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Generation of new TRAIL mutants DR5-A and DR5-B with improved selectivity to death receptor 5

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Abstract

TRAIL (tumor necrosis factor (TNF) related apoptosis-inducing ligand) has been introduced as an extrinsic pathway inducer of apoptosis that does not have the toxicities of Fas and TNF. However, the therapeutic potential of TRAIL is limited because of many primary tumor cells are resistant to TRAIL. Despite intensive investigations, little is known in regards to the mechanisms underlying TRAIL selectivity and efficiency. A major reason likely lies in the complexity of the interaction of TRAIL with its five receptors, of which only two DR4 and DR5 are death receptors. Binding of TRAIL with decoy receptors DcR1 and DcR2 or soluble receptor osteoprotegerin (OPG) fail to induce apoptosis. Here we describe design and expression in Escherichia coli of DR5-selective TRAIL variants DR5-A and DR5-B. The measurements of dissociation constants of these mutants with all five receptors show that they practically do not interact with DR4 and DcR1 and have highly reduced affinity to DcR2 and OPG receptors. These mutants are more effective than wild type TRAIL in induction of apoptosis in different cancer cell lines. In combination with the drugs targeted to cytoskeleton (taxol, cytochalasin D) the mutants of TRAIL induced apoptosis in resistant Hela cells overexpressing Bcl-2. The novel highly selective and effective DR5-A and DR5-B TRAIL variants will be useful in studies on the role of different receptors in TRAIL-induced apoptosis in sensitive and resistant cell lines.

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References

  1. Ashkenazi A, Pai RC, Fong S, Leung S, Lawrence DA, Marsters SA et al (1999) Safety and anti-tumor activity of recombinant soluble Apo2 ligand. J Clin Invest 104:155–162. doi:10.1172/JCI6926

    Article  PubMed  CAS  Google Scholar 

  2. LeBlanc HN, Ashkenazi A (2003) Apo2L/TRAIL and its death and decoy receptors. Cell Death Differ 10:66–75. doi:10.1038/sj.cdd.4401187

    Article  PubMed  CAS  Google Scholar 

  3. Ashkenazi A, Dixit VM (1998) Death receptors: signaling and modulation. Science 281:1305–1308. doi:10.1126/science.281.5381.1305

    Article  PubMed  CAS  Google Scholar 

  4. Simonet WS, Lacey DL, Dunstan CR, Kelley M, Chang MS, Luthy R et al (1997) Osteoprotegerin: a novel secreted protein involved in the regulation of bone density. Cell 89:159–161. doi:10.1016/S0092-8674(00)80209-3

    Article  Google Scholar 

  5. Bouralexis S, Findlay DM, Atkins GJ, Labrinidis A, Hay S, Evdokiou A (2003) Progressive resistance of BTK-143 osteosarcoma cells to Apo2L/TRAIL-induced apoptosis is mediated by acquisition of DcR2/TRAIL-R4 expression: resensitisation with chemotherapy. Br J Cancer 89:206–214. doi:10.1038/sj.bjc.6601021

    Article  PubMed  CAS  Google Scholar 

  6. Clancy L, Mruk K, Archer K, Woelfel M, Mongkolsapaya M, Screaton G et al (2005) Preligand assembly domain-mediated ligand independent association between TRAIL receptor 4 (TR4) and TR2 regulates TRAIL-induced apoptosis. Proc Natl Acad Sci USA 102:18099–18104. doi:10.1073/pnas.0507329102

    Article  PubMed  CAS  Google Scholar 

  7. Merino D, Lalaoui N, Morizot A, Schneider P, Solary E, Micheau O (2006) Differential inhibition of TRAIL-mediated DR5-DISC formation by decoy receptors 1 and 2. Mol Cell Biol 26:7046–7055. doi:10.1128/MCB.00520-06

    Article  PubMed  CAS  Google Scholar 

  8. Merino D, Lalaoui N, Morizot A, Solary E, Micheau O (2007) TRAIL in cancer therapy: present and future challenges. Expert Opin Ther Targets 11:1299–1314. doi:10.1517/14728222.11.10.1299

    Article  PubMed  CAS  Google Scholar 

  9. Kelley FR, Totpal K, Lindstrom SH, Mathieu M, Billeci K, de Forge L et al (2005) Receptor-selective mutants of Apo2L/TRAIL reveal a greater contribution of DR5 than DR4 to apoptosis signaling. J Biol Chem 280:2205–2212. doi:10.1074/jbc.M410660200

    Article  PubMed  CAS  Google Scholar 

  10. van der Sloot AM, Tur V, Szegezdi TE, Mullally MM, Cool RH, Samali A et al (2006) Designed tumor necrosis factor-related apoptosis-inducing ligand variants initiating apoptosis exclusively via the DR5 receptor. Proc Natl Acad Sci USA 103:8634–8639. doi:10.1073/pnas.0510187103

    Article  PubMed  Google Scholar 

  11. Gasparian ME, Ostapchenko VG, Yagolovich AV, Tsygannik IN, Chernyak BV, Dolgikh DA et al (2007) Overexpression and refolding of thioredoxin/TRAIL fusion from inclusion bodies and further purification of TRAIL after cleavage by enteropeptidase. Biotechnol Lett 29:1567–1573. doi:10.1007/s10529-007-9446-y

    Article  PubMed  CAS  Google Scholar 

  12. Kunkel TA (1985) Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci USA 82:488–492. doi:10.1073/pnas.82.2.488

    Article  PubMed  CAS  Google Scholar 

  13. Gasparian ME, Ostapchenko VG, Schulga AA, Dolgikh DA, Kirpichnikov MP (2003) Expression, purification and characterization of human enteropeptidase catalytic subunit in Escherichia coli. Protein Expr Purif 31:133–139. doi:10.1016/S1046-5928(03)00159-1

    Article  PubMed  CAS  Google Scholar 

  14. Schuck P (2000) Size-distribution analysis of macromolecules by sedimentation velocity ultracentrifugation and Lamm equation modeling. Biophys J 78:1606–1619. doi:10.1016/S0006-3495(00)76713-0

    Article  PubMed  CAS  Google Scholar 

  15. Agol VI, Belov GA, Bienz K, Egger D, Kolesnikova MS, Romanova LI et al (2000) Competing death programs in poliovirus-infected cells: commitment switch in the middle of the infectious cycle. J Virol 74:5534–5541. doi:10.1128/JVI.74.12.5534-5541.2000

    Article  PubMed  CAS  Google Scholar 

  16. Hymowitz SG, O’Connell MP, Ultsch MH, Hurst A, Totpal K, Ashkenazi A et al (2000) A unique zinc-binding site revealed by a high-resolution X-ray structure of homotrimeric Apo2L/TRAIL. Biochemistry 39:633–640. doi:10.1021/bi992242l

    Article  PubMed  CAS  Google Scholar 

  17. Wen J, Ramadevi N, Nguyen D, Perkins C, Worthington E, Bhalla K (2000) Antileukemic drugs increase death receptor 5 levels and enhance Apo-2L-induced apoptosis of human acute leukemia cells. Blood 96:3900–3906

    PubMed  CAS  Google Scholar 

  18. Phillips TA, Ni J, Pan G, Ruben SM, Wei Y, Pace JL et al (1999) TRAIL (Apo-2L) and TRAIL receptors in human placentas: Implications for immune privilege. J Immunol 162:6053–6059

    PubMed  CAS  Google Scholar 

  19. Mirandola P, Gobbi G, Ponti C, Sponzilli I, Cocco L, Vitale L (2006) PKCε control protection against TRAIL in erythroid progenitors. Blood 107:508–513. doi:10.1182/blood-2005-07-2676

    Article  PubMed  CAS  Google Scholar 

  20. Gasparian ME, Domnina LV, Ivanova OY, Izyumov DS, Lomakin AY, Popova EN et al (2008) Cytoskeleton inhibitors combined with TRAIL induce apoptosis in HeLa Carcinoma cells overexpressing antiapoptotic protein Bcl-2. Biochemistry (Mosc) 73:358–362

    CAS  Google Scholar 

  21. Ashkenazi A (2002) Targeting death and decoy receptors of the tumour-necrosis factor superfamily. Nat Rev Cancer 2:420–430. doi:10.1038/nrc821

    Article  PubMed  CAS  Google Scholar 

  22. MacFarlane M, Kohlhaas SL, Sutcliffe MJ, Dyer MJS, Cohen GM (2005) TRAIL receptor-selective mutants signal to apoptosis via TRAIL-R1 in primary lymphoid malignancies. Cancer Res 65:11265–11270. doi:10.1158/0008-5472.CAN-05-2801

    Article  PubMed  CAS  Google Scholar 

  23. Tur V, van der Sloot AM, Reis CR, Szegezdi E, Cool RH, Samali A et al (2008) DR4-selective tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) variants obtained by structure-based design. J Biol Chem 283:20560–20568. doi:10.1074/jbc.M800457200

    Article  PubMed  CAS  Google Scholar 

  24. Ashkenazi A, Dixit VM (1999) Apoptosis control by death and decoy receptors. Curr Opin Cell Biol 11:255–260. doi:10.1016/S0955-0674(99)80034-9

    Article  PubMed  CAS  Google Scholar 

  25. Griffith TS, Rauch ST, Smolak PJ, Waugh JY, Boiani N, Lynch DH et al (1999) Functional analysis of TRAIL receptors using monoclonal antibodies. J Immunol 162:2597–2605

    PubMed  CAS  Google Scholar 

  26. Ashkenazi A, Herbst RS (2008) To kill a tumor cell: the potential of proapoptotic receptor agonists. J Clin Invest 118:1979–1990. doi:10.1172/JCI34359

    Article  PubMed  CAS  Google Scholar 

  27. MacFarlane M, Inoue S, Kohlhaas SL, Majid A, Harper N, Kennedy DBJ et al (2005) Chronic lymphocytic leukemic cells exhibit apoptotic signaling via TRAIL-R1. Cell Death Differ 12:773–782. doi:10.1038/sj.cdd.4401649

    Article  PubMed  CAS  Google Scholar 

  28. Inoue S, MacFarlane M, Harper N, Wheat LMC, Dyer MJS, Cohen GM (2004) Histone deacetylase inhibitors potentiate TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in lymphoid malignancies. Cell Death Differ 11(Suppl 2):193–206. doi:10.1038/sj.cdd.4401535

    Article  Google Scholar 

  29. Pietro R, Secchiero P, Rana R, Gibellini D, Visani G, Bemis K et al (2001) Ionizing radiation sensitizes erythroleukemic cells but not normal erythroblasts to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated cytotoxicity by selective up-regulation of TRAIL-R1. Blood 97:2596–2603. doi:10.1182/blood.V97.9.2596

    Article  PubMed  Google Scholar 

  30. Perez-Cruz I, Carcamo JM, David W, Golde DW (2007) Caspase-8 dependent trail-induced apoptosis in cancer cell lines is inhibited by vitamin C and catalase. Apoptosis 12:225–234. doi:10.1007/s10495-006-0475-0

    Article  PubMed  CAS  Google Scholar 

  31. Gregorini A, Tomasetti M, Cinti C, Colomba D, Colomba S (2006) CD38 expression enhances sensitivity of lymphoma T and B cell lines to biochemical and receptor-mediated apoptosis. Cell Biol Int 30:727–732. doi:10.1016/j.cellbi.2006.05.004

    Article  PubMed  CAS  Google Scholar 

  32. Kim K, Fisher MJ, Xu SQ, El-Deiry WS (2000) Molecular determinants of response to TRAIL in killing of normal and cancer cells. Clin Cancer Res 6:335–346

    PubMed  CAS  Google Scholar 

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Acknowledgments

The authors would like to thank Natalia Magretova and Peter Kalmykov from A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University for help in analytical centrifugation. The work was supported by Federal Target Programme « Research and Development in Priority Fields of Scientific and Technologic Complex of Russia for 2007–2012» (contract # 02.522.11.2005).

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Authors and Affiliations

  1. Laboratory of Protein Engineering, Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, RAS, 16/10 Miklukho-Maklaya, 117997, Moscow, Russia

    Marine E. Gasparian, Dmitry A. Dolgikh, Anne V. Yagolovich & Mikhail P. Kirpichnikov

  2. A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, 119899, Moscow, Russia

    Boris V. Chernyak & Ekaterina N. Popova

  3. V. N. Orekhovich Institute of Biomedical Chemistry, RAMS, 119121, Moscow, Russia

    Anna M. Sycheva & Sergey A. Moshkovskii

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  1. Marine E. Gasparian
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  2. Boris V. Chernyak
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  3. Dmitry A. Dolgikh
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  4. Anne V. Yagolovich
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  5. Ekaterina N. Popova
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  7. Sergey A. Moshkovskii
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  8. Mikhail P. Kirpichnikov
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Correspondence to Marine E. Gasparian.

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Gasparian, M.E., Chernyak, B.V., Dolgikh, D.A. et al. Generation of new TRAIL mutants DR5-A and DR5-B with improved selectivity to death receptor 5. Apoptosis 14, 778–787 (2009). https://doi.org/10.1007/s10495-009-0349-3

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  • DOI: https://doi.org/10.1007/s10495-009-0349-3

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