Regulation Of Zinc Homeostasis Research Articles

Zinc dyshomeostasis in azoxymethane-induced colonic precancerous and cancerous lesions in Fischer rats.

Zinc is an essential micronutrient involved in various biological processes. It is also argued that tumors need zinc for maintenance and proliferation and tumor cell apoptosis. Zinc homeostasis is regulated by the gastrointestinal tract and involves interplay of host, dietary, environmental and social factors such as alcohol consumption. The DNA alkylation agent azoxymethane (AOM), which is primarily activated in the liver, induces a high incidence of initiation and promotion steps of precancerous lesions in the colon of rats. The altered expression of hepatic zinc transporters by AOM may lead to zinc dyshomeostasis in liver. Decreased serum zinc concentration, despite increased liver zinc also indicates altered liver zinc mobilization and failure to regulate zinc homeostasis. During the transformation from normal colonic mucosa to colonic epithelial hyperplasia and aberrant crypt formation, a reduction in zinc concentration is observed. It will be interesting to study further if the same trend continues throughout tumor progression towards adenocarcinomas. Lowered local zinc concentrations in the colon epithelium may not just reflect a bystander effect, but may induce cell proliferation and compromise DNA integrity due to impairment of zinc-containing proteins. In congruence with the tissue zinc concentrations, metallothionein levels were found to be less induced in AOM -administered colon compared to normal healthy colon. Lowered tissue zinc levels in small and large intestine were also associated with increased expression of mRNA and protein ZnT1. In this regard, the mode of zinc responsiveness to ZnT1 mimics that of metallothionein, albeit at a lower level for ZnT1.

Slc39a5-mediated zinc homeostasis plays an essential role in venous angiogenesis in zebrafish.

Angiogenesis is a precise process mediated by a variety of signals and the environmental niche. Although the essential trace element zinc and its homeostasis are essential for maintaining proper cellular functions, whether zinc plays a role in angiogenesis is currently unknown. Using zebrafish embryos as a model system, we found that zinc treatment significantly increased the expression of the slc39a5 gene, which encodes the zinc transporter Slc39a5. Moreover, knocking down slc39a5 expression using either a morpholino or CRISPR/Cas9-mediated gene editing led to cardiac ischaemia and an accumulation of red blood cells in the caudal vein plexus (CVP), as well as delayed venous sprouting and fewer vascular loops in the CVP region during early development. Further analysis revealed significantly reduced proliferation and delayed cell migration in the caudal vein of slc39a5 morphants. At the mechanistic level, we found increased levels of systemic zinc in slc39a5-deficient embryos, and chelating zinc restored CVP development. In addition, we found that zinc overload in wild-type embryos leads to impaired CVP formation. Taken together, these results indicate that Slc39a5 plays a critical role in endothelial sprouting and migration in venous angiogenesis by regulating zinc homeostasis.

Drosophila ZnT1 is essential in the intestine for dietary zinc absorption

Zinc is an essential trace element and participates in a variety of biological processes. ZnT (SLC30) family members are generally responsible for zinc efflux across the membrane regulating zinc homeostasis. In mammals, the only predominantly plasma membrane resident ZnT has been reported to be ZnT1, and ZnT1-/ZnT1- mice die at the embryonic stage. In Drosophila, knock down of ZnT1 homologue (dZnT1//ZnT63C/CG17723) results in growth arrest under zinc-limiting conditions. To investigate the essentiality of dZnT1 for zinc homeostasis, as well as its role in dietary zinc uptake especially under normal physiological conditions, we generated dZnT1 mutants by the CRISPER/Cas9 method. Homozygous mutant dZnT1 is lethal, with substantial zinc accumulation in the iron cell region, posterior midgut as well as gastric caeca. Expression of human ZnT1 (hZnT1), in the whole body or in the entire midgut, fully rescued the dZnT1 mutant lethality, whereas tissue-specific expression of hZnT1 in the iron cell region and posterior midgut partially rescued the developmental defect of the dZnT1 mutant. Supplementation of zinc together with clioquinol or hinokitiol conferred a limited but observable rescue upon dZnT1 loss. Our work demonstrated the absolute requirement of dZnT1 in Drosophila survival and indicated that the most essential role of dZnT1 is in the gut.

The S100B Alarmin Is a Dual-Function Chaperone Suppressing Amyloid-β Oligomerization through Combined Zinc Chelation and Inhibition of Protein Aggregation.

Amyloid beta (Aβ) aggregation and imbalance of metal ions are major hallmarks of Alzheimer's disease (AD). Indeed, amyloid plaques of AD patients are enriched in zinc and Aβ42, and AD related-cognitive decline is dependent on extracellular zinc concentration. In vitro, zinc induces the formation of polymorphic Aβ42 oligomers that delay the formation of amyloid fibers at the expense of increased cellular toxicity. S100B is an inflammatory alarmin and one of the most abundant proteins in the brain and is upregulated in AD and associated with amyloid plaques, where it exerts extracellular functions. Recent findings have uncovered novel neuroprotective functions for S100B as a suppressor of Aβ aggregation and toxicity and in the regulation of zinc homeostasis in neurons. Here we combine biophysical and kinetic approaches to demonstrate that such S100B protective functions converge, making the protein a dual-function chaperone capable of suppressing the formation of toxic Aβ oligomers through both chelation of zinc and inhibition of protein aggregation. From detailed kinetic analysis of Aβ42 aggregation monitoring ThT fluorescence, we show that substoichiometric S100B prevents the formation of toxic off-pathway oligomers that are formed by monomeric Aβ42 in the presence of zinc. Indeed, S100B is effective when added during the lag and transition phases of Aβ42 aggregation, and its action under these circumstances results from its ability to buffer zinc, as it perfectly mimics the effect obtained with the chelating agent EDTA. Further, bioimaging analysis combining transmission electron microscopy and atomic force microscopy confirms that catalytic amounts of S100B partly revert the formation of toxic oligomers. Taken together these results indicate a new role for S100B as a dual chaperone whose distinct functions are interrelated and depend on the relative levels of zinc, S100B, and Aβ, which dynamically evolve during AD.

The involvement of zinc transporters in the zinc accumulation in the Pacific oyster Crassostrea gigas.

Zinc transporters play vital roles in regulating zinc content and localization by mobilizing zinc across cellular and intracellular membranes. Pacific oyster Crassostrea gigas is one of the most zinc-rich animals, which has been regarded as an excellent food for zinc supplement. But the information about zinc transporters and their involvements in zinc accumulation in oysters is still limited. In the present study, a total of 28 zinc transporter genes, including nine Zinc transporter genes (CgZnTs) and 19 Zrt/Irt-like protein genes (CgZIPs), were identified in C. gigas genome using a genome-wide search strategy. There were five ZIP10 homologs in C. gigas, which were much more than those in mammals, fish and other mollusks. Among oyster zinc transporters, immense variations were detected in their gene structure, protein length and physicochemical properties. Phylogenetic analysis showed that most of these transporters were distinctly clustered with their homologs from Homo sapiens, Danio rerio and other mollusks, and the most closely related transporters shared similar motif compositions. The highest zinc content was detected in the oyster mantle and gill, while the lowest level was found in the adductor muscle. The mRNA of all tested CgZnTs and CgZIPs were constitutively expressed in oyster tissues, and most of them were highly expressed in the gill or hepatopancreas. The analysis of RNA-seq data from gill and hepatopancreas showed that all the transporters exhibited divergent response patterns under zinc stress, except for CgZIP4 whose expression was almost undetectable in the two tissues. The results indicated that zinc transporters played important roles in the regulation of zinc homeostasis in C. gigas, which provided a solid foundation for further functional analysis of zinc transporters in oysters and other mollusks.

One-carbon metabolism, folate, zinc and translation.

SummaryThe translation process, central to life, is tightly connected to the one‐carbon (1‐C) metabolism via a plethora of macromolecule modifications and specific effectors. Using manual genome annotations and putting together a variety of experimental studies, we explore here the possible reasons of this critical interaction, likely to have originated during the earliest steps of the birth of the first cells. Methionine, S‐adenosylmethionine and tetrahydrofolate dominate this interaction. Yet, 1‐C metabolism is unlikely to be a simple frozen accident of primaeval conditions. Reactive 1‐C species (ROCS) are buffered by the translation machinery in a way tightly associated with the metabolism of iron–sulfur clusters, zinc and potassium availability, possibly coupling carbon metabolism to nitrogen metabolism. In this process, the highly modified position 34 of tRNA molecules plays a critical role. Overall, this metabolic integration may serve both as a protection against the deleterious formation of excess carbon under various growth transitions or environmental unbalanced conditions and as a regulator of zinc homeostasis, while regulating input of prosthetic groups into nascent proteins. This knowledge should be taken into account in metabolic engineering.

Remodeling of Zn2+ homeostasis upon differentiation of mammary epithelial cells

Zinc is the second most abundant transition metal in humans and an essential nutrient required for growth and development of newborns. During lactation, mammary epithelial cells differentiate into a secretory phenotype, uptake zinc from blood circulation, and export it into mother's milk. At the cellular level, many zinc-dependent cellular processes, such as transcription, metabolism of nutrients, and proliferation are involved in the differentiation of mammary epithelial cells. Using mouse mammary epithelial cells as a model system, we investigated the remodeling of zinc homeostasis during differentiation induced by treatment with the lactogenic hormones cortisol and prolactin. RNA-Seq at different stages of differentiation revealed changes in global gene expression, including genes encoding zinc-dependent proteins and regulators of zinc homeostasis. Increases in mRNA levels of three zinc homeostasis genes, Slc39a14 (ZIP14) and metallothioneins (MTs) I and II were induced by cortisol but not by prolactin. The cortisol-induced increase was partially mediated by the nuclear glucocorticoid receptor signaling pathway. An increase in the cytosolic labile Zn2+ pool was also detected in lactating mammary cells, consistent with upregulation of MTs. We found that the zinc transporter ZIP14 was important for the expression of a major milk protein, whey acid protein (WAP), as knockdown of ZIP14 dramatically decreased WAP mRNA levels. In summary, our study demonstrated remodeling of zinc homeostasis upon differentiation of mammary epithelial cells resulting in changes in cytosolic Zn2+ and differential expression of zinc homeostasis genes, and these changes are important for establishing the lactation phenotype.

Protein kinase CK2 is involved in zinc homeostasis in breast and prostate cancer cells.

The intracellular zinc profiles of breast and prostate cancer cells are diametrically opposed, with hyper-accumulation of zinc in breast cancer, and low level in prostate cancer. This phenomenon is poorly understood. This study employs two breast and two prostate cancer cell lines to investigate the role of protein kinase CK2 in regulating zinc homeostasis. CK2 was targeted by its specific inhibitors 4,5,6,7-tetrabromobenzotriazole (TBB) and CX-4945, and by the specific siRNA against each of the three CK2 genes. The effect of zinc exposure after the above CK2 manipulation was observed by MTT [3-(4,5-dimethyliazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] cell viability assay and confocal microscopy for intracellular zinc level. The results demonstrate that CK2 is involved in regulating zinc homeostasis in breast and prostate cancer cells as both TBB and CX-4945 substantially decreased cell viability upon zinc exposure. siRNA-mediated knockdown of the three CK2 subunits (α, α' and β) revealed their discrete roles in regulating zinc homeostasis in breast and prostate cancer cells. Knockdown of CK2α' decreased the intracellular zinc level of breast cancer cells and in turn increased the cell viability while the opposite findings were obtained for the prostate cancer cells. Knockdown of CK2β expression substantially increased the zinc level in breast cancer cell lines whilst decreased the zinc level in prostate cancer cells. Taken together, this study shows that CK2 is involved in zinc homeostasis of breast and prostate cancer cells and opens a new avenue for research on these cancers.

Zn(II)-curcumin solid dispersion impairs hepatocellular carcinoma growth and enhances chemotherapy by modulating gut microbiota-mediated zinc homeostasis

Zinc(II) complexes of curcumin display moderate cytotoxicity towards cancer cells at low micromolar concentrations. However, the clinical use of zinc(II) complexes is hampered by hydrolytic insolubility and poor bioavailability and their anticancer mechanisms remain unclear. Here, we investigated the efficacy and mechanism of action of a polyvinylpyrrolidone (PVP-k30)-based solid dispersion of Zn(II)-curcumin (ZnCM-SD) against hepatocellular carcinoma (HCC) in vitro and in vivo. In vitro assays revealed ZnCM-SD not only reduced the viability of HepG2 cells and SK-HEP1 cells in a dose-dependent manner, but also potently and synergistically enhanced cell growth inhibition and cell death in response to doxorubicin by regulating cellular zinc homeostasis. ZnCM-SD was internalized into the cells via non-specific endocytosis and degraded to release curcumin and Zn2+ ions within cells. The anticancer effects also occur in vivo in animals following the oral administration of ZnCM-SD, without significantly affecting the weight of the animals. Interestingly, ZnCM-SD did not reduce tumor growth or affect zinc homeostasis in HepG2-bearing mice after gut microbiome depletion. Moreover, administration of ZnCM-SD alone or in combination with doxorubicin significantly attenuated gut dysbiosis and zinc dyshomeostasis in a rat HCC model. Notably, fecal microbiota transplantation revealed the ability of ZnCM-SD to regulate zinc homeostasis and act as a chemosensitizer for doxorubicin were dependent on the gut microbiota. The crucial role of the gut microbiota in the chemosensitizing ability of ZnCM-SD was confirmed by broad-spectrum antibiotic treatment. Collectively, ZnCM-SD could represent a simple, well-tolerated, safe, effective therapy and function as a novel chemosensitizing agent for cancer.

Knockdown of Zinc Transporter ZIP12 by shRNA Alters Genes Related to Mitochondria and Neuronal Differentiation in Neuro-2a Cells (P24-018-19)

ObjectivesZinc is vital for brain function and preservation of cognitive potential in aging. However, mechanisms by which the nervous system regulates zinc homeostasis are poorly understood. Hence, this study determined changes in gene expression due to the loss of ZIP12 in Neuro-2a neuroblastoma cells. MethodsWe used RNA-sequencing to uncover differentially expressed genes between cells transfected with a control short hairpin RNA (shRNA) and cells transfected with a shRNA targeting the ZIP12 mRNA sequence. Cells were transfected with NeuroMag (Oz Biosciences) (n = 6) in 6-well plates and differentiated with all-trans retinoic acid in reduced serum DMEM media for 48 hours. RNA libraries were prepared from total RNA (RNA integrity number >8.0) using the Illumina TruSeq Stranded mRNA Sample Preparation kit. Single-end 75 bp reads were generated on an Illumina NextSeq 500 sequencer. A transcriptome analysis pipeline including Trimmomatic, HISAT2, featureCounts, and DESeq2 was used to identify differentially expressed genes. The cutoff for differential expression was set at log2 fold change of 0.584962, corresponding to a 0.5x increase or decrease in expression relative to control. Results25 genes were found which met this cutoff, and all 25 genes had a false discovery rate (FDR) less than 1.0 × 10–9. Using Gene Set Enrichment Analysis (GSEA), we determined that differentially expressed genes (both upregulated and downregulated) were enriched for mitochondria-related function. These mitochondria-related genes include alcohol dehydrogenase-5, peroxiredoxin-3, and galectin-3, and translocase of inner mitochondrial membrane 8 homolog A, and these genes were more likely to be upregulated. Manual identification of genes important for neuronal differentiation in downregulated genes included dopamine beta hydroxylase, astrotactin-2, neural zinc finger-3, neuron specific gene family member-2, inhibitor of DNA binding-2, and inhibitor of DNA binding-3. ConclusionsThese findings indicate that changes in the transcriptome induced by loss of ZIP12 function may contribute impairments in neuronal function and reflect altered mitochondrial function. Funding SourcesOklahoma Center for the Advancement of Science & Technology; Oklahoma Agricultural Experiment Station. The RNA sequencing core is supported by grants from the National Science Foundation.

A conserved CCM complex promotes apoptosis non-autonomously by regulating zinc homeostasis

Apoptotic death of cells damaged by genotoxic stress requires regulatory input from surrounding tissues. The C. elegans scaffold protein KRI-1, ortholog of mammalian KRIT1/CCM1, permits DNA damage-induced apoptosis of cells in the germline by an unknown cell non-autonomous mechanism. We reveal that KRI-1 exists in a complex with CCM-2 in the intestine to negatively regulate the ERK-5/MAPK pathway. This allows the KLF-3 transcription factor to facilitate expression of the SLC39 zinc transporter gene zipt-2.3, which functions to sequester zinc in the intestine. Ablation of KRI-1 results in reduced zinc sequestration in the intestine, inhibition of IR-induced MPK-1/ERK1 activation, and apoptosis in the germline. Zinc localization is also perturbed in the vasculature of krit1−/− zebrafish, and SLC39 zinc transporters are mis-expressed in Cerebral Cavernous Malformations (CCM) patient tissues. This study provides new insights into the regulation of apoptosis by cross-tissue communication, and suggests a link between zinc localization and CCM disease.

Zn(ii)-Curcumin supplementation alleviates gut dysbiosis and zinc dyshomeostasis during doxorubicin-induced cardiotoxicity in rats.

Doxorubicin is a powerful anticancer agent used to treat a variety of human neoplasms. However, the clinical use of doxorubicin is hampered by cardiotoxicity and effective cardioprotective adjuvants do not exist. Dietary zinc, an essential nutrient, is required to maintain steady-state tissue zinc levels and intestinal homeostasis and may yield therapeutic benefits in diseases associated with zinc dysregulation or gut dysbiosis. Here, we investigated the effects of dietary Zn(ii)-curcumin (ZnCM) solid dispersions on gut dysbiosis and zinc dyshomeostasis during doxorubicin-induced cardiotoxicity in rats. Rats were injected with multiple low doses of doxorubicin and orally administered ZnCM daily over four weeks. Daily administration of ZnCM not only alleviated Dox-induced gut dysbiosis-as indicated by the increased Firmicutes-to-Bacteroidetes ratio and the maintenance of the relative abundances of major beneficial bacteria including Clostridium_XIVa, Clostridium_IV, Roseburia, Butyricicoccus and Akkermansia-but also maintained intestinal barrier integrity and decreased the lipopolysaccharide (LPS) contents of feces and plasma. ZnCM also significantly attenuated doxorubicin-induced zinc dyshomeostasis, which was mirrored by preservation of zinc levels and expression of zinc-related transporters. Furthermore, ZnCM significantly improved heart function and reduced cardiomyocyte apoptosis and myocardial injury in doxorubicin-treated rats. Notably, the regulation of zinc homeostasis and cardioprotective and microbiota-modulating effects of ZnCM were transmissible through horizontal feces transfer from ZnCM-treated rats to normal rats. Thus, ZnCM supplementation has potential as an effective therapeutic strategy to alleviate gut dysbiosis and zinc dyshomeostasis during doxorubicin-induced cardiotoxicity.

The Transcription Factor ZafA Regulates the Homeostatic and Adaptive Response to Zinc Starvation in Aspergillus fumigatus.

One of the most important features that enables Aspergillus fumigatus to grow within a susceptible individual and to cause disease is its ability to obtain Zn2+ ions from the extremely zinc-limited environment provided by host tissues. Zinc uptake from this source in A. fumigatus relies on ZIP transporters encoded by the zrfA, zrfB and zrfC genes. The expression of these genes is tightly regulated by the ZafA transcription factor that regulates zinc homeostasis and is essential for A. fumigatus virulence. We combined the use of microarrays, Electrophoretic Mobility Shift Assays (EMSA) analyses, DNase I footprinting assays and in silico tools to better understand the regulation of the homeostatic and adaptive response of A. fumigatus to zinc starvation. We found that under zinc-limiting conditions, ZafA functions mainly as a transcriptional activator through binding to a zinc response sequence located in the regulatory regions of its target genes, although it could also function as a repressor of a limited number of genes. In addition to genes involved in the homeostatic response to zinc deficiency, ZafA also influenced, either directly or indirectly, the expression of many other genes. It is remarkable that the expression of many genes involved in iron uptake and ergosterol biosynthesis is strongly reduced under zinc starvation, even though only the expression of some of these genes appeared to be influenced directly or indirectly by ZafA. In addition, it appears to exist in A. fumigatus a zinc/iron cross-homeostatic network to allow the adaptation of the fungus to grow in media containing unbalanced Zn:Fe ratios. The adaptive response to oxidative stress typically linked to zinc starvation was also mediated by ZafA, as was the strong induction of genes involved in gliotoxin biosynthesis and self-protection against endogenous gliotoxin. This study has expanded our knowledge about the regulatory and metabolic changes displayed by A. fumigatus in response to zinc starvation and has helped us to pinpoint new ZafA target genes that could be important for fungal pathogens to survive and grow within host tissues and, hence, for virulence.

Combinatorial effects of zinc deficiency and arsenic exposure on zebrafish (Danio rerio) development.

Zinc deficiency and chronic low level exposures to inorganic arsenic in drinking water are both significant public health concerns that affect millions of people including pregnant women. These two conditions can co-exist in the human population but little is known about their interaction, and in particular, whether zinc deficiency sensitizes individuals to arsenic exposure and toxicity, especially during critical windows of development. To address this, we utilized the Danio rerio (zebrafish) model to test the hypothesis that parental zinc deficiency sensitizes the developing embryo to low-concentration arsenic toxicity, leading to altered developmental outcomes. Adult zebrafish were fed defined zinc deficient and zinc adequate diets and were spawned resulting in zinc adequate and zinc deficient embryos. The embryos were treated with environmentally relevant concentrations of 0, 50, and 500 ppb arsenic. Arsenic exposure significantly reduced the amount of zinc in the developing embryo by ~7%. The combination of zinc deficiency and low-level arsenic exposures did not sensitize the developing embryo to increased developmental malformations or mortality. The combination did cause a 40% decline in physical activity of the embryos, and this decline was significantly greater than what was observed with zinc deficiency or arsenic exposure alone. Significant changes in RNA expression of genes that regulate zinc homeostasis, response to oxidative stress and insulin production (including zip1, znt7, nrf2, ogg1, pax4, and insa) were found in zinc deficient, or zinc deficiency and arsenic exposed embryos. Overall, the data suggests that the combination of zinc deficiency and arsenic exposure has harmful effects on the developing embryo and may increase the risk for developing chronic diseases like diabetes.

Culture conditions for zinc- and pH-regulated gene expression studies in Aspergillus fumigatus.

In Aspergillus fumigatus, the regulation of zinc homeostasis is strongly influenced by environmental pH. Thus, the study of zinc-regulated gene expression in A. fumigatus requires controlling variations in culture pH, as this may affect zinc availability. However, depending on the nitrogen source, the pH of the culture can change dramatically over time. In addition, due to the ubiquitous distribution of zinc and that it is an essential micronutrient required in minute amounts for optimal fungal growth, neither buffering of the culture media to prevent pH variations nor the use of chelating agents is advisable if mycelium is to be used for expression analyses. In this work, the growth of A. fumigatus in several culture media was examined in order to determine the conditions yielding mycelia suitable for gene expression analyses in acid and neutral media, regardless of zinc availability. Our results showed that a zinc-limiting synthetic basal medium could be readily converted into a zinc-replete one and subsequently into acid or neutral medium by using, respectively, ammonium or nitrate as nitrogen source.

The Expression of Zinc Transporters Changed in the Intestine of Weaned Pigs Exposed to Zinc Chitosan Chelate.

This study was conducted to investigate the effect of zinc chitosan chelate (CS-Zn) on zinc transporter expression and content of tissue zinc in weaned piglets. A total of 90 weaned pigs (Duroc × Landrace × Yorkshire) were randomly allocated to treatment groups with supplementation of 100mg/kg zinc as ZnSO4, 100mg/kg zinc as mixture of ZnSO4 and chitosan, or 100mg/kg zinc as CS-Zn, respectively. After 30days of trial, 18 piglets (six pigs per treatment) were killed and the samples of duodenal mucosa were taken for analysis of zinc transporter mRNA expressions and protein abundance. The results show that CS-Zn more effectively increases (p<0.05) the average daily gain (ADG) and serum zinc concentration. Zinc concentration in the liver and kidney did not differ between treatments. The mRNA expressions of ZnT1, ZIP4, and ZIP5 in CS-Zn treatment were all upregulated (p<0.05) than ZnSO4 or mixture of ZnSO4 and chitosan groups. ZnT1 abundance was greater (p<0.05) with CS-Zn as compared with ZnSO4 and mixture of ZnSO4 and chitosan treatments, whereas ZIP4 and ZIP5 abundance was higher (p<0.05) in ZnSO4 group. The results indicate that CS-Zn is more effective in serum zinc accumulation, and it might regulate zinc homeostasis by affecting zinc transporter mRNA expression and absorption mechanism might be different with ZnSO4.

Zinc Levels Modulate Lifespan through Multiple Longevity Pathways in Caenorhabditis elegans.

Zinc is an essential trace metal that has integral roles in numerous biological processes, including enzymatic function, protein structure, and cell signaling pathways. Both excess and deficiency of zinc can lead to detrimental effects on development and metabolism, resulting in abnormalities and disease. We altered the zinc balance within Caenorhabditis elegans to examine how changes in zinc burden affect longevity and healthspan in an invertebrate animal model. We found that increasing zinc levels in vivo with excess dietary zinc supplementation decreased the mean and maximum lifespan, whereas reducing zinc levels in vivo with a zinc-selective chelator increased the mean and maximum lifespan in C. elegans. We determined that the lifespan shortening effects of excess zinc required expression of DAF-16, HSF-1 and SKN-1 proteins, whereas the lifespan lengthening effects of the reduced zinc may be partially dependent upon this set of proteins. Furthermore, reducing zinc levels led to greater nuclear localization of DAF-16 and enhanced dauer formation compared to controls, suggesting that the lifespan effects of zinc are mediated in part by the insulin/IGF-1 pathway. Additionally, zinc status correlated with several markers of healthspan in worms, including proteostasis, locomotion and thermotolerance, with reduced zinc levels always associated with improvements in function. Taken together, these data support a role for zinc in regulating both development and lifespan in C. elegans, and that suggest that regulation of zinc homeostasis in the worm may be an example of antagonistic pleiotropy.

Influence of DNA-methylation on zinc homeostasis in myeloid cells: Regulation of zinc transporters and zinc binding proteins

The distribution of intracellular zinc, predominantly regulated through zinc transporters and zinc binding proteins, is required to support an efficient immune response. Epigenetic mechanisms such as DNA methylation are involved in the expression of these genes. In demethylation experiments using 5-Aza-2′-deoxycytidine (AZA) increased intracellular (after 24 and 48h) and total cellular zinc levels (after 48h) were observed in the myeloid cell line HL-60. To uncover the mechanisms that cause the disturbed zinc homeostasis after DNA demethylation, the expression of human zinc transporters and zinc binding proteins were investigated. Real time PCR analyses of 14 ZIP (solute-linked carrier (SLC) SLC39A; Zrt/IRT-like protein), and 9 ZnT (SLC30A) zinc transporters revealed significantly enhanced mRNA expression of the zinc importer ZIP1 after AZA treatment. Because ZIP1 protein was also enhanced after AZA treatment, ZIP1 up-regulation might be the mediator of enhanced intracellular zinc levels. The mRNA expression of ZIP14 was decreased, whereas zinc exporter ZnT3 mRNA was also significantly increased; which might be a cellular reaction to compensate elevated zinc levels. An enhanced but not significant chromatin accessibility of ZIP1 promoter region I was detected by chromatin accessibility by real-time PCR (CHART) assays after demethylation. Additionally, DNA demethylation resulted in increased mRNA accumulation of zinc binding proteins metallothionein (MT) and S100A8/S100A9 after 48h. MT mRNA was significantly enhanced after 24h of AZA treatment also suggesting a reaction of the cell to restore zinc homeostasis. These data indicate that DNA methylation is an important epigenetic mechanism affecting zinc binding proteins and transporters, and, therefore, regulating zinc homeostasis in myeloid cells.

Characterization of two cation diffusion facilitators NpunF0707 and NpunF1794 in Nostoc punctiforme.

To characterize genes involved in maintaining homeostatic levels of zinc in the cyanobacterium Nostoc punctiforme. Metal efflux transporters play a central role in maintaining homeostatic levels of trace elements such as zinc. Sequence analyses of the N. punctiforme genome identified two potential cation diffusion facilitator (CDF) metal efflux transporters, Npun_F0707 (Cdf31) and Npun_F1794 (Cdf33). Deletion of either Cdf31or Cdf33 resulted in increased zinc retention over 3 h. Interestingly, Cdf31(-) and Cdf33(-) mutants showed no change in sensitivity to zinc exposure in comparison with the wild type, suggesting some compensatory capacity for the loss of each other. Using qRT-PCR, a possible interaction was observed between the two cdf's, where the Cdf31(-) mutant had a more profound effect on cdf33 expression than Cdf33(-) did on cdf31. Over-expression of Cdf31 and Cdf33 in ZntA(-) - and ZitB(-) -deficient Escherichia coli revealed function similarities between the ZntA and ZitB of E. coli and the cyanobacterial transporters. The data presented shed light on the function of two important transporters that regulate zinc homeostasis in N. punctiforme. This study shows for the first time the functional characterization of two cyanobacterial zinc efflux proteins belonging to the CDF family.

Deficiency of metallothionein-1 and -2 genes shortens the lifespan of the 129/Sv mouse strain

Metallothionein (MT) family proteins are small molecular weight and cysteine-rich proteins that regulate zinc homeostasis and have potential protective effects against oxidative stress and toxic metals. To investigate whether MTs play a role in longevity determination in mammals, we measured the lifespans of wild-type (WT) and MT-1 and -2 gene knockout (MTKO) mice in a 129/Sv genetic background. MTKO mice of both sexes had shorter lifespans than WT mice. In particular, male MTKO mice living beyond the mean lifespan exhibited signs of weight loss, hunchbacked spines, lackluster fur and an absence of vigor. These results suggest that lifespan is shortened due to accelerated senescence in the absence of MT genes.