Mutated Atox 1 and its interactions with the anticancer drug, Cisplatin

Devulapally Praneeth Reddy Gaurav Dutta Dwivedi Mana Zamenzarrabi

Identifying role of cysteine residues in cisPt-Atox 1 binding, by investigating mutated Atox 1 (replacing three cysteines with alanine).

The aim of the project

Introduction• Cisplatin acts as an effective

chemotherapeutic agent in treating diverse class of solid tumors.

• Among cancer therapeutics, cisplatin carries out its antitumor activity by binding to DNA and interfering with replication and transcription process which leads to apoptosis.

• The primary response to this drug is strong; however, at later stage the tumor cell usually initiates the resistance towards cisplatin.

• Some copper transporting proteins play a role in cisplatin resistance.

Cisplatin

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

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.

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.

Since CisPt can bind with methionine , Histidine and even the third Cysteine residue of Atox 1, it may occupy another sites.

Results

Near-UV CD signal monitored for Atox 1 and Atox 1 + Cu in mutated and WT (left to wright) .

Palm M E et al. PNAS 2011;108:6951-6956

Near-UV CD-monitored titrations of CisPt to Atox1 +Cu , mutated and WT (left to wright) .

Palm M E et al. PNAS 2011;108:6951-6956

Near-UV CD-monitored titrations of Cu to CisPt +Atox1, mutated and WT (left to wright) .

Palm M E et al. PNAS 2011;108:6951-6956

Near-UV CD-monitored titrations of TClPt to Cu +3Cys 3Ala-Atox1

Far-UV CD spectra as a function of time for Mut-Atox1+CisPt and Cu-Mut-Atox1+CisPt (0-72h).

Far-UV CD spectra as a function of time (0 h to 14 d) for apo-Atox1 +5 eq cisPt .

Palm M E et al. PNAS 2011;108:6951-6956

CD changes at 220nm plotted as a function of time.

Thermal unfolding of Atox1 and Atox1 mixed with 5eq Cisplatin, WT and mutated (left to wright) .

Palm M E et al. PNAS 2011;108:6951-6956

Conclusion

• By replacing Cys, no metal binding was detected in the near-UV region.

• Time dependence experiment in far-UV; 1.Binding was recognized in both cases. 2.No binding . Signal is rather constant for Mut-Atox1+ CisPt. Free reactive Cu unfolds Cu+Mut-Atox1+CisPt. New set of Experiments including Mut-Atox1 should be considered.

• Thermal stability in Far-UV; Experiment should be repeated.

Acknowledgements

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