Milan Vašák

Possible role for metallothionein in protection against radiation-induced oxidative stress. Kinetics and mechanism of its reaction with superoxide and hydroxyl radicals

Rabbit liver metallothionein-1 (Mr 6500), which contains zinc and/or cadmium ions, appears to scavenge free hydroxyl (.OH) and superoxide (O-.2) radicals produced by the xanthine/xanthine oxidase reaction much more effectively than bovine serum albumin (Mr 65 000) which was used as a control. Kinetic competition studies between metallothionein and either a spin trap for .OH or ferricytochrome c for O-.2 radicals, gave bimolecular rate constants of the order of kOH/MT approximately equal to 10(12) M-1 X s-1 and kO-2/MT approximately equal to 5 X 10(5) M-1 X s-1, respectively. The former value suggests that all 20 cysteine sulfur atoms are involved in this quenching process and that they all act in the diffusion control limit. The aerobic radiolysis of an aqueous solution of metallothionein, generating O-.2 and .OH radicals, induced metal ion loss and thiolate oxidation. These effects could be reversed by incubation of the irradiated protein with reduced glutathione and the appropriate bivalent metal ion. Metallothionein appears to be an extraordinarily efficient .OH radical scavenger even when compared to proteins 10-50-times its molecular weight. Moreover, hydroxyl radical damage to metallothionein appears to occur at the metal-thiolate clusters, which may be repaired in the cell by reduced glutathione. Metallothionein has the characteristics of a sacrificial but renewable cellular target for .OH-mediated cellular damage.

Roles of the metallothionein family of proteins in the central nervous system.

Metallothioneins (MTs) constitute a family of proteins characterized by a high heavy metal [Zn(II), Cu(I)] content and also by an unusual cysteine abundance. Mammalian MTs are comprised of four major isoforms designated MT-1 trough MT-4. MT-1 and MT-2 are expressed in most tissues including the brain, whereas MT-3 (also called growth inhibitory factor) and MT-4 are expressed predominantly in the central nervous system and in keratinizing epithelia, respectively. All MT isoforms have been implicated in disparate physiological functions, such as zinc and copper metabolism, protection against reactive oxygen species, or adaptation to stress. In the case of MT-3, an additional involvement of this isoform in neuromodulatory events and in the pathogenesis of Alzheimers disease has also been suggested. It is essential to gain insight into how MTs are regulated in the brain in order to characterize MT functions, both in normal brain physiology, as well as in pathophysiological states. The focus of this review concerns the biology of the MT family in the context of their expression and functional roles in the central nervous system.

Metallothioneins: new functional and structural insights.

In the past few years, tissue-specific mammalian metallothioneins (i. e. metallothionein-3 and metallothionein-4) have been discovered that possess distinct functional properties. Other recent developments include an insight into the role of the widely expressed mammalian metallothionein-1 and metallothionein-2 isoforms in zinc homeostasis and apoptosis. The three-dimensional structure of the evolutionary distant sea urchin Cd(7)-metallothionein A, which contains two metal-thiolate clusters, supports previous conclusions regarding the functional importance of this structural motif. Despite correlated efforts involving techniques of structural biochemistry and molecular biology, the primary function of metallothioneins remains enigmatic.

Three-dimensional structure of rabbit liver [Cd7]metallothionein-2a in aqueous solution determined by nuclear magnetic resonance

In previous work the metal-polypeptide co-ordinative bonds in the major protein species of a reconstituted [113Cd7]metallothionein-2 preparation from rabbit liver in aqueous solution were determined, the secondary polypeptide structure was found to contain several half-turns and 3(10)-helical segments, and a preliminary characterization of the overall polypeptide backbone fold in the beta-domain containing the three-metal cluster, and the alpha-domain containing the four-metal cluster, was obtained. Using a new, more extensive set of nuclear magnetic resonance data these earlier structures were improved by new structure calculations. The new experimental data consist of distance constraints from measurements of nuclear Overhauser effects, and dihedral angle constraints derived from both coupling constants and nuclear Overhauser effects. The structure calculations were performed with the program DISMAN. Since no information on the orientation of the two domains relative to each other could be obtained, the structure calculations were performed separately for the alpha-domain and the beta-domain. The average of the pairwise root-mean-square distances among the 20 structures with the least residual violations of input constraints was 2.9 A for the beta-domain and 1.4 A for the alpha-domain (1 A = 0.1 nm). The overall chirality of the polypeptide fold is right-handed for the beta-domain and left-handed for the alpha-domain. For each of the seven metal ions the local chirality of the co-ordination of the four cysteinyl Sy atoms is clearly defined. The improved structures of both domains show the previously noted differences relative to the recently published crystal structure of metallothionein-2a from rat liver.

Activity of Metal-Responsive Transcription Factor 1 by Toxic Heavy Metals and H2O2 In Vitro Is Modulated by Metallothionein

ABSTRACT Metallothioneins are small, cysteine-rich proteins that avidly bind heavy metals such as zinc, copper, and cadmium to reduce their concentration to a physiological or nontoxic level. Metallothionein gene transcription is induced by several stimuli, notably heavy metal load and oxidative stress. Transcriptional induction of metallothionein genes is mediated by the metal-responsive transcription factor 1 (MTF-1), an essential zinc finger protein that binds to specific DNA motifs termed metal-response elements. In cell-free DNA binding reactions with nuclear extracts, MTF-1 requires elevated zinc concentrations for efficient DNA binding but paradoxically is inactivated by other in vivo inducers such as cadmium, copper, and hydrogen peroxide. Here we have developed a cell-free, MTF-1-dependent transcription system which accurately reproduces the activation of metallothionein gene promoters not only by zinc but also by these other inducers. We found that while transcriptional induction by zinc can be achieved by elevated zinc concentration alone, induction by cadmium, copper, or H2O2 additionally requires the presence of zinc-saturated metallothionein. This is explained by the preferential binding of cadmium or copper to metallothionein or its oxidation by H2O2; the concomitant release of zinc in turn leads to the activation of transcription factor MTF-1. Conversely, thionein, the metal-free form of metallothionein, inhibits activation of MTF-1. The release of zinc from cellular components, including metallothioneins, and the sequestration of zinc by newly produced apometallothionein might be a basic mechanism to regulate MTF-1 activity upon cellular stress.

Advances in the structure and chemistry of metallothioneins.

A low molecular weight (6-7 kDa) class of metalloproteins, designated as metallothioneins (MTs), exhibit repeated sequence motifs of either CxC or CxxC through which mono or divalent d(10) metal ions are bound in polymetallic-thiolate clusters. The preservation of metal-thiolate clusters in an increasing number of three-dimensional structures of these proteins signifies the importance of this structural motif. This review focuses on the recent developments regarding the versatile and striking chemical reactivity of MTs as well as on the existence of conformational/configurational dynamics within their structure. Both properties and their interplay are likely to be essential for the still elusive biological function of these proteins.

Chemistry and biology of mammalian metallothioneins

Metallothioneins (MTs) are a class of ubiquitously occurring low molecular mass, cysteine- and metal-rich proteins containing sulfur-based metal clusters formed with Zn(II), Cd(II), and Cu(I) ions. In mammals, four distinct MT isoforms designated MT-1 through MT-4 exist. The first discovered MT-1/MT-2 are widely expressed isoforms, whose biosynthesis is inducible by a wide range of stimuli, including metals, drugs, and inflammatory mediators. In contrast, MT-3 and MT-4 are noninducible proteins, with their expression primarily confined to the central nervous system and certain squamous epithelia, respectively. MT-1 through MT-3 have been reported to be secreted, suggesting that they may play different biological roles in the intracellular and extracellular space. Recent reports established that these isoforms play an important protective role in brain injury and metal-linked neurodegenerative diseases. In the postgenomic era, it is becoming increasingly clear that MTs fulfill multiple functions, including the involvement in zinc and copper homeostasis, protection against heavy metal toxicity, and oxidative damage. All mammalian MTs are monomeric proteins, containing two metal–thiolate clusters. In this review, after a brief summary of the historical milestones of the MT-1/MT-2 research, the recent advances in the structure, chemistry, and biological function of MT-3 and MT-4 are discussed.

Recent developments in quantification methods for metallothionein

The metallothioneins (MT), a family of proteins with relatively low molecular weight (6-7 kDa), are characterised by the intrinsic presence of 20 cysteinyl groups in their structure, which confers unique metal binding properties to the molecule. Since MT are involved in biological roles, quantification of MT remains an important task. To date, a large number of determination methods have been developed. In this paper recent developments, from 1995 to the present, in methodology employed in quantification studies of total MT and MT polymorphism are described. Different fields were taken into consideration, such as (i) separation techniques and hyphenated systems, (ii) electrochemical methods, (iii) immunological methods and (iv) quantification of MT mRNA. The data presented are based on our own and published results. A brief overview of the use of metallothionein as a biomarker is included as a relevant example of the importance of MT quantification. Finally, general problems associated with determination and evaluation of obtained results within the above four topics are mentioned.

Conformation of [Cd7]-metallothionein-2 from rat liver in aqueous solution determined by nuclear magnetic resonance spectroscopy.

The three-dimensional structure of [Cd7]-metallothionein-2 from rat liver was determined in aqueous solution, using nuclear magnetic resonance spectrometry and distance geometry calculations. The experimental data provided proton-proton distance constraints from measurements of nuclear Overhauser effects, constraints on the geometry of the metal-cysteine clusters determined by heteronuclear correlation spectroscopy, and dihedral angle constraints derived from both coupling constants and nuclear Overhauser effects. The structure calculations were performed with the program DISMAN. As in previous studies with rabbit liver metallothionein-2a, the structure calculations were performed separately for the alpha and beta-domains containing the 4 and 3-metal clusters, respectively, since no interdomain constraints were found. For both domains, the global polypeptide fold, the location of polypeptide secondary structure elements, the architecture of the metal-sulfur cluster and the local chirality of the metal co-ordination are very similar to the solution structure of rabbit metallothionein-2a, but show considerable difference relative to the crystal structure of rat metallothionein-2.

Metal swap between Zn7-metallothionein-3 and amyloid-|[beta]||[ndash]|Cu protects against amyloid-|[beta]| toxicity

Aberrant interactions of copper and zinc ions with the amyloid-beta peptide (Abeta) potentiate Alzheimers disease (AD) by participating in the aggregation process of Abeta and in the generation of reactive oxygen species (ROS). The ROS production and the neurotoxicity of Abeta are associated with copper binding. Metallothionein-3 (Zn(7)MT-3), an intra- and extracellularly occurring metalloprotein, is highly expressed in the brain and downregulated in AD. This protein protects, by an unknown mechanism, cultured neurons from the toxicity of Abeta. Here, we show that a metal swap between Zn(7)MT-3 and soluble and aggregated Abeta(1-40)-Cu(II) abolishes the ROS production and the related cellular toxicity. In this process, copper is reduced by the protein thiolates forming Cu(I)(4)Zn(4)MT-3, in which an air-stable Cu(I)(4)-thiolate cluster and two disulfide bonds are present. The discovered protective effect of Zn(7)MT-3 from the copper-mediated Abeta(1-40) toxicity may lead to new therapeutic strategies for treating AD.