Oxidation of Human Copper Chaperone Atox1 and Disulfide Bond Cleavage by Cisplatin and Glutathione.

Maria I Nardella,Fabio Arnesano,Rocco Caliandro,Benny D Belviso,Giovanni Natile,Antonio Rosato

doi:10.3390/ijms20184390

Abstract

Cancer cells cope with high oxidative stress levels, characterized by a shift toward the oxidized form (GSSG) of glutathione (GSH) in the redox couple GSSG/2GSH. Under these conditions, the cytosolic copper chaperone Atox1, which delivers Cu(I) to the secretory pathway, gets oxidized, i.e., a disulfide bond is formed between the cysteine residues of the Cu(I)-binding CxxC motif. Switching to the covalently-linked form, sulfur atoms are not able to bind the Cu(I) ion and Atox1 cannot play an antioxidant role. Atox1 has also been implicated in the resistance to platinum chemotherapy. In the presence of excess GSH, the anticancer drug cisplatin binds to Cu(I)-Atox1 but not to the reduced apoprotein. With the aim to investigate the interaction of cisplatin with the disulfide form of the protein, we performed a structural characterization in solution and in the solid state of oxidized human Atox1 and explored its ability to bind cisplatin under conditions mimicking an oxidizing environment. Cisplatin targets a methionine residue of oxidized Atox1; however, in the presence of GSH as reducing agent, the drug binds irreversibly to the protein with ammine ligands trans to Cys12 and Cys15. The results are discussed with reference to the available literature data and a mechanism is proposed connecting platinum drug processing to redox and copper homeostasis.

Highlights

Cancer cells have to cope with oxidative stress conditions, characterized by high levels of reactive oxygen species (ROS) [1]
With the aim to investigate the interaction of cisplatin with the disulfide form of the protein, we performed a structural characterization in solution and in the solid state of oxidized human Atox1 and explored its ability to bind cisplatin under conditions mimicking an oxidizing environment
The average interatomic distances between sulfur atoms of Cys residues in apoAtox1 indicate that an intramolecular disulfide bond can be formed between Cys12 and Cys15 of the CxxC motif, while Cys41 can still be modified by IAM (Figure 1A)

Summary

Introduction

Cancer cells have to cope with oxidative stress conditions, characterized by high levels of reactive oxygen species (ROS) [1]. Since many antineoplastic drugs used in chemotherapy contribute to increase the oxidative stress of target cells causing apoptosis [2], the ability to establish an imbalance between ROS and antioxidant levels may represent a useful strategy for cancer attack [3]. Atox is a 68-amino acids protein featuring a ferredoxin-like fold (β1α1β2β3α2β4) and a metal-binding motif CxxC (located in the β1α1 loop) able to bind a single Cu(I) ion [17]. This structure is highly conserved among metallochaperones and soluble domains of Cu-ATPases [18]. It has been shown that Atox can bind cisplatin in the cytosol and may prevent its transfer to the nucleus [19,20,21]

Methods

Results

Discussion

Conclusion