Skip to main content
PMC home page
The Journal of Experimental Medicine logoLink to The Journal of Experimental Medicine
. 1996 Nov 1;184(5):2067–2072. doi: 10.1084/jem.184.5.2067

Systemic injection of a tripeptide inhibits the intracellular activation of CPP32-like proteases in vivo and fully protects mice against Fas-mediated fulminant liver destruction and death

PMCID: PMC2192862  PMID: 8920897

Abstract

Mice injected with anti-Fas antibody die within a few hours with total liver destruction due to massive apoptosis of hepatocytes. We show that this is preceded and accompanied by the sequential activation of cysteine proteases of the interleukin 1 beta-converting enzyme (ICE) and CPP32 types in the cytosol of the hepatocytes, and that proCPP32 cleavage and enzymatic activity can be prevented by intravenous injections of the tripeptide N-benzyloxycarbonyl-Val-Ala-Asp- fluoromethylketone (Z-VAD.fmk), an inhibitor of ICE-like proteases. Four Z-VAD.fmk injections at 1-hour intervals abolished all signs of liver damage after anti-Fas antibody injection and resulted in 100% long-range recovery, without residual tissue damage, from a condition otherwise uniformly fatal within < 3 hours. This treatment was effective even when delayed until some liver DNA degradation was already detectable. Injections of the tetrapeptide Ac-YVAD.cmk, more specific for the ICE-like subfamily of cysteine proteases but less cell permeable, also gave protection, but at higher doses and when injections started before that of anti-Fas antibody. These observations afford a way of temporarily modulating a number of apoptotic processes in vivo and may have important therapeutic implications in some human diseases.

Full Text

The Full Text of this article is available as a PDF (684.7 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Boldin M. P., Goncharov T. M., Goltsev Y. V., Wallach D. Involvement of MACH, a novel MORT1/FADD-interacting protease, in Fas/APO-1- and TNF receptor-induced cell death. Cell. 1996 Jun 14;85(6):803–815. doi: 10.1016/s0092-8674(00)81265-9. [DOI] [PubMed] [Google Scholar]
  2. Cain K., Inayat-Hussain S. H., Couet C., Cohen G. M. A cleavage-site-directed inhibitor of interleukin-1 beta-converting enzyme-like proteases inhibits apoptosis in primary cultures of rat hepatocytes. Biochem J. 1996 Feb 15;314(Pt 1):27–32. doi: 10.1042/bj3140027. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chinnaiyan A. M., Dixit V. M. The cell-death machine. Curr Biol. 1996 May 1;6(5):555–562. doi: 10.1016/s0960-9822(02)00541-9. [DOI] [PubMed] [Google Scholar]
  4. Darmon A. J., Nicholson D. W., Bleackley R. C. Activation of the apoptotic protease CPP32 by cytotoxic T-cell-derived granzyme B. Nature. 1995 Oct 5;377(6548):446–448. doi: 10.1038/377446a0. [DOI] [PubMed] [Google Scholar]
  5. Dolle R. E., Hoyer D., Prasad C. V., Schmidt S. J., Helaszek C. T., Miller R. E., Ator M. A. P1 aspartate-based peptide alpha-((2,6-dichlorobenzoyl)oxy)methyl ketones as potent time-dependent inhibitors of interleukin-1 beta-converting enzyme. J Med Chem. 1994 Mar 4;37(5):563–564. doi: 10.1021/jm00031a003. [DOI] [PubMed] [Google Scholar]
  6. Duan H., Chinnaiyan A. M., Hudson P. L., Wing J. P., He W. W., Dixit V. M. ICE-LAP3, a novel mammalian homologue of the Caenorhabditis elegans cell death protein Ced-3 is activated during Fas- and tumor necrosis factor-induced apoptosis. J Biol Chem. 1996 Jan 19;271(3):1621–1625. doi: 10.1074/jbc.271.3.1621. [DOI] [PubMed] [Google Scholar]
  7. Enari M., Hug H., Nagata S. Involvement of an ICE-like protease in Fas-mediated apoptosis. Nature. 1995 May 4;375(6526):78–81. doi: 10.1038/375078a0. [DOI] [PubMed] [Google Scholar]
  8. Enari M., Talanian R. V., Wong W. W., Nagata S. Sequential activation of ICE-like and CPP32-like proteases during Fas-mediated apoptosis. Nature. 1996 Apr 25;380(6576):723–726. doi: 10.1038/380723a0. [DOI] [PubMed] [Google Scholar]
  9. Jacobsen M. D., Weil M., Raff M. C. Role of Ced-3/ICE-family proteases in staurosporine-induced programmed cell death. J Cell Biol. 1996 Jun;133(5):1041–1051. doi: 10.1083/jcb.133.5.1041. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Kägi D., Vignaux F., Ledermann B., Bürki K., Depraetere V., Nagata S., Hengartner H., Golstein P. Fas and perforin pathways as major mechanisms of T cell-mediated cytotoxicity. Science. 1994 Jul 22;265(5171):528–530. doi: 10.1126/science.7518614. [DOI] [PubMed] [Google Scholar]
  11. Lazebnik Y. A., Kaufmann S. H., Desnoyers S., Poirier G. G., Earnshaw W. C. Cleavage of poly(ADP-ribose) polymerase by a proteinase with properties like ICE. Nature. 1994 Sep 22;371(6495):346–347. doi: 10.1038/371346a0. [DOI] [PubMed] [Google Scholar]
  12. Liu X., Kim C. N., Pohl J., Wang X. Purification and characterization of an interleukin-1beta-converting enzyme family protease that activates cysteine protease P32 (CPP32). J Biol Chem. 1996 Jun 7;271(23):13371–13376. [PubMed] [Google Scholar]
  13. Los M., Van de Craen M., Penning L. C., Schenk H., Westendorp M., Baeuerle P. A., Dröge W., Krammer P. H., Fiers W., Schulze-Osthoff K. Requirement of an ICE/CED-3 protease for Fas/APO-1-mediated apoptosis. Nature. 1995 May 4;375(6526):81–83. doi: 10.1038/375081a0. [DOI] [PubMed] [Google Scholar]
  14. Martin S. J., Amarante-Mendes G. P., Shi L., Chuang T. H., Casiano C. A., O'Brien G. A., Fitzgerald P., Tan E. M., Bokoch G. M., Greenberg A. H. The cytotoxic cell protease granzyme B initiates apoptosis in a cell-free system by proteolytic processing and activation of the ICE/CED-3 family protease, CPP32, via a novel two-step mechanism. EMBO J. 1996 May 15;15(10):2407–2416. [PMC free article] [PubMed] [Google Scholar]
  15. Milligan C. E., Prevette D., Yaginuma H., Homma S., Cardwell C., Fritz L. C., Tomaselli K. J., Oppenheim R. W., Schwartz L. M. Peptide inhibitors of the ICE protease family arrest programmed cell death of motoneurons in vivo and in vitro. Neuron. 1995 Aug;15(2):385–393. doi: 10.1016/0896-6273(95)90042-x. [DOI] [PubMed] [Google Scholar]
  16. Miura M., Zhu H., Rotello R., Hartwieg E. A., Yuan J. Induction of apoptosis in fibroblasts by IL-1 beta-converting enzyme, a mammalian homolog of the C. elegans cell death gene ced-3. Cell. 1993 Nov 19;75(4):653–660. doi: 10.1016/0092-8674(93)90486-a. [DOI] [PubMed] [Google Scholar]
  17. Muzio M., Chinnaiyan A. M., Kischkel F. C., O'Rourke K., Shevchenko A., Ni J., Scaffidi C., Bretz J. D., Zhang M., Gentz R. FLICE, a novel FADD-homologous ICE/CED-3-like protease, is recruited to the CD95 (Fas/APO-1) death--inducing signaling complex. Cell. 1996 Jun 14;85(6):817–827. doi: 10.1016/s0092-8674(00)81266-0. [DOI] [PubMed] [Google Scholar]
  18. Nicholson D. W., Ali A., Thornberry N. A., Vaillancourt J. P., Ding C. K., Gallant M., Gareau Y., Griffin P. R., Labelle M., Lazebnik Y. A. Identification and inhibition of the ICE/CED-3 protease necessary for mammalian apoptosis. Nature. 1995 Jul 6;376(6535):37–43. doi: 10.1038/376037a0. [DOI] [PubMed] [Google Scholar]
  19. Nicholson D. W. ICE/CED3-like proteases as therapeutic targets for the control of inappropriate apoptosis. Nat Biotechnol. 1996 Mar;14(3):297–301. doi: 10.1038/nbt0396-297. [DOI] [PubMed] [Google Scholar]
  20. Ogasawara J., Watanabe-Fukunaga R., Adachi M., Matsuzawa A., Kasugai T., Kitamura Y., Itoh N., Suda T., Nagata S. Lethal effect of the anti-Fas antibody in mice. Nature. 1993 Aug 26;364(6440):806–809. doi: 10.1038/364806a0. [DOI] [PubMed] [Google Scholar]
  21. Quan L. T., Tewari M., O'Rourke K., Dixit V., Snipas S. J., Poirier G. G., Ray C., Pickup D. J., Salvesen G. S. Proteolytic activation of the cell death protease Yama/CPP32 by granzyme B. Proc Natl Acad Sci U S A. 1996 Mar 5;93(5):1972–1976. doi: 10.1073/pnas.93.5.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Ramage P., Cheneval D., Chvei M., Graff P., Hemmig R., Heng R., Kocher H. P., Mackenzie A., Memmert K., Revesz L. Expression, refolding, and autocatalytic proteolytic processing of the interleukin-1 beta-converting enzyme precursor. J Biol Chem. 1995 Apr 21;270(16):9378–9383. doi: 10.1074/jbc.270.16.9378. [DOI] [PubMed] [Google Scholar]
  23. Slee E. A., Zhu H., Chow S. C., MacFarlane M., Nicholson D. W., Cohen G. M. Benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethylketone (Z-VAD.FMK) inhibits apoptosis by blocking the processing of CPP32. Biochem J. 1996 Apr 1;315(Pt 1):21–24. doi: 10.1042/bj3150021. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Thornberry N. A., Bull H. G., Calaycay J. R., Chapman K. T., Howard A. D., Kostura M. J., Miller D. K., Molineaux S. M., Weidner J. R., Aunins J. A novel heterodimeric cysteine protease is required for interleukin-1 beta processing in monocytes. Nature. 1992 Apr 30;356(6372):768–774. doi: 10.1038/356768a0. [DOI] [PubMed] [Google Scholar]
  25. Thornberry N. A., Peterson E. P., Zhao J. J., Howard A. D., Griffin P. R., Chapman K. T. Inactivation of interleukin-1 beta converting enzyme by peptide (acyloxy)methyl ketones. Biochemistry. 1994 Apr 5;33(13):3934–3940. doi: 10.1021/bi00179a020. [DOI] [PubMed] [Google Scholar]
  26. Wong G. H., Elwell J. H., Oberley L. W., Goeddel D. V. Manganous superoxide dismutase is essential for cellular resistance to cytotoxicity of tumor necrosis factor. Cell. 1989 Sep 8;58(5):923–931. doi: 10.1016/0092-8674(89)90944-6. [DOI] [PubMed] [Google Scholar]
  27. Yuan J., Shaham S., Ledoux S., Ellis H. M., Horvitz H. R. The C. elegans cell death gene ced-3 encodes a protein similar to mammalian interleukin-1 beta-converting enzyme. Cell. 1993 Nov 19;75(4):641–652. doi: 10.1016/0092-8674(93)90485-9. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of Experimental Medicine are provided here courtesy of The Rockefeller University Press

RESOURCES