wild animals, e.g. woodcocks [38] and wild mice [39] highlighting the major role of MT in metal detoxification processes. We conclude that MT could be a useful biomarker for environmental metal contamination in free-living animals, even though, apart the lack of an official analytical method, several environmental and physiological factors can affect the protein expression in natural populations [40]. References [1] Margoshes M, Vallee BL. A cadmium protein from equine kidney cortex. J Am Chem Soc 1957;79:4813–4. [2] /http://www.bioc.unizh.ch/mtpage/intro.htmlS. [3] /http://www.expasy.org/cgi-bin/lists?metallo.txtS. [4] Braun W, Vašák M, Robbins AH, Stout CD, Wagner G, Kägi JH, et al. Comparison of the NMR solution structure and the X-ray crystal structure of rat metallothionein-2. Proc Natl Acad Sci USA 1992;89:10124–8. [5] Kägi JHR. 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