Zn2+ and Cu2+ Binding to the Extramembrane Loop of Zrt2, a Zinc Transporter of Candida albicans

Zrt2 is a zinc transporter of the ZIP family. It is predicted to be located in the plasma membrane and it is essential for Candida albicans zinc uptake and growth at acidic pH. Zrt2 from C. albicans is composed of 370 amino acids and contains eight putative transmembrane domains and an extra-membrane disordered loop, corresponding to the amino acid sequence 126–215. This protein region contains at least three possible metal binding motifs: HxHxHxxD (144–153), HxxHxxEHxD (181–193) and the Glu- and Asp- rich sequence DDEEEDxE (161–168). The corresponding model peptides, protected at their termini (Ac-GPHTHSHFGD-NH2, Ac-DDEEEDLE-NH2 and Ac-PSHFAHAQEHQDP-NH2), have been investigated in order to elucidate the thermodynamic and coordination properties of their Zn2+ and Cu2+ complexes, with the further aim to identify the most effective metal binding site among the three fragments. Furthermore, we extended the investigation to the peptides Ac-GPHTHAHFGD-NH2 and Ac-PAHFAHAQEHQDP-NH2, where serine residues have been substituted by alanines in order to check if the presence of a serine residue may favor the displacement of amidic protons by Cu2+. In the native Zrt2 protein, the Ac-GPHTHSHFGD-NH2 region of the Zrt2 loop has the highest metal binding affinity, showing that three alternated histidines separated by only one residue (-HxHxH-) bind Zn2+ and Cu2+ more strongly than the region in which three histidines are separated by two and three His residues (-HxxHxxxH- in Ac-PSHFAHAQEHQDP-NH2). All studied Zrt2 loop fragments have lower affinity towards Zn2+ than the zinc(II) binding site on the Zrt1 transporter; also, all three Zrt2 regions bind Zn2+ and Cu2+ with comparable affinity below pH 5 and, therefore, may equally contribute to the metal acquisition under the most acidic conditions in which the Zrt2 transporter is expressed.

The role of zinc transporter proteins as predictive and prognostic biomarkers of hepatocellular cancer

Identification of the key processes involved in the tumor progression, malignancy and the molecular factors which are responsible for the transition of the cirrhotic cells to the tumor cells, contribute to the detection of biomarkers for diagnosis of hepatocellular carcinoma (HCC) at an early stage. According to clinical data, HCC is mostly characterized by a significant decrease in zinc levels. It is strongly implied that zinc deficiency is the major event required in the early stages of tumor formation and development of malignancy. Due to this reason, the definition of the molecular players which have a role in zinc homeostasis and cellular zinc level could give us a clue about the transition state of the cirrhosis to hepatic tumor formation. Despite the well-known implications of zinc in the development of HCCthe correlation of the expression of zinc transporter proteins with tumor progression and malignancy remain largely unknown. In the present study, we evaluated in detail the relationship of zinc deficiency on the prognosis of early HCC patients. In this study, we aimed to test the potential zinc transporters which contribute tothe transformation of cirrhosis to HCCand the progression of HCC. Among the 24 zinc transporter proteins, the proteins to be examined were chosen by using Gene Expression Profiling Interactive Analysis (GEPIA) webpage and RNA-seq analysis using TCGA database. ZIP14 and ZIP5 transporters were found as common differentially expressed genes from both bioinformatic analyses. ZnT1, ZnT7 and ZIP7 transporters have been associated with tumor progression. Relative abundance of ZnT1, ZIP5 and ZIP14 protein level was determined by immunohistochemistry (IHC) in surgically resected liver specimens from 16 HCC patients at different stages. IHC staining intensity was analyzed by using ImageJ software and scored with the histological scoring (H-score) method. The staining of ZnT1 was significantly higher in Grade III comparing to Grade II and Grade I. On the contrary, ZIP14 staining decreased almost 10-foldcomparing to Grade Iand Grade II. ZIP5 staining was detected almost 2-fold higher in cirrhosis than HCC. But ZnT1 staining was observed almost 3-fold lower in cirrhosis comparing to HCC. Intracellular free zinc level was measured by flow cytometry in Hep40 and Snu398 cells using FluoZin-3 dye. The intracellular free zinc level was almost 9-fold decreased in poor differentiated Snu398 HCC cells comparing to well differentiated Hep40 HCC cells.This report establishes for the first time the correlation between the expression pattern of ZIP14, ZnT1 and ZIP5 and significant zinc deficiency which occurs concurrently with the advancing of malignancy. Our results provide new molecular insight into ZnT1, ZIP14 and ZIP5 mediated regulation of cellular zinc homeostasis and indicate that zinc transporters might be important factors and events in HCC malignancy, which can lead to the development of early biomarkers.

Zinc Transporter ZIP12 Maintains Zinc Homeostasis and Protects Spermatogonia From Oxidative Stress During Spermatogenesis

Background: Overwhelming evidences now suggest oxidative stress is a major cause of sperm dysfunction and male infertility. Zinc is an important non-enzyme antioxidant with a wide range of biological functions and plays a significant role in preserving male fertility. Notably, zinc trafficking through the cellular and intracellular membrane is endorsed by precise families of zinc transporters, i.e. SLC39s/ZIPs and SLC30s/ZnTs. However, the expression and function of zinc transporters in the male germ cells were rarely reported. The aim of this study is to determine the crucial zinc transporter responsible for the maintenance of spermatogenesis.Methods: In the present study, we investigated the expression of all fourteen ZIP members in mouse testis and further analyzed the characteristic of ZIP12 expression in testis and spermatozoa by qRT-PCR, immunoblot and immunohistochemistry analyses. To explore the antioxidant role of ZIP12 in spermatogenesis, an obese mouse model fed with high-fat-diet was employed to confirm the correlation between ZIP12 expression level and sperm quality. Furthermore, ZIP12 expression in response to oxidative stress in a spermatogonia cell line, C18-4 cells, was determined and its function involved in regulating cell viability and apoptosis was investigated by RNAi experiment. Results: We initially found that ZIP12 expression in mouse testis was significantly high compared to other members of ZIPs and its mRNA and protein were intensively expressed in testis rather than the other tissues. Importantly, ZIP12 was intensively abundant in spermatogonia and spermatozoa, both in mice and humans. Moreover, ZIP12 expression in testis significantly decreased in obese mice, which associated with reduced sperm zinc content, excessive sperm ROS, poor sperm quality and male subfertility. Similarly, its expression in C18-4 cells significantly declined in response to oxidative stress. Additionally, reduced ZIP12 expression by RNAi associated with a decline in zinc level subsequently caused low cell viability and high cell apoptosis in C18-4 cells. Conclusions: The zinc transporter ZIP12 is intensively expressed in testis, especially in spermatogonia and spermatozoa. ZIP12 may play a key role in maintaining intracellular zinc level in spermatogonia and spermatozoa, by which it resists oxidative stress during spermatogenesis and therefore preserves male fertility.

ZnT8 Deficiency Protects From APAP-Induced Acute Liver Injury by Reducing Oxidative Stress Through Upregulating Hepatic Zinc and Metallothioneins

Zinc transporter 8 (ZnT8) is an important zinc transporter highly expressed in pancreatic islets. Deficiency of ZnT8 leads to a marked decrease in islet zinc, which is thought to prevent liver diseases associated with oxidative stress. Herein, we aimed to investigate whether loss of islet zinc affects the antioxidant capacity of the liver and acute drug-induced liver injury. To address this question, we treated ZnT8 knockout (KO) or wild-type control mice with 300 mg/ kg acetaminophen (APAP) or phosphate-buffered saline (PBS). Unexpectedly, we found that loss of ZnT8 in mice ameliorated APAP-induced injury and was accompanied by inhibition of c-Jun N-terminal kinase (JNK) activation, reduced hepatocyte death, and decreased serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST). An increase in hepatic glutathione (GSH) was observed, corresponding to a decrease in malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) levels. APAP-induced inflammation and glycogen depletion were alleviated. In contrast, no significant changes were observed in cytochrome P450 family 2 subfamily E member 1 (CYP2E1), the main enzyme responsible for drug metabolism. Elevated levels of hepatic zinc and metallothionein (MT) were also observed, which may contribute to the hepatoprotective effect in ZnT8 KO mice. Taken together, these results suggest that ZnT8 deficiency protects the liver from APAP toxicity by attenuating oxidative stress and promoting hepatocyte proliferation. This study provides new insights into the functions of ZnT8 and zinc as key mediators linking pancreatic and hepatic functions.

Zinc transporter 8 autoantibody testing requires age-related cut-offs

IntroductionZinc transporter 8 autoantibodies (ZnT8A) are biomarkers of beta cell autoimmunity in type 1 diabetes that have become more widely available to clinicians in recent years. Robust control population-defined thresholds are essential to ensure high clinical specificity in islet autoantibody testing. We aimed to determine the optimal cut-offs for ZnT8A testing.Research design and methods97.5th and 99th centile cut-offs were determined using residual clinical sera from 1559 controls aged between 0 and 83 years with no history of diabetes and a hemoglobin A1c level of less than 6.0% (<42 mmol/mol). ZnT8A were measured by ELISA (RSR, Cardiff, UK) on a Dynex DS2 ELISA robot (Dynex, Preston, UK). We assessed the impact of age-related cut-offs in comparison with the manufacturer’s recommended threshold in a mixed cohort of young-onset (<age 30) diabetes (UNITED study (Using pharmacogeNetics to Improve Treatment in Early-onset Diabetes), n=145).ResultsUsing the manufacturer’s limit of detection, 6 WHO U/mL, 16.2% of people in the control cohort had detectable levels of ZnT8A and those who had detectable ZnT8A were much more likely to be younger (p<0.0001). The 97.5th and 99th centile thresholds were substantially higher in younger participants: 18 and 127 WHO U/mL (tested under 30 years) in comparison with 9 and 21 WHO U/mL (tested 30 years and over). In the UNITED cohort some of those found to be ZnT8A-positive by the manufacturer’s threshold but negative using the appropriate 99% centile cut-off (127 WHO U/mL) displayed characteristics suggestive of type 2 diabetes.ConclusionsAge-related thresholds are needed for ZnT8A testing. In those aged <30 years, use of manufacturers’ recommended cut-offs may result in low test specificity and potentially high rates of false positive test results in patients who do not have autoimmune diabetes.