Metallothionein

Metallothioneins (MTs) is a family of Cys-rich, low molecular weight (MW ranging from 3500 to 14000 Da) proteins. MTs have the capacity to bind both physiological (Zn, Cu, Se,...) and xenobiotic (Cd, Hg, Ag,...) heavy metals through the thiol group of its cysteine residues, which represents nearly the 30% of its amino acidic residues. They were first discovered in 1960 by Kägi & Vallee when they purified a Cd/binding protein from horse (equine) renal cortex [Fowler, B.A. et al, Nomenclature of Metallothionein, Metallothionein II: Experimentia Supplementum Vol. 52,1987, pg19]. It was characterised in 1970 by Kägi and others, and has been used since. Its function is not clear, but the experimental data relates MTs with protection against metal toxicity, regulation of physiological metals (Zn and Cu) and protection against oxidative stress. There are four main isoforms expressed in humans. In the human body, large quantities are synthesised primarily in the liver and kidneys. Their production is dependent on availability of the dietary minerals, as zinc, copper and selenium, and the amino acids histidine and cysteine.

Structure and classification
MTs are present in a vast range of taxonomic groups, ranging from prokaryotes (such as the cyanobacteria Syneccococus spp....), protozoa (p. ex. the ciliate Tetrahymena genera...), plants (such as Pisum sativum, Triticum durum, Zea mays, Quercus suber...), yeast (such as Saccharomyces cerevisiae, Candida albicans,...), invertebrates (such as the nematode Caenorhabditis elegans, the insect Drosophila melanogaster, the mollusc Mytilus edulis, or the echinoderm Strongylocentrotus purpuratus) and vertebrates (such as the chicken, Gallus gallus, or the mammalian Homo sapiens or Mus musculus).

The MTs from this diverse taxonomic range represent a high-heterogeneity sequence (regarding molecular weight and number and distribution of Cys residues) and do not show general homology; in spite of this, homology is found inside some taxonomic groups (such as vertebrate MTs).

From their primary structure, MTs have been classified by different methods. The first one dates from 1987, when Fowler et al., established three classes of MTs: Class I, including the MTs which show homology with horse MT, Class II, including the rest of the MTs with no homology with horse MT, and Class III, which includes Cadystins and Phytochelatins, Cys-rich enzymatically synthesised peptides which are no longer considered MTs. The second classification was performed by Binz and Kagi in 2001, and takes into account taxonomic parameters and the patterns of distribution of Cys residues along the MT sequence. It results in a classification of 15 families. Family 15 contains the plant MTs, which in 2002 have been further classified by Cobbet and Goldsbrough into 4 Types (1, 2, 3 and 4) depending on the distribution of their Cys residues and a Cys-devoid regions (called spacers) characteristic of plant MTs.

A table including the principal aspects of the two latter classifications is included. More data on this classification are disposable at the Expasy metallothionein page

Secondary structure elements have been observed in several MTs SmtA from Syneccochoccus, mammalian MT3, Echinoderma SpMTA, fish Notothenia Coriiceps MT, Crustacean MTH,, but until this moment, the content of such structures is considered to be poor in MTs, and its functional influence is not considered.

The tertiary level of structure show also a high hetereogeneity. While vertebrate, echinoderm and crustacean MTs show a bidominial structure with divalent metals as Zn(II) or Cd(II) (the protein is folded binding metals in two functionally independent domains, with a metallic cluster each), yeast and procariotyc MTs show a monodominial structure (only one domain as a receipt of a unique metallic cluster). In spite of no structural data is available for molluscan, nematoda and Drosophila MTs, it is commonly assumed that the formers are bidominial and the latter mondominial. No conclusive data are disposable for Plant MTs, but two possible tridimensional structures have been proposed: 1) a bidominial structure similar to that of vertebrate MTs; 2) a codominial structure, in which two Cys-rich domains interacts to form a unique metallic cluster.

Quaternary level of structure has been scarcely taken in account for MTs. Dimerization and oligomerization processes has been observed and attributed to several molecular mechanisms: 1) the formation of inter-molecular disulfide bridges by the oxidation of thiol residues. 2) the formation of a metallic bridge by a metal with bounds to Cys belonging to different MTs 3) the formation of a metallic bridge by a metal with bounds to His belonging to different MTs 4) the interaction through inorganic phosphate ions. In spite of dimeric and polymeric MTs have shown to acquire novel properties on metal detoxification, its physiological significance has been demonstrate only in the case of procaryotic Synechococcus SmtA, which forms an MT dimer able to form structures similar to Zn-fingers which develops Zn-regulatory activities.

Metallothioneins present also diverse metal-binding preferences, which have been associated to functional specificity. As an example, the mammalian Mus musculus MT1 presents a preference for binding divalent metal ions (Zn(II), Cd(II),...), while yeast CUP1 presents a preference for binding monovalent metal ions (Cu(I), Ag(I),...). Taking as a reference these functional preferences a novel functional classification of MTs which classifies MTs as Zn- or Cu-thioneins is currently being developed.

Metal binding
Metallothionein proteins participate in the uptake, transport, and regulation of zinc in biological systems. The zinc binding sites are typically cysteine-rich, and often bind three or four zinc ions. Cysteine is a sulfur-containing amino-acid, from there the name (thio means sulfur). However, the participation of inorganic sulfide and Chloride ions has been proposed for some MT forms. In some MTs, histidine also participates in zinc binding, playing also roles in the determination of the metal/binding preferences. By binding and releasing zinc, metallothioneins (MTs) regulate its level within the body. Zinc, in turn, is a key element for the activation and binding of certain transcription factors through its participation in (aptly-named) zinc finger region of the protein. Metallothionein also carries zinc ions (signals) from one part of the cell to another. When zinc enters a cell, it can be picked up by thionein (which thus becomes "metallothionein") and carried to another part of the cell where it is released to another organelle or protein. In this way the thionein-metallothionein becomes a key component of the zinc signaling system in cells. This system is particularly important in the brain, where zinc signaling is prominent both between and within nerve cells. It also seems to be important for the regulation of the tumor suppressor protein p53.

Metallothionein (MT) detoxifies mercury and heavy metals by binding to the metal before it can cause harm. It forms subcellular inclusions or crystals. The inclusions can accumulate within tissues (such as bone) over time.

Control of the oxidative stress
Cysteine residues from MTs can capture harmful oxidant radicals as hydroxide radical. From this reaction, cystein is oxidized to cystine, and the metal ions which were bound to cystine are liberated to the media. As explained in the Expression and regulation section, this Zn can activate the synthesis of more MTs. This mechanism has been proposed to be an important mechanism in the control of the oxidative stress by MTs. The role of MTs in oxidative stress has been confirmed by MT Knockout mutants, but some experiments propose also a prooxidant role form MTs.

Expression and regulation
Metallothionein gene expression is induced by a high variety of stimuli, as metal exposure, oxidative stress, glucocorticoids, hydric stress, etc... The level of the response to these inducers depends on the MT gene. MT genes present in their promotors specific sequences for the regulation of the expression, elements as Metal Response Elements (MRE), Glucocorticoid Response Elements (GRE),...

Cancer
Because MTs play an important role in transcription factor regulation, problems with MT function or expression may lead to malignant transformation of cells and ultimately cancer. Studies have found increased expression of MTs in some cancers of the breast, colon, kidney, liver, lung, nasopharynx, ovary, prostate, mouth, salivary gland, testes, thyroid and urinary bladder; they have also found lower levels of MT expression in hepatocellular carcinoma and liver adenocarcinoma.

There is evidence to suggest that higher levels of MT expression may also lead to resistance to chemotherapeutic drugs.

Autism
It has been hypothesized that a MT disorder explains several symptoms of autism, but a 2006 study found that autistic children did not differ significantly from normal children in levels of MT or antibodies to MT.