Download Metallothionein functions and structural characteristics

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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.
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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
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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].
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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.
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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].
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