The human zinc transporter ZnT8 provides the granules of pancreatic β-cells with zinc (II) ions for assembly of insulin hexamers for storage. Until recently, the structure and function of human ZnTs have been modelled on the basis of the 3D structures of bacterial zinc exporters, which form homodimers with each monomer having six transmembrane α-helices harbouring the zinc transport site and a cytosolic domain with an α,β structure and additional zinc-binding sites. However, there are important differences in function as the bacterial proteins export an excess of zinc ions from the bacterial cytoplasm, whereas ZnT8 exports zinc ions into subcellular vesicles when there is no apparent excess of cytosolic zinc ions. Indeed, recent structural investigations of human ZnT8 show differences in metal binding in the cytosolic domain when compared to the bacterial proteins. Two common variants, one with tryptophan (W) and the other with arginine (R) at position 325, have generated considerable interest as the R-variant is associated with a higher risk of developing type 2 diabetes. Since the mutation is at the apex of the cytosolic domain facing towards the cytosol, it is not clear how it can affect zinc transport through the transmembrane domain. We expressed the cytosolic domain of both variants of human ZnT8 and have begun structural and functional studies. We found that (i) the metal binding of the human protein is different from that of the bacterial proteins, (ii) the human protein has a C-terminal extension with three cysteine residues that bind a zinc(II) ion, and (iii) there are small differences in stability between the two variants. In this investigation, we employed nickel(II) ions as a probe for the spectroscopically silent Zn(II) ions and utilised colorimetric and fluorimetric indicators for Ni(II) ions to investigate metal binding. We established Ni(II) coordination to the C-terminal cysteines and found differences in metal affinity and coordination in the two ZnT8 variants. These structural differences are thought to be critical for the functional differences regarding the diabetes risk. Further insight into the assembly of the metal centres in the cytosolic domain was gained from potentiometric investigations of zinc binding to synthetic peptides corresponding to N-terminal and C-terminal sequences of ZnT8 bearing the metal-coordinating ligands. Our work suggests the involvement of the C-terminal cysteines, which are part of the cytosolic domain, in a metal chelation and/or acquisition mechanism and, as now supported by the high-resolution structural work, provides the first example of metal-thiolate coordination chemistry in zinc transporters.
- MeSH
- beta-buňky metabolismus MeSH
- diabetes mellitus 2. typu genetika patologie MeSH
- inzulin genetika metabolismus MeSH
- konformace proteinů, alfa-helix genetika MeSH
- lidé MeSH
- molekulární konformace MeSH
- nikl chemie MeSH
- proteinové domény genetika MeSH
- transportní proteiny chemie genetika ultrastruktura MeSH
- vztahy mezi strukturou a aktivitou * MeSH
- zinek chemie MeSH
- zinkový transportér 8 chemie genetika ultrastruktura MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Insulin is produced and stored inside the pancreatic β-cell secretory granules, where it is assumed to form Zn2+-stabilized oligomers. However, the actual storage forms of this hormone and the impact of zinc ions on insulin production in vivo are not known. Our initial X-ray fluorescence experiment on granules from native Langerhans islets and insulinoma-derived INS-1E cells revealed a considerable difference in the zinc content. This led our further investigation to evaluate the impact of the intra-granular Zn2+ levels on the production and storage of insulin in different model β-cells. Here, we systematically compared zinc and insulin contents in the permanent INS-1E and BRIN-BD11 β-cells and in the native rat pancreatic islets by flow cytometry, confocal microscopy, immunoblotting, specific messenger RNA (mRNA) and total insulin analysis. These studies revealed an impaired insulin production in the permanent β-cell lines with the diminished intracellular zinc content. The drop in insulin and Zn2+ levels was paralleled by a lower expression of ZnT8 zinc transporter mRNA and hampered proinsulin processing/folding in both permanent cell lines. To summarize, we showed that the disruption of zinc homeostasis in the model β-cells correlated with their impaired insulin and ZnT8 production. This indicates a need for in-depth fundamental research about the role of zinc in insulin production and storage.
- MeSH
- beta-buňky metabolismus ultrastruktura MeSH
- chemická frakcionace MeSH
- cytoplazmatická granula metabolismus MeSH
- exprese genu * MeSH
- glukosa metabolismus MeSH
- inzulin genetika metabolismus MeSH
- krysa rodu rattus MeSH
- Langerhansovy ostrůvky metabolismus MeSH
- messenger RNA genetika metabolismus MeSH
- průtoková cytometrie metody MeSH
- zinek metabolismus MeSH
- zinkový transportér 8 MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
BACKGROUND: Testing for autoantibodies against the zinc transporter ZnT8 (ZnTA) is becoming routine in pediatric diabetes. However, available data are inconclusive when focusing on adult-onset diabetes, including autoimmune diabetes, which does not require insulin at diagnosis (LADA). BASIC PROCEDURES: We examined the ZnTA prevalence and titers and matched them with the clinical phenotype and PTPN22 genotypes of Czech LADA patients who were positive for GADA and/or IA2A and had a fasting C-peptide level >200 pmol/L at diagnosis as well as HNF4A-, GCK- or HNF1A-MODY patients and healthy controls. MAIN FINDINGS: Most LADA patients were negative for ZnTA, and the sensitivity of the assay was only 18-20% for patients with LADA-like progression to insulinotherapy compared to healthy controls. In LADA patients, there was no association between the ZnTA and PTPN22 risk genotypes. LADA patients positive for ZnTA had a lower BMI than those positive for other autoantibodies alone. Importantly, MODY patients were completely negative for ZnTA, and the levels of ZnTA in MODY patients were similar to those in healthy controls. CONCLUSIONS: ZnTA quantification did not improve LADA diagnosis. However, positivity for ZnTA can be used as a negative MODY pre-diagnostic criterion even in the region of Central and East Europe, where other islet cell autoantibodies are common in MODY patients.
- MeSH
- autoprotilátky krev imunologie MeSH
- biologické markery krev MeSH
- diabetes mellitus 1. typu krev farmakoterapie genetika imunologie MeSH
- diabetes mellitus 2. typu krev farmakoterapie genetika imunologie MeSH
- fenotyp MeSH
- genotyp MeSH
- hypoglykemika terapeutické užití MeSH
- inzulin terapeutické užití MeSH
- lidé středního věku MeSH
- lidé MeSH
- progrese nemoci MeSH
- tyrosinfosfatasa nereceptorového typu 22 genetika MeSH
- zinkový transportér 8 krev imunologie MeSH
- Check Tag
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Česká republika MeSH
- MeSH
- autoprotilátky * analýza izolace a purifikace MeSH
- beta-buňky patologie MeSH
- časná diagnóza MeSH
- diabetes mellitus 1. typu * etiologie MeSH
- diabetes mellitus epidemiologie klasifikace MeSH
- ELISA využití MeSH
- insulinové protilátky analýza MeSH
- lidé MeSH
- radioligandová zkouška metody využití MeSH
- zinkový transportér 8 analýza MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- přehledy MeSH
- srovnávací studie MeSH
