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

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

Challenging the model for induction of metallothionein gene expression

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

金属结合蛋白基因及其在清除重金属污染中的应用

一些微生物和植物由于对毒性金属具有独特的抗性机制,使得利用它们来清除日益严重的环境污染已发展成为一种十分有效的技术——生物修复。研究表明,不同的金属结合蛋白（如MT 和PC）,在生物忍耐和降解过量重金属毒性机制中起重要作用。愈来愈多的MT 和PC基因被克隆,并已成功地应用于生物遗传转化,这些转基因生物在清除重金属污染方面已显示出潜在的应用价值。 Abstract:Heavy metal pollution has become a global environmental hazard.The use of microorganisms and plants for the decontamination of heavy metals is recognized as a low lost and high efficiency method for cleaning up metal contamination.It shows that various metal-binding proteins such as metallothioneins (MTs) or phytochelatines (PCs) play an important role in defense systems and detoxification to heavy metals in organisms.Many genes of MTs and PCs have been cloned and utilized successfully in genetically modified bacteria and plants for increasing remediation capacity.These transgenic organisms have been displayed a great potential in bioremediation and phytoremediation of heavy metals.     

Heavy metal and abiotic stress inducible metallothionein isoforms from <Emphasis Type="Italic">Prosopis juliflora</Emphasis> (SW) D.C. show differences in binding to heavy metals in vitro

Prosopis juliflora is a tree species that grows well in heavy metal laden industrial sites and accumulates heavy metals. To understand the possible contribution of metallothioneins (MTs) in heavy metal accumulation in P. juliflora, we isolated and compared the metal binding ability of three different types of MTs (PjMT1-3). Glutathione S-transferase fusions of PjMTs (GSTMT1-3) were purified from Escherichia coli cells grown in the presence of 0.3 mM cadmium, copper or zinc. Analysis of metal bound fusion proteins using atomic absorption spectrometry showed that PjMT1 bound higher levels of all three heavy metals as compared to PjMT2 and PjMT3. A comparative analysis of the genomic regions (including promoter for all three PjMTs) is also presented. All three PjMTs are induced by H₂O₂ and ABA applications. PjMT1 and PjMT2 are induced by copper and zinc respectively while PjMT3 is induced by copper, zinc and cadmium. Variation in induction of PjMTs in response to metal exposure and their differential binding to metals suggests that each MT has a specific role in P. juliflora. Of the three MTs analyzed, PjMT1 shows maximum heavy metal sequestration and is thus a potential candidate for use in heavy metal phytoremediation.     

Drosophila proteins interacting with metallothioneins: A metal‐dependent recognition

Metallothioneins (MTs) are ubiquitous, low‐molecular weight, cysteine‐rich proteins. Despite a well‐established protective role in metal excess detoxification, there is little data about their putative physiological functions, commonly assumed to be metal homeostasis and redox equilibrium. Protein–protein interactions should have provided useful information to unveil unsuspected functions, but reports on MT interactions are scarce. This is probably due to the MT metal‐dependent 3D structure, a fact that has been seldom taken into account when performing proteomic interaction assays. In the present work, we have detected that the two major D. melanogaster isoforms (MtnA and MtnB) interact with the peroxiredoxin (Prx) encoded by the gene Jafrac1, both in a clear metal‐dependent pattern. The MT–Prx interaction is further confirmed in Saccharomyces cerevisiae by assaying both yeast MTs (Crs5p and Cup1p) versus Tsa1p and Tsa2p, the Jafrac1 homologous Prxs in this organism. Thus, a new methodological approach to detect MT‐interacting proteins in different proteomes is established on the basis of assaying MTs in the form of different metal complexes. Furthermore, new perspectives to investigate the often hypothesized contribution of MTs to the redox physiological networks are open.     

Metal dealing at the origin of the chordata phylum: the metallothionein system and metal overload response in amphioxus

Non-vertebrate chordates, specifically amphioxus, are considered of the utmost interest for gaining insight into the evolutionary trends, i.e. differentiation and specialization, of gene/protein systems. In this work, MTs (metallothioneins), the most important metal binding proteins, are characterized for the first time in the cephalochordate subphylum at both gene and protein level, together with the main features defining the amphioxus response to cadmium and copper overload. Two MT genes (BfMT1 and BfMT2) have been identified in a contiguous region of the genome, as well as several ARE (antioxidant response element) and MRE (metal response element) located upstream the transcribed region. Their corresponding cDNAs exhibit identical sequence in the two lancelet species (B. floridae and B. lanceolatum), BfMT2 cDNA resulting from an alternative splicing event. BfMT1 is a polyvalent metal binding peptide that coordinates any of the studied metal ions (Zn, Cd or Cu) rendering complexes stable enough to last in physiological environments, which is fully concordant with the constitutive expression of its gene, and therefore, with a metal homeostasis housekeeping role. On the contrary, BfMT2 exhibits a clear ability to coordinate Cd(II) ions, while it is absolutely unable to fold into stable Cu (I) complexes, even as mixed species. This identifies it as an essential detoxification agent, which is consequently only induced in emergency situations. The cephalochordate MTs are not directly related to vertebrate MTs, neither by gene structure, protein similarity nor metal-binding behavior of the encoded peptides. The closest relative is the echinoderm MT, which confirm proposed phylogenetic relationships between these two groups. The current findings support the existence in most organisms of two types of MTs as for their metal binding preferences, devoted to different biological functions: multivalent MTs for housekeeping roles, and specialized MTs that evolve either as Cd-thioneins or Cu-thioneins, according to the ecophysiological needs of each kind of organisms.     

Erratum

The biochemical features of metallothioneins and their functional role in the cell are described. On this basis, the potential role of MTs as a biomarker of exposure in aquatic organisms, such as fishes and molluscs, is evaluated in the light of recent knowledge about MT gene regulation and inducibility. It appears that in fish MTs should be considered as a kind of stress protein which is particularly responsive to heavy metals. In molluscs, in particular in mussels, MTs seem more specifically involved in responses to heavy metals and they should therefore be considered a biomarker of exposure to heavy metal pollution. Common techniques for MT evaluation are listed and a simple spectrophotometric method recently developed is also reported. Finally, the correct approach to the use of MTs as a biomarker of exposure in biomonitoring programmes for an assessment of the physiological status of aquatic organisms is discussed.     

Metallothionein as a tool in biomonitoring programmes

The biochemical features of metallothioneins and their functional role in the cell are described. On this basis, the potential role of MTs as a biomarker of exposure in aquatic organisms, such as fishes and molluscs, is evaluated in the light of recent knowledge about MT gene regulation and inducibility. It appears that in fish MTs should be considered as a kind of stress protein which is particularly responsive to heavy metals. In molluscs, in particular in mussels, MTs seem more specifically involved in responses to heavy metals and they should therefore be considered a biomarker of exposure to heavy metal pollution. Common techniques for MT evaluation are listed and a simple spectrophotometric method recently developed is also reported. Finally, the correct approach to the use of MTs as a biomarker of exposure in biomonitoring programmes for an assessment of the physiological status of aquatic organisms is discussed.     

植物对重金属耐性的分子生态机理

植物适应重金属元素胁迫的机制包括阻止和控制重金属的吸收、体内螯合解毒、体内区室化分隔以及代谢平衡等。近年来,随着分子生物学技术在生态学研究中的深入应用,控制这些过程的分子生态机理逐渐被揭示出来。菌根、根系分泌物以及细胞膜是控制重金属进入植物根系细胞的主要生理单元。外生菌根能显著提高寄主植物的重金属耐性,根系分泌物通过改变根际pH、改变金属物质的氧化还原状态和形成络合物等机理减少植物对重金属的吸收。目前,控制菌根和根系分泌物重金属抗性的分子生态机理还不清楚。但细胞膜跨膜转运器已得到深入研究,相关金属离子转运器被鉴定和分离,一些控制基因如铁锌控制运转相关蛋白(ZIP)类、自然抵抗相关巨噬细胞蛋白(Nramp)类、P_1B-type ATPase类基因已被发现和克隆。金属硫蛋白(MTs)、植物螯合素(PCs)、有机酸及氨基酸等是植物体内主要的螯合物质,它们通过螯合作用固定金属离子,降低其生物毒性或改变其移动性。与MTs合成相关的MT-like基因已经被克隆,PCs合成必需的植物螯合素合酶(PCS), 即γ-Glu-Cys二肽转肽酶(γ-ECS) 的编码基因已经被克隆,控制麦根酸合成的氨基酸尼克烟酰胺(NA)在重金属耐性中的作用和分子机理也被揭示出来。ATP 结合转运器(ABC)和阳离子扩散促进器(CDF) 是植物体内两种主要膜转运器,通过它们和其它跨膜方式,重金属被分隔贮藏于液泡内。控制这些蛋白转运器合成的基因也已经被克隆,在植物中的表达证实其与重金属的体内运输和平衡有关。热休克蛋白(HSP)等蛋白类物质的产生是一种重要的体内平衡机制,其分子机理有待进一步研究。重金属耐性植物在这些环节产生了相关响应基因或功能蛋白质,分子克隆和转基因技术又使它们在污染治理上得到了初步的应用。     

Metallothionein induction and heavy metal accumulation in white shrimp Litopenaeus vannamei exposed to cadmium and zinc

Metallothioneins (MTs) have been widely considered for their potential use as specific biomarkers to reflect the existence of heavy metal pollution, because their induction has been observed to be obviously elevated after heavy metal exposure in a large number organism studied. However, relatively fewer efforts have been made in MT-related studies of prawn species, such as the white shrimp Litopenaeus vannamei, a globally important aquaculture species. With the results from gel filtration chromatography, we demonstrate the existence of MTs or MT-like proteins in L.vannamei. We further studied the relationship between MT induction and metals accumulation after long-term exposure to the heavy metals Cd and Zn. From our results, it is very clear that the response of L. vannamei to Cd differs from that to Zn, and this should be considered when using MTs in field applications to monitor metals contamination.     

Immunocytochemically detectable metallothionein in granulation tissue surrounding mucosal ulceration

Summary Metallothioneins (MTs) are low-molecular-weight heavy metal binding proteins which are effective free oxygen radical scavengersin vitro. Free oxygen radicals have been implicated in the pathogenesis of stress-induced acute gastric mucosal ulceration and ischaemic injury in rat and man. Experimentally, MTs can have a protective role in stress-induced ulceration in rats. The possible cytoprotective role of MTs in chronic mucosal ulceration in man has not been previously studied. Evidence for locally produced MTs in human chronic gastric and small bowel ulcers has been sought by immunocytochemical staining using a monoclonal antibody (E9) to MT. At the base of ulcers MT has been localized to spindle cells (fibroblasts) in granulation tissue. Labelling of macrophages with a pan-macrophage marker KP1, and double labelling with KP1 and E9 showed two distinct populations, and MT appeared to be localized primarily in fibroblast-like cells.     

Isoform‐specific responses of metallothioneins in a marine pollution biomonitor,the green‐lipped mussel Perna viridis,towards different stress stimulations

Metallothioneins (MTs) are commonly used as biomarker for metal pollution assessment in marine ecosystems. Using integrated genomic and proteomic analyses, this study characterized two types of MT isoform in the digestive gland of a common biomonitor, the green‐lipped mussel Perna viridis, towards the challenges of a metal (cadmium; Cd) and a non‐metal oxidant (hydrogen peroxide; H₂O₂) respectively. The two isoforms differed in their deduced protein sequences, with 73 amino acids for MT10‐I and 72 for MT10‐II (a novel type), but both consisted of a high percentage (27.4 to 29.2%) of cysteine. Two‐dimensional gel and Western blot showed that the MT proteins were present in multiple isoform spots, and they were further validated to be MT10‐I and MT10‐II using MS analysis coupled with unrestricted modifications searching. Expression of mRNA revealed that MT10‐I responded promptly to Cd but had a lagged induction to H₂O₂ treatments, while MT10‐II was exclusively induced by Cd treatment over the course of exposure. Expression of the MT proteins also showed a delayed response to H₂O₂, compared to Cd treatments. This study uncovered the potential different functional roles of various MTs isoforms in P. viridis and thus advances the resolution of using MTs as biomarkers in future applications.     

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. All the obtained Cu‐complexes share practically similar architectural features, if judging by their almost equivalent CD fingerprints, and they also share their capacity to restore copper tolerance in MT‐devoid yeast cells. Besides the analysis of the modular composition of these long fungal MTs, we evaluate the features of the Cys‐rich stretch spacer and flanking sequences that allow the construction of stable metal clusters by adjacent union of binding modules. Overall, our data support a mechanism by which some microbial MTs may have evolved to enlarge their original metal co‐ordination capacity under the specific selective pressure of counteracting the Cu‐based immunity mechanisms evolved by the infected hosts.     

Phylogenetic divergence of fish and mammalian metallothionein: relationships with structural diversification and organismal temperature

Metallothioneins (MTs) are nonenzymatic low molecular weight proteins, that play an important role in the homeostasis and detoxification of heavy metals in a large variety of organisms. These proteins are endowed with striking features, including an unusual amino acid composition characterized by the presence of 20 cysteines out of a total of 60 residues and absence of secondary structure elements. It is generally accepted that MTs underwent few modifications during evolution because of these structural and functional constraints. Such a conclusion is founded on the studies carried out mostly on MTs of mammalian origin. For such a reason, we have decided to compare the MTs of homeothermic and poikilothermic organisms, such as mammals and fish, with the specific aim to put in relation phylogenetic divergence and structural/functional adaptation to temperature. We have included in our analysis also Antarctic Notothenioids, a fish group characterized by genetic isolation and cold-adaptation to a particular harsh environment. We have determined the average hydropathic index of ancestral MT sequences and used them to infer the temperatures of the environment housing the hypothetical ancestor organisms. Finally, we have derived phylogenetic relationships of MT molecules from the pairwise comparison of their three-dimensional structures.     

Quantitative Proteomic Analysis of Biological Processes and Responses of the Bacterium Desulfovibrio desulfuricans ND132 upon Deletion of Its Mercury Methylation Genes

Recent studies of microbial mercury (Hg) methylation revealed a key gene pair, hgcAB, which is essential for methylmercury (MeHg) production in the environment. However, many aspects of the mechanism and biological processes underlying Hg methylation, as well as any additional physiological functions of the hgcAB genes, remain unknown. Here, quantitative proteomics are used to identify changes in potential functional processes related to hgcAB gene deletion in the Hg‐methylating bacterium Desulfovibrio desulfuricans ND132. Global proteomics analyses indicate that the wild type and ΔhgcAB strains are similar with respect to the whole proteome and the identified number of proteins, but differ significantly in the abundance of specific proteins. The authors observe changes in the abundance of proteins related to the glycolysis pathway and one‐carbon metabolism, suggesting that the hgcAB gene pair is linked to carbon metabolism. Unexpectedly, the authors find that the deletion of hgcAB significantly impacts a range of metal transport proteins, specifically membrane efflux pumps such as those associated with heavy metal copper (Cu) export, leading to decreased Cu uptake in the ΔhgcAB mutant. This observation indicates possible linkages between this set of proteins and metal homeostasis in the cell. However, hgcAB gene expression is not induced by Hg, as evidenced by similarly low abundance of HgcA and HgcB proteins in the absence or presence of Hg (500 nm ). Taken together, these results suggest an apparent link between HgcAB, one‐carbon metabolism, and metal homeostasis, thereby providing insights for further exploration of biochemical mechanisms and biological functions of microbial Hg methylation.     

Type 1 metallothionein (ZjMT) is responsible for heavy metal tolerance in <Emphasis Type="Italic">Ziziphus jujuba</Emphasis>

Metallothioneins (MTs) are a family of low molecular weight, cysteine-rich, metal-binding proteins that are able to make cells to uptake heavy metals from the environment. Molecular and functional characterization of this gene family improves understanding of the mechanisms underlying heavy metal tolerance in higher organisms. In this study, a cDNA clone, encoding 74-a.a. metallothionein type 1 protein (ZjMT), was isolated from the cDNA library of Ziziphus jujuba. At the N- and C-terminals of the deduced amino acid sequence of ZjMT, six cysteine residues were arranged in a CXCXXXCXCXXXCXC and CXCXXXCXCXXCXC structure, respectively, indicating that ZjMT is a type 1 MT. Quantitative PCR analysis of plants subjected to cadmium stress showed enhanced expression of ZjMT gene in Z. jujuba within 24 h upon Cd exposure. Escherichia coli cells expressing ZjMT exhibited enhanced metal tolerance and higher accumulation of metal ions compared with control cells. The results indicate that ZjMT contributes to the detoxification of metal ions and provides marked tolerance against metal stresses. Therefore, ZjMT may be a potential candidate for tolerance enhancement in vulnerable plants to heavy metal stress and E. coli cells containing the ZjMT gene may be applied to adsorb heavy metals in polluted wastewater.     

Antioxidant role of metallothioneins: a comparative overview.

Metallothioneins (MTs) are sulfhydryl-rich proteins binding essential and non-essential heavy metals. MTs display in vitro oxyradical scavenging capacity, suggesting that they may specifically neutralize hydroxyl radicals. Yet, this is probably an oversimplified view, as MTs represent a superfamily of widely differentiated metalloproteins. MT antioxidant properties mainly derive from sulfhydryl nucleophilicity, but also from metal complexation. Binding of transition metals displaying Fenton reactivity (Fe,Cu) can reduce oxidative stress, whereas their release exacerbates it. In vertebrates, MT gene promoters contain metal (MRE) and glucocorticoid response elements (GRE), Sp and AP sequences, but also antioxidant response elements (ARE). MT neosynthesis is induced by heavy metals, cytokines, hormones, but also by different oxidants and prooxidants. Accordingly, MT overexpression increases the resistance of tissues and cells to oxidative stress. As for invertebrates, data from the mussel show that MT can actually protect against oxidative stress, but is poorly inducible by oxidants. In yeast, there is a Cu(I)-MT that in contrast to mammalCu-MT exhibits antioxidant activity, possibly due to differences in metal binding domains. Finally, as the relevance of redox processes in cell signaling is becoming more and more evident, a search for MT effects on redox signaling could represent a turning point in the understanding of the functional role of these protein.