Expression of the PsMT A1 gene in white poplar engineered with the MAT system is associated with heavy metal tolerance and protection against 8-hydroxy-2′-deoxyguanosine mediated-DNA damage

Marker-free transgenic white poplar (Populus alba L., cv ‘Villafranca’) plants, expressing the PsMT _A1 gene from Pisum sativum for a metallothionein-like protein, were produced by Agrobacterium tumefaciens-mediated transformation. The 35SCaMV-PsMT _A1 -NosT cassette was inserted into the ipt-type vector pMAT22. The occurrence of the abnormal ipt-shooty phenotype allowed the visual selection of transformants, while the yeast site-specific recombination R/RS system was responsible for the excision of the undesired vector sequences with the consequent recovery of normal marker-free transgenic plants. Molecular analyses confirmed the presence of the 35SCaMV-PsMT _A1 -NosT cassette and transgene expression. Five selected lines were further characterized, revealing the ability to withstand heavy metal toxicity. They survived 0.1 mM CuCl₂, a concentration which strongly affected the nontransgenic plants. Moreover, root development was only slightly affected by the ectopic expression of the transgene. Reactive oxygen species were accumulated to a lower extent in leaf tissues of multi-auto-transformation (MAT)-PsMT_A1 plants exposed to copper and zinc, compared to control plants. Tolerance to photo-oxidative stress induced by paraquat was another distinctive feature of the MAT-PsMT_A1 lines. Finally, low levels of DNA damage were detected by quantifying the amounts of 8-hydroxy-2′-deoxyguanosine in leaf tissues of the transgenic plants exposed to copper.     

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.     

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.     

硒与金属硫蛋白及对小鼠肝损伤的防护作用研究

应用蛋白质芯片和RT-PCR技术研究硒对小鼠肝脏金属硫蛋白（Metallothionein, MT）的诱导表达;以蛋白质芯片技术为主要实验手段研究CCl4诱导的小鼠肝脏损伤后血清及肝脏蛋白质图谱变化,寻找小鼠血清及肝脏组织损伤标志物,同时观察有机硒(麦硒康,Organ-Se)对损伤的防护作用,结果表明与对照组相比给予有机硒后的小鼠肝脏MT表达明显;比较肝损伤组及用药组（Organ-Se）组的血清及组织蛋白质表达图谱变化,发现血清中存在3个具有统计学意义的标志物：5062.5Da 、5566.5Da、6358.5Da;肝脏组织中有4个具有统计学意义的标志物：5449.6Da、7131.5Da、9903.2Da和10767.3Da;与损伤组相比预先保护组（Organ-Se）的血清及肝脏标志物的表达水平接近正常组。本实验表明非金属元素硒,尤其是有机硒同金属元素（如锌）一样,能够有效诱导MT的表达,为今后进一步开展硒代金属硫蛋白的机制研究奠定了基础;同时在对小鼠肝损伤保护的研究中发现,有机硒具有明显的肝损伤保护作用,是一种较有前途的值得开发的肝损伤防护药。     

Interferonbeta-induced changes in metallothionein expression and subcellular distribution of zinc in HepG2 cells

We evaluated the changes of metallothionein induction and cellular zinc distribution in HepG2 cells by interferonbeta treatment. Immunohistochemical staining of metallothionein was observed in the cytoplasm and nuclei of hepatocytes; which was observed predominantly in the cells treated with interferon and zinc compared to those with zinc alone, interferon alone or the no-treated control. The cellular zinc level was higher in order of the interferon- and zinc-treated cells, the zinc-alone-treated cells, and the interferon-alone-treated cells. Flow cytometry showed that S-phase population increased in interferon-alone-treated cells and interferon- and zinc-treated cells, but not in zinc-alone-treated ones. Cellular elemental distribution was analyzed using in-air micro-particle induced X-ray emission. In zinc-alone-treated sample, X-ray spectra showed good consistency between the enhanced cellular zinc distribution and the phosphorous map. Localizations of bromine followed by interferon treatment were found accompanying a spatial correlation with the phosphorous map. The samples treated with interferon and zinc showed the marked accumulation of zinc and bromine. Discrete bromine accumulation sites were clearly visible with a strong spatial correlation followed by zinc accumulation. These findings suggest that interferonbeta in combination with zinc predominantly induces metallothionein expression in HepG2 cells. In addition, interferonbeta may promote the translocation of metallothionein-bound zinc from cytoplasm to S-phase nuclei.     

Study of the redox properties of metallothionein in vitro by reacting with DsbA protein

Mammalian metallothionein (MT) contains 20 cysteine residues involved in the two metal clusters without a disulfide bond. The redox reaction of the Cys thiols was proposed to be associated with the metal distribution of MT. The E. coli DsbA protein is extremely active in facilitating thiol/disulfide exchange both in vivo and in vitro. To further investigate the redox properties of MT, reaction between MT and DsbA was carried out in vitro by fluorescence detection. Equilibrium characterization indicates that the reaction is stoichiometric (1:1) under certain conditions. Kinetic study gives a rate constant of the redox reaction of 4.42 × 10⁵ sec^–1 M^–1, which is 10³-fold larger than that of glutathione reacting with DsbA. Metal-free MT (apo-MT) shows a higher equilibrium reduction potential than MT, but exhibits an indistinguishable kinetic rate. Oxidation of MT by DsbA leads to metal release from the clusters. The characteristic fluorescence increase during reduction of DsbA may provide a sensitive probe for exploring the redox properties of some reductants of biological interest. The result also implies that oxidation of Cys thiols may influence the metal release or delivery from MT.     

A differential assay for the reduced and oxidized states of metallothionein and thionein

In the cellular environment, the sulfur ligands in zinc/thiolate coordination sites of proteins can be oxidized with concomitant mobilization of zinc. The characterization of such "redox zinc switches" requires the determination of three species, i.e., the zinc-containing complex and the zinc-free complex with the thiolate ligands either reduced or oxidized. Differential chemical modification of thiol groups in the presence and absence of either reducing or chelating agents allows the analytical speciation of such systems as demonstrated here for the characterization of the redox and metal-binding states of mammalian metallothionein. Thiol derivatization with 6-iodoacetamidofluorescein in the presence and absence of the reducing agent tris(2-carboxyethyl)phosphine, high-performance liquid chromatographic separation, and photometric detection are employed to determine the reduced and oxidized protein. Because the holoprotein reacts only in the presence of a chelating agent such as ethylenediaminetetraacetate (EDTA) its amount can be determined as the difference between measurements in the presence and the absence of EDTA. This method is applied to the study of the chemical and enzymatic oxidation of metallothionein/thionein. It should also greatly facilitate the characterization of the redox and metal-binding properties of zinc/thiolate coordination environments of other proteins such as zinc finger proteins.     

Effects of soy protein isolate on LEC rats,a model of Wilson disease: mechanisms underlying enhancement of liver cell damage

Soy-protein isolate (SPI) enhances liver cell damage in Long-Evans rats with a cinnamon-like coat color (LEC rats), which have a defect in Atp7b, the Wilson disease gene. Animals administered an SPI-diet from an age of six weeks died significantly earlier than those administered a control-diet, AIN-93G, from severe liver cell damage associated with jaundice. Since the liver copper level was higher with the SPI-diet than the control-diet, one of the reasons for SPI-toxicity to LEC rats might be due to the higher uptake of copper into liver cells. In the present study, liver levels of glutathione, and liver and intestinal mRNA and protein levels were determined for metallothionein, MT-1 and MT-2. Furthermore, liver and intestinal mRNA expression for the high affinity copper transporter, Ctr1, was determined. None of the parameters showed any significant differences between the SPI-diet and control-diet groups, except for Ctr1 mRNA levels in the liver. It is thus suggested that SPI enhances liver cell copper uptake through induction of Ctr1 expression and this might be the mechanism underlying increased liver damage in LEC rats.     

Physiology and endocrinology of zinc accumulation during the female squirrelfish reproductive cycle

Females of the squirrelfish family (Holocentridae) accumulate higher levels of hepatic zinc than any other studied animal. This accumulation is accompanied by high expression of the zinc-binding protein, metallothionein (MT), and is strongly correlated to the onset of sexual maturity. In an attempt to further characterize the timeframe of this accumulation, and to possibly discern any potential mediators, we examined the physiology and endocrinology of the yearly reproductive cycle of mature female squirrelfish. There are two separate reproductive events during the year in December-January and again in March-April, as evidenced by peaks in ovarian growth, VTG production, steroid levels, zinc accumulation and redistribution. Increased hepatic zinc seems to be preceded by a necessary increase in MT, but this was not clearly correlated to plasma 17beta-estradiol, testosterone, or progesterone levels. The plasma zinc protein vitellogenin (VTG) is one, but probably not the predominant, vehicle for the transport of hepatic zinc to the ovary.