UMEÅ UNIVERSITY, Department of Chemistry, SE-901 87 Umeå Sweden

Mutated Atox 1 and its interactions with the anticancer drug Cisplatin

Protein Structure and Function, Autumn 2011

Group B

Devulapally Praneeth Reddy

Gaurav Dutta Dwivedi

Mana Zamenzarrabi

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Abbreviations:

Cys............................................................................................................................cysteine

Ala.............................................................................................................................Alanin

Cu..............................................................................................................................Copper

CisPt...................................................................Cisplatin (Cis-diamminedichloroplatinum)

TClPt.....................................................................................................Tetrachloroplatinate

WT......................................................................................................................Wild type

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Abstract:

Cisplatin acts as an effective chemotherapeutic agent in treating diverse class of solid tumors. The primary response to this drug is strong; however, at later stage the tumor cell usually initiates the resistance towards cisplatin.

In this study, we use spectroscopic methods to see the interaction of Cisplatin and discuss the role of cysteines in cisplatin binding to a mutated Atox 1 where all three cysteines have been replaced with alanine (3 Cys 3 Ala-Atox1) . We observed that by adding Cu to (3Cys 3Ala) Atox 1 no Cu binding was detected in near-UV CD region. In the thermal unfolding experiment, temperature facilitates the denaturation of both WT Apo and the mutated one. However in WT at 20°C Apo alone and with CisPt almost possess the same signals as associated to mutated Atox 1. Lacking of Cysteine residues interrupts the binding between the Atox 1 and metals (Cu, CisPt, and TClPt).

In future research Knowledge of these binding interactions should support in the development of future anticancer strategies. In addition, they may use the knowledge gained from this research to continue to look for Cisplatin analogs less eager to bind to proteins, avoiding side-effects and resistance.

Introduction:

It was found in 1965 that cisplatin inhibits the cell division (1); so, that is why nowadays platinum-based drugs become prominent in cancer treatment. Among cancer therapeutics, cisplatin carries out its antitumor activity by binding to DNA and interfering with replication and transcription process as a result of forming a stable chelate. (2)

It has been suggested that in cell, cisplatin (Cis-diamminedichloroplatinum) loses its chloride groups due to hydrolysation. Since water is a better leaving group than chloride, cisplatin can binds to DNA. (3)

Platinum-based drugs have limited effect and after sometime they lose their anticancer effect, namely, cells display resistance to them. (4)

Studies have shown that some copper transporting proteins play a role in cisplatin resistance. There are several pathways for uptake and transport of copper in the cell. Here, the pathway with Atox1 and ATP7A/B, linked to Golgi network is considered. ATP7A/B proteins contain domains which are structurally similar to Atox 1. (2) (4) (5)

It is believed that cisplatin interacts with the copper binding site of these proteins (Cys 12 and Cys 15 of Atox 1) and being transported out of the cell or accumulated by them. One evidence which supports Cu-transferring proteins involvement in cell resistance is the escalating number of these proteins in resistant cell. (6) Cisplatin binding to the Cu-binding site of Atox 1 has been shown in publications by focusing on crystal structure of these proteins and in the very recent article by obtaining in-cell NMR (7)

(8).

There is a possibility that cisplatin has other binding sites as well, since platinum can form a bond with methionine, histidine and even the third cysteine residue of Atox 1. (9) (10)

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The main goal of this project is to identify the role of cysteines in cisplatin binding to Atox 1. A mutated form of Atox 1 where all three cysteines have been exchange to alanine will be studied (3 Cys 3 Ala-Atox1). The results will be compared to wild type Atox 1.

Figure1: (A) Structure of Atox 1 (3IWL). The three cysteines are marked.

(B) Structure of Tetrachloroplatinum 12 and Cisplatin 13.

Materials and methods:

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Protein preparation: The mutated protein 3cys 3Ala- Atox1 was kindly provided by Maria Espling (Umea University).

CD Spectroscopy:

All the experiments were performed by JASCO J-810 CD-spectrophotometer at room temperature (20℃). Cu (20mM in H2O), CisPt (6.7mM in H2O), Buffer (50mM Nacl, 20mM MES, 0.25mM DTT, pH 6), 3Cys 3Ala-Atox 1 (2.2mM) and TClPt (24.1mM) were used as a stock solution. CisPt solution was freshly prepared. In titration experiments 0-5 fold of CisPt or TClPt was added to premixed Cu-3Cys 3Ala-Atox 1. In another set of titration experiments 0-5 folds of Cu was added to 1:1 CisPt-3Cys 3Ala- Atox 1 solution. CD signals were optimised by subtracting the buffer from each spectrum. Far- UV CD time dependences were performed for mut-Atox1 and Cu+mut- Atox 1 +5 eq CisPt for both in three days. Thermal unfolding experiment was done on mut-Atox 1 and for mut-Atox 1 +5 eq. CisPt (20-70 0C).

Results:

As it is demonstrated in fig. 2A no change in CD signal is observed by adding 1 eq.Cu to 3Cys 3Ala-Atox 1. While, changes in CD intensity of the wild type, apo and holo Atox1 are quite recognisable. Therefore by removing Cys residue, no Cu binding was detected in near-UV CD.

It is clear from fig. 2B that CisPt, even in higher concentrations does not bind to the mutated Atox 1 pre mixed with 1eq Cu. In contrast, CD signals become more negative by increasing the CisPt concentration in wild type.

Like the previous study on WT Atox 1, order of adding metals (Cu/Pt) to Atox 1 does not make any difference in CD signals intensity and overall result in near-UV titration (fig 2C) . Here there is no change in signal upon addition of Cu/Pt to the mutated protein.

Fig. 2D, indicates titrations of TClPt to 3Cys 3Ala-Atox1 (premixed with 1eq.Cu) in near-UV CD follows the same pattern as titration with CisPt and CuCl2, namely no metal binding was detected. Not even this highly reactive platinum substance induced new near-UV CD signals.

In fig. 3A and 3B which CD signals were detected over time (0-72h), are clearly shown that apo+5eq.CisPt and Cu+5eq.CisPt will be unfolded during time but unfolding in Cu samples are more significant compare to Apo based on far-UV detection. In both cases CisPt might bind to mutated protein (Atox1) which does not have cysteine residue; therefore, cysteine amino acids are not the only binding site for CisPt. With reservation for that 3Cys 3Ala Atox1+1eq.Cu or pure 3Cys 3Ala Atox1 without added CisPt were not tested. Cu that not has a site to bind is reactive and might be responsible for protein unfolding.

In the matter of thermal unfolding, CisPt mediates higher denaturation of WT. But for mutated protein alone and with CisPt presents nearly the same signal. (fig4)

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Figure 2: Near-UV CD-monitored titrations of Cu and Pt to 3Cys 3Ala-atox1(pH6,20°c).(A) 50 µM 3Cys 3Ala-Atox 1 (1:1), Cu; (B) CisPt (up to five fold excess as indicated) added to 50 µM 3Cys 3Ala-Atox 1 pre mixed with 50 µM Cu; (C) Cu (up to fivefold excess as indicated ) added to 50 µM 3Cys 3Ala-Atox 1 premixed with CisPt (1:1). Tetrachloroplatinum (TClPt) was used in one titration to further test the system because of its higher reactivity than cisplatin. 0-5 eq TClPt added to 1:1 premixed 3Cys 3Ala-atox1:Cu.

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Figure 3: Far-UV CD spectra as a function of time (0 h to 72h). Time points are (before) 0, 1, 2, 3, 5, 24, 48 and 72h. (a) 3cys 3 ala (50 μM) +5eq.cisplatin. (b) 3 cys 3 ala Atox1 (50 μM) pre mixed with 1 eq.Cu and added 5 eq cisPt and incubated at 20 °C.

Figure 4: (A) CD changes at 220nm (at 20°c) plotted as a function of time. (B) Thermal unfolding 3Cys3Ala-Atox1 (50 μM) alone and mixed with 5eq Cisplatin.

Discussion:

While there are diverse mechanisms which are involved in CisPt movement within the cell, we have a good reason to believe that there is a linkage between CisPt resistance and the human Cu transport system11. In the previous studies it was found that CisPt binds to apo-Atox1 in solution, as was expected from the crystal structure work.

We investigated interactions of CisPt with a mutated form of (3Cys 3Ala) Atox1 (both pure and premixed with Cu) looking at Cu and Pt binding in solution using the CD spectroscopy. Our solvent environment and parameters were selected to mimic the cell environment.

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It is also evident from the previous work that CisPt binds to the holo form (Cu WT-Atox1) without loss of Cu—in its place, both metals seems to bind to WT Atox 1 metal binding site, but it was not the same for mutated Atox1. Even by increasing the concentration of Cu in (3Cys 3Ala) Atox 1 no Cu binding was observed in near-UV CD. (fig 2) CisPt titration did not give new signals as for WT we reach to a conclusion that in near –UV CD changes for WT Atox 1depends on Cysteines.

In far-UV CD, both Apo/Holo Atox1+5eq.CisPt (previous study), Mut+5eq.CisPt , and Mut+1eq.Cu+5eq.CisPt were unfolded over time, mutated +5eq.CisPt in solution shows slow protein unfolding at 20 °C (Fig. 3) but stability for pure mut-Atox 1 is unknown while WT Apo was stable, So we cannot conclude stability of mutated protein from this result. Also we suspected about the involvement of other binding sites which may be Methionine or Histidine. Unfolding gets more extensive when adding Cu in both WT and mutant (Fig. 4A). Probably because Cu cannot bind and its high reactivity induces unfolding. Since pure Cu-3Cys3Ala Atox1 was not tested we cannot conclude CisPt binding, but suspected about some interactions with other amino acids. This has to be studied in the future.

We also performed thermal stability of (3Cys 3Ala) Atox 1 alone and with cisPt which was monitored by CD at 220 nm (Fig. 4). In the matter of thermal unfolding, CisPt mediates additional denaturation of WT apo and holo. In case of mutated Atox 1 there were rather the same but small changes in the CD signals can be detected which may be due to unfolding and exposure to the solvent cisPt has more chance to binds to other amino acids like Methionine/Histidine. Since there was some problem with CD temperature controller we could not continue this experiment up to 90℃. But from up and down scan we can say that mutated protein is not stable alone or with CisPt.

Acknowledgements:

We are extremely grateful to Maria Espling for supervising the project in Protein Structure and Function as well as reading the manuscript; she has given her valuable feedback throughout the project and necessary correction as and when needed. We are also deeply indebted to Magnus Wolf-Watz for his guidance and help during the project.

References:

1. Gilbert Chu, Cellular Responses to Cisplatin THE ROLES OF DNA-BINDING PROTEINS AND DNA REPAIR, Department of Medicine, Stanford University School of Medicine, Stanford, California 94305.

2. Fabio Arnesano, Lucia Banci and Giovanni Natile, Probing the interaction of Cisplatin with the human copper chaperone atox1 by solution and in-cell NMR spectroscopy, J Am Chem Soc 133(45):18361-18369 (2011).

3. Maria Kartalou, John M Essigmann, Mechanisms of resistance to cisplatin, Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, Volume 478, Issues 1-2, 1 July 2001, Pages 23-43.

4. Kuo MT, Chen HH, Song IS, Savaraj N, Ishikawa T., The roles of copper transporters in cisplatin resistance, Cancer Metastasis Rev. 2007 Mar;26(1):71-83.

5. Maria E. Palm,a Christoph F. Weise,a Christina Lundin,b Gunnar Wingsle,c Yvonne Nygren,a Erik Björn,a Peter Naredi,b Magnus Wolf-Watz,a and Pernilla Wittung-Stafshedea, Cisplatin binds human

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copper chaperone Atox1 and promotes unfolding in vitro, Proc Natl Acad Sci U S A. 2011 April 26; 108(17): 6951–6956.

6. Barnes N, Bartee MY, Braiterman L, Gupta A, Ustiyan V, Zuzel V, Kaplan JH, Hubbard AL, Lutsenko S., Cell-specific trafficking suggests a new role for renal ATP7B in the intracellular copper storage, Traffic. 2009 Jun; 10(6):767-79. 7. Boal AK, Rosenzweig AC, Crystal structures of cisplatin bound to a human copper chaperone. J Am Chem Soc. 2009 Oct 14; 131(40):14196-7.

8. Vrana O, Brabec V, L-methionine inhibits reaction of DNA with anticancer cis diamminedichloroplatinum(II), Biochemistry. 2002 Sep 10; 41(36):10994-9.

9. Andrei I. Ivanov,John Christodoulou, John A. Parkinson, Kevin J. Barnham,Alan Tucker, John Woodrowi, and Peter J. Sadler, JBC, Vol. 273, No. 24, Issue of June 12, 1998. pp. 14721–14730.

10. Y. Okamoto, A. Konno, K. Togawa, T. Kato, Y. Tamakawa, and Y. Amano, Arterial chemoembolization with cisplatin microcapsules, Br J Cancer. 1986 March; 53(3): 369–375.

11. Ishida S, Lee J, Thiele DJ, Herskowitz , Uptake of the anticancer drug cisplatin mediated by the copper transporter Ctr1 in yeast and mammals, Proc Natl Acad Sci U S A. 2002 Oct 29; 99(22):14298-302.12. http://www.lookchem.com/cas-134/13454-96-1.html, dated 12/16/2011.

13. http://spider.science.strath.ac.uk/platinum/showPage.php?page=Glossary%20of%20drugs,dated 12/16/2011.

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