Metallomics reveals a persisting impact of cadmium on the evolution of metal-selective snail metallothioneins.

The tiny contribution of cadmium (Cd) to the composition of the earth's crust contrasts with its high biological significance, owing mainly to the competition of Cd with the essential zinc (Zn) for suitable metal binding sites in proteins. In this context it was speculated that in several animal lineages, the protein family of metallothioneins (MTs) has evolved to specifically detoxify Cd. Although the multi-functionality and heterometallic composition of MTs in most animal species does not support such an assumption, there are some exceptions to this role, particularly in animal lineages at the roots of animal evolution.

The Solution Structure and Dynamics of Cd-Metallothionein from Reveal Optimization for Binding Cd over Zn.

Metallothioneins (MTs) are cysteine-rich polypeptides that are naturally found coordinated to monovalent and/or divalent transition metal ions. Three metallothionein isoforms from the Roman snail are known. They differ in their physiological metal load and in their specificity for transition metal ions such as Cd (HpCdMT isoform) and Cu (HpCuMT isoform) or in the absence of a defined metal specificity (HpCd/CuMT isoform).

Mouse metallothionein-1 and metallothionein-2 are not biologically interchangeable in an animal model of multiple sclerosis, EAE.

Mouse metallothionein-1 and 2 (MT1 and MT2) are often considered physiologically equivalent, because they are normally regulated coordinately by a wide range of stimuli, and it is assumed that in vivo they will be normally fully loaded with zinc(ii) (Zn7-MT1/2), although other metal ions, such as copper(i), may be eventually found as well. However, mouse MT2, in contrast to MT1, exhibits a preference for Zn(ii) coordination in comparison to that for Cu(i), which might underlie putatively different biological functions for these two mammalian isoforms. We have characterized the effects of exogenously administered mouse MT1 and MT2, and of transgenic Mt1 overexpression, in an animal model of multiple sclerosis (MS), experimental autoimmune encephalomyelitis (EAE), by active immunization with MOG35-55 peptide.

Copper redox chemistry of plant frataxins.

The presence of a conserved cysteine residue in the C-terminal amino acid sequences of plant frataxins differentiates these frataxins from those of other kingdoms and may be key in frataxin assembly and function. We report a full study on the ability of Arabidopsis (AtFH) and Zea mays (ZmFH-1 and ZmFH-2) frataxins to assemble into disulfide-bridged dimers by copper-driven oxidation and to revert to monomers by chemical reduction. We monitored the redox assembly-disassembly process by electrospray ionization mass spectrometry, electrophoresis, UV-Vis spectroscopy, and fluorescence measurements.

Biomphalaria glabrata Metallothionein: Lacking Metal Specificity of the Protein and Missing Gene Upregulation Suggest Metal Sequestration by Exchange Instead of through Selective Binding.

The wild-type metallothionein (MT) of the freshwater snail and a natural allelic mutant of it in which a lysine residue was replaced by an asparagine residue, were recombinantly expressed and analyzed for their metal-binding features with respect to Cd, Zn and Cu⁺, applying spectroscopic and mass-spectrometric methods. In addition, the upregulation of the gene was assessed by quantitative real-time detection PCR. The two recombinant proteins revealed to be very similar in most of their metal binding features.

Analysis of Metal-Binding Features of the Wild Type and Two Domain-Truncated Mutant Variants of Littorina littorea Metallothionein Reveals Its Cd-Specific Character.

After the resolution of the 3D structure of the Cd₉-aggregate of the metallothionein (MT), we report here a detailed analysis of the metal binding capabilities of the wild type MT, LlwtMT, and of two truncated mutants lacking either the N-terminal domain, Lltr2MT, or both the N-terminal domain, plus four extra flanking residues (SSVF), Lltr1MT. The recombinant synthesis and in vitro studies of these three proteins revealed that LlwtMT forms unique M₉-LlwtMT complexes with Zn(II) and Cd(II), while yielding a complex mixture of heteronuclear Zn,Cu-LlwtMT species with Cu(I). As expected, the truncated mutants gave rise to unique M₆-LltrMT complexes and Zn,Cu-LltrMT mixtures of lower stoichiometry with respect to LlwtMT, with the SSVF fragment having an influence on their metal binding performance.

Identification of two frataxin isoforms in Zea mays: Structural and functional studies.

Frataxin is a ubiquitous protein that plays a role in Fe-S cluster biosynthesis and iron and heme metabolism, although its molecular functions are not entirely clear. In non-photosynthetic eukaryotes, frataxin is encoded by a single gene, and the protein localizes to mitochondria. Here we report the presence of two functional frataxin isoforms in Zea mays, ZmFH-1 and ZmFH-2.

Structural Adaptation of a Protein to Increased Metal Stress: NMR Structure of a Marine Snail Metallothionein with an Additional Domain.

In this study, we present an NMR structure of the metallothionein (MT) from the snail Littorina littorea (LlMT) in complex with Cd . LlMT is capable of binding 9 Zn or 9 Cd ions. Sequence alignments with other snail MTs revealed that the protein is likely composed of three domains.

Metagenomics analysis reveals a new metallothionein family: Sequence and metal-binding features of new environmental cysteine-rich proteins.

Metallothioneins are cysteine-rich proteins, which function as (i) metal carriers in basal cell metabolism and (ii) protective metal chelators in conditions of metal excess. Metallothioneins have been characterized from different eukaryotic model and cultivable species. Presently, they are categorized in 15 families but evolutionary relationships between these metallothionein families remain unresolved.

The Fungus Tremella mesenterica Encodes the Longest Metallothionein Currently Known: Gene, Protein and Metal Binding Characterization.

Fungal Cu-thioneins, and among them, the paradigmatic Neurospora crassa metallothionein (MT) (26 residues), were once considered as the shortest MTs--the ubiquitous, versatile metal-binding proteins--among all organisms, and thus representatives of their primeval forms. Nowadays, fungal MTs of diverse lengths and sequence features are known, following the huge heterogeneity of the Kingdom of Fungi. At the opposite end of N.

Comparative Raman study of four plant metallothionein isoforms: Insights into their Zn(II) clusters and protein conformations.

Four Metallothioneins (MTs) from soybean (Glycine max) were heterologously synthesized and comparatively analysed by Raman spectroscopy. The participation of protein donor groups (S-thiol and N-imidazol) in Zn(II) chelation, as well as the presence of secondary structure elements was comparatively analysed. Metal clusters with different geometry can be hypothesised for the four GmMTs: a cubane-like or an adamantane-like metal cluster in Zn-GmMT1, and dinuclear Zn-S clusters in Zn-GmMT2, Zn-GmMT3 and Zn-GmMT4.

Does Variation of the Inter-Domain Linker Sequence Modulate the Metal Binding Behaviour of Helix pomatia Cd-Metallothionein?

Snail metallothioneins (MTs) constitute an ideal model to study structure/function relationships in these metal-binding polypeptides. Helix pomatia harbours three MT isoforms: the highly specific CdMT and CuMT, and an unspecific Cd/CuMT, which represent paralogous proteins with extremely different metal binding preferences while sharing high sequence similarity. Preceding work allowed assessing that, although, the Cys residues are responsible for metal ion coordination, metal specificity or preference is achieved by diversification of the amino acids interspersed between them.

Understanding the 7-Cys module amplification of C. neoformans metallothioneins: how high capacity Cu-binding polypeptides are built to neutralize host nutritional immunity.

Cryptococcus neoformans metallothioneins (MTs), CnMT1 and CnMT2, have been identified as essential infectivity and virulence factors of this pathogen. Both MTs are unusually long Cu-thioneins, exhibiting protein architecture and metal-binding abilities compatible with the hypothesis of resulting from three and five tandem repetitions of 7-Cys motives, respectively, each of them folding into Cu5-clusters. Through the study of the Zn(II)- and Cu(I)-binding capabilities of several CnMT1 truncated mutants, we show that a 7-Cys segment of CnMT1 folds into Cu5-species, of additive capacity when joined in tandem.

Hints for metal-preference protein sequence determinants: different metal binding features of the five tetrahymena thermophila metallothioneins.

The metal binding preference of metallothioneins (MTs) groups them in two extreme subsets, the Zn/Cd- and the Cu-thioneins. Ciliates harbor the largest MT gene/protein family reported so far, including 5 paralogs that exhibit relatively low sequence similarity, excepting MTT2 and MTT4. In Tetrahymena thermophila, three MTs (MTT1, MTT3 and MTT5) were considered Cd-thioneins and two (MTT2 and MTT4) Cu-thioneins, according to gene expression inducibility and phylogenetic analysis.

Sunflower metallothionein family characterisation. Study of the Zn(II)- and Cd(II)-binding abilities of the HaMT1 and HaMT2 isoforms.

Plant metallothioneins (MTs) constitute a family of small Cys-rich proteins capable of coordinating metal ions, significantly differing from microbial and animal MTs. They are divided into four subfamilies depending on the Cys pattern in their sequence. In this work, the MT system of the sunflower plant (Helianthus annuus) has been defined, with ten genes coding for MTs (HaMT) belonging to the four plant MT subfamilies; three HaMT1, four HaMT2, one HaMT3 and two HaMT4 isoforms.

Rhenium and technetium tricarbonyl, {M(CO)3} (+) (M = Tc, Re), binding to mammalian metallothioneins: new insights into chemical and radiopharmaceutical implications.

This paper deals with the binding of the four mammalian metallothioneins (MTs) to the organometallic metal fragment {fac-M(CO)3}(+) (M = (99)Tc, Re), which is highly promising for the preparation of second-generation radiopharmaceuticals. The study of the transmetallation reaction between zinc and rhenium in Zn7-MT1 by means of UV-vis and CD spectroscopy demonstrated the incorporation of the {fac-Re(CO)3}(+) fragment to the MTs. This reaction should be performed at 70 °C to accelerate the reaction rate, a result that is consistent with the reported reactivity of the rhenium fragment.

Chemically and biologically harmless versus harmful ferritin/copper-metallothionein couples.

The simultaneous measurement of the decrease of available Fe(II) ions and the increase of available Fe(III) ions allowed the analysis of the ferroxidase activity of two distinct apoferritins. Although recombinant human apoferritin (HuFtH) rapidly oxidizes Fe(II) to Fe(III) , this iron is not properly stored in the ferritin cavity, as otherwise occurs in horse-spleen H/L-apoferritin (HsFt; H=heavy subunit, L=light subunit). Iron storage in these apoferritins was also studied in the presence of two copper-loaded mammalian metallothioneins (MT2 and MT3), a scenario that occurs in different brain-cell types.

Understanding the interaction of an antitumoral platinum(II) 7-azaindolate complex with proteins and DNA.

The reactivity of the [Pt(dmba)(aza-N1)(dmso)] complex 1, (a potential antitumoral drug with lower IC50 than cisplatin in several tumoral cell lines) with different proteins and oligonucleotides is investigated by means of mass spectrometry (ESI-TOF MS). The results obtained show a particular binding behaviour of this platinum(II) complex. The interaction of 1 with the assayed proteins apparently takes place by Pt-binding to the most accessible coordinating amino acids, presumably at the surface of the protein -this avoiding protein denaturation or degradation- with the subsequent release of one or two ligands of 1.

Cantareus aspersus metallothionein metal binding abilities: the unspecific CaCd/CuMT isoform provides hints about the metal preference determinants in metallothioneins.

In Proteomics, gene/protein families including both specialized and non-specialized paralogs are an invaluable tool to study the evolution of structure/function relationships in proteins. Metallothioneins (MTs) of the pulmonate gastropod molluscs (snails) offer one of the best materials to study the metal-binding specificity of proteins, because they consist of a polymorphic system that includes members with extremely distinct metal preferences but with a high protein sequence similarity. Cantareus aspersus was the first snail where three paralogous MTs were isolated: the highly specific cadmium (CaCdMT) and copper (CaCuMT) isoforms, and an unspecific CaCd/CuMT isoform, so called because it was natively isolated as a mixed Cd and Cu complex.

His-containing plant metallothioneins: comparative study of divalent metal-ion binding by plant MT3 and MT4 isoforms.

Metallothioneins (MTs) are a superfamily of Cys-rich, low-molecular weight metalloproteins that bind heavy metal ions. These cytosolic metallopeptides, which exist in most living organisms, are thought to be involved in metal homeostasis, metal detoxification, and oxidative stress protection. In this work, we characterise the Zn(II)- and Cd(II)-binding abilities of plant type 3 and type 4 MTs identified in soybean and sunflower, both of them being His-containing peptides.

Cognate and noncognate metal ion coordination in metal-specific metallothioneins: the Helix pomatia system as a model.

The Helix pomatia metallothionein (MT) system, namely, its two highly specific forms, HpCdMT and HpCuMT, has offered once again an optimum model to study metal-protein specificity. The present work investigates the most unexplored aspect of the coordination behavior of MT polypeptides with respect to either cognate or noncognate metal ions, as opposed to the standard studies of cognate metal ion coordination. To this end, we analyzed the in vivo synthesis of the corresponding complexes with their noncognate metals, and we performed a detailed spectroscopic and spectrometric study of the Zn(2+)/Cd(2+) and Zn(2+)/Cu(+) in vitro replacement reactions on the initial Zn-HpMT species.

On the molecular relationships between high-zinc tolerance and aconitase (Aco1) in Saccharomyces cerevisiae.

Zinc is an essential metal for all organisms, as it participates in the structure and/or function of many proteins. However, zinc excess is as deleterious to cells as zinc deficiency. A genome-wide study of the transcriptomic response to high zinc in S.

In vivo-folded metal-metallothionein 3 complexes reveal the Cu-thionein rather than Zn-thionein character of this brain-specific mammalian metallothionein.

Metallothionein-3 (MT3) is one of the four mammalian metallothioneins (MT), and is constitutively synthesized in the brain. MT3 acts both intracellularly and extracellularly in this organ, performing functions related to neuronal growth and physiological metal (Zn and Cu) handling. It appears to be involved in the prevention of neurodegenerative disorders caused by insoluble Cu-peptide aggregates, as it triggers a Zn-Cu swap that may counteract the deleterious presence of copper in neural tissues.

Full characterization of the Cu-, Zn-, and Cd-binding properties of CnMT1 and CnMT2, two metallothioneins of the pathogenic fungus Cryptococcus neoformans acting as virulence factors.

We report here the full characterization of the metal binding abilities of CnMT1 and CnMT2, two Cryptococcus neoformans proteins recently identified as metallothioneins (MTs), which have been shown to play a crucial role in the virulence and pathogenicity of this human-infecting fungus. In this work, we first performed a thorough in silico study of the CnMT1 and CnMT2 genes, cDNAs and corresponding encoded products. Subsequently, the Zn(II)-, Cd(II)- and Cu(I) binding abilities of both proteins were fully determined through the analysis of the metal-to-protein stoichiometries and the structural features (determined by ESI-MS, CD, ICP-AES and UV-vis spectroscopies) of the corresponding recombinant Zn-, Cd- and Cu-MT preparations synthesized in metal-enriched media.

Mammalian MT1 and MT2 metallothioneins differ in their metal binding abilities.

Metallothioneins (MTs) constitute a universal family of polymorphic, ubiquitous small Cys-rich metal-binding polypeptides that in mammals are represented by four highly similar isoforms (MT1 to MT4). MT1 and MT2 have generally been considered as equivalent proteins, so that they are commonly referred to as MT1/MT2. However, transcription data have suggested a differential behavior for both gene products.