Intracellular Zn Research Articles

The Adc regulon mediates zinc homeostasis in Streptococcus mutans.

Zinc (Zn2+ ) is an essential divalent trace metal for living cells. Intracellular zinc homeostasis is critical to the survival and virulence of bacteria. Thus, the frequent fluctuations of salivary zinc, caused by the low physiological level and the frequent exogenous zinc introduction, present a serious challenge for bacteria colonizing the oral cavity. However, the regulation strategies to keep intracellular Zn2+ homeostasis in Streptococcus mutans, an important causative pathogen of dental caries, are unknown. Because zinc uptake is primarily mediated by an ATP-binding ABC transporter AdcABC in Streptococcus strains, we examined the function of AdcABC and transcription factor AdcR in S. mutans in this study. The results demonstrated that deletion of either adcA or adcCB gene impaired the growth but enhanced the extracellular polymeric matrix production in S. mutans, both of which could be relieved after excessive Zn2+ supplementation. Using RNA sequencing analysis, quantitative reverse transcription polymerase chain reaction examination, LacZ-reporter studies, and electrophoretic mobility shift assay, we showed that a MarR (multiple antibiotic resistance regulator) family transcription factor, AdcR, negatively regulates the expression of the genes adcR, adcC, adcB, and adcA by acting on the adcRCB and adcA promoters in response to Zn2+ concentration in their environmental niches. The deletion of adcR increases the sensitivity of S. mutans to excessive Zn2+ supply. Taken together, our findings suggest that Adc regulon, which consists of a Zn2+ uptake transporter AdcCBA and a Zn2+ -responsive repressor AdcR, plays a prominent role in the maintenance of intracellular zinc homeostasis of S. mutans.

TPEN attenuates amyloid-\u03b225\u201335-induced neuronal damage with changes in the electrophysiological properties of voltage-gated sodium and potassium channels

To understand the role of intracellular zinc ion (Zn2+) dysregulation in mediating age-related neurodegenerative changes, particularly neurotoxicity resulting from the generation of excessive neurotoxic amyloid-β (Aβ) peptides, this study aimed to investigate whether N, N, N′, N′-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN), a Zn2+-specific chelator, could attenuate Aβ25–35-induced neurotoxicity and the underlying electrophysiological mechanism. We used the 3-(4, 5-dimethyl-thiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay to measure the viability of hippocampal neurons and performed single-cell confocal imaging to detect the concentration of Zn2+ in these neurons. Furthermore, we used the whole-cell patch-clamp technique to detect the evoked repetitive action potential (APs), the voltage-gated sodium and potassium (K+) channels of primary hippocampal neurons. The analysis showed that TPEN attenuated Aβ25–35-induced neuronal death, reversed the Aβ25–35-induced increase in intracellular Zn2+ concentration and the frequency of APs, inhibited the increase in the maximum current density of voltage-activated sodium channel currents induced by Aβ25–35, relieved the Aβ25–35-induced decrease in the peak amplitude of transient outward K+ currents (IA) and outward-delayed rectifier K+ currents (IDR) at different membrane potentials, and suppressed the steady-state activation and inactivation curves of IA shifted toward the hyperpolarization direction caused by Aβ25–35. These results suggest that Aβ25–35-induced neuronal damage correlated with Zn2+ dysregulation mediated the electrophysiological changes in the voltage-gated sodium and K+ channels. Moreover, Zn2+-specific chelator-TPEN attenuated Aβ25–35-induced neuronal damage by recovering the intracellular Zn2+ concentration.

Disruption of Metallostasis in the Anaerobic Human Pathogen Fusobacterium nucleatum by the Zinc Ionophore PBT2.

The Gram-negative anaerobe Fusobacterium nucleatum is an opportunistic human pathogen, most frequently associated with periodontal disease through dental biofilm formation and, increasingly, with colorectal cancer development and progression. F. nucleatum infections are routinely treated by broad-spectrum β-lactam antibiotics and metronidazole. However, these antibiotics can negatively impact the normal microflora. Therefore, the development of novel narrow-spectrum antimicrobials active against anaerobic pathogens is of great interest. Here, we examined the antimicrobial Zn ionophore PBT2, an 8-hydroxyquinoline analogue with metal chelating properties, against a single type isolate F. nucleatum ATCC 25586. PBT2-Zn was a potent inhibitor of growth and exhibited synergistic bactericidal (>3-log10 killing) activity at 5× MIC in planktonic cells, and at the MIC in biofilms grown in vitro. Physiological and transcriptional analyses uncovered a strong cellular response relating to Zn and Fe homeostasis in PBT2-Zn treated cells across subinhibitory and inhibitory concentrations. At 1× MIC, PBT2 alone induced a 3.75-fold increase in intracellular Zn, whereas PBT2-Zn challenge induced a 19-fold accumulation of intracellular Zn after 2 h. A corresponding 2.1-fold loss of Fe was observed at 1× MIC. Transcriptional analyses after subinhibitory PBT2-Zn challenge (0.125 μg/mL and 200 μM ZnSO4) revealed significant differential expression of 15 genes at 0.5 h, and 12 genes at 1 h. Upregulated genes included those with roles in Zn homeostasis (e.g., a Zn-transporting ATPase and the Zn-sensing transcriptional regulator, smtB) and hemin transport (hmuTUV) to re-establish Fe homeostasis. A concentration-dependent protective effect was observed for cells pretreated with hemin (50 μg/mL) prior to PBT2-Zn challenge. The data presented here supports our proposal that targeting the disruption of metallostasis by Zn-translocating ionophores is a strategy worth investigating further for the treatment of Gram-negative anaerobic pathogens.

Selenoprotein P Regulates Synaptic Zinc and Reduces Tau Phosphorylation.

Selenoprotein P (SELENOP1) is a selenium-rich antioxidant protein involved in extracellular transport of selenium (Se). SELENOP1 also has metal binding properties. The trace element Zinc (Zn2+) is a neuromodulator that can be released from synaptic terminals in the brain, primarily from a subset of glutamatergic terminals. Both Zn2+ and Se are necessary for normal brain function. Although these ions can bind together with high affinity, the biological significance of an interaction of SELENOP1 with Zn2+ has not been investigated. We examined changes in brain Zn2+ in SELENOP1 knockout (KO) animals. Timm-Danscher and N-(6-methoxy-8-quinolyl)-p-toluenesulphonamide (TSQ) staining revealed increased levels of intracellular Zn2+ in the SELENOP1−/− hippocampus compared to wildtype (WT) mice. Mass spectrometry analysis of frozen whole brain samples demonstrated that total Zn2+ was not increased in the SELENOP1−/− mice, suggesting only local changes in Zn2+ distribution. Unexpectedly, live Zn2+ imaging of hippocampal slices with a selective extracellular fluorescent Zn2+ indicator (FluoZin-3) showed that SELENOP1−/− mice have impaired Zn2+ release in response to KCl-induced neuron depolarization. The zinc/metal storage protein metallothionein 3 (MT-3) was increased in SELENOP1−/− hippocampus relative to wildtype, possibly in response to an elevated Zn2+ content. We found that depriving cultured cells of selenium resulted in increased intracellular Zn2+, as did inhibition of selenoprotein GPX4 but not GPX1, suggesting the increased Zn2+ in SELENOP1−/− mice is due to a downregulation of antioxidant selenoproteins and subsequent release of Zn2+ from intracellular stores. Surprisingly, we found increased tau phosphorylation in the hippocampus of SELENOP1−/− mice, possibly resulting from intracellular zinc changes. Our findings reveal important roles for SELENOP1 in the maintenance of synaptic Zn2+ physiology and preventing tau hyperphosphorylation.

HIF-1α Dependent Upregulation of ZIP8, ZIP14, and TRPA1 Modify Intracellular Zn2+ Accumulation in Inflammatory Synoviocytes.

Intracellular free zinc ([Zn2+]i) is mobilized in neuronal and non-neuronal cells under physiological and/or pathophysiological conditions; therefore, [Zn2+]i is a component of cellular signal transduction in biological systems. Although several transporters and ion channels that carry Zn2+ have been identified, proteins that are involved in Zn2+ supply into cells and their expression are poorly understood, particularly under inflammatory conditions. Here, we show that the expression of Zn2+ transporters ZIP8 and ZIP14 is increased via the activation of hypoxia-induced factor 1α (HIF-1α) in inflammation, leading to [Zn2+]i accumulation, which intrinsically activates transient receptor potential ankyrin 1 (TRPA1) channel and elevates basal [Zn2+]i. In human fibroblast-like synoviocytes (FLSs), treatment with inflammatory mediators, such as tumor necrosis factor-α (TNF-α) and interleukin-1α (IL-1α), evoked TRPA1-dependent intrinsic Ca2+ oscillations. Assays with fluorescent Zn2+ indicators revealed that the basal [Zn2+]i concentration was significantly higher in TRPA1-expressing HEK cells and inflammatory FLSs. Moreover, TRPA1 activation induced an elevation of [Zn2+]i level in the presence of 1 μM Zn2+ in inflammatory FLSs. Among the 17 out of 24 known Zn2+ transporters, FLSs that were treated with TNF-α and IL-1α exhibited a higher expression of ZIP8 and ZIP14. Their expression levels were augmented by transfection with an active component of nuclear factor-κB P65 and HIF-1α expression vectors, and they could be abolished by pretreatment with the HIF-1α inhibitor echinomycin (Echi). The functional expression of ZIP8 and ZIP14 in HEK cells significantly increased the basal [Zn2+]i level. Taken together, Zn2+ carrier proteins, TRPA1, ZIP8, and ZIP14, induced under HIF-1α mediated inflammation can synergistically change [Zn2+]i in inflammatory FLSs.

Bacillus altitudinis MT422188: a potential agent for zinc bioremediation

Anthropogenic processes are one of the main contributors for the presence of heavy metals in air, land and water sources, leading to environmental pollution. In the present study, Bacillus altitudinis MT422188 was used for determining its potential role in zinc bioremediation. After the bacterium was isolated and characterized, its optimum growth conditions were determined. The growth parameters, antioxidative and biosorption potential, motility and chemotactic behavior, as well as physicochemical nature of the isolate was determined in the presence and absence of zinc. Optimum growth was observed at pH 7, 37 °C and at 1 mM phosphate. Minimum inhibitory concentration was found to be 20 mM for Zn2+ whereas metal resistance pattern for other heavy metal ions was Cu2+ (30 mM) > Ni2+ (25 mM) > Cr6+ (18 mM) > Pb2+ (12 mM) > Co2+ (8 mM) > Cd2+ (5 mM) > Hg2+ (3 mM). EC50 was found to be 3.4 mM Zn2+. Biosorption experiments in the presence of metal, DCCD and DNP in both live and killed cells demonstrated metal uptake, intracellular Zn2+ accumulation and adsorption by ATP independent efflux system. It was also found to effectively remove 81 mg/L and 87 mg/L after a period of 4 and 8 days, respectively. In the presence of Zn2+, positive chemotaxis was demonstrated where Zn2+ acted as a chemoattractant. Zn2+ induced elevated levels of GR, SOD and POX, while APOX and CAT activity were inhibited by it. The appearance of 92 kDa protein band in B. altitudinis under Zn2+ stress confirmed the induction of metallothionein. FTIR analysis revealed the active role of amide, hydroxyl, and carboxyl groups in combating Zn2+ stress. SEM and EDS analysis revealed surface adsorption of Zn2+ ions by bacterial cells. The experimental data obeyed the Freundlich isotherm model and pseudo second-order kinetics, indicating it to be an efficient biosorbent for Zn2+. This study revealed the potential application of B. altitudinis MT422188 in the bioremediation of Zn2+ from polluted wastewaters.

Magnetic field-induced Ca2+ intake by mesenchymal stem cells is mediated by intracellular Zn2+ and accompanied by a Zn2+ influx

Chronic exposure to magnetic fields (MFs) has a diverse range of effects on biological systems but definitive molecular mechanisms of the interaction remain largely unknown. One of the most frequently reported effects of MF exposure is an elevated concentration of intracellular Ca2+ through disputed pathways. Other prominent effects include increased oxidative stress and upregulation of neural markers through EGFR activation in stem cells. Further characterization of cascades triggered by MF exposure is hindered by the phenotype diversity of biological models used in the literature. In an attempt to reveal more mechanistic data in this field, we combined the most commonly used biological model and MF parameters with the most commonly reported effects of MFs.Based on clues from the pathways previously defined as sensitive to MFs (EGFR and Zn2+-binding enzymes), the roles of different types of channels (voltage gated Ca2+ channels, NMDA receptors, TRP channels) were inquired in the effects of 50 Hz MFs on bone marrow-derived mesenchymal stem cells. We report that, an influx of Zn2+ accompanies MF-induced Ca2+ intake, which is only attenuated by the broad-range inhibitor of TRP channels and store-operated Ca2+ entry (SOCE), 2-Aminoethoxydiphenyl borate (2-APB) among other blockers (memantine, nifedipine, ethosuximide and gabapentin). Interestingly, cation influx completely disappears when intracellular Zn2+ is chelated. Our results rule out voltage gated Ca2+ channels as a gateway to MF-induced Ca2+ intake and suggest Zn2+-related channels as a new focus in the field.

A highly selective turn-on fluorescent chemosensor for detecting zinc ions in living cells using symmetrical pyrene system

This manuscript presents a very simple receptor based on the combination of pyrene and malonohydrazide groups for the sensitive and selective detection of Zn2+. The sensor PYMH {N1’,N3’-bis(pyren-1-yl)ethylene)malonohydrazide} was synthesized in two simple steps involving synthesis of malonohydrazide followed by the synthesis of symmetrical pyrene malonohydrazide with two pyrene units on either side of the molecule. The synthesized PYMH was confirmed by FT-IR, 1H NMR, 13C NMR and Mass spectroanalytical techniques. The sensor was able to exhibit ‘turn-on’ fluorescence in the presence of Zn2+ with no significant response from other relevant ions including Na+, K+, Ca2+, Mg2+, Cd2+, Cu2+, Co2+, Ni2+, Mn2+, Hg2+, Pb2+, Fe2+, Al3+, Fe3+ and Cr3+. Moreover, the sensor was capable of selectively detecting Zn2+ in the presence of its most common interferent Cd2+. The mechanism of fluorescent enhancement was proposed based on DFT calculations and it was established to be photoinduced electron transfer (PET). 1H NMR titrations revealed that the Zn2+ binding sites to be imine NH group, besides FT-IR spectra confirmed the coordination of C = O with Zn2+. The sensor PYMH showed the LOD of 5.1 nM (3σ/S), which is well below the permitted drinking water level of 76 μM as recommended by WHO. Further, the probe PYMH is capable of detecting distribution of intracellular Zn2+ in HeLa cells.

Dehydroeffusol Pprevents Amyloid β1-42-mediated Hippocampal Neurodegeneration via Reducing Intracellular Zn2+ Toxicity.

Dehydroeffusol, a phenanthrene isolated from Juncus effusus, is a Chinese medicine. To explore an efficacy of dehydroeffusol administration for prevention and cure of Alzheimer's disease, here we examined the effect of dehydroeffusol on amyloid β1-42 (Aβ1-42)-mediated hippocampal neurodegeneration. Dehydroeffusol (15 mg/kg body weight) was orally administered to mice once a day for 6 days and then human Aβ1-42 was injected intracerebroventricularly followed by oral administration for 12 days. Neurodegeneration in the dentate granule cell layer, which was determined 2 weeks after Aβ1-42 injection, was rescued by dehydroeffusol administration. Aβ staining (uptake) was not reduced in the dentate granule cell layer by pre-administration of dehydroeffusol for 6 days, while increase in intracellular Zn2+ induced with Aβ1-42 was reduced, suggesting that pre-administration of dehydroeffusol prior to Aβ1-42 injection is effective for Aβ1-42-mediated neurodegeneration that was linked with intracellular Zn2+ toxicity. As a matter of fact, pre-administration of dehydroeffusol rescued Aβ1-42-mediated neurodegeneration. Interestingly, pre-administration of dehydroeffusol increased synthesis of metallothioneins, intracellular Zn2+-binding proteins, in the dentate granule cell layer, which can capture Zn2+ from Zn-Aβ1-42 complexes. The present study indicates that pre-administration of dehydroeffusol protects Aβ1-42-mediated neurodegeneration in the hippocampus by reducing intracellular Zn2+ toxicity, which is linked with induced synthesis of metallothioneins. Dehydroeffusol, a novel inducer of metallothioneins, may protect Aβ1-42-induced pathogenesis in Alzheimer's disease.

Zur: Zinc-Sensing Transcriptional Regulator in a Diverse Set of Bacterial Species.

Zinc (Zn) is the quintessential d block metal, needed for survival in all living organisms. While Zn is an essential element, its excess is deleterious, therefore, maintenance of its intracellular concentrations is needed for survival. The living organisms, during the course of evolution, developed proteins that can track the limitation or excess of necessary metal ions, thus providing survival benefits under variable environmental conditions. Zinc uptake regulator (Zur) is a regulatory transcriptional factor of the FUR superfamily of proteins, abundant among the bacterial species and known for its intracellular Zn sensing ability. In this study, we highlight the roles played by Zur in maintaining the Zn levels in various bacterial species as well as the fact that in recent years Zur has emerged not only as a Zn homeostatic regulator but also as a protein involved directly or indirectly in virulence of some pathogens. This functional aspect of Zur could be exploited in the ventures for the identification of newer antimicrobial targets. Despite extensive research on Zur, the insights into its overall regulon and its moonlighting functions in various pathogens yet remain to be explored. Here in this review, we aim to summarise the disparate functional aspects of Zur proteins present in various bacterial species.

Zn2+ influx activates ERK and Akt signaling pathways

Zinc (Zn2+) is an essential metal in biology, and its bioavailability is highly regulated. Many cell types exhibit fluctuations in Zn2+ that appear to play an important role in cellular function. However, the detailed molecular mechanisms by which Zn2+ dynamics influence cell physiology remain enigmatic. Here, we use a combination of fluorescent biosensors and cell perturbations to define how changes in intracellular Zn2+ impact kinase signaling pathways. By simultaneously monitoring Zn2+ dynamics and kinase activity in individual cells, we quantify changes in labile Zn2+ and directly correlate changes in Zn2+ with ERK and Akt activity. Under our experimental conditions, Zn2+ fluctuations are not toxic and do not activate stress-dependent kinase signaling. We demonstrate that while Zn2+ can nonspecifically inhibit phosphatases leading to sustained kinase activation, ERK and Akt are predominantly activated via upstream signaling and through a common node via Ras. We provide a framework for quantification of Zn2+ fluctuations and correlate these fluctuations with signaling events in single cells to shed light on the role that Zn2+ dynamics play in healthy cell signaling.

A comparative study of the effective response of di-potassium phosphate (K2HPO4) on physiological, biochemical and anatomical aspects of crops dwelling with zinc oxide nanoparticles toxicity.

The dipotassium phosphate (K2HPO4) is a source of phosphorus (P), which is an essential micronutrient for plant growth and reproduction and also acts as a stress alleviator against abiotic stresses. Therefore, it could also become a potential mineral to cope up with zinc oxide nanoparticles' (ZnONPs) toxicity in crops. This study primarily includes synthesis, characterization and differential toxic impacts of ZnONPs on two crop plantsThis study includes synthesis, characterization and differential toxic impacts of ZnONPs on two crop plants, i.e. Triticum aestivum and Solanum lycopersicum, as well as assuage the toxic impacts of ZnONPs through nutrient management approach implied via supplementation of P. The growth and physiological changes under toxic doses of ZnONPs and ameliorative potential of P in crop plants were examined by analysing growth, intracellular Zn accumulation, photosynthetic pigment contents, the kinetics of photosystem II (PS II) photochemistry, root cell anatomy and cell viability via histochemical staining 4',6-diamidino-2-phenylindole and propidium iodide. ZnONPs at 500 and 1000μM concentrations significantly affected the growth, photosynthetic pigment and PS II photochemistry and cell death in both the plants. It also caused deformation in root anatomy of T. aestivum and S. lycopersicum. Whereas supplementation of P caused significant improvement against ZnONPs stress by causing remarkable enhancement in growth, photosynthetic pigments and activity of PS II photochemistry and decreased cell death. Moreover, the study also discloses the tolerant nature of S. lycopersicum comparing with T. aestivum seedlings. Thus, P is comparatively more effective in managing the ZnONPs toxicity in S. lycopersicum than in T. aestivum.

ZIP8 mediates the extracellular matrix degradation of nucleus pulposus cells via NF-κB signaling pathway

The extracellular matrix (ECM) degradation of nucleus pulposus cells (NPCs) is mainly induced by metalloproteinases (MMPs). Zn2+ is an essential component of MMPs, but the effect of Zn2+ importers in controlling ECM metabolism remains unclear. The purpose of this research was to identify the involvement of Zn2+ importers in ECM degradation induced by inflammatory stimuli and excessive mechanical stressing. In this study, NPCs from Sprague-Dawley (SD) rats were separated and cultured. FluoZin-3 AM staining was applied to detect [Zn2+]i in NPCs treated with Interleukin-1β (IL-1β) or cyclic tensile strain (CTS) with a Flexcell Strain Unit. We found that intracellular Zn2+ concentration ([Zn2+]i) elevated dramatically, and ZIP8 is the predominant Zn2+ importer among all importers in senescent NPCs. The [Zn2+]i and MMP expression level both increased in IL-1β and CTS treated NPCs. Furthermore, the expression of ZIP8 was also markedly increased. However, knockdown of ZIP8 with siRNA alleviated ECM degradation induced by inflammatory stimuli and CTS. Both stimuli activated NF-κB signaling pathway, and knockdown of ZIP8 effectively inhibited NF-κB signaling pathway activation. In conclusion, knockdown of ZIP8 can alleviate NPCs’ ECM degradation caused by inflammatory stimuli and excessive mechanical stressing.

Effects of oxidative stress-induced increases in Zn2+ concentrations in human gingival epithelial cells.

Previous studies have reported that oxidative stress increases intracellular Zn2+ concentrations and induces cytotoxicity. However, no studies have investigated whether oxidative stress induces such changes in periodontal tissue cells. In the present study, we investigated the effect of oxidative stress on intracellular Zn2+ concentration in periodontium constituent cells and its potential relationship with periodontal disease. We analyzed changes in intracellular Zn2+ concentrations in human gingival epithelial (epi4) cells treated with hydrogen peroxide (H2 O2 ). The fluorescent probes FluoZin-3 AM and CellTracker Green CMFDA were used to detect intracellular Zn2+ and thiol groups, respectively. Western blot analyses, luciferase reporter assays, and real-time polymerase chain reaction (PCR) analyses were performed to examine the effect of intracellular Zn2+ on epi4 cells. H2 O2 treatment increased intracellular concentrations of Zn2+ in epi4 cells by facilitating the movement of Zn2+ from cellular nonprotein thiols to the cytoplasm and promoting cell membrane permeability to Zn2+ . Furthermore, H2 O2 -induced increases in intracellular Zn2+ activated the p38 cAMP response element-binding protein/mitogen-activated protein kinase (p38 CREB/MAPK) cascade, upregulated nuclear factor kappa B (NF-κB) DNA binding, and increased the expression of inflammatory cytokines and matrix metallopeptidase-9 (MMP-9). Increases in intracellular Zn2+ induced by oxidative stress activate signaling pathways involved in inflammation, potentially contributing to the progression of periodontal disease.

Low doses of zeolitic imidazolate framework-8 nanoparticles alter the actin organization and contractility of vascular smooth muscle cells

Zeolitic imidazolate framework-8 (ZIF-8) nanoparticles have emerged as a promising platform for drug delivery and controlled release. Considering most ZIF-8 nanoparticle drug carriers are designed to be administered intravenously, and thus would directly contact vascular smooth muscle cells (VSMCs) in many circumstances, the potential interactions of ZIF-8 nanoparticles with VSMCs require investigation. Here, the effects of low doses of ZIF-8 nanoparticles on VSMC morphology, actin organization, and contractility are investigated. Two nanoscale imaging tools, atomic force microscopy, and direct stochastic optical reconstruction microscopy, show that even at the concentrations (12.5 and 25 µg/ml) that were deemed “safe” by conventional biochemical cell assays (MTT and LDH assays), ZIF-8 nanoparticles can still cause changes in cell morphology and actin cytoskeleton organization at the cell apical and basal surfaces. These cytoskeletal structural changes impair the contractility function of VSMCs in response to Angiotensin II, a classic vasoconstrictor. Based on intracellular zinc and actin polymerization assays, we conclude that the increased intracellular Zn2+ concentration due to the uptake and dissociation of ZIF-8 nanoparticles could cause the actin cytoskeleton dis-organization, as the elevated Zn2+ directly disrupts the actin assembly process, leading to altered actin organization such as branches and networks. Since the VSMC phenotype change and loss of contractility are fundamental to the development of atherosclerosis and related cardiovascular diseases, it is worth noting that these low doses of ZIF-8 nanoparticles administered intravenously could still be a safety concern in terms of cardiovascular risks. Moving forward, it is imperative to re-consider the “safe” nanoparticle dosages determined by biochemical cell assays alone, and take into account the impact of these nanoparticles on the biophysical characteristics of VSMCs, including changes in the actin cytoskeleton and cell morphology.

Glutathionylation-dependent proteasomal degradation of wide-spectrum mutant p53 proteins by engineered zeolitic imidazolate framework-8

Point mutations within the DNA-binding domain of the TP53 gene occur in a significant percentage of human cancer, leading to cellular accumulation of highly stabilized mutant p53 proteins (mutp53) with tumor-promoting properties. Depletion of mutp53, through inducing either autophagic or proteasomal degradation, is an attractive strategy for the therapy of p53-mutated cancer, but the currently-known degradation inducers, almost exclusively small molecules, are inadequate. Here we show that pH-responsive zeolitic imidazolate framework-8 (ZIF-8) offers a novel solution to mutp53 degradation. ZIF-8 facilitated ubiquitination-mediated and glutathionylation-dependent proteasomal degradation of all of the nine mutp53 we tested, including six hot-spot mutp53, but not the wild-type p53 protein. Sustained elevation of intracellular Zn++ level, resulted from decomposition of the internalized ZIF-8 in the acidic endosomes, decreased the intracellular reduced glutathione (GSH): oxidized glutathione (GSSG) ratio and was essential for mutp53 glutathionylation and degradation. ZIF-8 modified with an Z1-RGD peptide, exhibiting enhanced cellular internalization and improved decomposition behavior, preferentially killed mutp53-expressing cancer cells and demonstrated remarkable therapeutic efficacy in a p53 S241F ES-2 ovarian cancer model as well as in a p53 Y220C patient-derived xenograft (PDX) breast cancer model. The ability to induce wide-spectrum mutp53 degradation gives ZIF-8 a clear advantage over other degradation-inducers, and engineered nanomaterials may be promising alternatives to small molecules for the development of mutp53-targeting drugs.

Methionine-chelated Zn promotes anabolism by integrating mTOR signal and autophagy pathway in juvenile yellow catfish

BackgroundAmino acid-chelated zinc (Zn) can increase anabolism of animals. However, the underlying mechanisms are unclear. We aimed to examine how autophagy impact anabolism following a diet containing methionine-chelated Zn (ZnMet) compared with inorganic Zn (ZnSO4). MethodsYellow catfish (weight: 4.02 ± 0.08 g) were fed two diets containing ZnSO4 or ZnMet for 8 wk. The differences in transcriptional responses and corresponding biological profiles were compared between the livers of fish fed the two Zn sources of diets. Hepatocytes of yellow catfish were incubated for 48 h in medium containing ZnSO4 (10 μM ZnSO4) or ZnMet (10 μM ZnMet) after 2 h pretreated with or without pathway inhibitors. Intracellular Zn, TG and protein contents, lipid droplet and autophagic vesicles were detected. Ultrastructural observation, enzymatic activities, qPCR assays, western blot and immunofluorescence analysis were conducted. ResultsZnMet up-regulated the expression of genes associated with anabolism and autophagy. The differentially expressed genes (DEG) analysis indicated that both mTOR and autophagy pathways were activated. ZnMet-induced activation of autophagy was mTOR-independent. In this process, forkhead box class O was deacetylated and activated, and induced autophagy, which provided substrates for energy generation. ConclusionsZnMet increased anabolism through integrating mTOR signal and autophagy pathway in yellow catfish. The present study unravels a novel mechanism for amino acid-chelated minerals improving anabolism.

Age-related vulnerability to nigral dopaminergic degeneration in rats via Zn2+-permeable GluR2-lacking AMPA receptor activation

On the basis of the evidence that extracellular Zn2+ influx induced with AMPA causes Parkinson's syndrome in rats that apomorphine-induced movement disorder emerges, here we used a low dose of AMPA, which does not increase intracellular Zn2+ level in the substantia nigra pars compacta (SNpc) of young adult rats, and tested whether intracellular Zn2+ dysregulation induced with AMPA is accelerated in the SNpc of aged rats, resulting in age-related vulnerability to Parkinson's syndrome. When AMPA (1 mM) was injected at the rate of 0.05 μl/min for 20 min into the SNpc, intracellular Zn2+ level was increased in the SNpc of aged rats followed by increase in turning behavior in response to apomorphine and nigral dopaminergic degeneration. In contrast, young adult rats do not show movement disorder and nigral dopaminergic degeneration, in addition to no increase in intracellular Zn2+. In aged rats, movement disorder and nigral dopaminergic degeneration were rescued by co-injection of either extracellular (CaEDTA) or intracellular (ZnAF-2DA) Zn2+ chelators. 1-Naphthyl acetyl spermine (NASPM), a selective blocker of Ca2+- and Zn2+-permeable GluR2-lacking AMPA receptors blocked increase in intracellular Zn2+ in the SNpc of aged rats followed by rescuing nigral dopaminergic degeneration. The present study indicates that intracellular Zn2+ dysregulation is accelerated by Ca2+- and Zn2+-permeable GluR2-lacking AMPA receptor activation in the SNpc of aged rats, resulting in age-related vulnerability to Parkinson's syndrome.

Alzheimer's disease pathogenesis focused on intracellular Zn2+ toxicity and its defense strategy

The basal levels of intracellular Zn2+ and extracellular Zn2+ are in the range of ~100 pM and ~10 nM, respectively, in the hippocampus. Extracellular Zn2+ dynamics, which serves bidirectionally and involved in cognitive activity and cognitive decline, is modified by extracellular glutamate signaling and the presence of amyloid-β1-42 (Aβ1-42), a causative peptide in Alzheimer's disease (AD) pathogenesis. When human Aβ1-42 reaches 100-500 pM in the extracellular compartment of the rat hippocampus, Zn-Aβ1-42 complexes are produced and readily taken up into dentate granule cells in a synaptic activity-independent manner. Furthermore, intracellular Zn-Aβ1-42 complexes release Zn2+ followed by intracellular Zn2+ dysregulation. Aβ1-42-mediated intracellular Zn2+ toxicity is accelerated with aging, because extracellular Zn2+ is age-relatedly increased. We have reported that Aβ1-42 released physiologically from neuron terminals disrupts intracellular Zn2+ homeostasis, resulting in age-related cognitive decline and neurodegeneration. Metallothioneins (MTs), zinc-binding proteins can capture Zn2+ released from intracellular Zn-Aβ1-42 complexes and serve for intracellular Zn2+-buffering under acute intracellular Zn2+ dysregulation. Aβ1-42-induced pathogenesis leads the AD development and its defense strategy may prevent the development. This review summarizes extracellular Zn2+-dependent Aβ1-42 neurotoxicity, which is accelerated with aging, and the potential defense strategy against AD.

Preventive effect of Ninjin-yoei-to, a Kampo medicine, on amyloid β1-42-induced neurodegeneration via intracellular Zn2+ toxicity in the dentate gyrus.

Ninjin-yoei-to (NYT), a Kampo medicine, has ameliorative effects on cognitive dysfunction via enhancing cholinergic neuron activity. To explore an efficacy of NYT administration for prevention and cure of Alzheimer’s disease, here we examined the effect of NYT on amyloid β1-42 (Aβ1-42)-induced neurodegeneration in the dentate gyrus. A diet containing 3% NYT was administered to mice for 2 weeks and human Aβ1-42 was intracerebroventricularly injected. Neurodegeneration in the dentate granule cell layer of the hippocampus, which was determined 2 weeks after the injection, was rescued by administration of the diet for 4 weeks. Aβ staining (uptake) was not modified in the dentate granule cell layer by pre-administration of the diet for 2 weeks, while Aβ1-42-induced increase in intracellular Zn2+ was reduced, suggesting that pre-administration of NYT prior to Aβ injection is effective for reducing Aβ1-42-induced Zn2+ toxicity in the dentate gyrus. As a matter of fact, Aβ1-42-induced neurodegeneration in the dentate gyrus was rescued by pre-administration of NYT. Interestingly, the level of metallothioneins, intracellular Zn2+-binding proteins, which can capture Zn2+ from Zn-Aβ1-42 complexes, was elevated in the dentate granule cell layer by pre-administration of NYT. The present study suggests that pre-administration of NYT prevents Aβ1-42-mediated neurodegeneration in the dentate gyurs by induced synthesis of metallothioneins, which reduces intracellular Zn2+ toxicity induced by Aβ1-42.