Spectroscopic characterization of a fruit-specific metallothionein: M. acuminata MT3

Metallothioneins (MTs) are ubiquitous low molecular mass, cysteine-rich proteins with the ability to bind d¹⁰ metal ions in the form of metal-thiolate clusters. In contrast to the vertebrate forms, knowledge about the properties of members of the plant metallothionein family is still scarce. The amino acid sequences of plant MTs are distinctively different to the sequences of other MT species. The protein under investigation, Musa acuminata (banana) MT3, belongs to the plant MT fruit-specific p3 subfamily. With a total of 10 cysteine residues, MT3 features a cysteine content and percentage that is more comparable to fungal and prokaryotic MTs than to the well characterized mammalian iso-forms. The gene sequence encoding MT3 was cloned into a suitable vector and the protein was recombinantly overexpressed in Escherichia coli. MT3 is able to coordinate a maximum of four divalent d¹⁰ metal ions under the formation of metal-thiolate clusters. The hitherto unknown spectroscopic behavior of MT3 in combination with the metal ions Zn²⁺, Cd²⁺, Pb²⁺, and Hg²⁺ will be presented and gives rise to the existence of a weaker metal ion coordination site. The pH stability of the investigated zinc and cadmium clusters is comparable to the values found for other plant metallothioneins though significantly lower than for the mammalian iso-forms. Possible metal-thiolate cluster structures will additionally be discussed.     

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.     

Advances in metallothionein structure and functions.

Metallothioneins (MTs) are a class of ubiquitously occurring low molecular weight cysteine- and metal-rich proteins containing sulfur-based metal clusters. The conservation of these clusters in an increasing number of three-dimensional structures of invertebrate, vertebrate and bacterial MTs signifies the importance of this structural motif. In the postgenomic era, it is becoming increasingly clear that MTs fulfil different functions. Increasing body of evidence show that diverse functions of the mammalian MT-1/MT-2 isoforms including involvement in zinc homeostasis, protection against heavy metal toxicity and oxidative damage are related to their clusters. In contrast, the biological properties of the brain-specific MT-3 isoform imply that the clusters in this protein play a structural role. The recent highlights of MT research are the subject of this review.     

Basal and copper-induced expression of metallothionein isoform 1,2 and 3 genes in epithelial cancer cells: The role of tumor suppressor p53

Metallothioneins (MTs) are a ubiquitous low-molecular weight, cysteine rich proteins with a high affinity for metal ions. The expression and induction of MTs have been associated with protection against DNA damage, oxidative stress, and apoptosis. Our past research had shown that p53 is an important factor in metal regulation of MTs. The present study was undertaken to explore further the interrelationship between p53 and MTs. We investigated whether silencing of p53 could affect expression pattern of basal and copper induced metallothioneins. The silencing of wild-type p53 (wt-p53) in epithelial breast cancer MCF7 cells affected the basal level of MT-2A RNA, whereas the levels of MT-1A and MT-1X RNA remained largely unchanged. The expression of MT-3 was undetectable in MCF7 with either functional or silenced p53. MCF7 cells with silenced wt-p53 failed to upregulate MT-2A in response to copper and showed a reduced sensitivity toward copper induced cell apoptotic death. Similarly in MCF7-E6 and MDA-MB-231 cells, the presence of inactive/mutated p53 halted MT-1A and MT-2A gene expression in response to copper. Constitutive expression of MT-3 RNA was detectable in the presence of mutated p53 (mtp53). Transient transfection of MDA-MB-231 cells with wt-p53 enabled copper induced upregulation of both MT-1A and MT-2A but not basal level of MT-2A, MT-1E, MT-1X and MT-3. Inactivation of p53 in HepG2 cells amplified the basal expression of studied MT isoforms, including MT-3, as well as copper-induced mRNA expression of MTs except MT-1H and MT-3. Presented data demonstrate a direct relation between p53 and MT-1A and MT-2A and they also indicate that wt-p53 might be a negative regulator of MT-3 in epithelial cancer cells.     

Induction,Regulation, Degradation,and Biological Significance of Mammalian Metallothioneins

MTs are small cysteine-rich metal-binding proteins found in many species and, although there are differences between them, it is of note that they have a great deal of sequence and structural homology. Mammalian MTs are 61 or 62 amino acid polypep-tides containing 20 conserved cysteine residues that underpin the binding of metals. The existence of MT across species is indicative of its biological demand, while the conservation of cysteines indicates that these are undoubtedly central to the function of this protein. Four MT isoforms have been found so far, MT-1, MT-2, MT-3, and MT-4, but these also have subtypes with 17 MT genes identified in man, of which 10 are known to be functional. Different cells express different MT isoforms with varying levels of expression perhaps as a result of the different function of each isoform. Even different metals induce and bind to MTs to different extents. Over 40 years of research into MT have yielded much information on this protein, but have failed to assign to it a definitive biological role. The fact that multiple MT isoforms exist, and the great variety of substances and agents that act as inducers, further complicates the search for the biological role of MTs. This article reviews the current knowledge on the biochemistry, induction, regulation, and degradation of this protein in mammals, with a particular emphasis on human MTs. It also considers the possible biological roles of this protein, which include participation in cell proliferation and apoptosis, homeostasis of essential metals, cellular free radical scavenging, and metal detoxification.     

Induction, regulation, degradation, and biological significance of mammalian metallothioneins

MTs are small cysteine-rich metal-binding proteins found in many species and, although there are differences between them, it is of note that they have a great deal of sequence and structural homology. Mammalian MTs are 61 or 62 amino acid polypeptides containing 20 conserved cysteine residues that underpin the binding of metals. The existence of MT across species is indicative of its biological demand, while the conservation of cysteines indicates that these are undoubtedly central to the function of this protein. Four MT isoforms have been found so far, MT-1, MT-2, MT-3, and MT-4, but these also have subtypes with 17 MT genes identified in man, of which 10 are known to be functional. Different cells express different MT isoforms with varying levels of expression perhaps as a result of the different function of each isoform. Even different metals induce and bind to MTs to different extents. Over 40 years of research into MT have yielded much information on this protein, but have failed to assign to it a definitive biological role. The fact that multiple MT isoforms exist, and the great variety of substances and agents that act as inducers, further complicates the search for the biological role of MTs. This article reviews the current knowledge on the biochemistry, induction, regulation, and degradation of this protein in mammals, with a particular emphasis on human MTs. It also considers the possible biological roles of this protein, which include participation in cell proliferation and apoptosis, homeostasis of essential metals, cellular free radical scavenging, and metal detoxification.     

The Properties of the Metal–Thiolate Clusters in Recombinant Mouse Metallothionein-4

Metallothioneins (MTs) are metal-binding proteins with low molecular weight and conservative cysteine residues. Metallothionein-4 (MT-4), one of MT isoforms, is first reported to be distributed in a tissue-specific manner, mainly in stratified squamous epithelia. Here, we compare the properties of metal–thiolate clusters in MT-4 to those in MT-1 and MT-3, including the stabilities toward both pH change and EDTA, as well as the exposure of thiolates to solvent. The metal–thiolate clusters in MT-3 show different property and activity to the reactions compared with MT-4 and MT-1. The structure of metal–thiolate clusters in MT-4 is similar to that of MT-1 from the UV and CD spectra. During pH titration and DTNB reaction, MT-4 and MT-1 exhibit comparable behavior. But while reacting with EDTA, the metal–thiolate clusters in MT-4 are more stable than those of MT-1. We suppose the negative charge of the β-domain of MT-4 prevents the EDTA attack to MT-4.     

Mammalian metallothionein

Chemical, spectroscopic, and structural studies have established the metallothioneins (MTs) to be a widely occurring family of polypeptidic bioinorganic structures. They are distinguished by an extremely high metal (Zn, Cd, Cu) and Cys content and by the arrangement of these components in metal-thiolate clusters. By structural criteria the MTs have recently been subdivided into three classes (Fowler et al.,Experientia Suppl. 52, 19–22, 1987). Class I MTs include mammalian MTs and related forms. Class II MTs display no such relationships, and Class III MTs are atypical polypeptides made up of repetitive γ-glutamylcysteinyl units. Amino acid sequences of over 50 MTs are now known. In mammals, over 55% of the residues, including the 20 Cys, are conserved. Mammalian MTs are genetically polymorphous. Thus, in human tissues and cell lines closely related structures of ten functional isoMTs have been determined either by amino acid or nucleotide sequencing. A comparable degree of polymorphism also exists in the rabbit. Mammalian MTs have been inferred to bind a total of seven bivalent metal ions (Me) through thiolate coordination in two separate clusters, i.e., Me(II)₃(Cys)₉ and Me(II)₄(Cys)₁₁. This two-cluster model has now fully been confirmed by the spatial structures of rat MT-2 and rabbit MT-2a determined by 2D NMR spectroscopy in aqueous solution.     

Comparative genomics analysis of metallothioneins in twelve Drosophila species

Metallothioneins (MTs) are a superfamily of Cys-rich polypeptides that bind heavy metal ions, both for physiological and detoxification purposes. They are present in all organisms, but their origin is probably polyphyletic, so that MT evolutionary studies are rather scarce. We present a thorough search and analysis of the MT coding sequences in the 12 Drosophila genomes completely sequenced, taking as reference the features reported for D. melanogaster, where four isogenes (MtnA to MtnD) are known and deeply characterized. We include a fifth isoform in this study, named MtnE, and recently annotated. The MTs polymorphism pattern is essentially the same for the 12 Drosophila species. Invariably, a MtnA form and an MtnB-cluster, comprising the MtnB-to-MtnE forms in tandem array, are observed. The whole set of genes are kept in the same synteny element (Muller E), but implicated in rearrangement events (mainly inversions), encompassing all or some of the isogenes. Gene exon/intron architecture, and cDNA and protein sequences appear highly conserved through Drosophila speciation, concordantly with an essential function for MT isoforms in flies, even for those previously considered as minor products. Data presented here will be comprehensively analyzed to provide a valuable guide for future MT evolutionary, structure and function studies.     

Fish and molluscan metallothioneins

Metallothioneins (MTs) are noncatalytic peptides involved in storage of essential ions, detoxification of nonessential metals, and scavenging of oxyradicals. They exhibit an unusual primary sequence and unique 3D arrangement. Whereas vertebrate MTs are characterized by the well-known dumbbell shape, with a beta domain that binds three bivalent metal ions and an alpha domain that binds four ions, molluscan MT structure is still poorly understood. For this reason we compared two MTs from aquatic organisms that differ markedly in primary structure: MT 10 from the invertebrate Mytilus galloprovincialis and MT A from Oncorhyncus mykiss. Both proteins were overexpressed in Escherichia coli as glutathione S-transferase fusion proteins, and the MT moiety was recovered after protease cleavage. The MTs were analyzed by gel electrophoresis and tested for their differential reactivity with alkylating and reducing agents. Although they show an identical cadmium content and a similar metal-binding ability, spectropolarimetric analysis disclosed significant differences in the Cd7-MT secondary conformation. These structural differences reflect the thermal stability and metal transport of the two proteins. When metal transfer from Cd7-MT to 4-(2-pyridylazo)resorcinol was measured, the mussel MT was more reactive than the fish protein. This confirms that the differences in the primary sequence of MT 10 give rise to peculiar secondary conformation, which in turn reflects its reactivity and stability. The functional differences between the two MTs are due to specific structural properties and may be related to the different lifestyles of the two organisms.     

Quantitation of Human Metallothionein Isoforms: A Family of Small,Highly Conserved,Cysteine-rich Proteins

Human metallothioneins (MTs) are important regulators of metal homeostasis and protectors against oxidative damage. Their altered mRNA expression has been correlated with metal toxicity and a variety of cancers. Current immunodetection methods lack the specificity to distinguish all 12 human isoforms. Each, however, can be distinguished by the mass of its acetylated, cysteine-rich, hydrophilic N-terminal tryptic peptides. These properties were exploited to develop a bottom-up MALDI-TOF/TOF-MS-based method for their simultaneous quantitation. Key features included enrichment of N-terminal acetylated peptides by strong cation exchange chromatography, optimization of C18 reversed-phase chromatography, and control of methionine oxidation. Combinations of nine isoforms were identified in seven cell lines and two tissues. Relative quantitation was accomplished by comparing peak intensities of peptides generated from pooled cytosolic proteins alkylated with ¹⁴N- or ¹⁵N-iodoacetamide. Absolute quantitation was achieved using ¹⁵N-iodoacetamide-labeled synthetic peptides as internal standards. The method was applied to the cadmium induction of MTs in human kidney HK-2 epithelial cells expressing recombinant MT-3. Seven isoforms were detected with abundances spanning almost 2 orders of magnitude and inductions up to 12-fold. The protein-to-mRNA ratio for MT-1E was one-tenth that of other MTs, suggesting isoform-specific differences in protein expression efficiency. Differential expression of MT-1G1 and MT-1G2 suggested tissue- and cell-specific alternative splicing for the MT-1G isoform. Protein expression of MT isoforms was also evaluated in human breast epithelial cancer cell lines. Estrogen-receptor-positive cell lines expressed only MT-2 and MT-1X, whereas estrogen-receptor-negative cell lines additionally expressed MT-1E. The combined expression of MT isoforms was 38-fold greater in estrogen-receptor-negative cell lines than in estrogen-receptor-positive cells. These findings demonstrate that individual human MT isoforms can be accurately quantified in cells and tissues at the protein level, complementing and expanding mRNA measurement as a means for evaluating MTs as potential biomarkers for cancers or heavy metal toxicity.The metallothioneins (MTs)¹ are a family of small, highly conserved proteins with the specific capacity to bind metal ions (1–3). Mammalian MTs, typically 61 to 68 amino acid residues in length, contain 20 invariant cysteine residues that form two distinct metal-binding domains. Up to seven or eight metal ions may be coordinated per MT. Many functions have been attributed to this redox-active protein, including zinc homeostasis; heavy metal detoxification; metal exchange; metal transfer; and protection against oxidative damage, inflammatory responses, and other cellular stresses (4–6). Changes in MT expression have been associated with human pathologies including cadmium-induced renal toxicity (7), neurodegeneration (8), and many forms of cancer (9, 10). The understanding of these changes is complicated by the 11 functional MT genes, seven pseudogenes, and four MT-like genes encoded in the genome, most of which contain only small differences in amino acid sequence (11). Seventeen of the 18 genes and pseudogenes are clustered together on chromosome 16, which is known to be enriched for intrachromosomal duplications (12). The various MT gene products differ in their patterns of mRNA and protein expression in human tissues and cell lines. Immunohistochemical detection using antibodies that do not discriminate between MT-1 and MT-2 isoforms indicates wide tissue and cell type distribution of MTs, as illustrated with the MT-1A entry of the Human Protein Atlas (13, 14). Measurements of individual MT mRNA levels, however, clearly demonstrate differential expression of specific MT-1 isoforms in human tissues and cell lines (15–17). The MT-3 (18, 19) and MT-4 (20) mRNAs are expressed in even narrower ranges of cell types.An abundance of immunohistochemical and mRNA measurements show that alteration of MT isoform expression is correlated with a variety of cancers (9, 10). For example, several studies show that the expression of specific MT isoforms is altered in invasive ductal breast carcinomas. Elevated MT-2A (21) or MT-1F (22) is correlated with increased proliferation or tumor grade, respectively. Expression of MT-3 is associated with poor prognosis (23, 24). The MT-1E isoform is found in estrogen-receptor-negative (ER⁻), but not estrogen-receptor-positive (ER⁺), tumors (25) and cell lines (26). Parallel assessment of changes in MT protein expression via immunohistochemistry supports the mRNA data up to a point. Except for antibodies specific for the MT-3 isoform (27), all commercially available MT antibodies are pan-specific for the MT-1, MT-2, and MT-4 protein isoforms (28). This is because epitopes recognized by antibodies raised against MT-1 or MT-2 are limited to the first five residues of the acetylated N terminus, which are invariant among all MT-1, MT-2, and MT-4 isoforms (29–31). This includes the commercially available E9 antibody that has been used to demonstrate the overexpression of MT in a wide variety of human cancers (28, 32, 33). In general, the overexpression of MT in various cancers has been associated with resistance to anticancer therapies and linked to a poor prognosis.The mounting evidence that specific MT isoforms may be useful prognostic and diagnostic markers for cancers highlights the need for alternative approaches to the assessment of MT isoform expression at the protein level. A few mass-spectrometry-based studies have succeeded in identifying the complement of MT isoforms in human cells (34, 35). Though top-down approaches hold promise for the quantitation of MTs based on the unique masses of intact isoforms (34, 36), this has yet to be exploited. Inductively coupled plasma MS has been used to quantify total metal-bound MTs in cells and tissues, but it cannot assign relative abundance values of MT isoforms because the proteins are reduced to their elemental composition with this technique. Thus far, MALDI-MS has been used in parallel with inductively coupled plasma MS for the qualitative identification of isoforms (35). Bottom-up quantitative approaches specifically targeting MTs have not yet been reported.The use of mass spectrometry to quantify MT isoforms is not straightforward. The N-terminal tryptic peptide of each human MT isoform encompasses the only sequence that distinguishes all 12 and therefore may be used for their identification and quantitation in complex biological samples from cells and tissues (34). Any attempt at quantitation of this family of small, highly conserved, cysteine-rich proteins therefore requires reproducible detection of these signature peptides.An optimized bottom-up proteomic method is presented here that is capable of identifying and quantifying all isoforms that constitute the human MT gene family in a single experiment. The approach is comparable in sensitivity and dynamic range to quantitative PCR methods used to measure mRNA levels. Quantitative and qualitative differences between mRNA and protein expression indicate that isoform-specific measurements of protein levels complement and extend our understanding of MT isoform expression in complex biological samples. The method was applied to the characterization of MT isoforms in ER⁺ and ER⁻ breast cancer cell lines. Protein and mRNA measurements showed the same complement of isoform expression, confirming differential MT expression between ER⁺ and ER⁻ cell lines. The mass spectrometry assay further showed dramatic differences in the abundance of protein and mRNA in specific isoforms, an observation that has not been previously reported.     

(Cu,Zn)-metallothioneins from fetal bovine liver. Chemical and spectroscopic properties

Two metallothioneins (MTs) from bovine fetal liver were purified by a combination of gel filtration and ion-exchange chromatography. The primary structures of the isoproteins MT-1 and MT-2 were elucidated by peptide and amino acid sequence analysis. The amino-terminal part was deduced from automated Edman degradations of the pyridylethylated CNBr-cleaved derivatives. The remaining part of the sequence was established by a comparison of the carboxamidomethylated tryptic peptides to those from equine liver MT-1A and MT-2B. Peptides differing in either amino acid composition or retention time from high pressure liquid chromatography were further subjected to manual Edman degradations or carboxypeptidase Y digestion. The two isoproteins consist of 61 amino acids and show a sequence identity of 90%. When compared with the primary structures of other mammalian MTs, the 20 cysteinyl residues are totally conserved, in agreement with their function as metal ligands. The two isoproteins contain Cu and Zn at a ratio of 3:4. Spectroscopic data reveal absorption properties typical for both Cu- and Zn-thiolate transitions. The marked differences of MT-1 and MT-2 in the Cu-thiolate CD features can be attributed to the six amino acid substitutions occurring exclusively in the amino-terminal parts of the molecules. It is proposed that in bovine fetal MTs also the three copper ions are preferentially bound to the first 9 cysteinyl residues (cluster B) and the four zinc ions to the remaining 11 cysteinyl residues (cluster A) suggested previously by 113Cd NMR spectroscopy of calf liver MTs (Briggs, R. W., and Armitage, I. M. (1982) J. Biol. Chem. 257, 1259-1262).     

Isolation of GIF from porcine brain and studies of its zinc transfer kinetics with apo-carbonic anhydrase

Neuronal growth inhibitory factor (GIF) of porcine brain, was isolated and purified by a similar procedure which was used on the isolation of human and bovine GIF. The native porcine protein with stoichiometry of 4Cu⁺, 3Zn²⁺ was obtained for the first time. The kinetics of zinc transfer from Cu₄Zn₃MT-3 to apo-carbonic anhydrase were studied, and zinc transfer rate constants and thermodynamic parameters were obtained. It is found that like other MTs, porcine Cu₄Zn₃MT-3 can also transfer its zinc atom to apoCA, even much easier than other MTs. A possible association mechanism has been proposed, the formation of Cu₄Zn₃MT3-apoCA complex may be the rate-determining step. The obtained data indicate besides its neuronal growth inhibitory function, GIF might play a role in cellular Zn homeostasis in brain.     

Metallothionein: a multifunctional protein from toxicity to cancer

The metallothionein (MT) family is a class of low molecular weight, intracellular and cysteine-rich proteins presenting high affinity for metal ions. Although the members of this family were discovered nearly 40 years ago, their functional significance remains obscure. Four major MT isoforms, MT-1, MT-2, MT-3 and MT-4, have been identified in mammals. MTs are involved in many pathophysiological processes such as metal ion homeostasis and detoxification, protection against oxidative damage, cell proliferation and apoptosis, chemoresistance and radiotherapy resistance. MT isoforms have been shown to be involved in several aspects of the carcinogenic process, cancer development and progression. MT expression has been implicated as a transient response to any form of stress or injury providing cytoprotective action. Although MT participates in the carcinogenic process, its use as a potential marker of tumor differentiation or cell proliferation, or as a predictor of poor prognosis remains unclear. In the present review the involvement of MT in defense mechanisms to toxicity and in carcinogenicity is discussed.     

Immunological and biochemical properties of metallothioneins of golden hamster,mouse and rat

Summary Golden hamster, mouse and rat hepatic cadmium metallothioneins (MT) were purified by Sephadex G-75 gel filtration, DEAE-Sephadex A-25 chromatography and activated Thiol-Sepharose 4B affinity chromatography. Metallothioneins were separated by DEAE-Sephadex A-25 chromatography into two forms: MT-1 and MT-2. In mouse and golden hamster liver, MT-1 was the major form. The purified proteins were homogeneous as judged by polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate. In non-denaturing polyacrylamide gel electrophoresis, migration of mouse, rat and golden hamster hepatic metallothioneins were found to be different. Antibodies to mouse hepatic MT-1 was raised in rabbits. The antiserum cross reacted with mouse and hamster MT-1 and MT-2 giving a single precipitin band. Mouse, rat and hamster hepatic MTs are immunologically identical but electrophoretically different. The kidney and pancreatic MTs of rat and golden hamster were purified by Sephadex G-75 gel filtration. They were immunologically distinct. Pancreas MT formed a line of partial identity with hepatic MTs. Kidney MTs form two precipitin band one identical with the pancreatic form and another of complete identity with the hepatic MTs. This indicates the presence of tissue specific MTs.     

Examining the specific contributions of individual Arabidopsis metallothioneins to copper distribution and metal tolerance

Metallothioneins (MTs) are small cysteine-rich proteins found in various eukaryotes. Plant MTs are classified into four types based on the arrangement of cysteine residues. To determine whether all four types of plant MTs function as metal chelators, six Arabidopsis (Arabidopsis thaliana) MTs (MT1a, MT2a, MT2b, MT3, MT4a, and MT4b) were expressed in the copper (Cu)- and zinc (Zn)-sensitive yeast mutants, Deltacup1 and Deltazrc1 Deltacot1, respectively. All four types of Arabidopsis MTs provided similar levels of Cu tolerance and accumulation to the Deltacup1 mutant. The type-4 MTs (MT4a and MT4b) conferred greater Zn tolerance and higher accumulation of Zn than other MTs to the Deltazrc1 Deltacot1 mutant. To examine the functions of MTs in plants, we studied Arabidopsis plants that lack MT1a and MT2b, two MTs that are expressed in phloem. The lack of MT1a, but not MT2b, led to a 30% decrease in Cu accumulation in roots of plants exposed to 30 mum CuSO(4). Ectopic expression of MT1a RNA in the mt1a-2 mt2b-1 mutant restored Cu accumulation in roots. The mt1a-2 mt2b-1 mutant had normal metal tolerance. However, when MT deficiency was combined with phytochelatin deficiency, growth of the mt1a-2 mt2b-1 cad1-3 triple mutant was more sensitive to Cu and cadmium compared to the cad1-3 mutant. Together these results provide direct evidence for functional contributions of MTs to plant metal homeostasis. MT1a, in particular, plays a role in Cu homeostasis in the roots under elevated Cu. Moreover, MTs and phytochelatins function cooperatively to protect plants from Cu and cadmium toxicity.     

Electrospray ionization mass spectrometry of zinc, cadmium, and copper metallothioneins: evidence for metal-binding cooperativity

Electrospray ionization (ESI) mass spectra of both well-characterized and novel metallothioneins (MTs) from various species were recorded to explore their metal-ion-binding modes and stoichiometries. The ESI mass spectra of the zinc- and cadmium-binding MTs showed a single main peak corresponding to metal-to-protein ratios of 4, 6, or 7. These findings combined with data obtained by other methods suggest that these MTs bind zinc or cadmium in a single predominant form and are consistent with the presence of three- and four-metal clusters. An unstable copper-specific MT isoform from Roman snails (Helix pomatia) could be isolated intact and was shown to preferentially bind 12 copper ions. To obtain additional information on the formation and relative stability of metal-thiolate clusters in MTs, a mass spectrometric titration study was conducted. One to seven molar equivalents of zinc or of cadmium were added to metal-free human MT-2 at neutral pH, and the resulting complexes were measured by ESI mass spectrometry. These experiments revealed that the formation of the four-metal cluster and of the thermodynamically less stable three-metal cluster is sequential and largely cooperative for both zinc and cadmium. Minor intermediate forms between metal-free MT, Me4MT, and fully reconstituted Me7MT were also observed. The addition of increasing amounts of cadmium to metal-free blue crab MT-I resulted in prominent peaks whose masses were consistent with apoMT, Cd3MT, and Cd6MT, reflecting the known structure of this MT with two Me3Cys9 centers. In a similar reconstitution experiment performed with Caenorhabditis elegans MT-II, a series of signals corresponding to apoMT and Cd3MT to Cd6MT species were observed.     

The Native Copper- and Zinc- Binding Protein Metallothionein Blocks Copper-Mediated Aβ Aggregation and Toxicity in Rat Cortical Neurons

Background

A major pathological hallmark of AD is the deposition of insoluble extracellular β-amyloid (Aβ) plaques. There are compelling data suggesting that Aβ aggregation is catalysed by reaction with the metals zinc and copper.

Methodology/Principal Findings

We now report that the major human-expressed metallothionein (MT) subtype, MT-2A, is capable of preventing the in vitro copper-mediated aggregation of Aβ_1–40 and Aβ_1–42. This action of MT-2A appears to involve a metal-swap between Zn₇MT-2A and Cu(II)-Aβ, since neither Cu₁₀MT-2A or carboxymethylated MT-2A blocked Cu(II)-Aβ aggregation. Furthermore, Zn₇MT-2A blocked Cu(II)-Aβ induced changes in ionic homeostasis and subsequent neurotoxicity of cultured cortical neurons.

Conclusions/Significance

These results indicate that MTs of the type represented by MT-2A are capable of protecting against Aβ aggregation and toxicity. Given the recent interest in metal-chelation therapies for AD that remove metal from Aβ leaving a metal-free Aβ that can readily bind metals again, we believe that MT-2A might represent a different therapeutic approach as the metal exchange between MT and Aβ leaves the Aβ in a Zn-bound, relatively inert form.     

The metallothionein genes of Mytilus galloprovincialis: genomic organization, tissue expression and evolution

Recently, increasing interest has been directed to the study of metallothioneins (MTs), which are small proteins that are able to bind metal ions. The induction of MT synthesis after exposure to metal or other environmental contaminants in a large number of aquatic invertebrates makes these proteins good biomarkers in water monitoring programs. Within bivalves, the species Mytilus galloprovincialis and Mytilus edulis represent model organisms for these types of studies, as well as for molecular studies regarding the expression and characterization of MT encoding genes. In the present paper, we focused on the genomic characterization, evolutionary, and tissue-expression analyses of the MT-10, MT-10 Intronless, and MT-20 genes in M. galloprovincialis. The comparison of the genomic sequences showed the presence of long nucleotide stretches within the introns of the MT genes that are conserved between M. galloprovincialis and M. edulis. These non-coding conserved sequences may contain regulatory motifs. Real-Time RT-PCR experiments revealed that, at the basal conditions, the MT-10 and MT-10 Intronless genes are expressed at levels considerably higher than the MT-20 gene, mainly in the digestive gland and gill tissue. The strong induction of the MT-20 gene expression detected in a field-collected sample is associated with the up-regulation of both the MT-10 and MT-10 Intronless genes. Evolutionary analysis revealed signals of localized positive selection that, together with the tissue-expression data, support a possible functional diversification between the MTs encoded by the MT-10 and MT-10 Intronless genes.     

Removal of MAP4 from microtubules in vivo produces no observable phenotype at the cellular level

Microtubule-associated protein 4 (MAP4) promotes MT assembly in vitro and is localized along MTs in vivo. These results and the fact that MAP4 is the major MAP in nonneuronal cells suggest that MAP4's normal functions may include the stabilization of MTs in situ. To understand MAP4 function in vivo, we produced a blocking antibody (Ab) to prevent MAP4 binding to MTs. The COOH-terminal MT binding domain of MAP4 was expressed in Escherichia coli as a glutathione transferase fusion protein and was injected into rabbits to produce an antiserum that was then affinity purified and shown to be monospecific for MAP4. This Ab blocked > 95% of MAP4 binding to MTs in an in vitro assay. Microinjection of the affinity purified Ab into human fibroblasts and monkey epithelial cells abolished MAP4 binding to MTs as assayed with a rat polyclonal antibody against the NH2-terminal projection domain of MAP4. The removal of MAP4 from MTs was accompanied by its sequestration into visible MAP4-Ab immunocomplexes. However, the MT network appeared normal. Tubulin photoactivation and nocodazole sensitivity assays indicated that MT dynamics were not altered detectably by the removal of MAP4 from the MTs. Cells progressed to mitosis with morphologically normal spindles in the absence of MAP4 binding to MTs. Depleting MAP4 from MTs also did not affect the state of posttranslational modifications of tubulin subunits. Further, no perturbations of MT- dependent organelle distribution were detected. We conclude that the association of MAP4 with MTs is not essential for MT assembly or for the MT-based functions in cultured cells that we could assay. A significant role for MAP4 is not excluded by these results, however, as MAP4 may be a component of a functionally redundant system.