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. 2000 May 1;347(Pt 3):669–677.

Serine protease inhibitors suppress cytochrome c-mediatedcaspase-9 activation and apoptosis during hypoxia-reoxygenation.

Z Dong 1, P Saikumar 1, Y Patel 1, J M Weinberg 1, M A Venkatachalam 1
PMCID: PMC1221002  PMID: 10769169

Abstract

We have shown that reoxygenation of hypoxic rat kidney proximaltubule cells leads to apoptosis. This is mediated by translocation ofBax from the cytosol to mitochondria, accompanied by release ofmitochondrial cytochrome c (cyt.c). The present studyhas examined the proteolytic mechanisms responsible for apoptosisduring hypoxia-reoxygenation. Caspases were activated duringhypoxia, as shown by cleavage of fluorogenic peptide substrates. By5 h caspase-3-like activity to cleave carbobenzoxy-Asp-Glu-Val-Asp-7-amino-4-trifluoromethyl coumarin was increased approx. 30-fold. Thiswas accompanied by specific processing of pro-caspase-3, -8 and -9 intoactive forms. Caspase activation during hypoxia was blocked bycarbobenzoxy-Val-Ala-Asp-fluoromethyl ketone and overexpression of Bcl-2. Of particular interest, caspase activation was also suppressed bythe chymotryptic inhibitors N-tosyl-L-phenylalaninechloromethyl ketone (TPCK) and Ala-Pro-Phe chloromethyl ketone (APF),and the general serine protease inhibitor 4-(2-aminoethyl)benzenesulphonyl fluoride. Inhibition of caspase activationby these compounds resulted in arrest of apoptosis. On the other hand,the serine protease inhibitors did not prevent release of mitochondrialcyt.c during hypoxia, suggesting that these compounds blockeda critical step in post-mitochondrial caspase activation. Furtherstudies using an in vitro reconstitution model showedthat cyt. c/dATP stimulated caspase-9 processing and downstreamcaspase activation were significantly suppressed in the presence ofTPCK and APF. Based on these results, we speculate that serineproteases may be involved in post-mitochondrial apoptotic events thatlead to activation of the initiator, caspase-9.

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Selected References

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  1. Ashkenazi A., Dixit V. M. Death receptors: signaling and modulation. Science. 1998 Aug 28;281(5381):1305–1308. doi: 10.1126/science.281.5381.1305. [DOI] [PubMed] [Google Scholar]
  2. Cain K., Brown D. G., Langlais C., Cohen G. M. Caspase activation involves the formation of the aposome, a large (approximately 700 kDa) caspase-activating complex. J Biol Chem. 1999 Aug 6;274(32):22686–22692. doi: 10.1074/jbc.274.32.22686. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. Cohen G. M. Caspases: the executioners of apoptosis. Biochem J. 1997 Aug 15;326(Pt 1):1–16. doi: 10.1042/bj3260001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cryns V., Yuan J. Proteases to die for. Genes Dev. 1998 Jun 1;12(11):1551–1570. doi: 10.1101/gad.12.11.1551. [DOI] [PubMed] [Google Scholar]
  6. Dong Z., Patel Y., Saikumar P., Weinberg J. M., Venkatachalam M. A. Development of porous defects in plasma membranes of adenosine triphosphate-depleted Madin-Darby canine kidney cells and its inhibition by glycine. Lab Invest. 1998 Jun;78(6):657–668. [PubMed] [Google Scholar]
  7. Fearnhead H. O., Rivett A. J., Dinsdale D., Cohen G. M. A pre-existing protease is a common effector of thymocyte apoptosis mediated by diverse stimuli. FEBS Lett. 1995 Jan 9;357(3):242–246. doi: 10.1016/0014-5793(94)01367-a. [DOI] [PubMed] [Google Scholar]
  8. Hara H., Friedlander R. M., Gagliardini V., Ayata C., Fink K., Huang Z., Shimizu-Sasamata M., Yuan J., Moskowitz M. A. Inhibition of interleukin 1beta converting enzyme family proteases reduces ischemic and excitotoxic neuronal damage. Proc Natl Acad Sci U S A. 1997 Mar 4;94(5):2007–2012. doi: 10.1073/pnas.94.5.2007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Harrison-Shostak D. C., Lemasters J. J., Edgell C. J., Herman B. Role of ICE-like proteases in endothelial cell hypoxic and reperfusion injury. Biochem Biophys Res Commun. 1997 Feb 24;231(3):844–847. doi: 10.1006/bbrc.1997.6129. [DOI] [PubMed] [Google Scholar]
  10. Jones R. A., Johnson V. L., Buck N. R., Dobrota M., Hinton R. H., Chow S. C., Kass G. E. Fas-mediated apoptosis in mouse hepatocytes involves the processing and activation of caspases. Hepatology. 1998 Jun;27(6):1632–1642. doi: 10.1002/hep.510270624. [DOI] [PubMed] [Google Scholar]
  11. Kumar S., Harvey N. L. Role of multiple cellular proteases in the execution of programmed cell death. FEBS Lett. 1995 Nov 20;375(3):169–173. doi: 10.1016/0014-5793(95)01186-i. [DOI] [PubMed] [Google Scholar]
  12. Li P., Nijhawan D., Budihardjo I., Srinivasula S. M., Ahmad M., Alnemri E. S., Wang X. Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell. 1997 Nov 14;91(4):479–489. doi: 10.1016/s0092-8674(00)80434-1. [DOI] [PubMed] [Google Scholar]
  13. Martin S. J., Green D. R. Protease activation during apoptosis: death by a thousand cuts? Cell. 1995 Aug 11;82(3):349–352. doi: 10.1016/0092-8674(95)90422-0. [DOI] [PubMed] [Google Scholar]
  14. Sadoul R., Fernandez P. A., Quiquerez A. L., Martinou I., Maki M., Schröter M., Becherer J. D., Irmler M., Tschopp J., Martinou J. C. Involvement of the proteasome in the programmed cell death of NGF-deprived sympathetic neurons. EMBO J. 1996 Aug 1;15(15):3845–3852. [PMC free article] [PubMed] [Google Scholar]
  15. Saikumar P., Dong Z., Patel Y., Hall K., Hopfer U., Weinberg J. M., Venkatachalam M. A. Role of hypoxia-induced Bax translocation and cytochrome c release in reoxygenation injury. Oncogene. 1998 Dec 31;17(26):3401–3415. doi: 10.1038/sj.onc.1202590. [DOI] [PubMed] [Google Scholar]
  16. Saikumar P., Dong Z., Weinberg J. M., Venkatachalam M. A. Mechanisms of cell death in hypoxia/reoxygenation injury. Oncogene. 1998 Dec 24;17(25):3341–3349. doi: 10.1038/sj.onc.1202579. [DOI] [PubMed] [Google Scholar]
  17. Salvesen G. S., Dixit V. M. Caspases: intracellular signaling by proteolysis. Cell. 1997 Nov 14;91(4):443–446. doi: 10.1016/s0092-8674(00)80430-4. [DOI] [PubMed] [Google Scholar]
  18. Shimizu S., Eguchi Y., Kamiike W., Akao Y., Kosaka H., Hasegawa J., Matsuda H., Tsujimoto Y. Involvement of ICE family proteases in apoptosis induced by reoxygenation of hypoxic hepatocytes. Am J Physiol. 1996 Dec;271(6 Pt 1):G949–G958. doi: 10.1152/ajpgi.1996.271.6.G949. [DOI] [PubMed] [Google Scholar]
  19. Shimizu T., Pommier Y. Camptothecin-induced apoptosis in p53-null human leukemia HL60 cells and their isolated nuclei: effects of the protease inhibitors Z-VAD-fmk and dichloroisocoumarin suggest an involvement of both caspases and serine proteases. Leukemia. 1997 Aug;11(8):1238–1244. doi: 10.1038/sj.leu.2400734. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. Stennicke H. R., Salvesen G. S. Biochemical characteristics of caspases-3, -6, -7, and -8. J Biol Chem. 1997 Oct 10;272(41):25719–25723. doi: 10.1074/jbc.272.41.25719. [DOI] [PubMed] [Google Scholar]
  22. Talanian R. V., Quinlan C., Trautz S., Hackett M. C., Mankovich J. A., Banach D., Ghayur T., Brady K. D., Wong W. W. Substrate specificities of caspase family proteases. J Biol Chem. 1997 Apr 11;272(15):9677–9682. doi: 10.1074/jbc.272.15.9677. [DOI] [PubMed] [Google Scholar]
  23. Thornberry N. A., Lazebnik Y. Caspases: enemies within. Science. 1998 Aug 28;281(5381):1312–1316. doi: 10.1126/science.281.5381.1312. [DOI] [PubMed] [Google Scholar]
  24. Thornberry N. A., Rano T. A., Peterson E. P., Rasper D. M., Timkey T., Garcia-Calvo M., Houtzager V. M., Nordstrom P. A., Roy S., Vaillancourt J. P. A combinatorial approach defines specificities of members of the caspase family and granzyme B. Functional relationships established for key mediators of apoptosis. J Biol Chem. 1997 Jul 18;272(29):17907–17911. doi: 10.1074/jbc.272.29.17907. [DOI] [PubMed] [Google Scholar]
  25. Weaver V. M., Lach B., Walker P. R., Sikorska M. Role of proteolysis in apoptosis: involvement of serine proteases in internucleosomal DNA fragmentation in immature thymocytes. Biochem Cell Biol. 1993 Sep-Oct;71(9-10):488–500. doi: 10.1139/o93-071. [DOI] [PubMed] [Google Scholar]
  26. Weis M., Schlegel J., Kass G. E., Holmström T. H., Peters I., Eriksson J., Orrenius S., Chow S. C. Cellular events in Fas/APO-1-mediated apoptosis in JURKAT T lymphocytes. Exp Cell Res. 1995 Aug;219(2):699–708. doi: 10.1006/excr.1995.1281. [DOI] [PubMed] [Google Scholar]
  27. Yaoita H., Ogawa K., Maehara K., Maruyama Y. Attenuation of ischemia/reperfusion injury in rats by a caspase inhibitor. Circulation. 1998 Jan 27;97(3):276–281. doi: 10.1161/01.cir.97.3.276. [DOI] [PubMed] [Google Scholar]
  28. Zhou Q., Salvesen G. S. Activation of pro-caspase-7 by serine proteases includes a non-canonical specificity. Biochem J. 1997 Jun 1;324(Pt 2):361–364. doi: 10.1042/bj3240361. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Zhu H., Dinsdale D., Alnemri E. S., Cohen G. M. Apoptosis in human monocytic THP.1 cells involves several distinct targets of N-tosyl-L-phenylalanyl chloromethyl ketone (TPCK). Cell Death Differ. 1997 Oct;4(7):590–599. doi: 10.1038/sj.cdd.4400284. [DOI] [PubMed] [Google Scholar]
  30. Zhuang J., Cohen G. M. Release of mitochondrial cytochrome c is upstream of caspase activation in chemical-induced apoptosis in human monocytic tumour cells. Toxicol Lett. 1998 Dec 28;102-103:121–129. doi: 10.1016/s0378-4274(98)00296-3. [DOI] [PubMed] [Google Scholar]
  31. Zou H., Li Y., Liu X., Wang X. An APAF-1.cytochrome c multimeric complex is a functional apoptosome that activates procaspase-9. J Biol Chem. 1999 Apr 23;274(17):11549–11556. doi: 10.1074/jbc.274.17.11549. [DOI] [PubMed] [Google Scholar]

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