Expression of plant metallothionein genes has been reported in a variety of senescing tissues, such as leaves and stems, ripening fruits, and wounded tissues, and has been proposed to function in both metal chaperoning and scavenging of reactive oxygen species. In this work, it is shown that MT is also associated with suberization, after identifying a gene actively transcribed in Quercus suber cork cells as a novel MT. This cDNA, isolated from a phellem cDNA library, encodes a MT that belongs to type 2 plant MTs (QsMT). Expression of the QsMT cDNA in E. coli grown in media supplemented with Zn, Cd, or Cu has yielded recombinant QsMT. Characterization of the respective metal aggregates agrees well with a copper-related biological role, consistent with the capacity of QsMT to restore copper tolerance to a MT-deficient, copper-sensitive yeast mutant. Furthermore, in situ hybridization results demonstrate that RNA expression of QsMT is mainly observed under conditions related to oxidative stress, either endogenous, as found in cork or in actively proliferating tissues, or exogenous, for example, in response to H₂O₂ or paraquat treatments. The putative role of QsMT in oxidative stress, both as a free radical scavenger via its sulphydryl groups or as a copper chelator is discussed.

Background: The degree of metal binding specificity in metalloproteins such as metallothioneins (MTs) can be crucial for their functional accuracy. Unlike most other animal species, pulmonate molluscs possess homometallic MT isoforms loaded with Cu⁺ or Cd²⁺. They have, so far, been obtained as native metal-MT complexes from snail tissues, where they are involved in the metabolism of the metal ion species bound to the respective isoform. However, it has not as yet been discerned if their specific metal occupation is the result of a rigid control of metal availability, or isoform expression programming in the hosting tissues or of structural differences of the respective peptides determining the coordinative options for the different metal ions. In this study, the Roman snail (Helix pomatia) Cu-loaded and Cd-loaded isoforms (HpCuMT and HpCdMT) were used as model molecules in order to elucidate the biochemical and evolutionary mechanisms permitting pulmonate MTs to achieve specificity for their cognate metal ion. Results: HpCuMT and HpCdMT were recombinantly synthesized in the presence of Cd²⁺, Zn²⁺ or Cu²⁺ and corresponding metal complexes analysed by electrospray mass spectrometry and circular dichroism (CD) and ultra violet-visible (UV-Vis) spectrophotometry. Both MT isoforms were only able to form unique, homometallic and stable complexes (Cd₆-HpCdMT and Cu₁₂-HpCuMT) with their cognate metal ions. Yeast complementation assays demonstrated that the two isoforms assumed metal-specific functions, in agreement with their binding preferences, in heterologous eukaryotic environments. In the snail organism, the functional metal specificity of HpCdMT and HpCuMT was contributed by metal-specific transcription programming and cell-specific expression. Sequence elucidation and phylogenetic analysis of MT isoforms from a number of snail species revealed that they possess an unspecific and two metal-specific MT isoforms, whose metal specificity was achieved exclusively by evolutionary modulation of non-cysteine amino acid positions. Conclusion: The Roman snail HpCdMT and HpCuMT isoforms can thus be regarded as prototypes of isoform families that evolved genuine metal-specificity within pulmonate molluscs. Diversification into these isoforms may have been initiated by gene duplication, followed by speciation and selection towards opposite needs for protecting copper-dominated metabolic pathways from nonessential cadmium. The mechanisms enabling these proteins to be metal-specific could also be relevant for other metalloproteins.

Four metallothionein genes are present in the Drosophila melanogaster genome, designated MtnA, MtnB, MtnC, MtnD, all of which are transcriptionally induced by heavy metals through the same metal‐responsive transcription factor, MTF‐1. Here we show, by targeted mutagenesis, that the four metallothionein genes exhibit distinct, yet overlapping, roles in heavy metal homeostasis and toxicity prevention. Among the individual metallothionein mutants, the most prominent distinction between them was that MtnA‐defective flies were the most sensitive to copper load, while MtnB‐defective flies were the most sensitive to cadmium. Using various reporter gene constructs and mRNA quantification, we show that the MtnA promoter is preferentially induced by copper, while the MtnB promoter is preferentially induced by cadmium. Such a metal preference is also observed at the protein level as the stoichiometric, spectrometric and spectroscopic features of the copper and cadmium complexes with MtnA and MtnB correlate well with a greater stability of copper‐MtnA and cadmium‐MtnB. Finally, MtnC and MtnD, both of which are very similar to MtnB, display lower copper and cadmium binding capabilities compared to either MtnA or MtnB. In accordance with these binding studies, Drosophila mutants of MtnC or MtnD have a near wild type level of resistance against copper or cadmium load. Furthermore, eye‐specific over‐expression of MtnA and MtnB, but not of MtnC or MtnD, can rescue a “rough eye” phenotype caused by copper load in the eye. Taken together, while the exact roles of MtnC and MtnD remain to be determined, the preferential protection against copper and cadmium toxicity by MtnA and MtnB, respectively, are the result of a combination of promoter preference and metal binding.

Metallothioneins (MTs) are a family of universal, small proteins, sharing a high cysteine content and an optimal capacity for metal ion coordination. They take part in a plethora of metal ion-related events (from detoxification to homeostasis, storage, and delivery), in a wide range of stress responses, and in different pathological processes (tumorigenesis, neurodegeneration, and inflammation). The information on both intracellular and extracellular interactions of MTs with other proteins is here comprehensively reviewed. In mammalian kidney, MT1/MT2 interact with megalin and related receptors, and with the transporter transthyretin. Most of the mammalian MT partners identified concern interactions with central nervous system (mainly brain) proteins, both through physical contact or metal exchange reactions. Physical interactions mainly involve neuronal secretion multimers. Regarding metal swap events, brain MT3 appears to control the metal ion load in peptides whose aggregation leads to neurodegenerative disorders, such as Aβ peptide, α-synuclein, and prion proteins (Alzheimer’s and Parkinson’s diseases, and spongiform encephalopathies, respectively). Interaction with ferritin and bovine serum albumin are also documented. The intercourse of MTs with zinc-dependent enzymes and transcription factors is capable to activate/deactivate them, thus conferring MTs the role of metabolic and gene expression regulators. As some of these proteins are involved in cell cycle and proliferation control (p53, nuclear factor κB, and PKCμ), they are considered in the context of oncogenesis and tumor progression. Only one non-mammalian MT interaction, involving Drosophila MtnA and MtnB major isoforms and peroxiredoxins, has been reported. The prospective use for biomedical applications of the MT-interaction information is finally discussed.