Mechanism Studies Of Atox1in Copper Transport And Interactions With Platinum Drugs

Copper is an essential trace metal element in living organisms. Because of the toxicity of free copper, the cellular copper transportation is achieved through protein-protein interactions. In humans, copper chaperone Atox1delivers copper to the metal-binding domains of ATP7A and ATP7B, which plays a central role in maintaining the copper homeostasis. Copper transfer occurs between the conserved CxxC metal-binding motifs of Atoxl to its target proteins through protein recognition. In addition to the copper binding motif, some conserved residues not directly involved in copper ligation could also determine the copper binding and transfer properties of Atox1. The K60A mutation enhances the copper release from Atoxl to copper chelate bicinchonic acid. Moreover, mutation of Lys60in Atoxl weakens the heterocomplex formation and diminishes copper transfer to its target protein. Recently, Atox1was revealed as a nuclear copper dependent transcription factor. This novel role of Atoxl has the biological implication both in the activation of cell proliferation and the moderation of oxidant stress in the cardiovascular system. The pleiotropic roles make Atox1be a potential therapeutic target. A metal agent tetrathiomolybdate can bind to CuI-Atoxl for treatment of disorders of copper metabolism. Additionally, increasing evidence indicates that copper transport system is involved in the cellular influx and efflux of cisplatin and refers to the drug resistance. Atoxl has been proved to bind cisplatin in vitro and in cells. This dissertation contributes to the mechanism studies of Atoxl in copper binding and transfer as well as its platination by platinum complexes.Chapter1reviews the roles of Atoxl in copper homeostasis at the outset, including cellular copper uptake, the structure and functions of Atoxl and the interactions of Atoxl with its target proteins. The roles of copper transport system in the mechanisms of platinum drugs have been discussed, including the research status of the mechanism of platinum agents, increased uptake of cisplatin through hCtrl, ATP7A and ATP7B mediated cisplatin efflux, and cisplatin bound to Atoxl.In chapter2, we carried out the structure determination and the dynamic analysis of Atoxl with the K60A mutation in order to elucidate the mechanism of the conserved residue Lys60in the copper transport. Results show that the K60A mutation results in subtle but crucial secondary structure rearrangements and side chain orientation alteration of metal binding residues in Atoxl. Protein dynamic studies reveal that the K60A mutation has led to increased overall flexibility, and much difference in dynamic property of metal binding sites. In contrast to the literature report, NMR titration reveals that both wild-type and K60A mutant Atoxl can deliver cuprous ions to the target protein ATPase in a fast exchange mode. This process is different from the slow exchange of free CuI binding to Atox1. In addition, the CuI mediated hetero-protein interactions are present on both Atoxl variants. However, different conformation changes occurred during copper transfer in the two Atoxl variants. Taken together, the residue Lys60modulates the copper binding and protein recognition of Atox1through maintaining its structure and dynamics especially for the copper binding region.In chapter3, we have performed detailed solution NMR and ESI-MS experiments to investigate the effect of copper binding on the reactions of Atox1with platinum agents, cisplatin, trans-EE and trans-PtTz. Results indicate that copper binding significantly enhances the platination of Atoxl. Tandem MS spectra analyses reveal that the copper coordination residues (Cys12and Cysl5) are the major binding sites for all three platinum complexes. Both apo-and Cu1-Atox1react faster with trans-platinum complexes than with cisplatin, however, more protein aggregation occurs in the reaction of cisplatin. These divergences could correlate to the different platination adducts formed in the reactions. The release of carrier ligands occurs only in the reaction of cisplatin. On the other hand, different effects of dithiothreitol (DTT) or glutathione (GSH) were observed in the reactions of platinum complexes with Atox1. In the reaction of cisplatin with Atox1, the presence of copper largely suppresses the platination of DTT or GSH, however, the copper coordination enhances the trans-platinum species transfer from Atoxl to DTT or GSH. These findings provide an insight into the mechanism of copper ions in the regulation of cellular trafficking of platinum drugs with different coordination configurations.