Effect of the N-terminal of the human high affinity copper transporter 1 (hCtr1) on cisplatin hydrolysis and uptake

Abstract

Cisplatin(cis-diamminedichloridoplatinum(II), cis-DDP) and other platinum complexes, such as carboplatin and oxaliplatin, have been widely used in the chemotherapy of different types of cancer [1]. By forming adducts with DNA, they can inhibitDNA replication and cause cell death. The entrance pathway of cisplatin was originally thought to be passive diffusion. However, recent in vivo and in vitro studies indicated that human copper transporter 1 (hCtr1)—an integral membrane protein necessary for high affinity copper uptake—could also be a transporter for cisplatin and other platinum complexes [2]. Yet, the mechanism of interaction between cisplatin and hCtr1 at the molecular level is currently unclear. In this work, we have successfully overexpressed, purified and characterized the N-terminal extracellular domain of hCtr1 (hCtr1_N, residues 1–55), which contains 2 methionine-rich and 2 histidine-rich regions.Asmall tag (GB1 with 8 kD) with no metalbinding- site is attached to hCtr1_N to improve its solubility and stability. 1H and 2D [1H, 15 N] HSQC NMR experiments of 15 N-labeled cisplatin and tagged hCtr1_N were carried out to examine the effect of the protein on cisplatin hydrolysis. Our results show that cisplatin hydrolyses rapidly by binding methionine residues of tagged hCtr1_N, following by the quickly loss of its ammonia ligand. Both NMR and ESI-MS results prove that the mutation of hCtr1_N methionine to alanine could reduce the effect of protein on cisplatin hydrolysis and, especially, the loss of ammonia ligand. The gel filtration work shows that cisplatin can cause oligomerization of protein, probably due to the cross-linking of the proteins by the anticancer drug. This work was supported by Research Grants Council of Hong Kong (HKU7512/05 M, HKU7043/06P, HKU2/06C, HKU1/07C, HKU7038/08P), National Science Foundation of China and the University of Hong Kong. References 1. Wang D, Lippard SJ (2005) Nat Rev Drug Discov 4:307–320 2. Ishida S, Lee J, Thiele SJ, Herskowitz I (2002) Proc Natl Acad Sci USA 99:14298–14302