Cellular and Molecular Life Sciences

, Volume 67, Issue 11, pp 1895–1905 | Cite as

Isolation of peptides blocking the function of anti-apoptotic Livin protein

  • Irena Crnković-Mertens
  • Julia Bulkescher
  • Christina Mensger
  • Felix Hoppe-Seyler
  • Karin Hoppe-Seyler
Research Article
First Online:
Received:
Accepted:

Abstract

Livin (ML-IAP) is a cancer-associated member of the inhibitor of apoptosis protein (IAP) family. By yeast two-hybrid screening of a randomized peptide expression library, we isolated short linear peptides that specifically bind to Livin, but not to other IAPs. Intracellular expression of the peptides sensitized livin-expressing cancer cells toward different pro-apoptotic stimuli. The bioactive peptides neither showed sequence homologies to Smac-derived IAP inhibitors, nor did they interfere with the binding of Livin to Smac. Intracellular expression of the peptides did not affect the levels or the subcellular distribution of Livin. Growth of livin-expressing tumor cells was inhibited in colony formation assays by the Livin-targeting peptides. These findings provide evidence that the targeted inhibition of Livin by peptides represents a viable approach for the apoptotic sensitization and growth inhibition of tumor cells. The inhibitory peptides isolated here could form a novel basis for the development of therapeutically useful Livin inhibitors.

Keywords

Cancer Apoptosis IAP Livin Peptides 
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Notes

Acknowledgments

We thank Drs. Bruce W. M. Jordan and Ulf R. Rapp for pPC97 vectors expressing cIAP-1, c-IAP-2, and XIAP, Hans-Walter Zentgraf for anti-Livin no. 6 and anti-His monoclonal antibodies, and Susanne Kintscher and Markus Moosmeier for helpful discussions. This work was supported by a grant from the Deutsche Krebshilfe to K.H.S. (108607).

References

  1. 1.
    Hanahan D, Weinberg RA (2000) The hallmarks of cancer. Cell 100:57–70CrossRefPubMedGoogle Scholar
  2. 2.
    Fulda S, Debatin KM (2006) Extrinsic versus intrinsic apoptosis pathways in anticancer chemotherapy. Oncogene 25:4798–4811CrossRefPubMedGoogle Scholar
  3. 3.
    Salvesen GS, Duckett CS (2002) IAP proteins: blocking the road to death’s door. Nat Rev Mol Cell Biol 3:401–410CrossRefPubMedGoogle Scholar
  4. 4.
    Crnkovic-Mertens I, Muley T, Meister M, Hartenstein B, Semzow J, Butz K, Hoppe-Seyler F (2006) The anti-apoptotic livin gene is an important determinant for the apoptotic resistance of non-small cell lung cancer cells. Lung Cancer 54:135–142CrossRefPubMedGoogle Scholar
  5. 5.
    Crnkovic-Mertens I, Wagener N, Semzow J, Grone EF, Haferkamp A, Hohenfellner M, Butz K, Hoppe-Seyler F (2007) Targeted inhibition of Livin resensitizes renal cancer cells towards apoptosis. Cell Mol Life Sci 64:1137–1144CrossRefPubMedGoogle Scholar
  6. 6.
    Chang H, Schimmer AD (2007) Livin/melanoma inhibitor of apoptosis protein as a potential therapeutic target for the treatment of malignancy. Mol Cancer Ther 6:24–30CrossRefPubMedGoogle Scholar
  7. 7.
    Wang L, Zhang Q, Liu B, Han M, Shan B (2008) Challenge and promise: roles for Livin in progression and therapy of cancer. Mol Cancer Ther 7:3661–3669CrossRefPubMedGoogle Scholar
  8. 8.
    Vucic D, Stennicke HR, Pisabarro MT, Salvesen GS, Dixit VM (2000) ML-IAP, a novel inhibitor of apoptosis that is preferentially expressed in human melanomas. Curr Biol 10:1359–1366CrossRefPubMedGoogle Scholar
  9. 9.
    Wagener N, Crnkovic-Mertens I, Vetter C, Macher-Goppinger S, Bedke J, Grone EF, Zentgraf H, Pritsch M, Hoppe-Seyler K, Buse S, Haferkamp A, Autschbach F, Hohenfellner M, Hoppe-Seyler F (2007) Expression of inhibitor of apoptosis protein Livin in renal cell carcinoma and non-tumorous adult kidney. Br J Cancer 97:1271–1276CrossRefPubMedGoogle Scholar
  10. 10.
    Crnkovic-Mertens I, Hoppe-Seyler F, Butz K (2003) Induction of apoptosis in tumor cells by siRNA-mediated silencing of the livin/ML-IAP/KIAP gene. Oncogene 22:8330–8336CrossRefPubMedGoogle Scholar
  11. 11.
    Jackson AL, Bartz SR, Schelter J, Kobayashi SV, Burchard J, Mao M, Li B, Cavet G, Linsley PS (2003) Expression profiling reveals off-target gene regulation by RNAi. Nat Biotechnol 21:635–637CrossRefPubMedGoogle Scholar
  12. 12.
    Marques JT, Williams BR (2005) Activation of the mammalian immune system by siRNAs. Nat Biotechnol 23:1399–1405CrossRefPubMedGoogle Scholar
  13. 13.
    Jackson AL, Burchard J, Schelter J, Chau BN, Cleary M, Lim L, Linsley PS (2006) Widespread siRNA “off-target” transcript silencing mediated by seed region sequence complementarity. RNA 12:1179–1187CrossRefPubMedGoogle Scholar
  14. 14.
    Aagaard L, Rossi JJ (2007) RNAi therapeutics: principles, prospects and challenges. Adv Drug Deliv Rev 59:75–86CrossRefPubMedGoogle Scholar
  15. 15.
    Sioud M (2004) Therapeutic siRNAs. Trends Pharmacol Sci 25:22–28CrossRefPubMedGoogle Scholar
  16. 16.
    Fulda S, Wick W, Weller M, Debatin KM (2002) Smac agonists sensitize for Apo2L/TRAIL- or anticancer drug-induced apoptosis and induce regression of malignant glioma in vivo. Nat Med 8:808–815PubMedGoogle Scholar
  17. 17.
    Zobel K, Wang L, Varfolomeev E, Franklin MC, Elliott LO, Wallweber HJ, Okawa DC, Flygare JA, Vucic D, Fairbrother WJ, Deshayes K (2006) Design, synthesis, and biological activity of a potent Smac mimetic that sensitizes cancer cells to apoptosis by antagonizing IAPs. ACS Chem Biol 1:525–533CrossRefPubMedGoogle Scholar
  18. 18.
    Baines IC, Colas P (2006) Peptide aptamers as guides for small-molecule drug discovery. Drug Discov Today 11:334–341CrossRefPubMedGoogle Scholar
  19. 19.
    Sun H, Nikolovska-Coleska Z, Yang CY, Qian D, Lu J, Qiu S, Bai L, Peng Y, Cai Q, Wang S (2008) Design of small-molecule peptidic and nonpeptidic Smac mimetics. Acc Chem Res 41:1264–1277CrossRefPubMedGoogle Scholar
  20. 20.
    Chevray PM, Nathans D (1992) Protein interaction cloning in yeast: identification of mammalian proteins that react with the leucine zipper of Jun. Proc Natl Acad Sci USA 89:5789–5793CrossRefPubMedGoogle Scholar
  21. 21.
    Butz K, Denk C, Ullmann A, Scheffner M, Hoppe-Seyler F (2000) Induction of apoptosis in human papillomaviruspositive cancer cells by peptide aptamers targeting the viral E6 oncoprotein. Proc Natl Acad Sci USA 97:6693–6697CrossRefPubMedGoogle Scholar
  22. 22.
    Dymalla S, Scheffner M, Weber E, Sehr P, Lohrey C, Hoppe-Seyler F, Hoppe-Seyler K (2009) A novel peptide motif binding to and blocking the intracellular activity of the human papillomavirus E6 oncoprotein. J Mol Med 87:321–331CrossRefPubMedGoogle Scholar
  23. 23.
    Jordan BW, Dinev D, LeMellay V, Troppmair J, Gotz R, Wixler L, Sendtner M, Ludwig S, Rapp UR (2001) Neurotrophin receptor-interacting mage homologue is an inducible inhibitor of apoptosis protein-interacting protein that augments cell death. J Biol Chem 276:39985–39989CrossRefPubMedGoogle Scholar
  24. 24.
    Schmitt M, Pawlita M (2009) High-throughput detection and multiplex identification of cell contaminations. Nucleic Acids Res 37:e119CrossRefPubMedGoogle Scholar
  25. 25.
    Chen C, Okayama H (1987) High-efficiency transformation of mammalian cells by plasmid DNA. Mol Cell Biol 7:2745–2752PubMedGoogle Scholar
  26. 26.
    Wender PA, Mitchell DJ, Pattabiraman K, Pelkey ET, Steinman L, Rothbard JB (2000) The design, synthesis, and evaluation of molecules that enable or enhance cellular uptake: peptoid molecular transporters. Proc Natl Acad Sci USA 97:13003–13008CrossRefPubMedGoogle Scholar
  27. 27.
    Crnkovic-Mertens I, Semzow J, Hoppe-Seyler F, Butz K (2006) Isoform-specific silencing of the Livin gene by RNA interference defines Livin beta as key mediator of apoptosis inhibition in HeLa cells. J Mol Med 84:232–240CrossRefPubMedGoogle Scholar
  28. 28.
    Butz K, Denk C, Fitscher B, Crnkovic-Mertens I, Ullmann A, Schroder CH, Hoppe-Seyler F (2001) Peptide aptamers targeting the hepatitis B virus core protein: a new class of molecules with antiviral activity. Oncogene 20:6579–6586CrossRefPubMedGoogle Scholar
  29. 29.
    Zentgraf H, Frey M, Schwinn S, Tessmer C, Willemann B, Samstag Y, Velhagen I (1995) Detection of histidine-tagged fusion proteins by using a high-specific mouse monoclonal anti-histidine tag antibody. Nucleic Acids Res 23:3347–3348CrossRefPubMedGoogle Scholar
  30. 30.
    Ma L, Huang Y, Song Z, Feng S, Tian X, Du W, Qiu X, Heese K, Wu M (2006) Livin promotes Smac/DIABLO degradation by ubiquitin-proteasome pathway. Cell Death Differ 13:2079–2088CrossRefPubMedGoogle Scholar
  31. 31.
    Varfolomeev E, Blankenship JW, Wayson SM, Fedorova AV, Kayagaki N, Garg P, Zobel K, Dynek JN, Elliott LO, Wallweber HJ, Flygare JA, Fairbrother WJ, Deshayes K, Dixit VM, Vucic D (2007) IAP antagonists induce autoubiquitination of c-IAPs, NF-kappaB activation, and TNFalpha-dependent apoptosis. Cell 131:669–681CrossRefPubMedGoogle Scholar
  32. 32.
    Vince JE, Wong WW, Khan N, Feltham R, Chau D, Ahmed AU, Benetatos CA, Chunduru SK, Condon SM, McKinlay M, Brink R, Leverkus M, Tergaonkar V, Schneider P, Callus BA, Koentgen F, Vaux DL, Silke J (2007) IAP antagonists target cIAP1 to induce TNFalpha-dependent apoptosis. Cell 131:682–693CrossRefPubMedGoogle Scholar
  33. 33.
    Tomai E, Butz K, Lohrey C, von Weizsacker F, Zentgraf H, Hoppe-Seyler F (2006) Peptide aptamer-mediated inhibition of target proteins by sequestration into aggresomes. J Biol Chem 281:21345–21352CrossRefPubMedGoogle Scholar
  34. 34.
    Johnston JA, Ward CL, Kopito RR (1998) Aggresomes: a cellular response to misfolded proteins. J Cell Biol 143:1883–1898CrossRefPubMedGoogle Scholar
  35. 35.
    Vucic D, Deshayes K, Ackerly H, Pisabarro MT, Kadkhodayan S, Fairbrother WJ, Dixit VM (2002) SMAC negatively regulates the anti-apoptotic activity of melanoma inhibitor of apoptosis (ML-IAP). J Biol Chem 277:12275–12279CrossRefPubMedGoogle Scholar
  36. 36.
    Hunter AM, LaCasse EC, Korneluk RG (2007) The inhibitors of apoptosis (IAPs) as cancer targets. Apoptosis 12:1543–1568CrossRefPubMedGoogle Scholar
  37. 37.
    Yuan D, Liu L, Xu H, Gu D (2009) The effects on cell growth and chemosensitivity by livin RNAi in non-small cell lung cancer. Mol Cell Biochem 320:133–140CrossRefPubMedGoogle Scholar
  38. 38.
    Wang R, Lin F, Wang X, Gao P, Dong K, Zou AM, Cheng SY, Wei SH, Zhang HZ (2008) Silencing Livin gene expression to inhibit proliferation and enhance chemosensitivity in tumor cells. Cancer Gene Ther 15:402–412CrossRefPubMedGoogle Scholar
  39. 39.
    Wu G, Chai J, Suber TL, Wu JW, Du C, Wang X, Shi Y (2000) Structural basis of IAP recognition by Smac/DIABLO. Nature 408:1008–1012CrossRefPubMedGoogle Scholar
  40. 40.
    Srinivasula SM, Hegde R, Saleh A, Datta P, Shiozaki E, Chai J, Lee RA, Robbins PD, Fernandes-Alnemri T, Shi Y, Alnemri ES (2001) A conserved XIAP-interaction motif in caspase-9 and Smac/DIABLO regulates caspase activity and apoptosis. Nature 410:112–116CrossRefPubMedGoogle Scholar
  41. 41.
    Nachmias B, Ashhab Y, Bucholtz V, Drize O, Kadouri L, Lotem M, Peretz T, Mandelboim O, Ben-Yehuda D (2003) Caspase-mediated cleavage converts Livin from an antiapoptotic to a proapoptotic factor: implications for drug-resistant melanoma. Cancer Res 63:6340–6349PubMedGoogle Scholar
  42. 42.
    Nachmias B, Lazar I, Elmalech M, Abed-El-Rahaman I, Asshab Y, Mandelboim O, Perlman R, Ben-Yehuda D (2007) Subcellular localization determines the delicate balance between the anti- and pro-apoptotic activity of Livin. Apoptosis 12:1129–1142CrossRefPubMedGoogle Scholar
  43. 43.
    Abd-Elrahman I, Hershko K, Neuman T, Nachmias B, Perlman R, Ben-Yehuda D (2009) The inhibitor of apoptosis protein Livin (ML-IAP) plays a dual role in tumorigenicity. Cancer Res 69:5475–5480CrossRefPubMedGoogle Scholar
  44. 44.
    Srinivasula SM, Ashwell JD (2008) IAPs: what’s in a name? Mol Cell 30:123–135CrossRefPubMedGoogle Scholar
  45. 45.
    Dubrez-Daloz L, Dupoux A, Cartier J (2008) IAPs: more than just inhibitors of apoptosis proteins. Cell Cycle 7:1036–1046PubMedGoogle Scholar
  46. 46.
    Torchilin VP, Lukyanov AN (2003) Peptide and protein drug delivery to and into tumors: challenges and solutions. Drug Discov Today 8:259–266CrossRefPubMedGoogle Scholar
  47. 47.
    Borghouts C, Kunz C, Groner B (2005) Current strategies for the development of peptide-based anti-cancer therapeutics. J Pept Sci 11:713–726CrossRefPubMedGoogle Scholar
  48. 48.
    Franklin MC, Kadkhodayan S, Ackerly H, Alexandru D, Distefano MD, Elliott LO, Flygare JA, Mausisa G, Okawa DC, Ong D, Vucic D, Deshayes K, Fairbrother WJ (2003) Structure and function analysis of peptide antagonists of melanoma inhibitor of apoptosis (ML-IAP). Biochemistry 42:8223–8231CrossRefPubMedGoogle Scholar

Copyright information

© Springer Basel AG 2010

Authors and Affiliations

  • Irena Crnković-Mertens
    • 1
  • Julia Bulkescher
    • 1
  • Christina Mensger
    • 1
  • Felix Hoppe-Seyler
    • 1
  • Karin Hoppe-Seyler
    • 1
  1. 1.Molecular Therapy of Virus-Associated Cancers (F065)German Cancer Research CenterHeidelbergGermany

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