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Special Issue "Metallothioneins in Bioinorganic Chemistry: Recent Developments"

A special issue of International Journal of Molecular Sciences (ISSN 1422-0067). This special issue belongs to the section "Bioinorganic Chemistry".

Deadline for manuscript submissions: closed (16 April 2017)

Special Issue Editor

Guest Editor
PD. Dr. Eva Freisinger

Department of Chemistry, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland
Website | E-Mail
Phone: +41 44 63 546 21
Interests: structures and properties of plant metallothioneins; gene cloning, protein over-expression, and purification; spectroscopic and biochemical characterization of MTs; determination of metal ion binding affinities and redox states; structure determination of MTs by X-ray and NMR

Special Issue Information

Dear Colleagues,

“Metallothioneins”—this is the name of a superfamily combining proteins with two rather basic characteristics, a high percentage of thiolate groups in form of cysteine residues and the ability to coordinate certain metal ions via these thiolate ligands. There is, however, no requirement for a specific function or structure. While the name was initially invented 60 years ago for a class of proteins found in mammalians, all having very similar sequences and a common three-dimensional structure involving metal-thiolate clusters, nowadays, proteins from nearly all kingdoms of life, among them vertebrates, molluscs, nematodes, prokaryotes, fungi, and plants are included. Often the classification as metallothionein is solely based on the respective gene sequence without further knowledge about the metal ion binding ability of the encoded protein. The elucidation of the metal ion binding modes including cluster formation, the spectroscopic properties and last not least the three-dimensional structures of the members of this very variable superfamily belongs to the classical research fields of Bioinorganic Chemistry. Beyond this basic characterisation, unravelling the diversity of functions including, but not being restricted to, metal ion homeostasis and detoxification as well as redox activities, investigating the role of MTs in certain diseases, and inventing biotechnological applications are hot topics in MT research.

It is the aim of this Special Issue to highlight the recent developments in the field in form of Topical Reviews, Articles, and Short Communications. I invite you all to make this Special Issue a valuable source of new and exciting information about this very unique protein superfamily.

PD. Dr. Eva Freisinger
Guest Editor

Manuscript Submission Information

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Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. International Journal of Molecular Sciences is an international peer-reviewed open access monthly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. The Article Processing Charge (APC) for publication in this open access journal is 1800 CHF (Swiss Francs). Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

Keywords

•    metallothioneins (MTs)
•    metal clusters
•    zinc
•    cadmium
•    copper
•    metal ion homeostasis
•    heavy metal detoxification
•    reactive oxygen species  (ROS) scavenging
•    spectroscopy

Published Papers (10 papers)

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Research

Jump to: Review

Open AccessArticle Metallothionein from Wild Populations of the African Catfish Clarias gariepinus: From Sequence, Protein Expression and Metal Binding Properties to Transcriptional Biomarker of Metal Pollution
Int. J. Mol. Sci. 2017, 18(7), 1548; doi:10.3390/ijms18071548
Received: 2 May 2017 / Revised: 3 July 2017 / Accepted: 13 July 2017 / Published: 18 July 2017
Cited by 1 | PDF Full-text (2363 KB) | HTML Full-text | XML Full-text | Supplementary Files
Abstract
Anthropogenic pollution with heavy metals is an on-going concern throughout the world, and methods to monitor release and impact of heavy metals are of high importance. With a view to probe its suitability as molecular biomarker of metal pollution, this study has determined
[...] Read more.
Anthropogenic pollution with heavy metals is an on-going concern throughout the world, and methods to monitor release and impact of heavy metals are of high importance. With a view to probe its suitability as molecular biomarker of metal pollution, this study has determined a coding sequence for metallothionein of the African sharptooth catfish Clarias gariepinus. The gene product was recombinantly expressed in Escherichia coli in presence of Zn(II), Cd(II), or Cu, and characterised by Electrospray Ionisation Mass Spectrometry and elemental analysis. C. gariepinus MT displays typical features of fish MTs, including 20 conserved cysteines, and seven bound divalent cations (Zn(II) or Cd(II)) when saturated. Livers from wild C. gariepinus fish collected in all three seasons from four different sites on the Kafue River of Zambia were analysed for their metal contents and for MT expression levels by quantitative PCR. Significant correlations were found between Zn and Cu levels and MT expression in livers, with MT expression clearly highest at the most polluted site, Chililabombwe, which is situated in the Copperbelt region. Based on our findings, hepatic expression of MT from C. gariepinus may be further developed as a major molecular biomarker of heavy metal pollution resulting from mining activities in this region. Full article
(This article belongs to the Special Issue Metallothioneins in Bioinorganic Chemistry: Recent Developments)
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Open AccessArticle Biomphalaria glabrata Metallothionein: Lacking Metal Specificity of the Protein and Missing Gene Upregulation Suggest Metal Sequestration by Exchange Instead of through Selective Binding
Int. J. Mol. Sci. 2017, 18(7), 1457; doi:10.3390/ijms18071457
Received: 30 May 2017 / Revised: 29 June 2017 / Accepted: 1 July 2017 / Published: 6 July 2017
Cited by 1 | PDF Full-text (2338 KB) | HTML Full-text | XML Full-text
Abstract
The wild-type metallothionein (MT) of the freshwater snail Biomphalaria glabrata 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 Cd2+,
[...] Read more.
The wild-type metallothionein (MT) of the freshwater snail Biomphalaria glabrata 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 Cd2+, Zn2+ and Cu+, applying spectroscopic and mass-spectrometric methods. In addition, the upregulation of the Biomphalaria glabrata MT 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. They lacked a clear metal-binding preference for any of the three metal ions assayed—which, to this degree, is clearly unprecedented in the world of Gastropoda MTs. There were, however, slight differences in copper-binding abilities between the two allelic variants. Overall, the missing metal specificity of the two recombinant MTs goes hand in hand with lacking upregulation of the respective MT gene. This suggests that in vivo, the Biomphalaria glabrata MT may be more important for metal replacement reactions through a constitutively abundant form, rather than for metal sequestration by high binding specificity. There are indications that the MT of Biomphalaria glabrata may share its unspecific features with MTs from other freshwater snails of the Hygrophila family. Full article
(This article belongs to the Special Issue Metallothioneins in Bioinorganic Chemistry: Recent Developments)
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Open AccessArticle Analysis of Metal-Binding Features of the Wild Type and Two Domain-Truncated Mutant Variants of Littorina littorea Metallothionein Reveals Its Cd-Specific Character
Int. J. Mol. Sci. 2017, 18(7), 1452; doi:10.3390/ijms18071452
Received: 31 May 2017 / Revised: 27 June 2017 / Accepted: 1 July 2017 / Published: 6 July 2017
Cited by 1 | PDF Full-text (2394 KB) | HTML Full-text | XML Full-text | Supplementary Files
Abstract
After the resolution of the 3D structure of the Cd9-aggregate of the Littorina littorea 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
[...] Read more.
After the resolution of the 3D structure of the Cd9-aggregate of the Littorina littorea 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. Our results also revealed a major specificity, and therefore a better metal-coordinating performance of the three proteins for Cd(II) than for Zn(II), although the analysis of the Zn(II)/Cd(II) displacement reaction clearly demonstrates a lack of any type of cooperativity in Cd(II) binding. Contrarily, the analysis of their Cu(I) binding abilities revealed that every LlMT domain is prone to build Cu4-aggregates, the whole MT working by modules analogously to, as previously described, certain fungal MTs, like those of C. neoformans and T. mesenterica. It is concluded that the Littorina littorea MT is a Cd-specific protein that (beyond its extended binding capacity through an additional Cd-binding domain) confers to Littorina littorea a particular adaptive advantage in its changeable marine habitat. Full article
(This article belongs to the Special Issue Metallothioneins in Bioinorganic Chemistry: Recent Developments)
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Open AccessArticle Function of Metallothionein-3 in Neuronal Cells: Do Metal Ions Alter Expression Levels of MT3?
Int. J. Mol. Sci. 2017, 18(6), 1133; doi:10.3390/ijms18061133
Received: 19 April 2017 / Revised: 16 May 2017 / Accepted: 16 May 2017 / Published: 25 May 2017
Cited by 1 | PDF Full-text (1448 KB) | HTML Full-text | XML Full-text | Supplementary Files
Abstract
A study of factors proposed to affect metallothionein-3 (MT3) function was carried out to elucidate the opaque role MT3 plays in human metalloneurochemistry. Gene expression of Mt2 and Mt3 was examined in tissues extracted from the dentate gyrus of mouse brains and in
[...] Read more.
A study of factors proposed to affect metallothionein-3 (MT3) function was carried out to elucidate the opaque role MT3 plays in human metalloneurochemistry. Gene expression of Mt2 and Mt3 was examined in tissues extracted from the dentate gyrus of mouse brains and in human neuronal cell cultures. The whole-genome gene expression analysis identified significant variations in the mRNA levels of genes associated with zinc homeostasis, including Mt2 and Mt3. Mt3 was found to be the most differentially expressed gene in the identified groups, pointing to the existence of a factor, not yet identified, that differentially controls Mt3 expression. To examine the expression of the human metallothioneins in neurons, mRNA levels of MT3 and MT2 were compared in BE(2)C and SH-SY5Y cell cultures treated with lead, zinc, cobalt, and lithium. MT2 was highly upregulated by Zn2+ in both cell cultures, while MT3 was not affected, and no other metal had an effect on either MT2 or MT3. Full article
(This article belongs to the Special Issue Metallothioneins in Bioinorganic Chemistry: Recent Developments)
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Open AccessArticle Regulatory Plasticity of Earthworm wMT-2 Gene Expression
Int. J. Mol. Sci. 2017, 18(6), 1113; doi:10.3390/ijms18061113
Received: 15 April 2017 / Revised: 16 May 2017 / Accepted: 21 May 2017 / Published: 24 May 2017
Cited by 1 | PDF Full-text (2158 KB) | HTML Full-text | XML Full-text
Abstract
Metallothioneins (MTs) are multifunctional proteins occurring throughout the animal kingdom. While the expression and transcriptional regulation of MTs is well-studied in vertebrates, the mechanism of MT activation is still unknown for most invertebrates. Therefore, we examined wMT-2 gene regulation and expression patterns in
[...] Read more.
Metallothioneins (MTs) are multifunctional proteins occurring throughout the animal kingdom. While the expression and transcriptional regulation of MTs is well-studied in vertebrates, the mechanism of MT activation is still unknown for most invertebrates. Therefore, we examined wMT-2 gene regulation and expression patterns in Lumbricus rubellus and L. terrestris. Transcription levels, the occupation of DNA binding sites, the expression of putative transcriptional regulators, and promotor DNA methylation were determined. We found that wMT-2 expression does not follow a circadian pattern. However, Cd-induced wMT-2 induction was observed, and was, interestingly, suppressed by physical injury. Moreover, the promotor region that is responsible for the wMT-2 gene regulation was elucidated. ATF, a putative transcriptional regulator, showed increased phosphorylation upon Cd exposure, suggesting that it plays a major role in wMT-2 gene activation. The promotor methylation of wMT-2, on the other hand, is probably not involved in transcriptional regulation. Elucidating the regulatory mechanism of the earthworm MT gene activation might provide insights into the molecular coordination of the environmental stress response in invertebrates, and might also reveal a link to wound repair and, in a broader sense, to immunity. Full article
(This article belongs to the Special Issue Metallothioneins in Bioinorganic Chemistry: Recent Developments)
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Open AccessArticle The Application of Curve Fitting on the Voltammograms of Various Isoforms of Metallothioneins–Metal Complexes
Int. J. Mol. Sci. 2017, 18(3), 610; doi:10.3390/ijms18030610
Received: 11 February 2017 / Revised: 3 March 2017 / Accepted: 7 March 2017 / Published: 11 March 2017
Cited by 3 | PDF Full-text (1380 KB) | HTML Full-text | XML Full-text | Supplementary Files
Abstract
The translation of metallothioneins (MTs) is one of the defense strategies by which organisms protect themselves from metal-induced toxicity. MTs belong to a family of proteins comprising MT-1, MT-2, MT-3, and MT-4 classes, with multiple isoforms within each class. The main aim of
[...] Read more.
The translation of metallothioneins (MTs) is one of the defense strategies by which organisms protect themselves from metal-induced toxicity. MTs belong to a family of proteins comprising MT-1, MT-2, MT-3, and MT-4 classes, with multiple isoforms within each class. The main aim of this study was to determine the behavior of MT in dependence on various externally modelled environments, using electrochemistry. In our study, the mass distribution of MTs was characterized using MALDI-TOF. After that, adsorptive transfer stripping technique with differential pulse voltammetry was selected for optimization of electrochemical detection of MTs with regard to accumulation time and pH effects. Our results show that utilization of 0.5 M NaCl, pH 6.4, as the supporting electrolyte provides a highly complicated fingerprint, showing a number of non-resolved voltammograms. Hence, we further resolved the voltammograms exhibiting the broad and overlapping signals using curve fitting. The separated signals were assigned to the electrochemical responses of several MT complexes with zinc(II), cadmium(II), and copper(II), respectively. Our results show that electrochemistry could serve as a great tool for metalloproteomic applications to determine the ratio of metal ion bonds within the target protein structure, however, it provides highly complicated signals, which require further resolution using a proper statistical method, such as curve fitting. Full article
(This article belongs to the Special Issue Metallothioneins in Bioinorganic Chemistry: Recent Developments)
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Review

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Open AccessReview Proteomic High Affinity Zn2+ Trafficking: Where Does Metallothionein Fit in?
Int. J. Mol. Sci. 2017, 18(6), 1289; doi:10.3390/ijms18061289
Received: 5 May 2017 / Revised: 7 June 2017 / Accepted: 9 June 2017 / Published: 17 June 2017
Cited by 1 | PDF Full-text (1299 KB) | HTML Full-text | XML Full-text
Abstract
The cellular constitution of Zn-proteins and Zn-dependent signaling depend on the capacity of Zn2+ to find specific binding sites in the face of a plethora of other high affinity ligands. The most prominent of these is metallothionein (MT). It serves as a
[...] Read more.
The cellular constitution of Zn-proteins and Zn-dependent signaling depend on the capacity of Zn2+ to find specific binding sites in the face of a plethora of other high affinity ligands. The most prominent of these is metallothionein (MT). It serves as a storage site for Zn2+ under various conditions, and has chemical properties that support a dynamic role for MT in zinc trafficking. Consistent with these characteristics, changing the availability of zinc for cells and tissues causes rapid alteration of zinc bound to MT. Nevertheless, zinc trafficking occurs in metallothionein-null animals and cells, hypothetically making use of proteomic binding sites to mediate the intracellular movements of zinc. Like metallothionein, the proteome contains a large concentration of proteins that strongly coordinate zinc. In this environment, free Zn2+ may be of little significance. Instead, this review sets forth the basis for the hypothesis that components of the proteome and MT jointly provide the platform for zinc trafficking. Full article
(This article belongs to the Special Issue Metallothioneins in Bioinorganic Chemistry: Recent Developments)
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Open AccessReview The Functions of Metamorphic Metallothioneins in Zinc and Copper Metabolism
Int. J. Mol. Sci. 2017, 18(6), 1237; doi:10.3390/ijms18061237
Received: 25 April 2017 / Revised: 2 June 2017 / Accepted: 3 June 2017 / Published: 9 June 2017
Cited by 3 | PDF Full-text (604 KB) | HTML Full-text | XML Full-text
Abstract
Recent discoveries in zinc biology provide a new platform for discussing the primary physiological functions of mammalian metallothioneins (MTs) and their exquisite zinc-dependent regulation. It is now understood that the control of cellular zinc homeostasis includes buffering of Zn2+ ions at picomolar
[...] Read more.
Recent discoveries in zinc biology provide a new platform for discussing the primary physiological functions of mammalian metallothioneins (MTs) and their exquisite zinc-dependent regulation. It is now understood that the control of cellular zinc homeostasis includes buffering of Zn2+ ions at picomolar concentrations, extensive subcellular re-distribution of Zn2+, the loading of exocytotic vesicles with zinc species, and the control of Zn2+ ion signalling. In parallel, characteristic features of human MTs became known: their graded affinities for Zn2+ and the redox activity of their thiolate coordination environments. Unlike the single species that structural models of mammalian MTs describe with a set of seven divalent or eight to twelve monovalent metal ions, MTs are metamorphic. In vivo, they exist as many species differing in redox state and load with different metal ions. The functions of mammalian MTs should no longer be considered elusive or enigmatic because it is now evident that the reactivity and coordination dynamics of MTs with Zn2+ and Cu+ match the biological requirements for controlling—binding and delivering—these cellular metal ions, thus completing a 60-year search for their functions. MT represents a unique biological principle for buffering the most competitive essential metal ions Zn2+ and Cu+. How this knowledge translates to the function of other families of MTs awaits further insights into the specifics of how their properties relate to zinc and copper metabolism in other organisms. Full article
(This article belongs to the Special Issue Metallothioneins in Bioinorganic Chemistry: Recent Developments)
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Open AccessReview Mammalian Metallothionein-3: New Functional and Structural Insights
Int. J. Mol. Sci. 2017, 18(6), 1117; doi:10.3390/ijms18061117
Received: 15 April 2017 / Revised: 12 May 2017 / Accepted: 15 May 2017 / Published: 24 May 2017
Cited by 2 | PDF Full-text (869 KB) | HTML Full-text | XML Full-text
Abstract
Metallothionein-3 (MT-3), a member of the mammalian metallothionein (MT) family, is mainly expressed in the central nervous system (CNS). MT-3 possesses a unique neuronal growth inhibitory activity, and the levels of this intra- and extracellularly occurring metalloprotein are markedly diminished in the brain
[...] Read more.
Metallothionein-3 (MT-3), a member of the mammalian metallothionein (MT) family, is mainly expressed in the central nervous system (CNS). MT-3 possesses a unique neuronal growth inhibitory activity, and the levels of this intra- and extracellularly occurring metalloprotein are markedly diminished in the brain of patients affected by a number of metal-linked neurodegenerative disorders, including Alzheimer’s disease (AD). In these pathologies, the redox cycling of copper, accompanied by the production of reactive oxygen species (ROS), plays a key role in the neuronal toxicity. Although MT-3 shares the metal-thiolate clusters with the well-characterized MT-1 and MT-2, it shows distinct biological, structural and chemical properties. Owing to its anti-oxidant properties and modulator function not only for Zn, but also for Cu in the extra- and intracellular space, MT-3, but not MT-1/MT-2, protects neuronal cells from the toxicity of various Cu(II)-bound amyloids. In recent years, the roles of zinc dynamics and MT-3 function in neurodegeneration are slowly emerging. This short review focuses on the recent developments regarding the chemistry and biology of MT-3. Full article
(This article belongs to the Special Issue Metallothioneins in Bioinorganic Chemistry: Recent Developments)
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Open AccessReview Residue Modification and Mass Spectrometry for the Investigation of Structural and Metalation Properties of Metallothionein and Cysteine-Rich Proteins
Int. J. Mol. Sci. 2017, 18(5), 913; doi:10.3390/ijms18050913
Received: 24 March 2017 / Revised: 18 April 2017 / Accepted: 20 April 2017 / Published: 26 April 2017
PDF Full-text (4227 KB) | HTML Full-text | XML Full-text
Abstract
Structural information regarding metallothioneins (MTs) has been hard to come by due to its highly dynamic nature in the absence of metal-thiolate cluster formation and crystallization difficulties. Thus, typical spectroscopic methods for structural determination are limited in their usefulness when applied to MTs.
[...] Read more.
Structural information regarding metallothioneins (MTs) has been hard to come by due to its highly dynamic nature in the absence of metal-thiolate cluster formation and crystallization difficulties. Thus, typical spectroscopic methods for structural determination are limited in their usefulness when applied to MTs. Mass spectrometric methods have revolutionized our understanding of protein dynamics, structure, and folding. Recently, advances have been made in residue modification mass spectrometry in order to probe the hard-to-characterize structure of apo- and partially metalated MTs. By using different cysteine specific alkylation reagents, time dependent electrospray ionization mass spectrometry (ESI-MS), and step-wise “snapshot” ESI-MS, we are beginning to understand the dynamics of the conformers of apo-MT and related species. In this review we highlight recent papers that use these and similar techniques for structure elucidation and attempt to explain in a concise manner the data interpretations of these complex methods. We expect increasing resolution in our picture of the structural conformations of metal-free MTs as these techniques are more widely adopted and combined with other promising tools for structural elucidation. Full article
(This article belongs to the Special Issue Metallothioneins in Bioinorganic Chemistry: Recent Developments)
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Planned Papers

The below list represents only planned manuscripts. Some of these manuscripts have not been received by the Editorial Office yet. Papers submitted to MDPI journals are subject to peer-review.

Article
Proteomic Zn2+ Trafficking:  Where Does Metallothionein Fit in?
Petering et al.

Review
Mammalian metallothionein-3: new functional and structural insights
Vasak et al.

Article
In depth analysis of the metal binding abilities of Ll-MT (Litorina littorea metallothionein) and two truncated mutants
Capdevila et al.

Article
In depth analysis of the metal binding abilities of BgMT (Biomphalaria glabrata metallothionein) and two crucial, natural  (CY and KN) mutants
Capdevila et al.

Article
cDNA sequence, protein expression and metal binding properties of metallothionein from wild populations of the catfish Clarias gariepinus: Potential as biomarker for metal pollution of Zambia’s Kafue River
Blindauer et al.

Review
The structures of human metallothioneins and their functions in human zinc metabolism
Maret et al.

Article
Regulation of MT3 by a variety of metal ions in human neuronal cell cultures
Austin et al.

Article
Earthworm MT
Höckner et al.

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