Dawson, Charmian Nicole; (2023) Disease-causing mutations in the human metal ion transporter ZIP14 alter its structure, ion uptake function and trafficking. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

ZIP14 is a member of the LIV-1 family, a sub-family of the Zrt-, Irt-like Protein (ZIP) family. These proteins import zinc into the cytosol, regulating zinc concentrations to control physiological processes. As a zinc transporter, ZIP14 regulates inflammation, the sensitivity of multiple signaling pathways, and in cancer, over-expression of ZIP14 in skeletal muscle causes cachexia. During iron overload, ZIP14 directs excess iron into the pancreas and liver. In the intestines, ZIP14 prevents excessive manganese absorption from the diet. Pathological mutations in human ZIP14 cause manganism or hyperostosis cranialis, due to effects on manganese and zinc transport, respectively. This is the first detailed study of ZIP14 structure and function, and the effects of the disease-causing mutations. Homology modelling was used to predict the structure of ZIP14, and further mutations were designed based on this structure. Epitope-tagged ZIP14 and ZIP14 mutants were expressed in HeLa cells and studied with a range of techniques, including western blotting to measure total cellular levels of the protein, immunofluorescence microscopy to assess intracellular localisation, flow cytometry to quantify total and cell surface protein levels, and Fe⁵⁵ uptake to assess ion transport. Mutations inhibiting dimerisation of the extracellular domain and subsequent formation of an inter-molecular disulphide bridge decreased localisation at the cell surface in a concentration- dependent manner, as well as preventing iron transport. Effects of mutations in the predicted metal transport pore differed: P379L primarily altered trafficking, G383R prevented iron uptake. A combination of the coevolution analysis of the transmembrane region, and the effects of the L441R and N469K mutations, showed that dimerisation of the transmembrane domain was also important for cell surface localisation, but not for metal transport. In summary, this research provides a convincing structure for the ZIP14 protein, and sheds light on how aspects of this structure relate to ZIP14 trafficking and function.

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