Cellular Zn Research Articles

Insight into brain metallothioneins from bidirectional Zn2+ signaling in synaptic dynamics.

The basal levels as the labile Zn2+ pools in the extracellular and intracellular compartments are in the range of ∼10nM and ∼100 pM, respectively. The influx of extracellular Zn2+ is used for memory via cognitive activity and is regulated for synaptic plasticity, a cellular mechanism of memory. When Zn2+ influx into neurons excessively occurs, however, it becomes a critical trigger for cognitive decline and neurodegeneration, resulting in acute and chronic pathogenesis. Aging, a biological process, generally accelerates vulnerability to neurodegenerative disorders such as Alzheimer's disease (AD) and Parkinson's disease (PD). The basal level of extracellular Zn2+ is age relatedly increased in the rat hippocampus, and the influx of extracellular Zn2+ contributes to accelerating vulnerability to the AD and PD pathogenesis in experimental animals with aging. Metallothioneins (MTs) are Zn2+-binding proteins for cellular Zn2+ homeostasis and involved in not only supplying functional Zn2+ required for cognitive activity, but also capturing excess (toxic) Zn2+ involved in cognitive decline and neurodegeneration. Therefore, it is estimated that regulation of MT synthesis is involved in both neuronal activity and neuroprotection. The present report provides recent knowledge regarding the protective/preventive potential of MT synthesis against not only normal aging but also the AD and PD pathogenesis in experimental animals, focused on MT function in bidirectional Zn2+ signaling in synaptic dynamics.

A Fluorometric Method for Zinc Estimation: Applications in the Estimation of Plasma Zinc and in Assessing Zinc Bioaccessibility from Rice.

Sensitive and precise methods for the estimation of zinc (Zn) in biological fluids and foods are important tools in understanding the various aspects related to Zn nutrition. Estimation of serum/plasma Zn was suggested for assessing the population Zn status while assessing the bioaccessible Zn following simulated gastrointestinal digestion of crop varieties such as rice helps in ranking the crops. Atomic absorption spectrometry (AAS) or inductively coupled plasma-mass spectrometry (ICP-MS) are widely used for Zn estimation. Zinquin, a Zn fluorophore, has been used for the localization of cellular Zn and labile Zn pools in biological fluids with extremely high sensitivity. However, it was not tested for its use in Zn estimation in serum/plasma or in assessing the Zn bioaccessibility from foods. In the current study, we demonstrate a sensitive method for Zn estimation in human plasma and validate it against the reference method (AAS) by comparing the paired measurements on the same samples. The method-related bias between zinquin with AAS was negligible (0.48µg/dL), and the precision (CV) of the assay was < 5% across different Zn concentrations. In addition, we also demonstrated the utility of zinquin assay in estimating the bioaccessibility of Zn from rice varieties and showed that the method is again comparable to AAS. The zinquin method is capable of discriminating the differences in zinc bioaccessibility between polished and unpolished rice varieties. In the context of required lowplasma volume (100µL Vs 400µL), excellent comparability of the results with the reference method and analytical simplicity could be particularly useful.

Phytochelatins Bind Zn(II) with Micro- to Picomolar Affinities without the Formation of Binuclear Complexes, Exhibiting Zinc Buffering and Muffling Rather than Storing Functions.

Phytochelatins (PCs) are poly-Cys peptides containing a repeating γ-Glu-Cys motif synthesized in plants, algae, certain fungi, and worms by PC synthase from reduced glutathione. It has been shown that an excess of toxic metal ions induces their biosynthesis and that they are responsible for the detoxification process. Little is known about their participation in essential metal binding under nontoxic, basal conditions under which PC synthase is active. This study presents spectroscopic and thermodynamic interactions with the PC2-PC5 series, mainly focusing on the relations between Zn(II) complex stability and cellular Zn(II) availability. The investigations employed mass spectrometry, UV-vis spectroscopy, potentiometry, competition assays with zinc probes, and isothermal titration calorimetry (ITC). All peptides form ZnL complexes, while ZnL2 was found only for PC2, containing two to four sulfur donors in the coordination sphere. Binuclear species typical of Cd(II)-PC complexes are not formed in the case of Zn(II). Results demonstrate that the affinity for Zn(II) increases linearly from PC2 to PC4, ranging from micro- to low-picomolar. Further elongation does not significantly increase the stability. Stability elevation is driven mainly by entropic factors related to the chelate effect and conformational restriction rather than enthalpic factors related to the increasing number of sulfur donors. The affinity of the investigated PCs falls within the range of exchangeable Zn(II) concentrations (hundreds of pM) observed in plants, supporting for the first time a role of PCs both in buffering and in muffling cytosolic Zn(II) concentrations under normal conditions, not exposed to zinc excess, where short PCs have been identified in numerous studies. Furthermore, we found that Cd(II)-PC complexes demonstrate significantly higher metal capacities due to the formation of polynuclear species, which are lacking for Zn(II), supporting the role of PCs in Cd(II) storage (detoxification) and Zn(II) buffering and muffling. Our results on phytochelatins' coordination chemistry and thermodynamics are important for zinc biology and understanding the molecular basis of cadmium toxicity, leaving room for future studies.

Zinc and manganese homeostasis closely interact in mammalian cells.

Understanding the homeostatic interactions among essential trace metals is important for explaining their roles in cellular systems. Recent studies in vertebrates suggest that cellular Mn metabolism is related to Zn metabolism in multifarious cellular processes. However, the underlying mechanism remains unclear. In this study, we examined the changes in the expression of proteins involved in cellular Zn and/or Mn homeostatic control and measured the Mn as well as Zn contents and Zn enzyme activities to elucidate the effects of Mn and Zn homeostasis on each other. Mn treatment decreased the expression of the Zn homeostatic proteins metallothionein (MT) and ZNT1 and reduced Zn enzyme activities, which were attributed to the decreased Zn content. Moreover, loss of Mn efflux transport protein decreased MT and ZNT1 expression and Zn enzyme activity without changing extracellular Mn content. This reduction was not observed when supplementing with the same Cu concentrations and in cells lacking Cu efflux proteins. Furthermore, cellular Zn homeostasis was oppositely regulated in cells expressing Zn and Mn importer ZIP8, depending on whether Zn or Mn concentration was elevated in the extracellular milieu. Our results provide novel insights into the intricate interactions between Mn and Zn homeostasis in mammalian cells and facilitate our understanding of the physiopathology of Mn, which may lead to the development of treatment strategies for Mn-related diseases in the future.

Mis-regulation of Zn and Mn homeostasis is a key phenotype of Cu stress in Streptococcus pyogenes.

All bacteria possess homeostastic mechanisms that control the availability of micronutrient metals within the cell. Cross-talks between different metal homeostasis pathways within the same bacterial organism have been reported widely. In addition, there have been previous suggestions that some metal uptake transporters can promote adventitious uptake of the wrong metal. This work describes the cross-talk between Cu and the Zn and Mn homeostasis pathways in Group A Streptococcus (GAS). Using a ∆copA mutant strain that lacks the primary Cu efflux pump and thus traps excess Cu in the cytoplasm, we show that growth in the presence of supplemental Cu promotes downregulation of genes that contribute to Zn or Mn uptake. This effect is not associated with changes in cellular Zn or Mn levels. Co-supplementation of the culture medium with Zn or, to a lesser extent, Mn alleviates key Cu stress phenotypes, namely bacterial growth and secretion of the fermentation end-product lactate. However, neither co-supplemental Zn nor Mn influences cellular Cu levels or Cu availability in Cu-stressed cells. In addition, we provide evidence that the Zn or Mn uptake transporters in GAS do not promote Cu uptake. Together, the results from this study strengthen and extend our previous proposal that mis-regulation of Zn and Mn homeostasis is a key phenotype of Cu stress in GAS.

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.

A turn-on fluorescence sensor based on water-soluble amino-pyridyl-benzothiazole-derivatives for measuring cellular Zn2+

Novel amino-pyridyl-benzothiazole-derivatives (PBT) were synthesized through cycloaddition/nucleophilic aromatic substitution reactions for the development of a turn-on fluorescence chemosensor that accurately determines Zn2+ in polar media. The selectivity, water-solubility, and overall analytical response were systematically optimized through incorporation of different aliphatic amines as substituents: pyrrolidine (PBT-Pyn), ethylenediamine (PBT-Et) or 1,3-propylenediamine (PBT-Pr). Those ligands presented intense dual radiative emissions in the blue/green region upon activation with a 395 nm LED, which were attributed to the local excitation and internal energy transfer states. Although no variations were observed upon the interaction of Zn2+ with PBT-Pyn, both turn-off and turn-on responses were observed upon its interaction with PBT-Et. These responses depended on the ligand concentration and the associated inner filter effects. Thereby, PBT-Pr was selected for a deeper evaluation towards photoluminescence determination of Zn2+ due to its exclusive turn-on response and major sensibility obtained at pH 7.0, making it very convenient for analysis in physiological media. Under those conditions, the detection limits were 1.9 µM and 4.7 µM for measurements in aqueous or ethanolic solutions, respectively, with linear ranges up to 40 and 80 μM. The selectivity and good biocompatibility of the chemosensor were demonstrated through cell-viability assays with MCF-7, 3T3-L1 and HT-29 cell lines. The probe was then applied for the real-time epifluorescence imaging of Zn2+, observing emission intensities and distribution profiles that correlated with the concentration and location of the target within living cells.

New insights into bacterial Zn homeostasis and molecular architecture of the metal resistome in soil polluted with nano zinc oxide

Accumulation of nano ZnO (nZnO) in soils could be toxic to bacterial communities through disruption of Zn homeostasis. Under such conditions, bacterial communities strive to maintain cellular Zn levels by accentuation of appropriate cellular machinery. In this study, soil was exposed to a gradient (50–1000 mg Zn kg-1) of nZnO for evaluating their effects on genes involved in Zn homeostasis (ZHG). The responses were compared with similar levels of its bulk counterpart (bZnO). It was observed that ZnO (as nZnO or bZnO) induced a plethora of influx and efflux transporters as well as metallothioneins (MTs) and metallochaperones mediated by an array of Zn sensitive regulatory proteins. Major influx system identified was the ZnuABC transporter, while important efflux transporters identified were CzcCBA, ZntA, YiiP and the major regulator was Zur. The response of communities was dose- dependent at lower concentrations (<500 mg Zn kg-1 as nZnO or bZnO). However, at 1000 mg Zn kg-1, a size-dependent threshold of gene/gene family abundances was evident. Under nZnO, a poor adaptation to toxicity induced anaerobic conditions due to deployment of major influx and secondary detoxifying systems as well as poor chelation of free Zn ions was evident. Moreover, Zn homeostasis related link with biofilm formation and virulence were accentuated under nZnO than bZnO. While these findings were verified by PCoA and Procrustes analysis, Network analysis and taxa vs ZHG associations also substantiated that a stronger Zn shunting mechanism was induced under nZnO due to higher toxicity. Molecular crosstalks with systems governing Cu and Fe homeostasis were also evident. Expression analysis of important resistance genes by qRT-PCR showed good alignment with the predictive metagenome data, thereby validating our findings. From the study it was evident that the induction of detoxifying and resistant genes was greatly lowered under nZnO, which markedly hampered Zn homeostasis among the soil bacterial communities.

Interplay between zinc and cell proliferation and implications for the growth of livestock.

Zinc (Zn) plays a critical role in the growth of livestock, which depends on cell proliferation. In addition to modifying the growth associated with its effects on food intake, mitogenic hormones, signal transduction and gene transcription, Zn also regulates body weight gain through mediating cell proliferation. Zn deficiency in animals leads to growth inhibition, along with an arrest of cell cycle progression at G0/G1 and S phase due to depression in the expression of cyclin D/E and DNA synthesis. Therefore, in the present study, the interplay between Zn and cell proliferation and implications for the growth of livestock were reviewed, in which Zn regulates cell proliferation in several ways, especially cell cycle progression at the G0/G1 phase DNA synthesis and mitosis. During the cell cycle, the Zn transporters and major Zn binding proteins such as metallothioneins are altered with the requirements of cellular Zn level and nuclear translocation of Zn. In addition, calcium signaling, MAPK pathway and PI3K/Akt cascades are also involved in the process of Zn-interfering cell proliferation. The evidence collected over the last decade highlights the necessity of Zn for normal cell proliferation, which suggests Zn supplementation should be considered for the growth and health of poultry.

The role of manganese in morphogenesis and pathogenesis of the opportunistic fungal pathogen Candida albicans.

Metals such as Fe, Cu, Zn, and Mn are essential trace nutrients for all kingdoms of life, including microbial pathogens and their hosts. During infection, the mammalian host attempts to starve invading microbes of these micronutrients through responses collectively known as nutritional immunity. Nutritional immunity for Zn, Fe and Cu has been well documented for fungal infections; however Mn handling at the host-fungal pathogen interface remains largely unexplored. This work establishes the foundation of fungal resistance against Mn associated nutritional immunity through the characterization of NRAMP divalent metal transporters in the opportunistic fungal pathogen, Candida albicans. Here, we identify C. albicans Smf12 and Smf13 as two NRAMP transporters required for cellular Mn accumulation. Single or combined smf12Δ/Δ and smf13Δ/Δ mutations result in a 10-80 fold reduction in cellular Mn with an additive effect of double mutations and no losses in cellular Cu, Fe or Zn. As a result of low cellular Mn, the mutants exhibit impaired activity of mitochondrial Mn-superoxide dismutase 2 (Sod2) and cytosolic Mn-Sod3 but no defects in cytosolic Cu/Zn-Sod1 activity. Mn is also required for activity of Golgi mannosyltransferases, and smf12Δ/Δ and smf13Δ/Δ mutants show a dramatic loss in cell surface phosphomannan and in glycosylation of proteins, including an intracellular acid phosphatase and a cell wall Cu-only Sod5 that is key for oxidative stress resistance. Importantly, smf12Δ/Δ and smf13Δ/Δ mutants are defective in formation of hyphal filaments, a deficiency rescuable by supplemental Mn. In a disseminated mouse model for candidiasis where kidney is the primary target tissue, we find a marked loss in total kidney Mn during fungal invasion, implying host restriction of Mn. In this model, smf12Δ/Δ and smf13Δ/Δ C. albicans mutants displayed a significant loss in virulence. These studies establish a role for Mn in Candida pathogenesis.

Characterizing Mammalian Zinc Transporters Using an In Vitro Zinc Transport Assay.

Transition metals such as Zn2+ ions must be tightly regulated due to their cellular toxicity. Previously, the activity of Zn2+ transporters was measured indirectly by determining the expression level of the transporter under different concentrations of Zn2+. This was done by utilizing immunohistochemistry, measuring mRNA in the tissue, or determining the cellular Zn2+ levels. With the development of intracellular Zn2+ sensors, the activities of zinc transporters are currently primarily determined by correlating changes in intracellular Zn2+, detected using fluorescent probes, with the expression of the Zn2+ transporters. However, even today, only a few labs monitor dynamic changes in intracellular Zn2+ and use it to measure the activity of zinc transporters directly. Part of the problem is that out of the 10 zinc transporters of the ZnT family, except for ZnT10 (transports manganese), only zinc transporter 1 (ZnT1) is localized at the plasma membrane. Therefore, linking the transport activity to changes in the intracellular Zn2+ concentration is hard. This article describes a direct way to determine the zinc transport kinetics using an assay based on a zinc-specific fluorescent dye, FluoZin-3. This dye is loaded into mammalian cells in its ester form and then trapped in the cytosol due to cellular di-esterase activity. The cells are loaded with Zn2+ by utilizing the Zn2+ ionophore pyrithione. The ZnT1 activity is assessed from the linear part of the reduction in fluorescence following the cell washout. The fluorescence measured at an excitation of 470 nm and emission of 520 nm is proportional to the free intracellular Zn2+. Selecting the cells expressing ZnT1 tagged with the mCherry fluorophore allows for monitoring only the cells expressing the transporter. This assay is used to investigate the contribution of different domains of ZnT1 protein to the transport mechanism of human ZnT1, a eukaryotic transmembrane protein that extrudes excess zinc from the cell.

From methodological limitations to the function of metallothioneins - A guide to approaches for determining weak, moderate, and tight affinity zinc sites.

Mammalian metallothioneins (MTs) are small cysteine-rich proteins whose primary role is participation in zinc and copper homeostasis. Ever since their discovery, MTs have been investigated in terms of metal-binding affinity. The initial concept of seven Zn(II) ions (Zn7MT) bound with the same, undifferentiated low picomolar affinity in the α and β domains prevailed for many years and derived from spectroscopic studies. The application of fluorescent zinc probes has changed the perception of MTs, showing that they function in nano- to subnanomolar free zinc concentrations due to the presence of tight, moderate, and weak binding sites. The discovery of Zn(II)-depleted MTs in many tissues and determination of cellular free Zn(II) concentrations with differentiated zinc affinity sites revealed the critical importance of partially saturated Zn4-6MTs species in cellular zinc buffering in a wide pico- to nanomolar range of free Zn(II) concentrations. Until today, there is no clear agreement on the presence of differentiated or only tight zinc sites. Here, we present a series of spectroscopic, mass spectrometry-based, and enzymatic competition experiments which reveal how weak, moderate, or high affinity ligands interact with human MT2, with special attention to the determination of Zn(II) affinities. The results show that the simplification of the stability model is the major reason for determining significantly different stability data that obscured the actual MTs function. Therefore, we emphasize that different metal affinities are the single most reason for their presumed function, which changed over the years from tight binding and, thus, storage to one that is highly dynamic.

Zinc Protects against Tumor Necrosis Factor-Induced Disruption of Porcine Endothelial Cell Monolayer Integrity

Some nutrients influence the metabolic response of cytokines such as tumor necrosis factor. Inadequate levels of the essential trace element zinc may play a role in tumor necrosis factor-induced disruption of the vascular endothelial barrier function. To test this hypothesis, endothelial cells cultured on polycarbonate filters or culture plates were exposed to six different treatments for 3 d: medium 199 enriched with 5% fetal bovine serum (control), control+two levels of supplemental zinc (7.7 and 12.3 mumol/L medium), tumor necrosis factor (5 x 10(5) U/L) and tumor necrosis factor+the two levels of Zn as noted previously. Endothelial barrier function, expressed as albumin transfer across cultured endothelial monolayers, was not affected by Zn enrichment alone. Tumor necrosis factor treatment significantly increased albumin transfer compared with control cultures. The lower concentration of Zn partially and the higher concentration totally prevented the tumor necrosis factor-induced increase in albumin transfer. The increase in cytosolic release of [3H]adenine (marker of cell injury) induced by tumor necrosis factor was prevented by added Zn. Tumor necrosis factor treatment significantly decreased angiotensin-converting enzyme activity, and tumor necrosis factor also decreased activities of two other membrane-bound enzymes, total ATPase and Ca(2+)-ATPase. These activities all were restored by Zn enrichment. Tumor necrosis factor treatment caused a decrease in cellular Zn concentration, which was prevented when the culture media were enriched with Zn. These data suggest that an important relationship exists between Zn status and tumor necrosis factor-induced endothelial cell dysfunction.

The Impact of Zinc on Manganese Bioavailability and Cytotoxicity in HepG2 Cells.

Despite their essentiality, several studies have shown that either manganese (Mn) or zinc (Zn) overexposure may lead to detrimental health effects. Although Mn is transported by some of the SLC family transporters that translocate Zn, the role of Zn in hepatocellular Mn transport and Mn-induced toxicity have yet to be fully characterized. The human hepatoma cell line, HepG2, is utilized. Total cellular Mn and Zn amounts are determined after cells are treated with Zn 2 or 24h prior to Mn incubation for additional 24h with inductively coupled plasma-based spectrometry and labile Zn is assessed with the fluorescent probe FluoZin-3. Furthermore, mRNA expression of genes involved in metal homeostasis, and mechanistic endpoints associated with Mn-induced cytotoxicity are addressed. These results suggest that Zn protects against Mn-induced cytotoxicity and impacts Mn bioavailability to a great extent when cells are preincubated with higher Zn concentrations for longer duration as characterized by decreased activation of caspase-3 as well as lactate dehydrogenase (LDH) release. Zn protects against Mn-induced cytotoxicity in HepG2 cells possibly due to decreased Mn bioavailability. Additionally, mRNA expression of metal homeostasis-related genes indicates possible underlying pathways that should to be addressed in future studies.

Dexamethasone leads to Zn2+ accumulation and increased unbound Zn2+ in C2C12 muscle and 3T3-L1 adipose cells.

Skeletal muscle atrophy is associated with increases in circulating glucocorticoid levels and insulin resistance. Zinc accumulates in atrophic muscle, but the relationship between atrophy, insulin resistance, and Zn2+ homeostasis remains unclear. In this study, the effect of the glucocorticoid dexamethasone (DEX) on insulin and Zn2+ homeostasis was explored. Treatment of differentiated C2C12 skeletal myotubes and 3T3-L1 adipocytes with DEX significantly increased mRNA expression of the metal-binding proteins Mt1 and 2 and altered energy storage as shown by the increased size of lipid droplets in 3T3-L1 cells. In C2C12 cells the total cellular Zn2+ was higher after DEX treatment, and in both C2C12 and 3T3-L1 adipocytes, free unbound Zn2+ was increased. Insulin treatment led to a gradual increase in free Zn2+ in C2C12 cells, and no significant change in DEX-treatedcells such that concentrations were similar 10 min after insulin treatment. These data demonstrate that DEX disturbs Zn2+ homeostasis in muscle and fat cells. Further study of the molecular pathways involved to identify novel therapeutic targets for treatment of skeletal muscle atrophy is warranted.

Knocking-Out OsPDR7 Triggers Up-Regulation of OsZIP9 Expression and Enhances Zinc Accumulation in Rice

Zinc (Zn) is an essential trace mineral that is required for plant growth and development. A number of protein transporters, which are involved in Zn uptake, translocation and distribution, are finely regulated to maintain Zn homeostasis in plant. In this study, we functionally characterized an ATP-binding cassette (ABC) transporter gene, OsPDR7, which is involved in Zn homeostasis. OsPDR7 encodes a plasma membrane-localized protein that is expressed mainly in the exodermis and xylem in the rice root. ospdr7 mutants resulted in higher Zn accumulation compared with the wild type. Heterogeneous expression of OsPDR7 in a yeast mutant rescued the Zn-deficiency phenotype, implying transport activity of OsPDR7 to Zn in yeast. However, no ZIP genes except for OsZIP9 showed change in expression profile in the ospdr7 mutants, which suggested that OsPDR7 maintains cellular Zn homeostasis through regulating OsZIP9 expression. RNA-Seq analysis further revealed a set of differentially expressed genes between the wild type and ospdr7 mutants that allowed us to propose a possible OsPDR7-associated signaling network involving transporters, hormone responsive genes, and transcription factors. Our results revealed a novel transporter involved in the regulation of Zn homeostasis and will pave the way toward a better understanding of the fine-tuning of gene expression in the network of transporter genes.

Metal retention and replacement in QueD2 protect queuosine-tRNA biosynthesis in metal-starved Acinetobacter baumannii

In response to bacterial infection, the vertebrate host employs the metal-sequestering protein calprotectin (CP) to withhold essential transition metals, notably Zn(II), to inhibit bacterial growth. Previous studies of the impact of CP-imposed transition-metal starvation in A. baumannii identified two enzymes in the de novo biosynthesis pathway of queuosine-transfer ribonucleic acid (Q-tRNA) that become cellularly abundant, one of which is QueD2, a 6-carboxy-5,6,7,8-tetrahydropterin (6-CPH4) synthase that catalyzes the initial, committed step of the pathway. Here, we show that CP strongly disrupts Q incorporation into tRNA. As such, we compare the AbQueD2 "low-zinc" paralog with a housekeeping, obligatory Zn(II)-dependent enzyme QueD. The crystallographic structure of Zn(II)-bound AbQueD2 reveals a distinct catalytic site coordination sphere and assembly state relative to QueD and possesses a dynamic loop, immediately adjacent to the catalytic site that coordinates a second Zn(II) in the structure. One of these loop-coordinating residues is an invariant Cys18, that protects QueD2 from dissociation of the catalytic Zn(II) while maintaining flux through the Q-tRNA biosynthesis pathway in cells. We propose a "metal retention" model where Cys18 introduces coordinative plasticity into the catalytic site which slows metal release, while also enhancing the metal promiscuity such that Fe(II) becomes an active cofactor. These studies reveal a complex, multipronged evolutionary adaptation to cellular Zn(II) limitation in a key Zn(II) metalloenzyme in an important human pathogen.

Zinc trafficking to apo-Zn-proteins 2. Cellular interplay of proteome, metallothionein, and glutathione.

A recent study investigated the impact of glutathione (GSH) on the transfer of zinc (Zn) from proteome to apo-carbonic anhydrase. Here, we probed the requirement of glutathione for zinc trafficking in LLC-PK1 pig kidney epithelial cells. Depletion of GSH by at least 95% left cells viable and able to divide and synthesize Zn-proteins at the control rate over a 48-h period. Loss of GSH stimulated the accumulation of 2.5x the normal concentration of cellular Zn. According to gel filtration chromatography, differential centrifugal filtration, and spectrofluorimetry with TSQ, the extra Zn was distributed between the proteome and metallothionein (MT). To test the functionality of proteome and/or MT as sources of Zn for the constitution of Zn-proteins, GSH-deficient cells were incubated with CaEDTA to isolate them from their normal source of nutrient Zn. Control cells plus CaEDTA stopped dividing; GSH-depleted cells plus CaEDTA continued to divide at ∼40% the rate of GSH deficient cells. Evidently, proteome and/or MT served as a functional source of Zn for generating Zn-proteins. In vitro insertion of Zn bound to proteome into apo-carbonic anhydrase occurred faster at larger concentrations of Zn bound to proteome. These results support the hypothesis that enhanced transport of Zn into cells drives the conversion of apo-Zn-proteins to Zn-proteins by mass action. Similar results were also obtained with human Jurkat T lymphocyte epithelial cells. This study reveals a powerful new model for studying the chemistry of Zn trafficking, including transport processes, involvement of intermediate binding sites, and constitution of Zn-proteins.

The Zinc Ionophore Clioquinol Reduces Parkinson's Disease Patient-Derived Brain Extracts-Induced Neurodegeneration.

Parkinson's disease (PD) is pathologically characterized by intracellular α-synuclein-rich protein aggregates, named Lewy bodies (LB), and by the progressive loss of dopaminergic neurons in the substantia nigra. Several heavy metals, including zinc (Zn), have been suggested to play a role in PD progression, although the exact role of Zn in neurodegeneration remains to be fully elucidated. To address this gap, we investigated the effects of Zn modulation on the progression of degeneration in mice injected with PD patient-derived LB-extracts carrying toxic α-synuclein aggregates. Zn modulation was achieved using either a clioquinol-enriched diet, a Zn ionophore that redistributes cellular Zn, or a Zn-enriched diet that increases Zn levels. Clioquinol treatment significantly prevented dopaminergic neurodegeneration and reduced α-synuclein-associated pathology in LB-injected mice, while no differences were observed with Zn supplementation. Biochemical analyses further demonstrate that the expression levels of vesicle-specific Zn transporter ZnT3 in the striatum of LB-injected mice treated with clioquinol were decreased, suggesting an intracellular redistribution of Zn. Additionally, we found that clioquinol modulates the autophagy-lysosomal pathway by enhancing lysosomal redistribution within the neuronal compartments. Collectively, we found that in vivo pharmacological chelation of Zn, by dampening Zn-mediated cytotoxicity, can result in an overall attenuation of PD-linked lysosomal alterations and dopaminergic neurodegeneration. The results support zinc chelation as a disease-modifying strategy for treating PD.

551 Murine mast cells, express zip7

Skin is the third most zinc (Zn)-abundant tissue in the body, In the skin, mast cell (MC) granules are enriched in Zn. Cellular Zn levels are strictly regulated by two groups of Zn transporters: Zn transporters (ZnTs) and Zrt-, Irt-like protein transporters (ZIPs). ZnT expressions were previously determined in the bone-marrow derived MCs (BMMCs). In contrast, little is known about ZIP expressions. Thus, the aim of this study is to determine ZIP expressions in murine MCs. Rodent MCs are categorized into connective tissue MCs (CTMCs) and mucosal MCs (MMCs).