Skip to main content
SpringerLink
Log in

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

  • Research Article
  • Published:
Cellular and Molecular Life Sciences Aims and scope Submit manuscript

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  1. Hanahan D, Weinberg RA (2000) The hallmarks of cancer. Cell 100:57–70

    Article  CAS  PubMed  Google Scholar 

  2. Fulda S, Debatin KM (2006) Extrinsic versus intrinsic apoptosis pathways in anticancer chemotherapy. Oncogene 25:4798–4811

    Article  CAS  PubMed  Google Scholar 

  3. Salvesen GS, Duckett CS (2002) IAP proteins: blocking the road to death’s door. Nat Rev Mol Cell Biol 3:401–410

    Article  CAS  PubMed  Google Scholar 

  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 54:135–142

    Article  PubMed  Google Scholar 

  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 64:1137–1144

    Article  CAS  PubMed  Google Scholar 

  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 6:24–30

    Article  CAS  PubMed  Google Scholar 

  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 7:3661–3669

    Article  CAS  PubMed  Google Scholar 

  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 10:1359–1366

    Article  CAS  PubMed  Google Scholar 

  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 97:1271–1276

    Article  CAS  PubMed  Google Scholar 

  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 22:8330–8336

    Article  CAS  PubMed  Google Scholar 

  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 21:635–637

    Article  CAS  PubMed  Google Scholar 

  12. Marques JT, Williams BR (2005) Activation of the mammalian immune system by siRNAs. Nat Biotechnol 23:1399–1405

    Article  CAS  PubMed  Google Scholar 

  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 12:1179–1187

    Article  CAS  PubMed  Google Scholar 

  14. Aagaard L, Rossi JJ (2007) RNAi therapeutics: principles, prospects and challenges. Adv Drug Deliv Rev 59:75–86

    Article  CAS  PubMed  Google Scholar 

  15. Sioud M (2004) Therapeutic siRNAs. Trends Pharmacol Sci 25:22–28

    Article  CAS  PubMed  Google Scholar 

  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 8:808–815

    CAS  PubMed  Google Scholar 

  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 1:525–533

    Article  CAS  PubMed  Google Scholar 

  18. Baines IC, Colas P (2006) Peptide aptamers as guides for small-molecule drug discovery. Drug Discov Today 11:334–341

    Article  CAS  PubMed  Google Scholar 

  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 41:1264–1277

    Article  CAS  PubMed  Google Scholar 

  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 89:5789–5793

    Article  CAS  PubMed  Google Scholar 

  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 97:6693–6697

    Article  CAS  PubMed  Google Scholar 

  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 87:321–331

    Article  CAS  PubMed  Google Scholar 

  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 276:39985–39989

    Article  CAS  PubMed  Google Scholar 

  24. Schmitt M, Pawlita M (2009) High-throughput detection and multiplex identification of cell contaminations. Nucleic Acids Res 37:e119

    Article  PubMed  Google Scholar 

  25. Chen C, Okayama H (1987) High-efficiency transformation of mammalian cells by plasmid DNA. Mol Cell Biol 7:2745–2752

    CAS  PubMed  Google Scholar 

  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 97:13003–13008

    Article  CAS  PubMed  Google Scholar 

  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 84:232–240

    Article  CAS  PubMed  Google Scholar 

  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 20:6579–6586

    Article  CAS  PubMed  Google Scholar 

  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 23:3347–3348

    Article  CAS  PubMed  Google Scholar 

  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 13:2079–2088

    Article  CAS  PubMed  Google Scholar 

  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 131:669–681

    Article  CAS  PubMed  Google Scholar 

  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 131:682–693

    Article  CAS  PubMed  Google Scholar 

  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 281:21345–21352

    Article  CAS  PubMed  Google Scholar 

  34. Johnston JA, Ward CL, Kopito RR (1998) Aggresomes: a cellular response to misfolded proteins. J Cell Biol 143:1883–1898

    Article  CAS  PubMed  Google Scholar 

  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 277:12275–12279

    Article  CAS  PubMed  Google Scholar 

  36. Hunter AM, LaCasse EC, Korneluk RG (2007) The inhibitors of apoptosis (IAPs) as cancer targets. Apoptosis 12:1543–1568

    Article  CAS  PubMed  Google Scholar 

  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 320:133–140

    Article  CAS  PubMed  Google Scholar 

  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 15:402–412

    Article  CAS  PubMed  Google Scholar 

  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 408:1008–1012

    Article  CAS  PubMed  Google Scholar 

  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 410:112–116

    Article  CAS  PubMed  Google Scholar 

  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 63:6340–6349

    CAS  PubMed  Google Scholar 

  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 12:1129–1142

    Article  CAS  PubMed  Google Scholar 

  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 69:5475–5480

    Article  CAS  PubMed  Google Scholar 

  44. Srinivasula SM, Ashwell JD (2008) IAPs: what’s in a name? Mol Cell 30:123–135

    Article  CAS  PubMed  Google Scholar 

  45. Dubrez-Daloz L, Dupoux A, Cartier J (2008) IAPs: more than just inhibitors of apoptosis proteins. Cell Cycle 7:1036–1046

    CAS  PubMed  Google Scholar 

  46. Torchilin VP, Lukyanov AN (2003) Peptide and protein drug delivery to and into tumors: challenges and solutions. Drug Discov Today 8:259–266

    Article  CAS  PubMed  Google Scholar 

  47. Borghouts C, Kunz C, Groner B (2005) Current strategies for the development of peptide-based anti-cancer therapeutics. J Pept Sci 11:713–726

    Article  CAS  PubMed  Google Scholar 

  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 42:8223–8231

    Article  CAS  PubMed  Google Scholar 

Download references

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).

Author information

Authors and Affiliations

  1. Molecular Therapy of Virus-Associated Cancers (F065), German Cancer Research Center, Im Neuenheimer Feld 242, 69120, Heidelberg, Germany

    Irena Crnković-Mertens, Julia Bulkescher, Christina Mensger, Felix Hoppe-Seyler & Karin Hoppe-Seyler

Authors
  1. Irena Crnković-Mertens
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Julia Bulkescher
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Christina Mensger
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Felix Hoppe-Seyler
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Karin Hoppe-Seyler
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Karin Hoppe-Seyler.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Crnković-Mertens, I., Bulkescher, J., Mensger, C. et al. Isolation of peptides blocking the function of anti-apoptotic Livin protein. Cell. Mol. Life Sci. 67, 1895–1905 (2010). https://doi.org/10.1007/s00018-010-0300-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00018-010-0300-3

Keywords

Search

Navigation