Metallothionein and associated systems

Metallothionein is a peculiar protein with an extremely high affinity for free metal ions such as Zn2 +, Cd2 +, and Cu +. In the presence of sufficient metallothionein the free concentrations of these metals in solution are reduced to the picomolar range. Interestingly, iron is not bound by metal-lothionein but instead has its own pathways of uptake and intracellular transport, involving ferro-transferrin and ferritin. As we will see below, changes in iron trafficking are part of the general stress response, but the regulation of these changes does not appear to interact with metallothionein and therefore we leave iron out of consideration here.

As the name indicates, metallothionein contains not only a high amount of metal but also of sulphur. This is due to an extraordinarily high percentage of cysteine residues (around 30%). These cysteines participate in the formation of metal-thiolate clusters, in which the thiol groups of several cysteines coordinate with a group of metal atoms. In the metallothionein of mammals there are two clusters, one binding four metal ions using eleven cysteines, the other binding three metal ions with nine cysteines. These two clusters appear as two separate protein domains, a C-terminal a-domain (four-metal cluster) and an N-terminal b-domain (three-metal cluster), which are separated by a short linker sequence (Fig. 6.6). There are no aromatic amino acids in metallothionein; the whole protein is very hydrophilic.

The two-cluster structure of metallothionein has also been found in other vertebrates, invertebrates, and plants; however, in some species the a and b clusters are in reversed configuration with respect to their N- and C-terminal positions. Other species have two three-metal clusters. The metallothionein genes (Mts) of Drosophila are an oddity among animals. Drosophila has four Mt genes, each encoding a small metallothionein with one metal-binding domain (Valls et al. 2000; Egli et al. 2003). Such single-cluster Mts have not been found in other animals up to now, but they are present in fungi. In plants the Mt genes encode proteins in which the two metal-thiolate clusters are connected by a very long linker of non-cysteine amino acids (Cobbett and Goldsbrough 2002). So it appears that the evolution of metallothionein, quite unlike the stress proteins and the components

Metallothionein
Figure 6.6 Model, based on crystallography data, of rat liver metallothionein with five cadmium and two zinc atoms, bound in two metal-thiolate clusters. The molecule can be viewed in three dimensions by stereoscopy. From Robbins et al. (1991), with permission from Elsevier.

of signalling pathways discussed above, has come with a considerable reshuffling of the molecule (Van Straalen et al. 2006).

When metallothionein was first described, the function attributed to it was to regulate the cellular concentrations of essential metals. This was assumed to involve donating metals to specific metal-requiring ligands (enzymes, zinc fingers, structural proteins), while preventing aspecific binding to macromolecules by keeping the free concentrations of metals very low. In addition some metallothioneins turned out to be highly inducible by non-essential heavy metals (e.g. Cd) and this suggested a detoxification role. This classical, dual role of metallothionein has come under fire recently. The following issues describe the more complicated situation today.

First, it turned out that metallothionein is induced not only by metal ions, but also by a variety of other stresses, including changes in redox state, oxidative stress and stress hormone signals. This suggests that the protein might be a member of the integrated stress response and has other roles as well. A function as a scavenger of free radicals is often suggested. Second, metal-lothionein should not be considered a single protein. Many organisms have more than one Mt gene; the human genome has no less than 16 Mt genes. Most invertebrates investigated so far have two genes, one strongly inducible by cadmium and encoding a cadmium-binding protein, the other not inducible and encoding a copper-binding protein. The presence of a specific zinc-binding metallothionein in invertebrates is doubtful; the copper- and cadmium-binding metallothioneins of Drosophila, nematodes, earthworms, and snails are not inducible by zinc. Third, other metal-chelating substances have been found; this happened initially in plants, hence the name phytochelatins. They are the main zinc-binding ligands in plant cytoplasm and could play a similar role in invertebrates. Phytochelatins are peptides of variable length with the general formula (g-Glu-Cys)n-Gly, where n varies from 2 to 5. The peptides are synthesized from glutathione by the enzyme phyto-chelatin synthase. The gene encoding this enzyme is not only found in plants but also in nematodes, earthworms, and chironomids (Cobbett and Goldsbrough 2002). Phytochelatin synthase is also present in the genome of the tunicate Ci. intesti-nalis, as mentioned in Section 3.3.5, but it is absent from vertebrates.

Induction of metallothionein by exposure to heavy metals has been studied extensively, but the mechanism is not yet clear. As in the case of antioxidant enzymes, inducibility is due to the presence of specific sequences in the promoter of the gene, which bind a transcription factor activated by metals. In the case of metallothionein these sequences are called metal-responsive elements (Table 6.1). Such elements are not only present in the promoters of Mt genes, but also in those of genes encoding membrane-bound zinc transporters and enzymes associated with glutathione synthesis (Andrews 2001). The best-characterized transcription factor binding to these sequences is metal-responsive element-binding transcription factor (MTF). Induction by metals takes place by activation of MTF in the cytoplasm, followed by translocation to the nucleus; however, how MTF is activated by metals is not clear. One model suggests that in uninduced circumstances MTF is inhibited by a factor called metallothionein transcription inhibitor (MTI). This MTI has possible binding sites for zinc, which if occupied would result in the release of MTF (Palmiter 1994; Roesijadi 1996; Haq et al. 2003). Under this model, activation of MTF by cadmium is explained by an increase in the free zinc concentration in the cell, brought about by cadmium displacing zinc from cellular binding sites (Fig. 6.7).

The validity of the model for MTF activation by free zinc may be limited to mammals and cannot be extrapolated without modification to fish or invertebrates. In rainbow trout it was shown that silver could activate metallothionein expression without mediation by zinc (Mayer et al. 2003). In Drosophila, zinc itself does not activate MTF, although cadmium does. In earthworms and nematodes induction must involve an entirely different mechanism because metal-responsive elements seem to be completely absent; yet induction of the Mt gene by cadmium is possible (Sturzenbaum et al. 2001). Another, non-MTF/ metal-responsive element-like induction mechanism

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