Cisplatin binds human copper chaperone Atox1 and promotes unfolding in vitro

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 108; no. 17; pp. 6951 - 6956
• Main Authors: Palm, Maria E, Weise, Christoph F, Lundin, Christina, Wingsle, Gunnar, Nygren, Yvonne, Björn, Erik, Naredi, Peter, Wolf-Watz, Magnus, Wittung-Stafshede, Pernilla
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 26-04-2011; National Acad Sciences
• Subjects: Adducts; ammonia; Antineoplastic Agents; Antineoplastic Agents - chemistry; Antineoplastic Agents - pharmacokinetics; Binding Sites; Biological Sciences; Biological Transport; Biological Transport - drug effects; Cancer; Cation Transport Proteins; Cation Transport Proteins - chemistry; Cation Transport Proteins - genetics; Cation Transport Proteins - metabolism; Cell lines; chemistry; circular dichroism spectroscopy; Cisplatin; Cisplatin - chemistry; Cisplatin - pharmacokinetics; Copper; Copper - metabolism; Correlation analysis; Crystal structure; Cytoplasm; dechlorination; DNA; DNA - chemistry; DNA - metabolism; drug effects; Drug Resistance; Drug Resistance, Neoplasm - drug effects; drug therapy; Forest Science; genetics; Hepatolenticular degeneration; Humans; Ligands; metabolism; Metallochaperones; Molecular Chaperones; Molecular Chaperones - chemistry; Molecular Chaperones - genetics; Molecular Chaperones - metabolism; Molecules; Neoplasm; Neoplasms; Neoplasms - drug therapy; Neoplasms - metabolism; NMR; Nuclear magnetic resonance; nuclear magnetic resonance spectroscopy; pharmacokinetics; Protein Binding; Protein Binding - drug effects; Protein Folding; Protein Structure; Protein Structure, Tertiary; Proteins; Skogsvetenskap; Tertiary; Titration; Tumor cell line
• ISSN: 0027-8424; 1091-6490; 1091-6490
• DOI: 10.1073/pnas.1012899108

Cisplatin (cisPt), Pt(NH₃)₂Cl₂, is a cancer drug believed to kill cells via DNA binding and damage. Recent work has implied that the cellular copper (Cu) transport machinery may be involved in cisPt cell export and drug resistance. Normally, the Cu chaperone Atox1 binds Cu(I) via two cysteines and delivers the metal to metal-binding domains of ATP7B; the ATP7B domains then transfer the metal to the Golgi lumen for loading on cuproenzymes. Here, we use spectroscopic methods to test if cisPt interacts with purified Atox1 in solution in vitro. We find that cisPt binds to Atox1's metal-binding site regardless of the presence of Cu or not: When Cu is bound to Atox1, the near-UV circular dichroism signals indicate Cu-Pt interactions. From NMR data, it is evident that cisPt binds to the folded protein. CisPt-bound Atox1 is however not stable over time and the protein begins to unfold and aggregate. The reaction rates are limited by slow cisPt dechlorination. CisPt-induced unfolding of Atox1 is specific because this effect was not observed for two unrelated proteins that also bind cisPt. Our study demonstrates that Atox1 is a candidate for cisPt drug resistance: By binding to Atox1 in the cytoplasm, cisPt transport to DNA may be blocked. In agreement with this model, cell line studies demonstrate a correlation between Atox1 expression levels, and cisplatin resistance.