Challenging the model for induction of metallothionein gene expression

Metallothioneins (MTs) are low-molecular-weight, cysteine-rich metal-binding proteins found in a wide variety of organisms including bacteria, fungi and all eukaryotic plant and animal species. MTs bind essential and non-essential heavy metals. In mammalian cells MT genes are highly inducible by many heavy metals including Zn, Cd, Hg, and Cu. Aquatic systems are contaminated by different pollutants, including metals, as a result of man's activities. Bivalve molluscs are known to accumulate high concentrations of heavy metals in their tissue and are widely used as bioindicators for pollution in marine and freshwater environments, with MTs frequently used as a valuable marker of metal contamination. We here describe the MT isoform gene expression patterns of marine and freshwater molluscs and fish species after Cd or Zn contamination. Contamination was carried out at a river site polluted by a zinc ore extraction plant or in the laboratory at low, environmentally relevant metal concentrations. A comparison for each species based on the accumulated MT protein levels often shows discrepancies between gene expression and protein level. In addition, several differences observed in the pattern of MT gene expression between mollusc and mammalian species enable us to discuss and challenge a model for the induction of MT gene expression.     

Channa punctata brain metallothionein is a potent scavenger of superoxide radicals and prevents hydroxyl radical-induced in vitro DNA damage

Mammalian brain metallothioneins (MTs) have been shown to scavenge free radicals. However, a similar role for fish brain MT has not been established yet. Previously, we have reported that MT from the liver of a freshwater fish, Channa punctata Bloch, had free-radical-scavenging activity in vitro. In this study, we report on the induction of MT in brain and other tissues of C. punctata treated with a low concentration of zinc chloride. We partially purified MT (Zn-MT)-rich fraction from the brain and studied its free-radical-scavenging and DNA damage attenuating effects. Zinc exposure showed significant MT induction in brain, gill, kidney, and liver. C. punctata brain MT efficiently scavenged superoxide radicals and also attenuated hydroxyl radical-mediated DNA damage. These findings suggest that fish brain MT has a free-radical-scavenging activity, and its expression may be regulated in response to stress and chemical exposure. C. punctata has been identified as a potent biomarker fish species. It is suggested that this fish species may be a good model for the study of MTs with regard to their regulatory and biomarker functions.     

Zinc-induced metallothionein synthesis by Caco-2 cells

Caco-2 cells possess many morphological and biochemical characteristics of intestinal absorptive cells, including the ability to transport zinc. In the present study, metallothionein (MT) synthesis in response to increased levels of zinc was examined. Increased incorporation of [³⁵S]cysteine into MTs was observed when excess ZnCl₂ was added to the medium. The rate of MT synthesis was found to be concentration dependent. Also, induction of MT synthesis was greater early in the culture, before the cells were fully differentiated. Incubation of the monolayers with ⁶⁵Zn and 200 μm zinc revealed that approximately 50% of the zinc incorporated into the cells was associated with MTs. The remainder was associated with large proteins as well as amino acids and small peptides. Actinomycin D and cycloheximide both inhibited the induction of MT synthesis, suggesting that the newly synthesized MTs are a result of expression of MT genes. Hence, Caco-2 cells, a model of intestinal absorptive cells, may be used to examine the role of MTs in zinc absorption.     

Zinc-regulating proteins, ZnT-1, and metallothionein I/II are present in different cell populations in the mouse testis.

Zinc ions play an important role in testis development and spermatogenesis. Thus, nutritional zinc deficiency leads to aberrant testicular development, reduced spermatogenesis, and male sterility. The precise actions of zinc in mediating these functions and the mechanisms by which zinc is itself regulated in the testis, however, have not been adequately elucidated. We have assessed the distribution of the zinc-regulating proteins ZnT-1 and metallothionein I/II (MT I/II) in the mouse seminiferous tubule. Co-labeling for ZnT-1 and MT I/II demonstrated unique patterns of distribution for these proteins, with ZnT-1 present in Sertoli cells in addition to luminal spermatozoa and MT I/II restricted to spermatocytes. These findings were confirmed by dual-label immunofluorescence for ZnT-1 and the Sertoli cell marker, vimentin, and by immunoelectron microscopy. The differential expression patterns of ZnT-1 and MTs support the hypothesis that ZnT-1 and MTs play different roles in the regulation of intracellular zinc in this organ. The specific expression of ZnT-1 in the Sertoli cells, moreover, is consistent with their role in maintaining a nurturing, closely regulated environment for spermatogenesis.     

Kif18A uses a microtubule binding site in the tail for plus-end localization and spindle length regulation

The mitotic spindle is a macromolecular structure utilized to properly align and segregate sister chromatids to two daughter cells. During mitosis, the spindle maintains a constant length, even though the spindle microtubules (MTs) are constantly undergoing polymerization and depolymerization [1]. Members of the kinesin-8 family are important for the regulation of spindle length and for chromosome positioning [2-9]. Kinesin-8 proteins are length-specific, plus-end-directed motors that are proposed to be either MT depolymerases [3, 4, 8, 10, 11] or MT capping proteins [12]. How Kif18A uses its destabilization activity to control spindle morphology is not known. We found that Kif18A controls spindle length independently of its role in chromosome positioning. The ability of Kif18A to control spindle length is mediated by an ATP-independent MT binding site at the C-terminal end of the Kif18A tail that has a strong affinity for MTs in?vitro and in cells. We used computational modeling to ask how modulating the motility or binding properties of Kif18A would affect its activity. Our modeling predicts that both fast motility and a low off rate from the MT end are important for Kif18A function. In addition, our studies provide new insight into how depolymerizing and capping enzymes can lead to MT destabilization.     

Induction of two major isoforms of metallothionein in crucian carp (Carassius cuvieri) by air-pumping stress,dexamethasone, and metals

The induction of metallothionein (MT) by physical and chemical stress was assessed using the fresh-water fish, crucian carp (Carassius cuvieri Temminck et Schlegel). The fish exposed to violent air-pumping stress for 6 days revealed time-dependent induction of MT-like metal-binding proteins in both their livers and kidneys. Their hepatic contents after exposure to stress were elevated to twice the basal level with 24 h, resulting in more than a 3-fold increase at 144 h, whereas their renal contents gradually increased after 24 h and reached the same level as that in the liver around 96 h. Two major inducible proteins were purified from livers of fish exposed to stress and were shown to be MT based upon their chromatographic behavior, UV absorption spectra and their molecular weights. Consequently, they were termed ccMT-1 and ccMT-2, according to their elution sequence upon anion-exchange chromatography. Both proteins mainly bound zinc in their endogenous forms and showed different immunogenicity to rat and rabbit MTs. Dexamethasone, a potent inducer for MT synthesis in mammals, induced the production of both isoforms in crucian carp, whereas cadmium and zinc ions prominently induced the synthesis of ccMT-2. These results indicate that crucian carp have the ability to produce MTs in response to various kinds of environmental stress and that violent air-pumping stress in crucian carp may induce MT synthesis, in part, via the release of endogenous factor(s), such as glucocorticoids.     

From heavy metal‐binders to biosensors: Ciliate metallothioneins discussed

Metallothioneins (MTs) are ubiquitous proteins with the capacity to bind heavy metal ions (mainly Cd, Zn or Cu), and they have been found in animals, plants, eukaryotic and prokaryotic micro‐organisms. We have carried out a comparative analysis of ciliate MTs (Tetrahymena species) to well‐known MTs from other organisms, discussing their exclusive features, such as the presence of aromatic amino acid residues and almost exclusive cysteine clusters (CCC) present in cadmium‐binding metallothioneins (CdMTs), higher heavy metal‐MT stoichiometry values, and a strictly conserved modular–submodular structure. Based on this last feature and an extensive gene duplication, we propose a possible model for the evolutionary history of T. thermophila MTs. We also suggest possible functions for these MTs from consideration of their differential gene expressions and discuss the potential use of these proteins and/or their gene promoters for designing molecular or whole‐cell biosensors for a fast detection of heavy metals in diverse polluted ecosystems.     

Redox biochemistry of mammalian metallothioneins

Metallothionein (MT) is a generic name for certain families of structurally rather variable metal-binding proteins. While purely chemical or biological approaches failed to establish a single physiologic function for MTs in any species, a combination of chemical and biological approaches and recent progress in defining the low but significant concentrations of cytosolic free zinc(II) ions have demonstrated that mammalian MTs function in cellular zinc metabolism in specific ways that differ from conventional knowledge about any other metalloprotein. Their thiolate coordination environments make MTs redox-active zinc proteins that exist in different molecular states depending on the availability of cellular zinc and the redox poise. The zinc affinities of MTs cover a range of physiologic zinc(II) ion concentrations and are modulated. Oxidative conditions make more zinc available, while reductive conditions make less zinc available. MTs move from the cytosol to cellular compartments, are secreted from cells, and are taken up by cells. They provide cellular zinc ions in a chemically available form and participate in cellular metal muffling: the combination of physiologic buffering in the steady state and the cellular redistribution and compartmentalization of transiently elevated zinc(II) ion concentrations in the pre-steady state. Cumulative evidence indicates that MTs primarily have a redox-dependent function in zinc metabolism, rather than a zinc-dependent function in redox metabolism.     

Microtubule-driven multimerization recruits ase1p onto overlapping microtubules

Microtubule (MT) crosslinking proteins of the ase1p/PRC1/Map65 family play a major role in the construction of MT networks such as the mitotic spindle. Most homologs in this family have been shown to localize with a remarkable specificity to sets of MTs that overlap with an antiparallel relative orientation [1-4]. Regulatory proteins bind to ase1p/PRC1/Map65 and appear to use the localization to set up precise spatial signals [5-10]. Here, we present evidence for a mechanism of localized protein multimerization underlying the specific targeting of ase1p, the fision yeast homolog. In controlled in vitro experiments, dimers of ase1-GFP diffused along the surface of single MTs and, at concentrations above a certain threshold, assembled into static multimeric structures. We observed that this threshold was significantly lower on overlapping MTs. We also observed diffusion and multimerization of ase1-GFP on MTs inside living cells, suggesting that a multimerization-driven localization mechanism is relevant in vivo. The domains responsible for MT binding and multimerization were identified via a series of ase1p truncations. Our findings show that cells use a finely tuned cooperative localization mechanism that exploits differences in the geometry and concentration of ase1p binding sites along single and overlapping MTs.     

Ciliate metallothioneins: unique microbial eukaryotic heavy-metal-binder molecules

This article represents an updated review of ciliate metallothioneins (Tetrahymena species) including a comparative analysis with regard to well-known metallothioneins (MTs) from other organisms and discussion of their exclusive features. It opens with an introduction to ciliates, summarizing the main characteristics of these eukaryotic microorganisms and their use as cellular models to study metallothioneins and metal–eukaryotic cell interactions. It has been experimentally proved that at least three different metal resistance mechanisms exist in ciliates, of which bioaccumulation is the most studied. Structural comparative analysis reveals that Tetrahymena MTs have unique characteristics, such as longer length, a considerably higher cysteine content, different metal–MT stoichiometry values, the presence of new cysteine clusters, and a strictly conserved modular–submodular structure. Gene expression analysis reveals a multistress and differential response to diverse metals and other environmental stressors, which corroborates the classification of these MTs. An in silico analysis of the promoter sequences of some MT genes reveals the presence of conserved motifs that are probably involved in gene expression regulation. We also discuss the great advantages of the first ciliate whole-cell biosensors based on MT promoters from Tetrahymena thermophila to detect heavy metal ions in environmental samples.     

Effects of cadmium and zinc on solar-simulated light-irradiated cells: potential role of zinc-metallothionein in zinc-induced genoprotection

Zinc is an essential oligoelement for cell growth and cell survival and has been demonstrated to protect cells from oxidative stress induced by UVA or from genotoxic stress due to UVB. In a recent work we demonstrated that the antioxidant role of zinc could be related to its ability to induce metallothioneins (MTs). In this study we identified the mechanism of zinc protection against solar-simulated light (SSL) injury. Cultured human keratinocytes (HaCaT) were used to examine MTs expression and localization in response to solar-simulated radiation. We found translocation to the nucleus, with overexpression of MTs in irradiated cells, a novel observation. The genoprotective effect of zinc was dependent on time and protein synthesis. DNA damage was significantly decreased after 48 h of ZnCl(2) (100 microM) treatment and is inhibited by actinomycin D. ZnCl(2) treatment (100 microM) led to an intense induction, redistribution, and accumulation of MT in the nucleus of irradiated cells. MT expression correlated with the time period of ZnCl(2) treatment. CdCl(2), a potent MT inducer, did not show any genoprotection, although the MTs were expressed in the nucleus. Overall our findings demonstrate that MTs could be a good candidate for explaining the genoprotection mediated by zinc on irradiated cells.     

Effect of metallothionein on cell viability and its interactions with cadmium and zinc in HEK293 cells

Metallothioneins (MTs) are thought to participate in a wide variety of physiological roles, but the mechanisms involved are still unclear. The study was designed to examine the possible factors related to these mechanisms. Methods, including transfection, MTT assay and flow cytometry, were used to investigate the effect of MTs on cell viability and their interactions with cadmium and zinc in HEK293 cells. The results showed that transient overexpression of human MT1A, MT2 and MT3 genes dynamically affected cell viability, and the effect was influenced by zinc and cadmium ions. Overexpressed MTs with added zinc showed a greater inhibitory effect on cell viability. Overexpressed MTs protected cells against low concentrations of cadmium ions (10 microM), but increased cell death in response to high concentrations (20-50 microM). Out of the three MTs, MT1A was more efficient than MT2 and MT3 in its resistance to cadmium (10 microM), and MT3 together with zinc showed more cell growth inhibition than MT1 and MT2. These results indicate that both of the divalent metal ions that could bind MTs, as well as the individual MT isoforms, affect the role of MTs on cell viability, which may explain in part why the comprehensive effect of MTs on the cells was elusive.     

Metal-binding functions and antioxidant properties in human thyroid gland under iodine deficient nodular colloidal goiter

Copper and zinc levels in the tissue of thyroid gland (TG) and their metal-binding proteins metallothioneins (MT) as well as state of the antioxidant system in persons that had no thyroid disease and patients with endemic iodine deficiency nodular colloidal goiter has been investigated. In the patients with thyroid disease, oxidative damage was indicated despite elevated levels of MT-SH and glutathione, and elevated copper and decreased zinc concentration in TG tissue. MTs partly bound the excess of copper but its concentration in the unbound to MT form was two-fold compared to the control value.     

Providing Positional Information with Active Transport on Dynamic Microtubules

Microtubules (MTs) are dynamic protein polymers that change their length by switching between growing and shrinking states in a process termed dynamic instability. It has been suggested that the dynamic properties of MTs are central to the organization of the eukaryotic intracellular space, and that they are involved in the control of cell morphology, but the actual mechanisms are not well understood. Here, we present a theoretical analysis in which we explore the possibility that a system of dynamic MTs and MT end-tracking molecular motors is providing specific positional information inside cells. We compute the MT length distribution for the case of MT-length-dependent switching between growing and shrinking states, and analyze the accumulation of molecular motors at the tips of growing MTs. Using these results, we show that a transport system consisting of dynamic MTs and associated motor proteins can deliver cargo proteins preferentially to specific positions within the cell. Comparing our results with experimental data in the model organism fission yeast, we propose that the suggested mechanisms could play important roles in setting length scales during cellular morphogenesis.     

Regulation of metallothionein synthesis in HeLa cells by heavy metals and glucocorticoids

Metallothioneins (MTs) are low molecular weight, cysteine-rich proteins that bind heavy metals. MT induction occurs in liver in response to either heavy metal (Zn++ or Cd++) administration or stress. The synthesis of MT can also be induced by either heavy metals or glucocorticoid hormones in HeLa cells cultured in serum-free medium. Induction of MT by zinc is subject to "desensitization." In contrast, dexamethasone (dex) induction results in a continued elevation in the rate of MT synthesis. The stability of MT is dependent on the availability of metal; consequently, MT induced by dex is degraded much more rapidly (half-life of 11 to 12 hours) than MT induced by elevated zinc levels (half-life of 36 to 38 hours). Removal of either inducer results in biphasic degradation curves, as apothionein and zinc come into balance. In contrast, deinduction kinetics for MT synthesis following removal of the two inducers (zinc and dex) are the same, with a half-life of two and one-half hours. Inhibition of RNA synthesis blocks deinduction after removal of inducer. Induction of MT occurs in a wide variety of species, from blue-green algae to man. This system should provide an excellent model for the comparative biochemistry of regulation of gene expression.