The Functions of Metallothionein and ZIP and ZnT Transporters: An Overview and Perspective

Around 3000 proteins are thought to bind zinc in vivo, which corresponds to ~10% of the human proteome. Zinc plays a pivotal role as a structural, catalytic, and signaling component that functions in numerous physiological processes. It is more widely used as a structural element in proteins than any other transition metal ion, is a catalytic component of many enzymes, and acts as a cellular signaling mediator. Thus, it is expected that zinc metabolism and homeostasis have sophisticated regulation, and elucidating the underlying molecular basis of this is essential to understanding zinc functions in cellular physiology and pathogenesis. In recent decades, an increasing amount of evidence has uncovered critical roles of a number of proteins in zinc metabolism and homeostasis through influxing, chelating, sequestrating, coordinating, releasing, and effluxing zinc. Metallothioneins (MT) and Zrt- and Irt-like proteins (ZIP) and Zn transporters (ZnT) are the proteins primarily involved in these processes, and their malfunction has been implicated in a number of inherited diseases such as acrodermatitis enteropathica. The present review updates our current understanding of the biological functions of MTs and ZIP and ZnT transporters from several new perspectives.     

Effects of Cadmium Exposure on Gut Villi in Danio rerio

In aquatic organisms, cadmium exposure occurs from ovum to death and the route of absorption is particularly wide, being represented by skin, gills and gastrointestinal tract, through which contaminated water and/or preys are ingested. It is known that cadmium interferes with the gut; however, less information is available on cadmium effects on an important component of the gut, namely goblet cells, specialized in mucus synthesis. In the present work, we studied the effects of two sublethal cadmium concentrations on the gut mucosa of Danio rerio. Particular attention was paid to changes in the distribution of glycan residues, and in metallothionein expression in intestinal cells. The results show that cadmium interferes with gut mucosa and goblet cells features. The effects are dose- and site-dependent, the anterior gut being more markedly affected than the midgut. Cadmium modifies the presence and/or distribution of glycans in the brush border and cytoplasm of enterocytes and in the goblet cells’ cytoplasm and alters the metallothionein expression and localization. The results suggest a significant interference of cadmium with mucosal efficiency, representing a health risk for the organism in direct contact with contamination and indirectly for the trophic chain.     

An adsorptive transfer technique coupled with brdicka reaction to reveal the importance of metallothionein in chemotherapy with platinum based cytostatics

The drugs based on platinum metals represent one of the oldest, but also one of the most effective groups of chemotherapeutic agents. Thanks to many clinical studies it is known that resistance of tumor cells to drugs is a frequent cause of chemotherapy failure. With regard to platinum based drugs, multidrug resistance can also be connected with increased expression of low-molecular weight protein metallothionein (MT). This study aimed at investigating the interactions of MT with cisplatin or carboplatin, using the adsorptive transfer technique coupled with differential pulse voltammetry Brdicka reaction (AdTS DPV Brdicka reaction), and a comparison of in vitro results with results obtained in vivo. The results obtained from the in vitro study show a strong affinity between platinum based drugs and MT. Further, we analyzed extracts of neuroblastoma cell lines treated with cisplatin or carboplatin. It is clear that neuroblastoma UKF-NB-4 cisplatin-resistant and cisplatin-sensitive cell lines unlikely respond to the presence of the platinum-based cytostatics cisplatin and carboplatin. Finally, we determined the level of MT in samples from rabbits treated with carboplatin and patients with retinoblastoma treated with the same drug.     

Direct-binding quartz crystal microbalance immunosensor to detect carp metallothionein

Metallothionein (MT) is a valuable biomarker against xenobiotic heavy metal contamination in organs and blood of fish. In this study, MT was induced in carp blood by cadmium injection and was purified to homogeneity by DEAE-cellulose ion exchange column chromatography to be used dually as a standard and immunogen. A sensitive detection on carp MT was then followed using a batch-type direct-binding quartz crystal microbalance immunosensor system installed with a well cell. Among antibody immobilization methods tested, two methods that were carried out via a heterobifunctional thiolation cross-linker, and via the cross-linker and protein G showed their respective advantages in simplicity and sensitivity. When analyzed using the immunosensor for carp MT in the concentration range of 50-4000 ng/mL, the limit of detection around 250 ng/mL was obtained. Based on its reasonable sensitivity and real-time presentation for the sensor signal, the immunosensor of this study was presumed as a screening tool to monitor possible heavy metal contamination in a fish farm and imported fish.     

Vectors for Cu2+-inducible production of glutathione S-transferase-fusion proteins for single-step purification from yeast

We describe six new yeast episomal vectors which encode glutathione S-transferase (GST) affinity tags. These allow for the production of GST-fusion proteins in Saccharomyces cerevisiae under the control of the CUP1 promoter. Affinity chromatography with glutathione-Sepharose permits convenient purification of the fusion protein from a yeast lysate. The presence of a protease cleavage site facilitates subsequent removal of the GST tag. The expression and single-step purification of both GST and a functional GST-metallothionein fusion from yeast are shown as an example of the application of these vectors.     

Polyphenol-Rich Beverages Enhance Zinc Uptake and Metallothionein Expression in Caco-2 Cells

ABSTRACT: The effect of red wine (RW), red grape juice (RGJ), green tea (GT), and representative polyphenols on Caco-2 cell ⁶⁵Zn uptake was explored. RW, RGJ, and GT enhanced the uptake of zinc from rice matrix. Fractionation of RW revealed that enhancing activity of zinc uptake was exclusively resided in the polyphenol fraction. Among the polyphenols tested, only tannic acid and quercitin stimulated the uptake of zinc while others did not influence the uptake. In tune with these results, only tannic acid and quercitin competed with zinquin (a zinc selective fluorophore) for zinc in vitro. Although all the polyphenols tested appear to enhance the expression of metallothionein (MT), the induction was higher with tannic acid, quercitin, and RW extract. Furthermore, phytic acid abrogated the tannic acid-induced MT expression. These results suggest that polyphenol-rich beverages, tannic acid, and quercitin bind and stimulate the zinc uptake and MT expression in Caco-2 cells.     

The Metallothionein/Thionein System: An Oxidoreductive Metabolic Zinc Link

Metallothioneins (MTs) were discovered more than 50 years ago and identified as low‐molecular weight, sulfhydryl‐rich proteins that were subsequently found to bind zinc predominantly. The binding of seemingly redox inactive zinc ions allows MT to play a central role in oxidoreductive cellular metabolism, cellular zinc distribution and homeostasis. In this interpretive study, we discuss the interaction of MT with physiologically relevant molecules and its effect on zinc? thiolate bonds. These interactions are linked to recent progress in the functional role of MT in cellular zinc transport, energy production, and protection of the organism against oxidative stress and neurodegenerative diseases.     

Trace elements bioaccumulation in Porcellionides pruinosus (Brandt, 1833) and related biomarkers of exposure

In the last years, the increase of anthropogenic activities has led to a progressive pollution of ecosystems. Oniscidean isopods are a group of terrestrial animals known as excellent bio‐indicators and bio‐accumulators, because of their capacity to accumulate contaminants and to implement appropriate strategies of detoxification. For this study, we collected Porcellionides pruinosus from two different sites in the Corinth region (Greece), a polluted site and a control site, to assess the heavy metal pollution status. We conducted an inductively coupled plasma mass spectrometry (ICP‐MS) analysis for the detection of trace elements in animals' hepatopancreas and soil; we performed also immunohistochemistry for detection of metallothioneins 1 (MT1) and heat shock protein 70 (HSP70s). The results suggest a great capacity of the species in trace elements bioaccumulation and the high degree of pollution of the area near Agioi Theodoroi where the metal pollution index (MPI) is threefolds higher than the control site, due to the presence of significantly higher concentration of arsenic, cadmium, cobalt, chromium, manganese, molybdenum, and lead found in hepatopancreas and soil. The environmental stress is confirmed by the expression of the relative biomarkers of exposure MT1 and HSP70. Our study, with a multimarker approach, consents getting a complete analysis of environmental quality and the effect of pollution on organisms and shows for the first time the heavy metals contamination status in certain Greece area. Furthermore, P. pruinosus proved to be an appropriate indicator of pollution in terrestrial ecosystems.