Metallothionein Functions and Structural Characteristics Emilio Carpene` Ã, Giulia Andreani, Gloria Isani

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 Carpene` Ã, 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 fields, which range from analytical chemistry and structural spectroscopy to molecular biology and Metallothioneins (MTs), first isolated in the equine medicine. There is currently no simple quantification kidney [1], are ubiquitous low molecular weight proteins method to detect tissue concentrations of MT whereas and polypeptides of extremely high metal and cysteine sophisticated 2D NMR spectroscopy shows that despite content which give rise to metal-thiolate clusters. Any their different aminoacid sequences MTs have similar protein or polypeptide resembling mammalian MTs can spatial structure with two metal-thiolate clusters con- be classified as MT [2]. MTs constitute a protein taining three and four bivalent metal ions, respectively superfamily of 15 families comprising many sequences [4]. Increasing evidence shows that mammalian MT-1/ inferred from both aminoacid and polynucleotide MT-2 isoforms are involved in zinc homeostasis and sequences obtained from all animal phyla examined to protection against heavy metal toxicity and oxidative date and also from certain fungi, plants and cyanobac- stress. MT-3 is expressed mainly in neurons but also in teria [3]. The MT gene family in mammals consists of glia; MT-4 is mostly present in differentiating stratified four subfamilies designated MT-1 through MT-4. The squamous epithelial cells. Many reports have described study of MTs includes the competences in different MT structure, functions [5–7] and gene expression [8], but despite the increasing data several topics await ÃCorresponding author. clarification and the true function of this elusive protein E-mail address: [email protected] (E. Carpene` ). [9] has yet to be disclosed.

0946-672X/$ - see front matter r 2007 Elsevier GmbH. All rights reserved. doi:10.1016/j.jtemb.2007.09.011 ARTICLE IN PRESS 36 E. Carpene` 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 factor-1 (MTF-1) plays an important role in MT are considered valid biomarkers in the medical and transcription. Several lines of evidence suggest that the environmental fields. Here, we will discuss only few topics highly conserved six-zinc finger DNA-binding domain taken mostly from recent papers among the 7960 reported of MTF1 functions as a zinc-sensing domain and the in Pub Med. The same site gives the following references linkers between the six different fingers can actively when entering MT and the link-word in brackets: 2852 participate in modulating MTF1 translocation to the (zinc), 2781 (cadmium), 1689 (copper), 1133 (cancer), 415 nucleus and binding to the MT1 gene promoter [8]. (carcinoma), 55 (Alzheimer-AD), 33 (Amyotrophic Lat- eral Sclerosis-ALS), 15 (Parkinson), and 5 (Prion). Function (1) MT as scavenger of free radicals: Since the classical Structure work of Thornalley and Vasak [15] on the scaven- (1) Thionein: Despite the wealth of information available ging activity of MT toward free hydroxyl (1OH) and for the metallated mammalian MTs, the exact superoxide (O21À) radicals produced by the xantine/ mechanism of the initial metal ion chelation remains xantine oxidase reaction much more evidence has unsettled as do the kinetics of removal and subsequent accumulated on the antioxidant activity of MT by protein unfolding. Apo-MT has recently been reported in vivo and in vitro experiments [16]. Several animal in the cell in quantities equal to those of the metallated and cell models and free radical generating systems protein, which might indicate a potential role for MT have been investigated. Using an epithelioma cell in the absence of metals. Calculations carried out on line from a piscine species (Cyprinus carpio)we the demetallation of CdMT1a indicate the metal free demonstrated a protective effect of MT in radical protein is structurally stable [7]. scavenging when cells were treated with the redox (2) Metallation: After the thionein has been synthesized at cycling diquat and menadione after MT levels had ribosome level it will be saturatedwithdifferentmetal been pre-induced by Cd exposure [17]. Despite the ions according to the specific isoform or to the number large body of literature on this topic the exact of different concentrations of the available metals. reaction involving the protein and the different Electrospray ionization time-of-flight mass spectro- radicals remains unsettled. More recent in vitro ex- metry (ESI–TOF–MS) probing of reconstituted MT-3 periments with a NO donor showed that S-nitro- demonstrates that MT-3 binds Zn and Cd ions more sothiols formed in the b domain of mouse Cd7MT1 weakly than MT-2 but exposes higher metal-binding with a subsequent random formation of disulfide capacity and plasticity. The lower metal-binding bonds [18]. Because zinc is preferentially bound in affinity may be connected with its hexapeptide insert, the b domain under natural conditions, the amount and at the same time this acidic insert could be of zinc released can be fine-tuned. In turn, the involved in binding additional metals [10]. released zinc can suppress the inducible NO synthase (3) Dimerization: MT dimerization has been observed lowering NO production. by several authors and is very evident in marine (2) MT and metal detoxication and homeostasis:MTwas mussel exposed to cadmium [11,12]. However, it has primarily considered a protein involved in detoxifica- only recently been demonstrated in mammals that tion of non-essential and excess essential metals. This under metal excess, the N-terminal domain is role is still claimed by most authors working in the MT responsible for the formation of non-oxidative field and often supported by data from species ranging metal-bridged dimers, whereas under aerobic condi- from fungi to mammals, which could explain the wide tions, a specific intermolecular disulfide is formed variety of MT isoforms. Drosophila melanogaster has between the C terminal domains. Both forms of four MT genes, which are transcriptionally induced by dimers exhibit radical differences in the reactive heavy metals through the same metal-responsive properties of their respective cluster bound metal transcription factor, MTF-1. Targeted mutagenesis ions [13]. The oxidation of cysteines has been demonstrated that the four MT genes exhibit distinct, involved in the dissociative mechanism controlling yet overlapping, roles in heavy metal homeostasis and free zinc fluctuations and modulation of cellular toxicity prevention [19]. A copper-specific MT isoform signalling pathways [14]. 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 There is a growing amount of exciting information on the metal responsive element-binding transcription the genome changes from fertilization through the different ARTICLE IN PRESS E. Carpene` et al. / Journal of Trace Elements in Medicine and Biology 21 (2007) S1, 35–39 37 stages of the individual life span and the ongoing Cancer: Recent studies have indicated a strong phenotype modifications caused by diseases and environ- relation between the mutated zinc protein p53 and MT mental changes. Misregulation of gene repression and in tumors. MT could regulate the DNA binding of p53 activation can be related to the etiology of animal diseases through zinc transfer reaction, while the apo-MT can in a very large number of cases, and many transcription sequester zinc and thereby reduce the transcriptional factors belong to the zinc-finger protein family [21]. activity of p53 [26]. In vitro a complex between MT and Considering that metal metabolism is dysregulated and p53 was observed in breast cancer epithelial cells with reactive oxygen species (ROS) are produced during most both wild and inactive type p53. In addition, experi- pathological disorders, including neurodegenerative dis- ments based on p53 attached to glutathione-Sepharose eases [22] and senescence [23]; it is not surprising that MT showed that only apo-MT1 and not MT1 forms a expression varies extensively in several diseases. complex with p53. This interaction may prevent binding Neurodegenerative diseases: Cu(II)-binding amyloid-b of p53 to DNA so that p53 may not be able to act as a peptides and the production of ROS have been reported transcriptional factor and modulate gene transcription to play a significant role in the progression of Alzheimer and apoptosis in tumor cells [27]. disease (AD) [22]. In vitro experiments performed with a Myocardial hypertrophies: Pressure and volume over- recombinant human MT-3 expressed in Escherichia coli load produce distinct forms of cardiac hypertrophy. and reconstituted as Zn7MT-3 gave rise to a novel Gene expression profiled in rat hearts subjected to hypothesis in the redox silencing of copper. ESI–MS pressure overload showed that MT was one of the genes spectra suggest the formation of Cu(I)4Zn4 MT-3, the with the highest level of up-regulation. MT could be source of electrons for the reduction of Cu(II), is associated with caspase-3 activity suggesting that MT furnished by CysS ligands in a process concomitant inhibits the apoptosis of cardiomyocytes thereby con- with the formation of disulfide bridges and zinc release ferring a protective effect against heart failure [28]. (Fig. 1). Titration of Zn7MT-3, with increasing Cu(II) Prion: The prion protein contains several octapeptide concentrations, revealed the progressive disappearance repeats sequences toward the N-terminus, which have of the mass peak of ZnMT and the concomitant binding affinity for metals such as copper, zinc and occurrence of the Cu4Zn4-MT3 species, confirming its manganese. Therefore, an imbalanced metal home- cooperative formation. At higher Cu(II)/protein stoi- ostasis was claimed to generate the pathological isoform chiometries, the simultaneous presence of a number of [29], even if we failed to confirm these findings. different mass peaks was detected (5 major mass peaks) Relations between MT and the prion protein have been [22]. Oxidation of a cytosolic factor irrespective of MT investigated in cattle with BSE and marked astrocytic oxidation has been implicated in the nuclear trafficking MTi/II immunolabelling was seen in all BSE affected of MT [24]. Comparing the MT profiles from samples of animals [30]. Using a cell line expressing a deoxycycline- AD and control brains, it was found that without inducible PrPC gene it was demonstrated that PrPC dithiothreitol (DTT) the copper and zinc levels of the expression in turn induces MT expression [31].In MT were lower in AD. With DTT, the difference conclusion, these studies suggest that MT expression between AD and control brains was no more significant. could be a useful biomarker of the disease phase and This shows that a comparable amount of MT was prognosis. 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 MT quantification and MT as biomarker of indicates that a significant difference between AD and environmental metal exposure control brains is not the amount of MT, but the relative part of oxidised MT. There seem to be more oxidative It is generally accepted that MT is an important processes taking place in AD brains [25]. 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. Carpene` 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 [8] Li Y, Kimura T, Laity JH, Andrews GK. The zinc- trace metals, namely cadmium, in different species makes sensing mechanism of mouse MTF-1 involves linker this protein a potential and biomarker useful to assess the peptides between the zinc fingers. 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