Dataset Information

Heterologous Expression and Biochemical Characterization of the Human Zinc Transporter 1 (ZnT1) and Its Soluble C-Terminal Domain.

ABSTRACT: Human zinc transporter 1 (hZnT1) belongs to the cation diffusion facilitator (CDF) family. It plays a major role in transporting zinc (Zn²⁺) from the cytoplasm across the plasma membrane and into the extracellular space thereby protecting cells from Zn²⁺ toxicity. Through homology with other CDF family members, ZnT1 is predicted to contain a transmembrane region and a soluble C-terminal domain though little is known about its biochemistry. Here, we demonstrate that human ZnT1 and a variant can be produced by heterologous expression in Saccharomyces cerevisiae cells and purified in the presence of detergent and cholesteryl hemisuccinate. We show that the purified hZnT1 variant has Zn²⁺/H⁺ antiporter activity. Furthermore, we expressed, purified and characterized the soluble C-terminal domain of hZnT1 (hZnT1-CTD) in a bacterial expression system. We found that the hZnT1-CTD melting temperature increases at acidic pH, thus, we used an acetate buffer at pH 4.5 for purifications and concentration of the protein up to 12 mg/mL. Small-angle X-ray scattering analysis of hZnT1-CTD is consistent with the formation of a dimer in solution with a V-shaped core.

SUBMITTER: Cotrim CA

PROVIDER: S-EPMC8120272 | biostudies-literature | 2021

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Heterologous Expression and Biochemical Characterization of the Human Zinc Transporter 1 (ZnT1) and Its Soluble C-Terminal Domain.

Cotrim Camila A CA Jarrott Russell J RJ Whitten Andrew E AE Choudhury Hassanul G HG Drew David D Martin Jennifer L JL

Frontiers in chemistry 20210430

Human zinc transporter 1 (hZnT1) belongs to the cation diffusion facilitator (CDF) family. It plays a major role in transporting zinc (Zn<sup>2+</sup>) from the cytoplasm across the plasma membrane and into the extracellular space thereby protecting cells from Zn<sup>2+</sup> toxicity. Through homology with other CDF family members, ZnT1 is predicted to contain a transmembrane region and a soluble C-terminal domain though little is known about its biochemistry. Here, we demonstrate that human Zn ...[more]

PMID: 33996761

Similar Datasets

Project description: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.

Dataset Information

Heterologous Expression and Biochemical Characterization of the Human Zinc Transporter 1 (ZnT1) and Its Soluble C-Terminal Domain.

Publications

Heterologous Expression and Biochemical Characterization of the Human Zinc Transporter 1 (ZnT1) and Its Soluble C-Terminal Domain.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets