Intracellular Zinc Homeostasis Research Articles

Xanthomonas axonopodis pv. glycines Zur is involved in pathogenicity in host and hypersensitive responses in nonhosts

The zinc uptake regulator (Zur) has highly conserved sequences in the plant pathogen Xanthomonas, while its functions are diverse in different strains or races. To elucidate the functions of Zur in Xanthomonas axonopodis pv. glycines (Xag), we constructed a zur-deleted mutant (Δzur) by homologous recombination. Compared with the wild type, Δzur demonstrated reduced pathogenicity in the host soybean and reduced ability to trigger hypersensitive responses (HR) in nonhosts such as tobacco, tomato, chili pepper, and eggplant. Additionally, the deletion of zur significantly enhanced Xag's sensitivity to Zn2+, Fe3+, and Cu2+, induced an imbalance in intracellular zinc homeostasis, decreased extracellular polysaccharide (EPS) production, and down-regulated the expression of extracellular hydrolases (cellulase, endo-glucanase, amylase, and protease). Functional complementation restored the defective properties of Δzur to the wild-type levels. The qRT-PCR results showed that zur expression was remarkably induced by Zn2+. Moreover, the deletion of zur evidently reduced the expression levels of hrp representative genes (hrpB1, hrpD6, hrpE, hrcV, and hrcC), extracellular hydrolase encoding genes (engXCA, egl2, pro1, pro2, pro8, pro11, and alpha1), and EPS synthesis genes (gumB, gumD, gumK, gumM, gumG, and gumH) relative to the wild type. In summary, the results suggested that Zur may be involved in pathogenicity in the host soybean and in triggering HR in nonhosts of Xag by regulating the synthesis of virulence factors and the expression of hrp genes. This laid a foundation for further analysis of the mechanism of Zur in Xanthomonas-plant interaction.

Duhuo Jisheng Decoction regulates intracellular zinc homeostasis by enhancing autophagy via PTEN/Akt/mTOR pathway to improve knee cartilage degeneration.

Articular cartilage and cartilage matrix degradation are key pathological changes occurring in the early stage of knee osteoarthritis (KOA). However, currently, there are limited strategies for early prevention and treatment of KOA. Duhuo Jisheng Decoction (DHJSD) is a formula quoted in Bei Ji Qian jin Yao Fang, which was compiled by Sun Simiao in the Tang Dynasty of China. As a complementary therapy, it is widely used to treat early-stage KOA in China; however, its mechanism has not been completely elucidated. This study investigated the potential role of DHJSD in preventing cartilage degradation and the underlying mechanism. A rat model of KOA model was established via the Hulth method. Subsequently, 25 rats were randomized into sham (saline), model control (saline), high-DHJSD (1.9g/mL of DHJSD), medium-DHJSD (1.2g/mL of DHJSD), and low-DHJSD groups (0.6g/mL of DHJSD). After 4 weeks of treatment, all rats were sacrificed and the severity of the cartilage degeneration was evaluated by a series of histological methods. The autophagosome was observed using transmission electron microscopy, and the related functional proteins were detected by the western blotting and real-time polymerase chain reaction. Next, the mechanism by which DHJSD improves knee cartilage degeneration was further clarified the in vitro by gene silencing technology combined with a series of functional experiments. The proteins levels of PTEN, Akt, p-Akt, mTOR, and p-mTOR, as well as the marker proteins of autophagy and apoptosis were determined. Zinc levels in chondrocytes were determined using inductively coupled plasma mass spectrometry. Histopathological staining revealed that DHJSD had a protective effect on the cartilage. DHJSD increased autophagosome synthesis and the expression of autophagy proteins LC3 and Beclin-1 in chondrocytes. Moreover, it reduced the phosphorylation levels of Akt and mTOR and the levels of zinc, MMP-13, Bax, and Bcl-2. Following PTEN silencing, this DHJSD-mediated reduction in Akt and mTOR phosphorylation and Bax, Bcl-2, and zinc levels were further decreased; in addition, DHJSD-mediated increase in LC3 and Beclin-1 levels was decreased. DHJSD inhibits the Akt/mTOR signaling pathway by targeting PTEN to promote autophagy in chondrocytes, which may help reduce MMP-13 production by regulating zinc levels in chondrocytes.

Galvanized material is a promising approach to control Amyloodinium ocellatum infection in fishes

Amyloodinium ocellatum is a worldwide distributed dinoflagellate that infects marine and brackish water fish, causing mass mortality and economic loss. This study evaluated the efficacy of the seawater immersed with galvanized materials (IGM seawater) in controlling A. ocellatum reinfection in susceptible host Trachinotus ovatus. Furthermore, the toxicity of IGM seawater to the dinospores infectivity, tomonts development, and trophonts parasitism was determined in Acanthopagrus latus. The expression of zinc transporters in A. latus gills and livers was also detected after A. latus exposure to IGM seawater. The results revealed that parasite abundances on infected T. ovatus were significantly decreased in the seawater immersed with 0.1 m2/m3 of galvanized iron mesh for 2 days ([0.1–2 d] group) or galvanized iron plates with a 56.25% tank coverage for 1 day ([56.25% - 1 d] group). Besides, the survival rates of the infected T. ovatus in the [0.1–2 d] and [56.25% - 1 d] group were 100% on the 14th-day post-infection, while 0% in the control group. The infectivity of dinospores was significantly inhibited in the seawater immersed with 0.4 m2/m3 of galvanized iron mesh for 2 days ([0.4–2 d] group) by 89.50%. In addition, the reduction of trophonts in the [0.4–2 d] group was 46.24% compared to the control group. However, the hatching rate of tomonts in the seawater immersed with 0.4 m2/m3 of galvanized iron mesh for either 1, 2, or 3 days was 100% at 48th hr post-treatment. At the same time, the zinc ion concentrations in the IGM seawater group were continuously increased. On the contrary, the mRNA levels of zinc transporters ZIP7 and ZIP11 in the liver of A. latus exposure to IGM seawater were significantly down-regulated. Therefore, A. latus acclimatizes to a high‑zinc environment by reducing zinc ions influx to maintain intracellular zinc homeostasis. These results imply that using galvanized material is a promising approach to controlling A. ocellatum infection in fish.

A Responsive Nanorobot Modulates Intracellular Zinc Homeostasis to Amplify Mitochondria-Targeted Phototherapy.

Zinc has been proven to interweave with many critical cell death pathways, and not only exhibits potent anticancer activity solely, but sensitizes cancer cells to anticancer treatment, making zinc supplementation ideal for boosting odds against malignancy. Herein, a smart nanorobot (termed as Zinger) is developed, composed of iRGD-functionalized liposome encapsulating black phosphorus nanosheet (BPNs) doped zeolite imidazole framework-8 (BPN@ZIF-8), for advancing zinc-promoted photodynamic therapy (PDT). Zinger exhibits photo-triggered sequential mitochondria-targeting ability, and can induce zinc overload-mediated mitochondrial stress, which consequently sensitized tumor to PDT through synergistically modulating reactive oxygen species (ROS) production and p53 pathway. It is identified that Zinger selectively triggered intracellular zinc overload and photodynamic effect in cancer cells, which together enhanced PDT treatment outcomes. Importantly, Zinger shows high efficacy in overcoming various treatment barriers, allowing for effectively killing cancer cells in the complex circumstances. Particularly, Zinger exhibits good tumor accumulation, penetration, and even cell uptake, and can respond to light stimulation to eliminate tumors while avoiding normal tissues, thereby prolonging survival of tumor-bearing mice. Therefore, the study provides a novel insight in the development of novel zinc-associated therapy for advancing cancer treatment approaches.

Annexin A1 is a cell-intrinsic metalloregulator of zinc in human ILC2s

Group 2 innate lymphoid cells (ILC2s) produce large amounts of type 2 cytokines including interleukin-5 (IL-5) and IL-13 in response to various stimuli, causing allergic and eosinophilic diseases. However, the cell-intrinsic regulatory mechanisms of human ILC2s remain unclear. Here, we analyze human ILC2s derived from different tissues and pathological conditions and identify ANXA1, encoding annexin A1, as a commonly highly expressed gene in non-activated ILC2s. The expression of ANXA1 decreases when ILC2s activate, but it increases autonomously as the activation subsides. Lentiviral vector-based gene transfer experiments show that ANXA1 suppresses the activation of human ILC2s. Mechanistically, ANXA1 regulates the expression of the metallothionein family genes, including MT2A, which modulate intracellular zinc homeostasis. Furthermore, increased intracellular zinc levels play an essential role in the activation of human ILC2s by promoting the mitogen-activated protein kinase (MAPK) and nuclear factor κB (NF-κB) pathways and GATA3 expression. Thus, the ANXA1/MT2A/zinc pathway is identified as a cell-intrinsic metalloregulatory mechanism for human ILC2s.

Selective Metal Chelation by a Thiosemicarbazone Derivative Interferes with Mitochondrial Respiration and Ribosome Biogenesis in Candida albicans.

ABSTRACTMetal chelation is generally considered as a promising antifungal approach but its specific mechanisms are unclear. Here, we identify 13 thiosemicarbazone derivatives that exert broad-spectrum antifungal activity with potency comparable or superior to that of fluconazole in vitro by screening a small compound library comprising 89 thiosemicarbazone derivatives as iron chelators. Among the hits, 19ak exhibits minimal cytotoxicity and potent activity against either azole-sensitive or azole-resistant fungal pathogens. Mechanism investigations reveal that 19ak inhibits mitochondrial respiration mainly by retarding mitochondrial respiratory chain complex I activity through iron chelation, and further reduces mitochondrial membrane potential and ATP synthesis in Candida albicans. In addition, 19ak inhibits fungal ribosome biogenesis mainly by disrupting intracellular zinc homeostasis. 19ak also stimulates the activities of antioxidant enzymes and decreases reactive oxygen species formation in C. albicans, resulting in an increase in detrimental intracellular reductive stress. However, 19ak has minor effects on mammalian cells in depleting intracellular iron and zinc. Moreover, 19ak exhibits low capacity to induce drug resistance and in vivo efficacy in a Galleria mellonella infection model. These findings uncover retarded fungal mitochondrial respiration and ribosome biogenesis as downstream effects of disruption of iron and zinc homeostasis in C. albicans and provide a basis for the thiosemicarbazone 19ak in antifungal application.IMPORTANCE The increasing incidence of fungal infections and resistance to existing antifungals call for the development of broad-spectrum antifungals with novel mechanisms of action. In this study, we demonstrate that a thiosemicarbazone derivative 19ak selectively inhibits mitochondrial respiration mainly by retarding mitochondrial respiratory chain complex I activity through iron chelation and inhibits ribosome biogenesis mainly by disrupting intracellular zinc homeostasis in C. albicans. In addition, 19ak exhibits low capacity to induce fungal resistance, minimal cytotoxicity, and in vivo antifungal efficacy. This study provides the basis of thiosemicarbazone derivative 19ak as a metal chelator for the treatment of fungal infections.

Loss of MBD2 ameliorates LPS-induced alveolar epithelial cell apoptosis and ALI in mice via modulating intracellular zinc homeostasis.

Apoptosis of alveolar epithelial cells is a critical initial link in the pathogenesis of acute lung injury (ALI), recent studies have revealed that Methyl-CpG binding domain protein 2 (MBD2) was involved in the execution of apoptosis, yet its role in ALI remained unclear. In the present study, we aim to explore the role and mechanism of MBD2 in the pathogenesis of ALI. We have found that MBD2 expression, in parallel to apoptosis, increased in alveolar epithelial cells of mice treated with LPS, knockout of MBD2 reduced apoptosis and protected mice from LPS-induced ALI. In MLE-12 cells, a cell line of murine alveolar epithelial cells, LPS induced MBD2 expression and apoptosis in a dose- and time-dependent manner. Knockdown of MBD2 with shRNA alleviated, while overexpression of MBD2 increased LPS-induced apoptosis. Mechanistically, intracellular zinc level decreased when MLE-12 cells were treated with LPS. MBD2knockdown restored intracellular zinc level after LPS treatment, and MBD2 overexpression further aggravated LPS-induced intracellular zinc loss. Metal transcription factor 1 (MTF1) is a critical transcription factor in charge of intracellular zinc efflux. LPS treatment induced MTF1 expression both in vivo and in vitro. Inhibition of MTF1 reduced LPS-induced apoptosis in MLE-12 cells. MBD2 could bind to the promoter region of MTF1 and promote MTF1 expression. Collectively, these data indicated that loss of MBD2-ameliorated LPS-induced alveolar epithelial cell apoptosis and ALI in mice via modulating intracellular zinc homeostasis by upregulating MTF1.

Dengue virus replication enhances labile zinc pools by modulation of ZIP8

Zinc-dependent viral proteins rely on intracellular zinc homeostasis for successful completion of infectious life-cycle. Here, we report that the intracellular labile zinc levels were elevated at early stages of dengue virus (DENV) infection in hepatic cells and this increase in free zinc was abolished in cells infected with UV-inactivated virus or with a DENV replication inhibitor implicating a role for zinc homeostasis in viral RNA replication. This change in free zinc was mediated by zinc transporter, ZIP8, as siRNA-mediated knockdown of ZIP8 resulted in abrogation of increase in free zinc levels leading to significant reduction in DENV titers suggesting a crucial role for ZIP8 in early stages of DENV replication. Furthermore, elevated free zinc levels correlated with high copy numbers of dengue genome in peripheral blood leukocytes obtained from dengue patients compared to healthy controls suggesting a critical role for zinc homeostasis in dengue infection.Take AwaysDengue virus utilises cellular zinc homeostasis during replication of its RNA.ZIP8 upregulates free zinc levels during dengue virus replication.Enhanced viremia associates with elevated intracellular free zinc in dengue.

Fear-of-intimacy-mediated zinc transport controls fat body cell dissociation through modulating Mmp activity in Drosophila

Matrix metalloproteinases (Mmps) are pivotal extracellular proteinases that have been implicated in tumour invasion and metastasis. Drosophila fat body is important for energy storage and utilization, as well as biosynthetic and metabolic activities. The fat body undergoes remodelling during metamorphosis which is characterized by the dissociation of the fat body into individual cells. Mmps play important roles in the regulation of fat body cell dissociation. Here we show that a zinc transporter fear-of-intimacy (foi) is necessary for the cell dissociation of fat body in Drosophila. The progression of fat body cell dissociation was delayed by fat body-specific foi knockdown while it was accelerated by foi overexpression (OE). In essence, these phenotypes are closely associated with intracellular zinc homeostasis, which can be modulated by dietary zinc intervention or genetic modulation of other zinc transporters. Further study indicated that Mmp1 and Mmp2 levels could be transcriptionally regulated by zinc in vivo. Consistently, the retarded fat body cell dissociation caused by Mmp1 or Mmp2 RNAi could be regulated by modulating the expression of foi. Further, by using Drosophila models of malignant tumour RafGOFscrib−/− and RasV12lgl−/−, we showed that the tumour growth, invasion and migration could be markedly inhibited by foi knockdown. These findings demonstrate a close connection between zinc levels and cell dissociation in vivo, and also suggest that manipulation of zinc levels may provide a novel therapeutic strategy for cancer.

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.

A zinc transporter, transmembrane protein 163, is critical for the biogenesis of platelet dense granules

Lysosome-related organelles (LROs) are a category of secretory organelles enriched with ions such as calcium, which are maintained by ion transporters or channels. Homeostasis of these ions is important for LRO biogenesis and secretion. Hermansky-Pudlak syndrome (HPS) is a recessive disorder with defects in multiple LROs, typically platelet dense granules (DGs) and melanosomes. However, the underlying mechanism of DG deficiency is largely unknown. Using quantitative proteomics, we identified a previously unreported platelet zinc transporter, transmembrane protein 163 (TMEM163), which was significantly reduced in BLOC-1 (Dtnbp1sdy and Pldnpa)–, BLOC-2 (Hps6ru)–, or AP-3 (Ap3b1pe)–deficient mice and HPS patients (HPS2, HPS3, HPS5, HPS6, or HPS9). We observed similar platelet DG defects and higher intracellular zinc accumulation in platelets of mice deficient in either TMEM163 or dysbindin (a BLOC-1 subunit). In addition, we discovered that BLOC-1 was required for the trafficking of TMEM163 to perinuclear DG and late endosome marker–positive compartments (likely DG precursors) in MEG-01 cells. Our results suggest that TMEM163 is critical for DG biogenesis and that BLOC-1 is required for the trafficking of TMEM163 to putative DG precursors. These new findings suggest that loss of TMEM163 function results in disruption of intracellular zinc homeostasis and provide insights into the pathogenesis of HPS or platelet storage pool deficiency.

TMEM163 (ZNT11) protein interacts with distinct ZNT efflux transporters

Our recent work showed that TMEM163 is a zinc efflux transporter that belongs to the cation diffusion facilitator (CDF) family of proteins. We propose that the TMEM163 be now called ZNT11 as a new member of the Group 1 CDF family ZNT efflux proteins that include 10 mammalian zinc transporters, ZNT1‐ZNT10. Accordingly, we hypothesize that TMEM163/ZNT11 interacts with at least one of the ZNT proteins, namely ZNT1 and ZNT2, based on its structural and functional characteristics. To show that TMEM163/ZNT11 interacts with either ZNT1 or ZNT2, we cloned their cDNAs into mammalian expression constructs containing either Myc‐DDK or HA peptide tag. Using HEK‐293 cells, we individually transfected TMEM163/ZNT11, ZNT1, and ZNT2 constructs as negative controls, while we co‐transfected TMEM163/ZNT11 and ZNT1, or TMEM163/ZNT11 and ZNT2. As a positive control, we co‐transfected HA‐tagged TMEM163/ZNT11 with a Myc‐DDK‐tagged TMEM163/ZNT11 construct in HEK‐293 cells, based on a previous report that its rodent counterpart exists as a homodimer. We then used a co‐immunoprecipitation assay using either anti‐HA‐ or anti‐DDK‐bound agarose beads to pull down one of the target proteins. Western blot analysis showed that TMEM163/ZNT11 physically bound ZNT1 or ZNT2 protein. To determine the functional relevance of the interaction, we performed zinc flux assays using two zinc‐specific fluorescence dyes, Fluozin‐3 (high affinity, membrane impermeable) and Newport Green (low affinity, membrane permeable) following single‐ and co‐transfection of TMEM163/ZNT11, ZNT1, and ZNT2 constructs in HeLa cells. Our results confirmed that homodimers of TMEM163/ZNT11, ZNT1, and ZNT2 proteins transport zinc out of the cells, but that the efflux activity of homodimer TMEM163/ZNT11 proteins varied slightly in magnitude when compared with heterodimers of TMEM163/ZNT11 and ZNT1, or TMEM163/ZNT11 and ZNT2 proteins. These results suggest that the interaction between TMEM163/ZNT11 and distinct ZNT proteins is physiologically relevant and may serve to modify the transport activity of ZNT protein interactor. Overall, our investigations showed for the first time that TMEM163/ZNT11 forms functional heterodimers with ZNT1 and ZNT2 proteins. Thus, TMEM163/ZNT11 by itself, or in combination with one of these specific ZNT proteins, may play a crucial role in maintaining intracellular zinc homeostasis in specific cell types.Support or Funding InformationThis work is funded by NIH R15 NS101594.

Azoxystrobin at sub-cytotoxic concentrations disrupts intracellular zinc homeostasis: A flow cytometric analysis with rat thymic lymphocytes and fluorescent probes

Azoxystrobin at sub-cytotoxic concentrations disrupts intracellular zinc homeostasis: A flow cytometric analysis with rat thymic lymphocytes and fluorescent probes

Role of Tmem163 in zinc-regulated insulin storage of MIN6 cells: Functional exploration of an Indian type 2 diabetes GWAS associated gene

Genome wide association study for type 2 diabetes discovered TMEM163 as a risk locus. Perturbations in TMEM163 expression was reported to be associated with impaired intracellular zinc homeostasis. Physiological concentration of zinc is instrumental to maintain insulin storage and functionality in pancreatic β cells. We found abundant TMEM163 expression in human pancreas, both at transcriptional and translational levels. Knockdown of endogenous Tmem163 in MIN6 cells resulted in increased intracellular zinc and total insulin content, coupled with compromised insulin secretion at high glucose stimuli. Furthermore, Tmem163 knockdown led to enhanced cellular glucose uptake. Upon next generation sequencing, one-third of the studied T2D patients were found to have a novel missense variant in TMEM163 gene. Study participants harboring this missense variant displayed a trend of higher glycemic indices. This is the first report on exploring the biological role of TMEM163 in relation to T2D pathophysiology.

Alterations in ZnT1 expression and function lead to impaired intracellular zinc homeostasis in cancer

Zinc is vital for the structure and function of ~3000 human proteins and hence plays key physiological roles. Consequently, impaired zinc homeostasis is associated with various human diseases including cancer. Intracellular zinc levels are tightly regulated by two families of zinc transporters: ZIPs and ZnTs; ZIPs import zinc into the cytosol from the extracellular milieu, or from the lumen of organelles into the cytoplasm. In contrast, the vast majority of ZnTs compartmentalize zinc within organelles, whereas the ubiquitously expressed ZnT1 is the sole zinc exporter. Herein, we explored the hypothesis that qualitative and quantitative alterations in ZnT1 activity impair cellular zinc homeostasis in cancer. Towards this end, we first used bioinformatics to analyze inactivating mutations in ZIPs and ZNTs, catalogued in the COSMIC and gnomAD databases, representing tumor specimens and healthy population controls, respectively. ZnT1, ZnT10, ZIP8, and ZIP10 showed extremely high rates of loss of function mutations in cancer as compared to healthy controls. Analysis of the putative functional impact of missense mutations in ZnT1-ZnT10 and ZIP1-ZIP14, using homologous protein alignment and structural predictions, revealed that ZnT1 displays a markedly increased frequency of predicted functionally deleterious mutations in malignant tumors, as compared to a healthy population. Furthermore, examination of ZnT1 expression in 30 cancer types in the TCGA database revealed five tumor types with significant ZnT1 overexpression, which predicted dismal prognosis for cancer patient survival. Novel functional zinc transport assays, which allowed for the indirect measurement of cytosolic zinc levels, established that wild type ZnT1 overexpression results in low intracellular zinc levels. In contrast, overexpression of predicted deleterious ZnT1 missense mutations did not reduce intracellular zinc levels, validating eight missense mutations as loss of function (LoF) mutations. Thus, alterations in ZnT1 expression and LoF mutations in ZnT1 provide a molecular mechanism for impaired zinc homeostasis in cancer formation and/or progression.

Abstract 2665: Zinc dysregulation as an emerging molecular target in epithelial cancers

Abstract Zinc plays an established role in structure and function of enzymes and transcription factors, and zinc signaling has now been recognized as a molecular determinant of epithelial differentiation and cell fate. Intracellular zinc homeostasis is regulated by zinc importers (ZIPs encoded by the SLC39A gene family). Cumulative evidence suggests that dysruption of zinc homeostasis is an emerging hallmark of various tumor types, characterized by downregulation of ZIP expression upstream of intracellular zinc depletion. In nonmelanoma skin cancer (NMSC) examined in TMA format we have observed downregulation of ZIP2 (involved in epidermal differentiation) that occurs with zinc depletion and impaired differentiation. Likewise, in pancreatic ductal adenocarcinoma (PDAC) tumor tissue, downregulation of ZIP3 and zinc depletion are detectable. We therefore tested feasibility of pharmacological induction of intracellular zinc overload for chemotherapeutic intervention. Using the small molecule ionophore ZnPT (zinc pyrithione) as an experimental chemotherapeutic, rapid induction of zinc overload followed by metal stress response signaling and cell death were observable in a panel of malignant epithelial cell lines (NMSC: SCC-25; HaCaT-ras II-4; PDAC: BxPC-3, MIA PaCa-2, PANC-1). Remarkably, PDAC cell lines displayed nanomolar sensitivity to ZnPT-induced cytotoxicity. In BxPC-3 cells, pronounced upregulation of stress response gene expression (MT2A, HSPA6, HMOX1) and signaling (p-p38, p-eIF2α, HO-1) was detectable within one hour of ZnPT exposure. Accumulation of protein-ubiquitin conjugates further substantiated the rapid occurrence of ZnPT-induced proteotoxic stress and UPR, consistent with a role of ZnPT in the inhibition of proteasome-associated deubiquitinating enzymes (DUBs). Next, we confirmed NMSC-directed anti-tumor activity of ZnPT in a photocarcinogenesis SKH1 mouse model, followed by exploratory chemotherapeutic intervention targeting BxPC-3 PDAC xenografts. Taken together, our data suggest that ZIP-based dysruption of intracellular zinc homeostasis may be a valid molecular target in specific epithelial cancers. [This research was funded in part by NCI: P30 CA023074 & R03 CA230949.] Citation Format: Jessica Perer, Rebecca Justiniano, Anh Hua, Georg T. Wondrak. Zinc dysregulation as an emerging molecular target in epithelial cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2665.

Specificity of the Metallothionein-1 Response by Cadmium-Exposed Normal Human Urothelial Cells.

Occupational and environmental exposure to cadmium is associated with the development of urothelial cancer. The metallothionein (MT) family of genes encodes proteins that sequester metal ions and modulate physiological processes, including zinc homeostasis. Little is known about the selectivity of expression of the different MT isoforms. Here, we examined the effect of cadmium exposure on MT gene and isoform expression by normal human urothelial (NHU) cell cultures. Baseline and cadmium-induced MT gene expression was characterized by next-generation sequencing and RT-PCR; protein expression was assessed by Western blotting using isoform-specific antibodies. Expression of the zinc transporter-1 (SLC30A1) gene was also assessed. NHU cells displayed transcription of MT-2A, but neither MT-3 nor MT-4 genes. Most striking was a highly inducer-specific expression of MT-1 genes, with cadmium inducing transcription of MT-1A, MT-1G, MT-1H, and MT-1M. Whereas MT-1G was also induced by zinc and nickel ions and MT-1H by iron, both MT-1A and MT-1M were highly cadmium-specific, which was confirmed for protein using isoform-specific antibodies. Protein but not transcript endured post-exposure, probably reflecting sequestration. SLC30A1 transcription was also affected by cadmium ion exposure, potentially reflecting perturbation of intracellular zinc homeostasis. We conclude that human urothelium displays a highly inductive profile of MT-1 gene expression, with two isoforms identified as highly specific to cadmium, providing candidate transcript and long-lived protein biomarkers of cadmium exposure.

Functional Characterization of Single Nucleotide Polymorphisms in the TMEM163 Gene

Mucolipidosis type IV (MLIV) is a human lysosomal storage disorder caused by a loss or dysfunctional Mucolipin‐1 (TRPML1) ion channel protein. Abnormal levels of zinc accumulation have been found in the brain tissues of MLIV mouse model and cells from MLIV patients. We previously identified a putative zinc transporter called Transmembrane 163 protein (TMEM163) through a yeast two‐hybrid screen in search of interaction partners for TRPML1. We found both proteins to co‐localize on the plasma membrane and lysosomal compartments, and we proposed that both proteins play a role in intracellular zinc homeostasis. The function of TMEM163 has yet to be fully elucidated; however, it is predicted to have six transmembrane domains (TMD) and recent evidence suggests that it transports zinc. To further study TMEM163's function and how it might be involved in certain human diseases like MLIV, we used the single nucleotide polymorphism database (dbSNP) from the National Center for Biotechnology Information (NCBI) website to investigate specific nucleotide sequence variations within the TMEM163 gene. We identified SNPs that produce amino acid substitution when compared with the wild‐type (WT) TMEM163 protein sequence. We then used bioinformatics to predict the topology of TMEM163 protein, and found that some SNP‐associated amino acid substitutions are located in areas of the protein that could affect post‐translational modification and change its structure and function. Thus, we hypothesize that SNPs that result in amino acid substitution dramatically alters the zinc transport function of TMEM163. To accomplish this project, we performed site‐directed mutagenesis using In‐Fusion homologous recombination cloning technique to systematically replace specific nucleotides corresponding to the SNPs located within or in proximity of N‐ and C‐termini, as well as TMD‐1 to TMD‐6. Upon sequence verification, we tested the effect of each SNP‐associated mutant by transfecting them into cultured HeLa cells. Twenty‐four hours post transfection, the cells were exposed to zinc chloride (100 μM) and assayed for intracellular zinc transport using the zinc specific FluoZin‐3 AM dye. We observed that the SNP‐associated mutants have marked decrease in intracellular zinc fluorescence following exposure to exogenous zinc. These observations indicate that certain SNP‐associated amino acid substitution located within critical areas of TMEM163 protein results in abnormal zinc transport. Our preliminary data suggest that TMEM163 is indeed a zinc transporter. We are currently conducting additional experiments using different zinc specific dyes to support this result. Understanding the nature of TMEM163‐TRPML1 interaction is necessary to further support our findings of cellular zinc dyshomeostasis in MLIV cells.Support or Funding InformationThis work was funded by CSUPERB Research and Development Grant 2016, and partly funded by NIH R15 NS101594This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Differential Gene Expression Analysis of Brain Tissue RNA From Mucolipidosis IV Knockout Mice

Mucolipidosis IV (MLIV) is an autosomal recessive disorder that is characterized by progressive neuronal and retinal degeneration. MLIV is caused by loss of function mutations in the human Mucolipin‐1 (MCOLN1) gene, whose protein product, Transient Receptor Potential Mucolipin‐1 (TRPML1), serves as a lysosomal non‐selective cation channel. We have previously reported abnormal zinc levels in MLIV patient fibroblasts, human MCOLN1‐knockdown cells, and Mcoln1‐knockout (KO) mouse brain tissues. Collectively, these observations suggest that the loss of cellular zinc homeostasis may play an underlying role in MLIV pathogenesis. To investigate whether these irregularities are reflected in the gene expression patterns of zinc buffering and transport proteins, we performed RNA sequencing (RNA‐seq) to analyze the brain transcriptome of three Mcoln1 KO mice and three wild‐type (WT) littermate controls. Gene ontology data revealed significant differential gene expression in the category of “Zinc Ion Transmembrane Transporter Activity.” Gene cluster analyses of zinc transport and zinc buffering genes revealed a distinct reduction in brain transcript levels of solute carrier 30a3 (Slc30a3; also known as the efflux zinc transporter, ZnT3) and transmembrane (Tmem)‐163 genes in KO mice, both of which facilitate zinc transport within neuronal synaptic vesicles. Comparison of RNA‐seq data between individual assembly and combined assembly of KO and WT sequencing reads showed distinct transcript‐level variability that could be explained by inter‐subject differences. Validation using real‐time quantitative polymerase chain reaction (qPCR) confirmed this effect. Nevertheless, both RNA‐seq and qPCR results consistently showed a decrease in ZnT3 transcript levels in KO mice compared to WT littermate control mice. These data imply that abnormal ZnT3 expression could be a mitigating factor for the observed zinc dyshomeostasis in MLIV cells. Overall, our findings suggest that the loss of human TRPML1 protein function perturbs the expression of specific genes necessary for intracellular zinc homeostasis. Future studies will investigate whether the reduction of ZnT3 transcript levels also reflect decreased protein levels in brain tissues of MLIV KO versus control mice.Support or Funding InformationThis work was funded by NIH R15 NS101594 and NIH R25 MD010397.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Implications of impaired zinc homeostasis in diabetic cardiomyopathy and nephropathy.

Impaired zinc homeostasis is observed in diabetes mellitus (DM2) and its complications. Zinc has a specific role in pancreatic β-cells via insulin synthesis, storage, and secretion. Intracellular zinc homeostasis is tightly controlled by zinc transporters (ZnT and Zip families) and metallothioneins (MT) which modulate the uptake, storage, and distribution of zinc. Several investigations in animal models demonstrate the protective role of MT in DM2 and its cardiovascular or renal complications, while a copious literature shows that a common polymorphism (R325W) in ZnT8, which affects the protein's zinc transport activity, is associated with increased DM2 risk. Emerging studies highlight a role of other zinc transporters in β-cell function, suggesting that targeting them could make a possible contribution in managing the hyperglycemia in diabetic patients. This article summarizes the current findings concerning the role of zinc homeostasis in DM2 pathogenesis and development of diabetic cardiomyopathy and nephropathy and suggests novel therapeutic targets. © 2017 BioFactors, 43(6):770-784, 2017.