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. 1997 Nov;17(11):6502–6507. doi: 10.1128/mcb.17.11.6502

Requirements for proteolysis during apoptosis.

D L Vaux 1, S Wilhelm 1, G Häcker 1
PMCID: PMC232503  PMID: 9343413

Abstract

The key effector proteins of apoptosis are a family of cysteine proteases termed caspases. Following activation of caspases, biochemical events occur that lead to DNA degradation and the characteristic morphological changes associated with apoptosis. Here we show that cytoplasmic extracts activated in vitro by proteinase K were able to cleave the caspase substrate DEVD-7-amino-4-methylcoumarin, while neither proteinase K nor nonactivated extracts were able to do so alone. Caspase-like activity was inhibited by the specific caspase inhibitor DEVD-aldehyde and by the protease inhibitor iodoacetamide, but not by N-ethylmaleimide. When added to isolated nuclei, the activated extracts caused internucleosomal DNA degradation and morphological changes typical of apoptosis. As DNA cleavage and morphological changes could be inhibited by N-ethylmaleimide but not by iodoacetamide, we conclude that during apoptosis, caspase activation causes activation of another cytoplasmic enzyme that can be inhibited by N-ethylmaleimide. Activity of this enzyme is necessary for activation of endonucleases, DNA cleavage, and changes in nuclear morphology.

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Selected References

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  1. Ahmad M., Srinivasula S. M., Wang L., Talanian R. V., Litwack G., Fernandes-Alnemri T., Alnemri E. S. CRADD, a novel human apoptotic adaptor molecule for caspase-2, and FasL/tumor necrosis factor receptor-interacting protein RIP. Cancer Res. 1997 Feb 15;57(4):615–619. [PubMed] [Google Scholar]
  2. Alnemri E. S. Mammalian cell death proteases: a family of highly conserved aspartate specific cysteine proteases. J Cell Biochem. 1997 Jan;64(1):33–42. doi: 10.1002/(sici)1097-4644(199701)64:1<33::aid-jcb6>3.0.co;2-0. [DOI] [PubMed] [Google Scholar]
  3. Boldin M. P., Varfolomeev E. E., Pancer Z., Mett I. L., Camonis J. H., Wallach D. A novel protein that interacts with the death domain of Fas/APO1 contains a sequence motif related to the death domain. J Biol Chem. 1995 Apr 7;270(14):7795–7798. doi: 10.1074/jbc.270.14.7795. [DOI] [PubMed] [Google Scholar]
  4. Chinnaiyan A. M., O'Rourke K., Lane B. R., Dixit V. M. Interaction of CED-4 with CED-3 and CED-9: a molecular framework for cell death. Science. 1997 Feb 21;275(5303):1122–1126. doi: 10.1126/science.275.5303.1122. [DOI] [PubMed] [Google Scholar]
  5. Chinnaiyan A. M., O'Rourke K., Tewari M., Dixit V. M. FADD, a novel death domain-containing protein, interacts with the death domain of Fas and initiates apoptosis. Cell. 1995 May 19;81(4):505–512. doi: 10.1016/0092-8674(95)90071-3. [DOI] [PubMed] [Google Scholar]
  6. Chow S. C., Weis M., Kass G. E., Holmström T. H., Eriksson J. E., Orrenius S. Involvement of multiple proteases during Fas-mediated apoptosis in T lymphocytes. FEBS Lett. 1995 May 8;364(2):134–138. doi: 10.1016/0014-5793(95)00370-o. [DOI] [PubMed] [Google Scholar]
  7. Deiss L. P., Galinka H., Berissi H., Cohen O., Kimchi A. Cathepsin D protease mediates programmed cell death induced by interferon-gamma, Fas/APO-1 and TNF-alpha. EMBO J. 1996 Aug 1;15(15):3861–3870. [PMC free article] [PubMed] [Google Scholar]
  8. Drexler H. C. Activation of the cell death program by inhibition of proteasome function. Proc Natl Acad Sci U S A. 1997 Feb 4;94(3):855–860. doi: 10.1073/pnas.94.3.855. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Duan H., Dixit V. M. RAIDD is a new 'death' adaptor molecule. Nature. 1997 Jan 2;385(6611):86–89. doi: 10.1038/385086a0. [DOI] [PubMed] [Google Scholar]
  10. Fernandes-Alnemri T., Litwack G., Alnemri E. S. Mch2, a new member of the apoptotic Ced-3/Ice cysteine protease gene family. Cancer Res. 1995 Jul 1;55(13):2737–2742. [PubMed] [Google Scholar]
  11. Fernandes-Alnemri T., Takahashi A., Armstrong R., Krebs J., Fritz L., Tomaselli K. J., Wang L., Yu Z., Croce C. M., Salveson G. Mch3, a novel human apoptotic cysteine protease highly related to CPP32. Cancer Res. 1995 Dec 15;55(24):6045–6052. [PubMed] [Google Scholar]
  12. Fujita E., Mukasa T., Tsukahara T., Arahata K., Omura S., Momoi T. Enhancement of CPP32-like activity in the TNF-treated U937 cells by the proteasome inhibitors. Biochem Biophys Res Commun. 1996 Jul 5;224(1):74–79. doi: 10.1006/bbrc.1996.0986. [DOI] [PubMed] [Google Scholar]
  13. Grimm L. M., Goldberg A. L., Poirier G. G., Schwartz L. M., Osborne B. A. Proteasomes play an essential role in thymocyte apoptosis. EMBO J. 1996 Aug 1;15(15):3835–3844. [PMC free article] [PubMed] [Google Scholar]
  14. Katunuma N., Kominami E. Structure, properties, mechanisms, and assays of cysteine protease inhibitors: cystatins and E-64 derivatives. Methods Enzymol. 1995;251:382–397. doi: 10.1016/0076-6879(95)51142-3. [DOI] [PubMed] [Google Scholar]
  15. Lazebnik Y. A., Cole S., Cooke C. A., Nelson W. G., Earnshaw W. C. Nuclear events of apoptosis in vitro in cell-free mitotic extracts: a model system for analysis of the active phase of apoptosis. J Cell Biol. 1993 Oct;123(1):7–22. doi: 10.1083/jcb.123.1.7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Lazebnik Y. A., Takahashi A., Moir R. D., Goldman R. D., Poirier G. G., Kaufmann S. H., Earnshaw W. C. Studies of the lamin proteinase reveal multiple parallel biochemical pathways during apoptotic execution. Proc Natl Acad Sci U S A. 1995 Sep 26;92(20):9042–9046. doi: 10.1073/pnas.92.20.9042. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Liu X., Zou H., Slaughter C., Wang X. DFF, a heterodimeric protein that functions downstream of caspase-3 to trigger DNA fragmentation during apoptosis. Cell. 1997 Apr 18;89(2):175–184. doi: 10.1016/s0092-8674(00)80197-x. [DOI] [PubMed] [Google Scholar]
  18. Lugasi H., Hajos S., Murphy J. R., Strom T. B., Nichols J., Peñarroja C., Naor D. Murine spontaneous T-cell leukemia constitutively expressing IL-2 receptor--a model for human T-cell malignancies expressing IL-2 receptor. Int J Cancer. 1990 Jan 15;45(1):163–167. doi: 10.1002/ijc.2910450129. [DOI] [PubMed] [Google Scholar]
  19. Nagata S. Apoptosis by death factor. Cell. 1997 Feb 7;88(3):355–365. doi: 10.1016/s0092-8674(00)81874-7. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. Rawlings N. D., Barrett A. J. Families of cysteine peptidases. Methods Enzymol. 1994;244:461–486. doi: 10.1016/0076-6879(94)44034-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Sarin A., Adams D. H., Henkart P. A. Protease inhibitors selectively block T cell receptor-triggered programmed cell death in a murine T cell hybridoma and activated peripheral T cells. J Exp Med. 1993 Nov 1;178(5):1693–1700. doi: 10.1084/jem.178.5.1693. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Shinohara K., Tomioka M., Nakano H., Toné S., Ito H., Kawashima S. Apoptosis induction resulting from proteasome inhibition. Biochem J. 1996 Jul 15;317(Pt 2):385–388. doi: 10.1042/bj3170385. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Squier M. K., Cohen J. J. Calpain, an upstream regulator of thymocyte apoptosis. J Immunol. 1997 Apr 15;158(8):3690–3697. [PubMed] [Google Scholar]
  25. Ucker D. S., Obermiller P. S., Eckhart W., Apgar J. R., Berger N. A., Meyers J. Genome digestion is a dispensable consequence of physiological cell death mediated by cytotoxic T lymphocytes. Mol Cell Biol. 1992 Jul;12(7):3060–3069. doi: 10.1128/mcb.12.7.3060. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Williams M. S., Henkart P. A. Apoptotic cell death induced by intracellular proteolysis. J Immunol. 1994 Nov 1;153(9):4247–4255. [PubMed] [Google Scholar]
  27. Wyllie A. H. Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation. Nature. 1980 Apr 10;284(5756):555–556. doi: 10.1038/284555a0. [DOI] [PubMed] [Google Scholar]
  28. Yoshida A., Takauji R., Inuzuka M., Ueda T., Nakamura T. Role of serine and ICE-like proteases in induction of apoptosis by etoposide in human leukemia HL-60 cells. Leukemia. 1996 May;10(5):821–824. [PubMed] [Google Scholar]
  29. 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]

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