Skip to main content

Advertisement

SpringerLink
Log in

A flavivirus protein M-derived peptide directly permeabilizes mitochondrial membranes, triggers cell death and reduces human tumor growth in nude mice

  • Original Paper
  • Published:
Apoptosis Aims and scope Submit manuscript

Abstract

Dengue viruses belong to the Flavivirus family and are responsible for hemorrhagic fever in Human. Dengue virus infection triggers apoptosis especially through the expression of the small membrane (M) protein. Using isolated mitochondria, we found that synthetic peptides containing the C-terminus part of the M ectodomain caused apoptosis-related mitochondrial membrane permeabilization (MMP) events. These events include matrix swelling and the dissipation of the mitochondrial transmembrane potential (ΔΨm). Protein M Flavivirus sequence alignments and helical wheel projections reveal a conserved distribution of charged residues. Moreover, when combined to the cell penetrating HIV-1 Tat peptide transduction domain (Tat-PTD), this sequence triggers a caspase-dependent cell death associated with ΔΨm loss and cytochrome c release. Mutational approaches coupled to functional screening on isolated mitochondria resulted in the selection of a protein M derived sequence containing nine residues with potent MMP-inducing properties on isolated mitochondria. A chimeric peptide composed of a Tat-PTD linked to the 9-mer entity triggers MMP and cell death. Finally, local administration of this chimeric peptide induces growth inhibition of xenograft prostate PC3 tumors in immuno-compromised mice, and significantly enhances animal survival. Together, these findings support the notion of using viral genomes as valuable sources to discover mitochondria-targeted sequences that may lead to the development of new anticancer compounds.

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

Abbreviations

AIF:

Apoptosis-inducing factor

ANT:

Adenine nucleotide translocator

Biot:

Biotin

Boc:

Di-tert-butyl dicarbonate

mClCCP:

Carbonyl cyanide m-chlorophenylhydrazone

CsA:

Cyclosporin A

ΔΨm :

Mitochondrial transmembrane potential

DiOC(6)(3):

3,3′-Dihexyloxacarbocyanine iodide

FBS:

Fetal bovine serum

FITC:

Fluorescein isothiocyanate

Fmoc:

Fluorenylmethyloxycarbonyl

FSC:

Forward scatter

HPLC:

High-performance liquid chromatography

IM:

Inner membrane

JC-1:

5,5′,6,6′-Tetracholoro-1,1,3,3′-tetraethylbenzimidazolylcarbocyanine iodide

LC/MS:

Liquid chromatography–mass spectrometry

MMP:

Mitochondrial membrane permeabilization

MOPS:

3-[N-morpholino]-propanesulfonic acid

OM:

Outer membrane

PBS:

Phosphate buffer saline

PMP:

Plasma membrane permeabilization

PTP:

Permeability transition pore

Q-VD-OPH:

Quinolin-carbonyl-Val-Asp-(OMe)-CH2-O-F2-Ph

Rh123:

Rhodamine 123

RT:

Room temperature

RP-HPLC:

Reverse phase HPLC

SAR:

Structure activity relationships

SEM:

Standard error of the mean

SSC:

Side scatter

z-VAD-fmk:

N-benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluromethylketone

References

  1. Kerr JF, Wyllie AH, Currie AR (1972) Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer 26:239–257

    PubMed  CAS  Google Scholar 

  2. Kroemer G, Galluzzi L, Brenner C (2007) Mitochondrial membrane permeabilization in cell death. Physiol Rev 87:99–163

    Article  PubMed  CAS  Google Scholar 

  3. Adams JM, Cory S (2002) Apoptosomes: engines for caspase activation. Curr Opin Cell Biol 14:715–720

    Article  PubMed  CAS  Google Scholar 

  4. Costantini P, Jacotot E, Decaudin D, Kroemer G (2000) Mitochondrion as a novel target of anticancer chemotherapy. J Natl Cancer Inst 92:1042–1053

    Article  PubMed  CAS  Google Scholar 

  5. Dias N, Bailly C (2005) Drugs targeting mitochondrial functions to control tumor cell growth. Biochem Pharmacol 70:1–12

    Article  PubMed  CAS  Google Scholar 

  6. Galluzzi L, Larochette N, Zamzami N, Kroemer G (2006) Mitochondria as therapeutic targets for cancer chemotherapy. Oncogene 25:4812–4830

    Article  PubMed  CAS  Google Scholar 

  7. Jacotot E, Deniaud A, Borgne-Sanchez A et al (2006) Therapeutic peptides: targeting the mitochondrion to modulate apoptosis. Biochim Biophys Acta 1757:1312–1323

    Article  PubMed  CAS  Google Scholar 

  8. Debatin KM, Poncet D, Kroemer G (2002) Chemotherapy: targeting the mitochondrial cell death pathway. Oncogene 21:8786–8803

    Article  PubMed  CAS  Google Scholar 

  9. Cesura AM, Pinard E, Schubenel R et al (2003) The voltage-dependent anion channel is the target for a new class of inhibitors of the mitochondrial permeability transition pore. J Biol Chem 278:49812–49818

    Article  PubMed  CAS  Google Scholar 

  10. Green DR, Kroemer G (2004) The pathophysiology of mitochondrial cell death. Science 305:626–629

    Article  PubMed  CAS  Google Scholar 

  11. Hagland H, Nikolaisen J, Hodneland LI, Gjertsen BT, Bruserud O, Tronstad KJ (2007) Targeting mitochondria in the treatment of human cancer: a coordinated attack against cancer cell energy metabolism and signalling. Expert Opin Ther Targets 11:1055–1069

    Article  PubMed  CAS  Google Scholar 

  12. Kroemer G, Pouyssegur J (2008) Tumor cell metabolism: cancer’s Achilles’ heel. Cancer Cell 13:472–482

    Article  PubMed  CAS  Google Scholar 

  13. Green DR, Kroemer G (2005) Pharmacological manipulation of cell death: clinical applications in sight? J Clin Invest 115:2610–2617

    Article  PubMed  CAS  Google Scholar 

  14. Hail N Jr (2005) Mitochondria: a novel target for the chemoprevention of cancer. Apoptosis 10:687–705

    Article  PubMed  CAS  Google Scholar 

  15. Bouchier-Hayes L, Lartigue L, Newmeyer DD (2005) Mitochondria: pharmacological manipulation of cell death. J Clin Invest 115:2640–2647

    Article  PubMed  CAS  Google Scholar 

  16. Letai A (2005) Pharmacological manipulation of Bcl-2 family members to control cell death. J Clin Invest 115:2648–2655

    Article  PubMed  CAS  Google Scholar 

  17. Oltersdorf T, Elmore SW, Shoemaker AR et al (2005) An inhibitor of Bcl-2 family proteins induces regression of solid tumours. Nature 435:677–681

    Article  PubMed  CAS  Google Scholar 

  18. Boya P, Pauleau AL, Poncet D, Gonzalez-Polo RA, Zamzami N, Kroemer G (2004) Viral proteins targeting mitochondria: controlling cell death. Biochim Biophys Acta 1659:178–189

    Article  PubMed  CAS  Google Scholar 

  19. Everett H, Barry M, Sun X et al (2002) The myxoma poxvirus protein, M11L, prevents apoptosis by direct interaction with the mitochondrial permeability transition pore. J Exp Med 196:1127–1139

    Article  PubMed  CAS  Google Scholar 

  20. Everett H, McFadden G (2001) Viruses and apoptosis: meddling with mitochondria. Virology 288:1–7

    Article  PubMed  CAS  Google Scholar 

  21. D’Agostino DM, Bernardi P, Chieco-Bianchi L, Ciminale V (2005) Mitochondria as functional targets of proteins coded by human tumor viruses. Adv Cancer Res 94:87–142

    Article  PubMed  Google Scholar 

  22. Jacotot E, Ravagnan L, Loeffler M et al (2000) The HIV-1 viral protein R induces apoptosis via a direct effect on the mitochondrial permeability transition pore. J Exp Med 191:33–46

    Article  PubMed  CAS  Google Scholar 

  23. Jacotot E, Ferri KF, El Hamel C et al (2001) Control of mitochondrial membrane permeabilization by adenine nucleotide translocator interacting with HIV-1 viral protein rR and Bcl-2. J Exp Med 193:509–519

    Article  PubMed  CAS  Google Scholar 

  24. Borgne-Sanchez A, Dupont S, Langonne A et al (2007) Targeted Vpr-derived peptides reach mitochondria to induce apoptosis of alphaVbeta3-expressing endothelial cells. Cell Death Differ 14:422–435

    Article  PubMed  CAS  Google Scholar 

  25. Zamarin D, Garcia-Sastre A, Xiao X, Wang R, Palese P (2005) Influenza virus PB1–F2 protein induces cell death through mitochondrial ANT3 and VDAC1. PLoS Pathog 1:e4

    Article  PubMed  CAS  Google Scholar 

  26. Chanturiya AN, Basanez G, Schubert U, Henklein P, Yewdell JW, Zimmerberg J (2004) PB1–F2, an influenza A virus-encoded proapoptotic mitochondrial protein, creates variably sized pores in planar lipid membranes. J Virol 78:6304–6312

    Article  PubMed  CAS  Google Scholar 

  27. Gibbs JS, Malide D, Hornung F, Bennink JR, Yewdell JW (2003) The influenza A virus PB1–F2 protein targets the inner mitochondrial membrane via a predicted basic amphipathic helix that disrupts mitochondrial function. J Virol 77:7214–7224

    Article  PubMed  CAS  Google Scholar 

  28. Everett H, Barry M, Lee SF et al (2000) M11L: a novel mitochondria-localized protein of myxoma virus that blocks apoptosis of infected leukocytes. J Exp Med 191:1487–1498

    Article  PubMed  CAS  Google Scholar 

  29. Wang G, Barrett JW, Nazarian SH et al (2004) Myxoma virus M11L prevents apoptosis through constitutive interaction with Bak. J Virol 78:7097–7111

    Article  PubMed  CAS  Google Scholar 

  30. Wang HW, Sharp TV, Koumi A, Koentges G, Boshoff C (2002) Characterization of an anti-apoptotic glycoprotein encoded by Kaposi’s sarcoma-associated herpesvirus which resembles a spliced variant of human survivin. EMBO J 21:2602–2615

    Article  PubMed  CAS  Google Scholar 

  31. Deniaud A, Brenner C, Kroemer G (2004) Mitochondrial membrane permeabilization by HIV-1 Vpr. Mitochondrion 4:223–233

    Article  PubMed  CAS  Google Scholar 

  32. Chambers TJ, Hahn CS, Galler R, Rice CM (1990) Flavivirus genome organization, expression, and replication. Annu Rev Microbiol 44:649–688

    Article  PubMed  CAS  Google Scholar 

  33. Gubler DJ, Meltzer M (1999) Impact of dengue/dengue hemorrhagic fever on the developing world. Adv Virus Res 53:35–70

    Article  PubMed  CAS  Google Scholar 

  34. Courageot MP, Catteau A, Despres P (2003) Mechanisms of dengue virus-induced cell death. Adv Virus Res 60:157–186

    Article  PubMed  CAS  Google Scholar 

  35. Marianneau P, Flamand M, Deubel V, Despres P (1998) Apoptotic cell death in response to dengue virus infection: the pathogenesis of dengue haemorrhagic fever revisited. Clin Diagn Virol 10:113–119

    Article  PubMed  CAS  Google Scholar 

  36. Lindenbach BD, Rice CM (2003) Molecular biology of flaviviruses. Adv Virus Res 59:23–61

    Article  PubMed  CAS  Google Scholar 

  37. Despres P, Flamand M, Ceccaldi PE, Deubel V (1996) Human isolates of dengue type 1 virus induce apoptosis in mouse neuroblastoma cells. J Virol 70:4090–4096

    PubMed  CAS  Google Scholar 

  38. Duarte dos Santos CN, Frenkiel MP, Courageot MP et al (2000) Determinants in the envelope E protein and viral RNA helicase NS3 that influence the induction of apoptosis in response to infection with dengue type 1 virus. Virology 274:292–308

    Article  PubMed  CAS  Google Scholar 

  39. Catteau A, Roue G, Yuste VJ, Susin SA, Despres P (2003) Expression of dengue ApoptoM sequence results in disruption of mitochondrial potential and caspase activation. Biochimie 85:789–793

    Article  PubMed  CAS  Google Scholar 

  40. Dawson PE, Muir TW, Clark-Lewis I, Kent SB (1994) Synthesis of proteins by native chemical ligation. Science 266:776–779

    Article  PubMed  CAS  Google Scholar 

  41. Hojo Y, Satomi Y (1991) In vivo nephrotoxicity induced in mice by chromium(VI). Involvement of glutathione and chromium(V). Biol Trace Elem Res 31:21–31

    Article  PubMed  CAS  Google Scholar 

  42. Neimark J, Briand JP (1993) Development of a fully automated multichannel peptide synthesizer with integrated TFA cleavage capability. Pept Res 6:219–228

    PubMed  CAS  Google Scholar 

  43. King DS, Fields CG, Fields GB (1990) A cleavage method which minimizes side reactions following Fmoc solid phase peptide synthesis. Int J Pept Protein Res 36:255–266

    PubMed  CAS  Google Scholar 

  44. Lecoeur H, Langonne A, Baux L et al (2004) Real-time flow cytometry analysis of permeability transition in isolated mitochondria. Exp Cell Res 294:106–117

    Article  PubMed  CAS  Google Scholar 

  45. Piccotti L, Marchetti C, Migliorati G, Roberti R, Corazzi L (2002) Exogenous phospholipids specifically affect transmembrane potential of brain mitochondria and cytochrome c release. J Biol Chem 277:12075–12081

    Article  PubMed  CAS  Google Scholar 

  46. Cossarizza A, Ceccarelli D, Masini A (1996) Functional heterogeneity of an isolated mitochondrial population revealed by cytofluorometric analysis at the single organelle level. Exp Cell Res 222:84–94

    Article  PubMed  CAS  Google Scholar 

  47. Zhang L, Yu J, Park BH, Kinzler KW, Vogelstein B (2000) Role of BAX in the apoptotic response to anticancer agents. Science 290(5493):989–992

    Article  PubMed  CAS  Google Scholar 

  48. Subramanian T, Chinnadurai G (2003) Pro-apoptotic activity of transiently expressed BCL-2 occurs independent of BAX and BAK. J Cell Biochem 89(6):1102–1114

    Article  PubMed  CAS  Google Scholar 

  49. Smiley ST, Reers M, Mottola-Hartshorn C et al (1991) Intracellular heterogeneity in mitochondrial membrane potentials revealed by a J-aggregate-forming lipophilic cation JC-1. Proc Natl Acad Sci USA 88:3671–3675

    Article  PubMed  CAS  Google Scholar 

  50. Lecoeur H, Chauvier D, Langonne A et al (2004) Dynamic analysis of apoptosis in primary cortical neurons by fixed- and real-time cytofluorometry. Apoptosis 9:157–169

    Article  PubMed  CAS  Google Scholar 

  51. Zhang W, Chipman PR, Corver J et al (2003) Visualization of membrane protein domains by cryo-electron microscopy of dengue virus. Nat Struct Biol 10:907–912

    Article  PubMed  CAS  Google Scholar 

  52. Jacotot E, Costantini P, Laboureau E, Zamzami N, Susin SA, Kroemer G (1999) Mitochondrial membrane permeabilization during the apoptotic process. Ann N Y Acad Sci 887:18–30

    PubMed  CAS  Google Scholar 

  53. Brooks H, Lebleu B, Vives E (2005) Tat peptide-mediated cellular delivery: back to basics. Adv Drug Deliv Rev 57:559–577

    Article  PubMed  CAS  Google Scholar 

  54. Vives E, Brodin P, Lebleu B (1997) A truncated HIV-1 Tat protein basic domain rapidly translocates through the plasma membrane and accumulates in the cell nucleus. J Biol Chem 272:16010–16017

    Article  PubMed  CAS  Google Scholar 

  55. Chauvier D, Ankri S, Charriaut-Marlangue C, Casimir R, Jacotot E (2007) Broad-spectrum caspase inhibitors: from myth to reality? Cell Death Differ 14:387–391

    Article  PubMed  CAS  Google Scholar 

  56. Despres P, Frenkiel MP, Ceccaldi PE, Duarte Dos Santos C, Deubel V (1998) Apoptosis in the mouse central nervous system in response to infection with mouse-neurovirulent dengue viruses. J Virol 72:823–829

    PubMed  CAS  Google Scholar 

  57. Marianneau P, Cardona A, Edelman L, Deubel V, Despres P (1997) Dengue virus replication in human hepatoma cells activates NF-kappaB which in turn induces apoptotic cell death. J Virol 71:3244–3249

    PubMed  CAS  Google Scholar 

  58. Thongtan T, Panyim S, Smith DR (2004) Apoptosis in dengue virus infected liver cell lines HepG2 and Hep3B. J Med Virol 72:436–444

    Article  PubMed  CAS  Google Scholar 

  59. Shafee N, AbuBakar S (2003) Dengue virus type 2 NS3 protease and NS2B-NS3 protease precursor induce apoptosis. J Gen Virol 84:2191–2195

    Article  PubMed  CAS  Google Scholar 

  60. Avirutnan P, Malasit P, Seliger B, Bhakdi S, Husmann M (1998) Dengue virus infection of human endothelial cells leads to chemokine production, complement activation, and apoptosis. J Immunol 161:6338–6346

    PubMed  CAS  Google Scholar 

  61. Jan JT, Chen BH, Ma SH et al (2000) Potential dengue virus-triggered apoptotic pathway in human neuroblastoma cells: arachidonic acid, superoxide anion, and NF-kappaB are sequentially involved. J Virol 74:8680–8691

    Article  PubMed  CAS  Google Scholar 

  62. Parquet MC, Kumatori A, Hasebe F, Morita K, Igarashi A (2001) West Nile virus-induced bax-dependent apoptosis. FEBS Lett 500:17–24

    Article  PubMed  CAS  Google Scholar 

  63. Su HL, Lin YL, Yu HP et al (2001) The effect of human bcl-2 and bcl-X genes on dengue virus-induced apoptosis in cultured cells. Virology 282:141–153

    Article  PubMed  CAS  Google Scholar 

  64. Catteau A, Kalinina O, Wagner MC, Deubel V, Courageot MP, Desprès P (2003) Dengue virus M protein contains a proapoptotic sequence referred to as ApoptoM. J Gen Virol 84:2781–2793

    Article  PubMed  CAS  Google Scholar 

  65. Ferri KF, Jacotot E, Blanco J, Esté JA, Kroemer G (2000) Mitochondrial control of cell death induced by HIV-1-encoded proteins. Ann N Y Acad Sci 926:149–164

    Article  PubMed  CAS  Google Scholar 

  66. Galluzzi L, Brenner C, Morselli E, Touat Z, Kroemer G (2008) Viral control of mitochondrial apoptosis. PLoS Pathog 4:e1000018

    Article  PubMed  CAS  Google Scholar 

  67. Ellerby HM, Arap W, Ellerby LM et al (1999) Anti-cancer activity of targeted pro-apoptotic peptides. Nat Med 5:1032–1038

    Article  PubMed  CAS  Google Scholar 

  68. Arap W, Haedicke W, Bernasconi M et al (2002) Targeting the prostate for destruction through a vascular address. Proc Natl Acad Sci USA 99:1527–1531

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

We thank Prof. Cornelis Lucas for critical reading of the manuscript and helpful suggestions. We are grateful to Dr. Peter Daniel for kindly providing Bax (+/−) and Bax/Bak (−/−) colon cancer cell lines generated by Prof. Bert Vogelstein (Johns Hopkins University) and Prof. Govindaswamy Chinnadurai (Saint Louis University School of Medicine). This work was supported by grants from the French Ministry of Research (GenHomme) to E.J. (No. 01H0476), C.B. (N°01H0477) and S.M. (No. 01H0478), by Agence Nationale pour la Valorisation de la Recherche (ANVAR) to E.J. (No. R0209333Q and No. A0404096Q), by Sidaction and Centre National pour la Recherche Scientifique (CNRS) to S.M. D.R. was supported by ANVAR (No. K0109377Q), O.C. by Sidaction and A.L. by Centre Régional d’Innovation et de Transfert de Technologie (CRITT) d’Ile de France.

Author information

Authors and Affiliations

  1. Theraptosis Research Laboratory, Theraptosis S.A., Pasteur BioTop, 28 rue du Dr Roux, 75015, Paris, France

    Magali Brabant, Ludwig Baux, Richard Casimir, Mathieu Porceddu, Nelly Buron, David Chauvier, Myriam Lassalle, Hervé Lecoeur, Alain Langonné, Sylvie Dupont, Olivier Déas, Dominique Rebouillat, Annie Borgne-Sanchez & Etienne Jacotot

  2. Theraptosis R&D Laboratories, Theraptosis S.A., Biocitech Technology Park, 93230, Romainville, France

    Magali Brabant, Richard Casimir, Mathieu Porceddu, Nelly Buron, David Chauvier, Annie Borgne-Sanchez & Etienne Jacotot

  3. Immunologie et Chimie Thérapeutiques CNRS UPR9021, 67000, Strasbourg, France

    Richard Casimir, Jean Paul Briand, Olivier Chaloin & Sylviane Muller

  4. University of Versailles/SQY, PRES UniverSud Paris, CNRS UMR 8159, Versailles, France

    Catherine Brenner

  5. Department of Reproductive Biology, Faculty of Medicine, Imperial College London, Hammersmith Hospital Campus, Du Cane Road, London, W12 0NN, UK

    Etienne Jacotot

Authors
  1. Magali Brabant
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ludwig Baux
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Richard Casimir
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jean Paul Briand
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Olivier Chaloin
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Mathieu Porceddu
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Nelly Buron
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. David Chauvier
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Myriam Lassalle
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Hervé Lecoeur
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Alain Langonné
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Sylvie Dupont
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Olivier Déas
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Catherine Brenner
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Dominique Rebouillat
    View author publications

    You can also search for this author in PubMed Google Scholar

  16. Sylviane Muller
    View author publications

    You can also search for this author in PubMed Google Scholar

  17. Annie Borgne-Sanchez
    View author publications

    You can also search for this author in PubMed Google Scholar

  18. Etienne Jacotot
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Etienne Jacotot.

Additional information

Magali Brabant and Ludwig Baux contributed equally to this work.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Brabant, M., Baux, L., Casimir, R. et al. A flavivirus protein M-derived peptide directly permeabilizes mitochondrial membranes, triggers cell death and reduces human tumor growth in nude mice. Apoptosis 14, 1190–1203 (2009). https://doi.org/10.1007/s10495-009-0394-y

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10495-009-0394-y

Keywords

Search

Navigation