Interactions between zinc and NRF2 in vascular redox signalling.

Recent evidence highlights the importance of trace metal micronutrients such as zinc (Zn) in coronary and vascular diseases. Zn2+ plays a signalling role in modulating endothelial nitric oxide synthase and protects the endothelium against oxidative stress by up-regulation of glutathione synthesis. Excessive accumulation of Zn2+ in endothelial cells leads to apoptotic cell death resulting from dysregulation of glutathione and mitochondrial ATP synthesis, whereas zinc deficiency induces an inflammatory phenotype, associated with increased monocyte adhesion. Nuclear factor-E2-related factor 2 (NRF2) is a transcription factor known to target hundreds of different genes. Activation of NRF2 affects redox metabolism, autophagy, cell proliferation, remodelling of the extracellular matrix and wound healing. As a redox-inert metal ion, Zn has emerged as a biomarker in diagnosis and as a therapeutic approach for oxidative-related diseases due to its close link to NRF2 signalling. In non-vascular cell types, Zn has been shown to modify conformations of the NRF2 negative regulators Kelch-like ECH-associated Protein 1 (KEAP1) and glycogen synthase kinase 3ß (GSK3ß) and to promote degradation of BACH1, a transcriptional suppressor of select NRF2 genes. Zn can affect phosphorylation signalling, including mitogen-activated protein kinases (MAPK), phosphoinositide 3-kinases and protein kinase C, which facilitate NRF2 phosphorylation and nuclear translocation. Notably, several NRF2-targeted proteins have been suggested to modify cellular Zn concentration via Zn exporters (ZnTs) and importers (ZIPs) and the Zn buffering protein metallothionein. This review summarises the cross-talk between reactive oxygen species, Zn and NRF2 in antioxidant responses of vascular cells against oxidative stress and hypoxia/reoxygenation.

Chemistry meets biology in the coordination dynamics of metalloproteins.

Metal sites in proteins are often presented in an idealized way that does not capture the intrinsic dynamic behavior of the protein or the extrinsic factors that affect changes in the coordination of the metal ion in biological space and time. The bioinorganic chemistry possible in healthy and diseased living organisms is limited by prevailing pH values, redox potentials, and availability and concentrations of metal ions and ligands. Changes in any of these parameters and protein-protein or protein-ligand interactions can result in differences in the type of metal ion bound, metal occupancy, and coordination number or geometry. This article addresses the plasticity and complexity of metal coordination in proteins when these parameters are considered. It uses three examples of zinc sites with sulfur donor atoms from cysteines in mammalian proteins: alcohol dehydrogenases, metallothioneins, and zinc transporters of the ZnT (SLC30A) family. Coordination dynamics of the metal sites in these proteins has different purposes; in alcohol dehydrogenases for the metal ion to perform its different roles in the catalytic cycle, in metallothioneins for serving as a metal buffer, and in ZnT zinc transporters for sensing metal ions and moving them through the protein and thus biological membranes. Defining the biological and chemical parameters that determine and affect coordination dynamics of metal ions in proteins will inform future investigations of metalloproteins.

GPR39: An orphan receptor begging for ligands.

Progress in the understanding of the receptor GPR39 is held up by inconsistent pharmacological data. First, the endogenous ligand(s) remain(s) contentious. Data pointing to zinc ions (Zn2+) and/or eicosanoids as endogenous ligands are a matter of debate. Second, there are uncertainties in the specificity of the widely used synthetic ligand (agonist) TC-G 1008. Third, activation of GPR39 has been often proposed as a novel treatment strategy, but new data also support that inhibition might be beneficial in certain disease contexts. Constitutive activity/promiscuous signaling suggests the need for antagonists/inverse agonists in addition to (biased) agonists. Here, we scrutinize data on the signaling and functions of GPR39 and critically assess factors that might have contributed to divergent outcomes and interpretations of investigations on this important receptor.

Aryl hydrocarbon receptor utilises cellular zinc signals to maintain the gut epithelial barrier.

Zinc and plant-derived ligands of the aryl hydrocarbon receptor (AHR) are dietary components affecting intestinal epithelial barrier function. Here, we explore whether zinc and the AHR pathway are linked. We show that dietary supplementation with an AHR pre-ligand offers protection against inflammatory bowel disease in a mouse model while protection fails in mice lacking AHR in the intestinal epithelium. AHR agonist treatment is also ineffective in mice fed zinc depleted diet. In human ileum organoids and Caco-2 cells, AHR activation increases total cellular zinc and cytosolic free Zn2+ concentrations through transcription of genes for zinc importers. Tight junction proteins are upregulated through zinc inhibition of nuclear factor kappa-light-chain-enhancer and calpain activity. Our data show that AHR activation by plant-derived dietary ligands improves gut barrier function at least partly via zinc-dependent cellular pathways, suggesting that combined dietary supplementation with AHR ligands and zinc might be effective in preventing inflammatory gut disorders.

Vascular protection afforded by zinc supplementation in human coronary artery smooth muscle cells mediated by NRF2 signaling under hypoxia/reoxygenation.

Zinc (Zn) has antioxidant, anti-inflammatory and anti-proliferative actions, with Zn dysregulation associated with coronary ischemia/reperfusion injury and smooth muscle cell dysfunction. As the majority of studies concerning Zn have been conducted under non-physiological hyperoxic conditions, we compare the effects of Zn chelation or supplementation on total intracellular Zn content, antioxidant NRF2 targeted gene transcription and hypoxia/reoxygenation-induced reactive oxygen species generation in human coronary artery smooth muscle cells (HCASMC) pre-adapted to hyperoxia (18 kPa O2) or normoxia (5 kPa O2). Expression of the smooth muscle marker SM22-α was unaffected by lowering pericellular O2, whereas calponin-1 was significantly upregulated in cells under 5 kPa O2, indicating a more physiological contractile phenotype under 5 kPa O2. Inductively coupled plasma mass spectrometry established that Zn supplementation (10 µM ZnCl2 + 0.5 µM pyrithione) significantly increased total Zn content in HCASMC under 18 but not 5 kPa O2. Zn supplementation increased metallothionein mRNA expression and NRF2 nuclear accumulation in cells under 18 or 5 kPa O2. Notably, NRF2 regulated HO-1 and NQO1 mRNA expression in response to Zn supplementation was only upregulated in cells under 18 but not 5 kPa. Furthermore, whilst hypoxia increased intracellular glutathione (GSH) in cells pre-adapted to 18 but not 5 kPa O2, reoxygenation had negligible effects on GSH or total Zn content. Reoxygenation-induced superoxide generation in cells under 18 kPa O2 was abrogated by PEG-superoxide dismutase but not by PEG-catalase, and Zn supplementation, but not Zn chelation, attenuated reoxygenation-induced superoxide generation in cells under 18 but not 5kPaO2, consistent with a lower redox stress under physiological normoxia. Our findings highlight that culture of HCASMC under physiological normoxia recapitulates an in vivo contractile phenotype and that effects of Zn on NRF2 signaling are altered by oxygen tension.

Redox and metal profiles in human coronary endothelial and smooth muscle cells under hyperoxia, physiological normoxia and hypoxia: Effects of NRF2 signaling on intracellular zinc.

Zinc is an important component of cellular antioxidant defenses and dysregulation of zinc homeostasis is a risk factor for coronary heart disease and ischemia/reperfusion injury. Intracellular homeostasis of metals, such as zinc, iron and calcium are interrelated with cellular responses to oxidative stress. Most cells experience significantly lower oxygen levels in vivo (2-10 kPa O2) compared to standard in vitro cell culture (18kPa O2). We report the first evidence that total intracellular zinc content decreases significantly in human coronary artery endothelial cells (HCAEC), but not in human coronary artery smooth muscle cells (HCASMC), after lowering of O2 levels from hyperoxia (18 kPa O2) to physiological normoxia (5 kPa O2) and hypoxia (1 kPa O2). This was paralleled by O2-dependent differences in redox phenotype based on measurements of glutathione, ATP and NRF2-targeted protein expression in HCAEC and HCASMC. NRF2-induced NQO1 expression was attenuated in both HCAEC and HCASMC under 5 kPa O2 compared to 18 kPa O2. Expression of the zinc efflux transporter ZnT1 increased in HCAEC under 5 kPa O2, whilst expression of the zinc-binding protein metallothionine (MT) decreased as O2 levels were lowered from 18 to 1 kPa O2. Negligible changes in ZnT1 and MT expression were observed in HCASMC. Silencing NRF2 transcription reduced total intracellular zinc under 18 kPa O2 in HCAEC with negligible changes in HCASMC, whilst NRF2 activation or overexpression increased zinc content in HCAEC, but not HCASMC, under 5 kPa O2. This study has identified cell type specific changes in the redox phenotype and metal profile in human coronary artery cells under physiological O2 levels. Our findings provide novel insights into the effect of NRF2 signaling on Zn content and may inform targeted therapies for cardiovascular diseases.

The first trimester plasma copper-zinc ratio is independently related to pregnancy-specific psychological distress symptoms throughout pregnancy.

OBJECTIVES: High plasma copper (Cu) and low zinc (Zn) levels have been associated with depression. However, most studies used low sample sizes and a cross-sectional design, and perinatal data are scarce. We investigated the possible association between pregnancy-specific psychological distress and the plasma CuZn ratio using a prospective design. METHODS: Pregnancy-specific distress symptoms were assessed at each trimester by means of the Tilburg Pregnancy Distress Scale, negative affect subscale, in 2036 pregnant women. Cu and Zn were assessed at 12 wk of gestation in plasma samples by inductively coupled plasma mass spectrometry. Growth mixture modeling determined trajectories of women's pregnancy-specific negative affect (P-NA) symptoms, which were entered in a multiple logistic regression analysis as dependent variable and the CuZn ratio as independent variable. RESULTS: Two P-NA symptom classes were found: 1) persistently low (n = 1820) and 2) persistently high (n = 216). A higher CuZn ratio was independently associated with persistently high P-NA symptom scores (odds ratio = 1.52; 95% confidence interval, 1.13-2.04) after adjustment for confounders. A sensitivity analysis was performed excluding all women with high P-NA scores at 12 wk of gestation (>1 SD above the mean P-NA score). In the 1719 remaining women, a higher CuZn ratio significantly predicted the development of increasing P-NA symptom scores after adjustment for confounders (odds ratio = 1.40; 95% confidence interval, 1.04-1.95). CONCLUSIONS: A higher CuZn plasma ratio is an independent determinant of developing pregnancy-specific distress symptoms throughout pregnancy, suggesting that micronutrients could be used as novel biomarkers for psychological distress research of perinatal mood disorders.

The quintessence of metallomics: a harbinger of a different life science based on the periodic table of the bioelements.

This year marks the 20th anniversary of the field of metallomics. As a landmark in time, it is an occasion to reflect on the past, present, and future of this integrated field of biometal sciences. A fundamental bias is one reason for having metallomics as a scientific discipline. The focus of biochemistry on the six non-metal chemical elements, collectively known with the acronym SPONCH (sulphur, phosphorus, oxygen, nitrogen, carbon, hydrogen), glosses over the fact that the lower quantities of many other elements have qualities that made them instrumental in the evolution of life and pivotal in numerous life processes. The metallome, alongside the genome, proteome, lipidome, and glycome, should be regarded as a fifth pillar of elemental-vis-à-vis molecular-building blocks in biochemistry. Metallomics as 'global approaches to metals in the biosciences' considers the biological significance of most chemical elements in the periodic table, not only the ones essential for life, but also the non-essential ones that are present in living matter-some at higher concentrations than the essential ones. The non-essential elements are bioactive with either positive or negative effects. Integrating the significance of many more chemical elements into the life sciences requires a transformation in learning and teaching with a focus on elemental biology in addition to molecular biology. It should include the dynamic interactions between the biosphere and the geosphere and how the human footprint is changing the ecology globally and exposing us to many additional chemical elements that become new bioelements.

Teaching the chemical elements in biochemistry: Elemental biology and metallomics.

Biochemistry primarily focuses on the non-metal chemical elements carbon, oxygen, nitrogen, hydrogen, sulfur, and phosphorus in the four groups of building blocks (sugars, lipids, amino acids, and nucleotides) and the corresponding macromolecules. However, at least 10 essential chemical elements of life are metals. This article discusses the consequences of such a bias, presents current knowledge that over 20 chemical elements are required for life, and makes a case for-and suggests benefits of-teaching elemental biology alongside molecular biology and biochemistry, and inorganic chemistry in addition to organic chemistry. A relatively new interdisciplinary field, metallomics, has the potential to be a platform for integration when added to glycomics, lipidomics, proteomics, and genomics. It would fill a major gap in contemporary education, be relevant for many areas of science, and facilitate the teaching of important principles of chemistry in the biological sciences, thus helping students to gain a broader understanding of life processes from the molecular to the systemic biology level.

Bert Vallee-A 20th Century Adventure(r) in Zincology.

Prelude [...].

The Bioinorganic Chemistry of Mammalian Metallothioneins.

The functions, purposes, and roles of metallothioneins have been the subject of speculations since the discovery of the protein over 60 years ago. This article guides through the history of investigations and resolves multiple contentions by providing new interpretations of the structure-stability-function relationship. It challenges the dogma that the biologically relevant structure of the mammalian proteins is only the one determined by X-ray diffraction and NMR spectroscopy. The terms metallothionein and thionein are ambiguous and insufficient to understand biological function. The proteins need to be seen in their biological context, which limits and defines the chemistry possible. They exist in multiple forms with different degrees of metalation and types of metal ions. The homoleptic thiolate coordination of mammalian metallothioneins is important for their molecular mechanism. It endows the proteins with redox activity and a specific pH dependence of their metal affinities. The proteins, therefore, also exist in different redox states of the sulfur donor ligands. Their coordination dynamics allows a vast conformational landscape for interactions with other proteins and ligands. Many fundamental signal transduction pathways regulate the expression of the dozen of human metallothionein genes. Recent advances in understanding the control of cellular zinc and copper homeostasis are the foundation for suggesting that mammalian metallothioneins provide a highly dynamic, regulated, and uniquely biological metal buffer to control the availability, fluctuations, and signaling transients of the most competitive Zn(II) and Cu(I) ions in cellular space and time.

Comparative outcomes of exposing human liver and kidney cell lines to tungstate and molybdate.

Tungsten has no known function in humans and is a relatively new contaminant, whereas molybdenum, its congener in the periodic table, is a nutritionally essential element. In addition to early studies on molybdosis in ruminants, their toxic effects in the form of tungstate and molybdate have been addressed primarily in rodents and are predominantly mediated by inducing oxidative stress in various tissues. The purpose of this study was to evaluate the differences between tungstate and molybdate in human liver (HepG2) and kidney (HEK293) cell lines in terms of retention in cells, effect on reactive oxygen species, and activities of xanthine oxidase and phosphatases. The cell lines were exposed to tungstate or molybdate (1 µM to 10 mM) for 24 h, lysed and analyzed for the above biochemical parameters. Despite the chemical similarity of the two anions, cell-specific differential effects were observed. At all concentrations, tungstate was retained more in HEK293 cells while molybdate was retained more in HepG2 cells. HepG2 cells were more sensitive to tungstate than molybdate, showing reduced viability at concentrations as low as 10 µM. Exposure to either anion resulted in the inhibition of protein tyrosine phosphatases at 1 mM and an increased production of reactive oxygen species (ROS) at 100 µM despite their inhibition of the ROS-producing molybdenum enzyme xanthine oxidase. In conclusion, the results indicate that excess of nutritionally essential molybdate or non-essential tungstate causes toxicity by affecting ROS- and phosphorylation-dependent signaling pathways and ensuing gene expression.

Probing the Structure and Function of the Cytosolic Domain of the Human Zinc Transporter ZnT8 with Nickel(II) Ions.

The human zinc transporter ZnT8 provides the granules of pancreatic ß-cells with zinc (II) ions for assembly of insulin hexamers for storage. Until recently, the structure and function of human ZnTs have been modelled on the basis of the 3D structures of bacterial zinc exporters, which form homodimers with each monomer having six transmembrane α-helices harbouring the zinc transport site and a cytosolic domain with an α,ß structure and additional zinc-binding sites. However, there are important differences in function as the bacterial proteins export an excess of zinc ions from the bacterial cytoplasm, whereas ZnT8 exports zinc ions into subcellular vesicles when there is no apparent excess of cytosolic zinc ions. Indeed, recent structural investigations of human ZnT8 show differences in metal binding in the cytosolic domain when compared to the bacterial proteins. Two common variants, one with tryptophan (W) and the other with arginine (R) at position 325, have generated considerable interest as the R-variant is associated with a higher risk of developing type 2 diabetes. Since the mutation is at the apex of the cytosolic domain facing towards the cytosol, it is not clear how it can affect zinc transport through the transmembrane domain. We expressed the cytosolic domain of both variants of human ZnT8 and have begun structural and functional studies. We found that (i) the metal binding of the human protein is different from that of the bacterial proteins, (ii) the human protein has a C-terminal extension with three cysteine residues that bind a zinc(II) ion, and (iii) there are small differences in stability between the two variants. In this investigation, we employed nickel(II) ions as a probe for the spectroscopically silent Zn(II) ions and utilised colorimetric and fluorimetric indicators for Ni(II) ions to investigate metal binding. We established Ni(II) coordination to the C-terminal cysteines and found differences in metal affinity and coordination in the two ZnT8 variants. These structural differences are thought to be critical for the functional differences regarding the diabetes risk. Further insight into the assembly of the metal centres in the cytosolic domain was gained from potentiometric investigations of zinc binding to synthetic peptides corresponding to N-terminal and C-terminal sequences of ZnT8 bearing the metal-coordinating ligands. Our work suggests the involvement of the C-terminal cysteines, which are part of the cytosolic domain, in a metal chelation and/or acquisition mechanism and, as now supported by the high-resolution structural work, provides the first example of metal-thiolate coordination chemistry in zinc transporters.

Plasma mineral (selenium, zinc or copper) concentrations in the general pregnant population, adjusted for supplement intake, in relation to thyroid function.

The present study reports on first-trimester reference ranges of plasma mineral Se/Zn/Cu concentration in relation to free thyroxine (FT4), thyrotropin (TSH) and thyroid peroxidase antibodies (TPO-Ab), assessed at 12 weeks' gestation in 2041 pregnant women, including 544 women not taking supplements containing Se/Zn/Cu. The reference range (2·5th-97·5th percentiles) in these 544 women was 0·72-1·25 µmol/l for Se, 17·15-35·98 µmol/l for Cu and 9·57-16·41 µmol/l for Zn. These women had significantly lower mean plasma Se concentration (0·94 (sd 0·12) µmol/l) than those (n 1479) taking Se/Zn/Cu supplements (1·03 (sd 0·14) µmol/l; P < 0·001), while the mean Cu (26·25 µmol/l) and Zn (12·55 µmol/l) concentrations were almost identical in these sub-groups. Women with hypothyroxinaemia (FT4 below reference range with normal TSH) had significantly lower plasma Zn concentrations than euthyroid women. After adjusting for covariates including supplement intake, plasma Se (negatively), Zn and Cu (positively) concentrations were significantly related to logFT4; Se and Cu (but not Zn) were positively and significantly related to logTSH. Women taking additional Se/Zn/Cu supplements were 1·46 (95 % CI 1·09, 2·04) times less likely to have elevated titres of TPO-Ab at 12 weeks of gestation. We conclude that first-trimester Se reference ranges are influenced by Se-supplement intake, while Cu and Zn ranges are not. Plasma mineral Se/Zn/Cu concentrations are associated with thyroid FT4 and TSH concentrations. Se/Zn/Cu supplement intake affects TPO-Ab status. Future research should focus on the impact of trace mineral status during gestation on thyroid function.

The ZIP6/ZIP10 heteromer is essential for the zinc-mediated trigger of mitosis.

Zinc has been known to be essential for cell division for over 40 years but the molecular pathways involved remain elusive. Cellular zinc import across biological membranes necessitates the help of zinc transporters such as the SLC39A family of ZIP transporters. We have discovered a molecular process that explains why zinc is required for cell division, involving two highly regulated zinc transporters, as a heteromer of ZIP6 and ZIP10, providing the means of cellular zinc entry at a specific time of the cell cycle that initiates a pathway resulting in the onset of mitosis. Crucially, when the zinc influx across this heteromer is blocked by ZIP6 or ZIP10 specific antibodies, there is no evidence of mitosis, confirming the requirement for zinc influx as a trigger of mitosis. The zinc that influxes into cells to trigger mitosis additionally changes the phosphorylation state of STAT3 converting it from a transcription factor to a protein that complexes with this heteromer and pS38Stathmin, the form allowing microtubule rearrangement as required in mitosis. This discovery now explains the specific cellular role of ZIP6 and ZIP10 and how they have special importance in the mitosis process compared to other ZIP transporter family members. This finding offers new therapeutic opportunities for inhibition of cell division in the many proliferative diseases that exist, such as cancer.

Zinc and Cadmium in the Aetiology and Pathogenesis of Osteoarthritis and Rheumatoid Arthritis.

Osteoarthritis (OA) and rheumatoid arthritis (RA) are inflammatory articular conditions with different aetiology, but both result in joint damage. The nutritionally essential metal zinc (Zn2+) and the non-essential metal cadmium (Cd2+) have roles in these arthritic diseases as effectors of the immune system, inflammation, and metabolism. Despite both metal ions being redox-inert in biology, they affect the redox balance. It has been known for decades that zinc decreases in the blood of RA patients. It is largely unknown, however, whether this change is only a manifestation of an acute phase response in inflammation or relates to altered availability of zinc in tissues and consequently requires changes of zinc in the diet. As a cofactor in over 3000 human proteins and as a signaling ion, zinc affects many pathways relevant for arthritic disease. How it affects the diseases is not just a question of zinc status, but also an issue of mutations in the many proteins that maintain cellular zinc homoeostasis, such as zinc transporters of the ZIP (Zrt-/Irt-like protein) and ZnT families and metallothioneins, and the multiple pathways that change the expression of these proteins. Cadmium interferes with zinc's functions and there is increased uptake under zinc deficiency. Remarkably, cadmium exposure through inhalation is now recognized in the activation of macrophages to a pro-inflammatory state and suggested as a trigger of a specific form of nodular RA. Here, we discuss how these metal ions participate in the genetic, metabolic, and environmental factors that lead to joint destruction. We conclude that both metal ions should be monitored routinely in arthritic disease and that there is untapped potential for prognosis and treatment.

Total Iron Measurement in Human Serum With a Novel Smartphone-Based Assay.

Background: Abnormally low or high blood iron levels are common health conditions worldwide and can seriously affect an individual's overall well-being. A low-cost point-of-care technology that measures blood iron markers with a goal of both preventing and treating iron-related disorders represents a significant advancement in medical care delivery systems. Methods: A novel assay equipped with an accurate, storable, and robust dry sensor strip, as well as a smartphone mount and (iPhone) app is used to measure total iron in human serum. The sensor strip has a vertical flow design and is based on an optimized chemical reaction. The reaction strips iron ions from blood-transport proteins, reduces Fe(III) to Fe(II), and chelates Fe(II) with ferene, with the change indicated by a blue color on the strip. The smartphone mount is robust and controls the light source of the color reading App, which is calibrated to obtain output iron concentration results. The real serum samples are then used to assess iron concentrations from the new assay, and validated through intra-laboratory and inter-laboratory experiments. The intra-laboratory validation uses an optimized iron detection assay with multi-well plate spectrophotometry. The inter-laboratory validation method is performed in a commercial testing facility (LabCorp). Results: The novel assay with the dry sensor strip and smartphone mount, and App is seen to be sensitive to iron detection with a dynamic range of 50 - [Formula: see text]/dL, sensitivity of 0.00049 a.u/[Formula: see text]/dL, coefficient of variation (CV) of 10.5%, and an estimated detection limit of [Formula: see text]/dL These analytical specifications are useful for predicting iron deficiency and overloads. The optimized reference method has a sensitivity of 0.00093 a.u/[Formula: see text]/dL and CV of 2.2%. The correlation of serum iron concentrations (N = 20) between the optimized reference method and the novel assay renders a slope of 0.95, and a regression coefficient of 0.98, suggesting that the new assay is accurate. Last, a spectrophotometric study of the iron detection reaction kinetics is seen to reveal the reaction order for iron and chelating agent. Conclusion: The new assay is able to provide accurate results in intra- and inter- laboraty validations, and has promising features of both mobility and low-cost manufacturing suitable for global healthcare settings.