Date Published: July 30, 2013
Publisher: Public Library of Science
Author(s): Maria E. Palm-Espling, C. David Andersson, Erik Björn, Anna Linusson, Pernilla Wittung-Stafshede, Eugene A. Permyakov.
Cisplatin (CisPt) is an anticancer agent that has been used for decades to treat a variety of cancers. CisPt treatment causes many side effects due to interactions with proteins that detoxify the drug before reaching the DNA. One key player in CisPt resistance is the cellular copper-transport system involving the uptake protein Ctr1, the cytoplasmic chaperone Atox1 and the secretory path ATP7A/B proteins. CisPt has been shown to bind to ATP7B, resulting in vesicle sequestering of the drug. In addition, we and others showed that the apo-form of Atox1 could interact with CisPt in vitro and in vivo. Since the function of Atox1 is to transport copper (Cu) ions, it is important to assess how CisPt binding depends on Cu-loading of Atox1. Surprisingly, we recently found that CisPt interacted with Cu-loaded Atox1 in vitro at a position near the Cu site such that unique spectroscopic features appeared. Here, we identify the binding site for CisPt in the Cu-loaded form of Atox1 using strategic variants and a combination of spectroscopic and chromatographic methods. We directly prove that both metals can bind simultaneously and that the unique spectroscopic signals originate from an Atox1 monomer species. Both Cys in the Cu-site (Cys12, Cys15) are needed to form the di-metal complex, but not Cys41. Removing Met10 in the conserved metal-binding motif makes the loop more floppy and, despite metal binding, there are no metal-metal electronic transitions. In silico geometry minimizations provide an energetically favorable model of a tentative ternary Cu-Pt-Atox1 complex. Finally, we demonstrate that Atox1 can deliver CisPt to the fourth metal binding domain 4 of ATP7B (WD4), indicative of a possible drug detoxification mechanism.
Cisplatin (cis-PtCl2(NH3)2; here abbreviated CisPt) is a commonly used anticancer agent for treatment of a variety of cancers, including; testicular, head, neck, bladder and lung , . Its anticancer activity arises from forming stable adducts with DNA in the nucleus, thus interfering with replication and transcription. Initial results of CisPt treatment are often good but can decline over time due to development of resistance. The resistance can be either acquired or intrinsic and seems to be multifactorial . One of the proposed mechanisms for CisPt resistance is involvement of the cells own copper (Cu) transporting system.
Cisplatin, dithiothreitol (DTT), CuCl2 and 2-(N-morpholino)ethanesulfonic acid (MES) were purchased from Sigma-Aldrich (Sweden). CisPt was dissolved in water, 2 mg/ml, and was shortly heated in the microwave to enhance solubility. The CisPt stock was incubated and prehydrolyzed for three days and used within five days to avoid degradation products. The protein sample buffer was 50 mM NaCl, 20 mM MES, pH = 6 and the reducing agent DTT. The DTT concentration in samples was kept fivefold higher than protein concentration. Copper was added to the samples as CuCl2 (20 mM in water) and immediately reduced to Cu1 by the DTT in the buffer.