Elucidating the role of N-terminus of Human Copper Transporter (hCTR1) in ligand binding and trafficking

Dutta, Raunak (2019) Elucidating the role of N-terminus of Human Copper Transporter (hCTR1) in ligand binding and trafficking. Masters thesis, Indian Institute of Science Education and Research Kolkata.

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Abstract

Copper is an essential micronutrient for development and survival of eukaryotes. Copper is an essential cofactor for several enzymes which catalyse biochemical reactions critical to cellular homeostasis like electron transport chain, free radical scavanging, iron metabolism, catecholamine and collagen synthesis. The N-terminus of Human Copper Transporter (hCTR1) is the key domain for import of dietary copper in cells. It is 67 amino acid long and contains 8 histidine residues that are crucial for binding of Cu(II) on the extracellular side. The N-terminus of hCTR1 also binds to cisplatin, a potent chemotherapeutic agent and helps in the entry of the drug inside cell through hCTR1. The regulation of copper uptake by hCTR1 occurs through vesicularisation of the transporter from cell membrane. In other words, when there is abundant copper inside cell and there is no need to import copper, hCTR1 gets endocytosed and exists as vesicle. Our hypothesis is N- terminus of hCTR1 might act as a regulatory lid on the trimeric channel present on the plasma membrane. As Cu (II) binds to the N-terminus the lid might get lifted and copper transport might start. If copper fails to bind to the N-terminus then it cannot move through the channel pore and hence after a threshold level of copper entry the channel will not be capable of endocytosis. Histidine has high affinity for Cu (II) thus histidine residues in N-terminus might be crucial for Cu (II) binding. In order to study the binding of N-terminus with Cu (II), it was cloned in pGEX vector and expressed in BL21 E. coli cell. The GST-tagged protein was purified using glutathione affinity column. However, the affinity tag was couldn’t be separated by the action of Prescission Protease which cleaves at HRV 3C site. As a change in strategy, the GST tag was separately purified to be used as a control for binding experiments with the GST-tagged protein. The N-terminus of hCTR1 binds to Cu(II) and cisplatin for their entry inside the cell. A novel ruthenium base cisplatin like molecule was synthesised in Prof. Arindam Mukherjee’s laboratory and a sequestration assay in bacteria was performed as an indirect method to examine the sequestration of the novel drug by the N-terminus of hCTR1. In the assay the length of filament formed by bacteria was measured as an indicator of stress experienced by the bacteria upon drug treatment. It could be seen that there is a significant decrease in the length of the filament in presence of the drug when the N-terminus of hCTR1 is expressed in the bacteria. To elucidate the role of histidines in N-terminus in trafficking of hCTR1, three deletion mutants were constructed, each corresponding to the deletion of one of the three histidine stretch in the N-terminus. The deleted stretches were Δ3HSHH6, Δ22HHH24 and Δ31HSH33. Among the three deletions, the first two were transfected in HEK 293T cells and the localisation of mutant hCTR1 in plasma membrane was examined in presence of Cu(II) and TTM (intra-cellular copper chelator). Molecular dynamics data from our lab suggested that histidines 5 and 6 are critical histidines in N-terminus which binds Cu(II). To understand further the role of these histidines, two substitution mutants of hCTR1 was made - H5A and H5AH6A. H5A was transfected in HEK 293T cells and its localisation in plasma membrane was examined in presence of Cu(II) and TTM (copper chelator). In the mutants no significant change in the plasma membrane localisation of the transporter was noticed between basal copper level, high Cu (II) and TTM.

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