Internally quenched fluorescent peptide substrates disclose the subsite preferences of human caspases 1, 3, 6, 7 and 8 (original) (raw)

. 2000 Sep 1;350(Pt 2):563–568.

Abstract

Subsite interactions are considered to define the stringent specificity of proteases for their natural substrates. To probe this issue in the proteolytic pathways leading to apoptosis we have examined the P(4), P(1) and P(1)' subsite preferences of human caspases 1, 3, 6, 7 and 8, using internally quenched fluorescent peptide substrates containing o-aminobenzoyl (also known as anthranilic acid) and 3-nitro-tyrosine. Previous work has demonstrated the importance of the S(4) subsite in directing specificity within the caspase family. Here we demonstrate the influence of the S(1) and S(1)' subsites that flank the scissile peptide bond. The S(1) subsite, the major specificity-determining site of the caspases, demonstrates tremendous selectivity, with a 20000-fold preference for cleaving substrates containing aspartic acid over glutamic acid at this position. Thus caspases are among the most selective of known endopeptidases. We find that the caspases show an unexpected degree of discrimination in the P(1)' position, with a general preference for small amino acid residues such as alanine, glycine and serine, with glycine being the preferred substituent. Large aromatic residues are also surprisingly well-tolerated, but charged residues are prohibited. While this describes the general order of P(1)' subsite preferences within the caspase family, there are some differences in individual profiles, with caspase-3 being particularly promiscuous. Overall, the subsite preferences can be used to predict natural substrates, but in certain cases the cleavage site within a presumed natural substrate cannot be predicted by looking for the preferred peptide cleavage sites. In the latter case we conclude that second-site interactions may overcome otherwise sub-optimal cleavage sequences.

Full Text

The Full Text of this article is available as a PDF (192.9 KB).

Selected References

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

  1. Blanchard H., Kodandapani L., Mittl P. R., Marco S. D., Krebs J. F., Wu J. C., Tomaselli K. J., Grütter M. G. The three-dimensional structure of caspase-8: an initiator enzyme in apoptosis. Structure. 1999 Sep 15;7(9):1125–1133. doi: 10.1016/s0969-2126(99)80179-8. [DOI] [PubMed] [Google Scholar]
  2. Bode W., Papamokos E., Musil D., Seemueller U., Fritz H. Refined 1.2 A crystal structure of the complex formed between subtilisin Carlsberg and the inhibitor eglin c. Molecular structure of eglin and its detailed interaction with subtilisin. EMBO J. 1986 Apr;5(4):813–818. doi: 10.1002/j.1460-2075.1986.tb04286.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Breddam K., Meldal M. Substrate preferences of glutamic-acid-specific endopeptidases assessed by synthetic peptide substrates based on intramolecular fluorescence quenching. Eur J Biochem. 1992 May 15;206(1):103–107. doi: 10.1111/j.1432-1033.1992.tb16906.x. [DOI] [PubMed] [Google Scholar]
  4. Casciola-Rosen L., Nicholson D. W., Chong T., Rowan K. R., Thornberry N. A., Miller D. K., Rosen A. Apopain/CPP32 cleaves proteins that are essential for cellular repair: a fundamental principle of apoptotic death. J Exp Med. 1996 May 1;183(5):1957–1964. doi: 10.1084/jem.183.5.1957. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. 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]
  6. Ding L., Coombs G. S., Strandberg L., Navre M., Corey D. R., Madison E. L. Origins of the specificity of tissue-type plasminogen activator. Proc Natl Acad Sci U S A. 1995 Aug 15;92(17):7627–7631. doi: 10.1073/pnas.92.17.7627. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Garcia-Calvo M., Peterson E. P., Leiting B., Ruel R., Nicholson D. W., Thornberry N. A. Inhibition of human caspases by peptide-based and macromolecular inhibitors. J Biol Chem. 1998 Dec 4;273(49):32608–32613. doi: 10.1074/jbc.273.49.32608. [DOI] [PubMed] [Google Scholar]
  8. Garcia-Calvo M., Peterson E. P., Rasper D. M., Vaillancourt J. P., Zamboni R., Nicholson D. W., Thornberry N. A. Purification and catalytic properties of human caspase family members. Cell Death Differ. 1999 Apr;6(4):362–369. doi: 10.1038/sj.cdd.4400497. [DOI] [PubMed] [Google Scholar]
  9. Germain M., Affar E. B., D'Amours D., Dixit V. M., Salvesen G. S., Poirier G. G. Cleavage of automodified poly(ADP-ribose) polymerase during apoptosis. Evidence for involvement of caspase-7. J Biol Chem. 1999 Oct 1;274(40):28379–28384. doi: 10.1074/jbc.274.40.28379. [DOI] [PubMed] [Google Scholar]
  10. Gervais F. G., Xu D., Robertson G. S., Vaillancourt J. P., Zhu Y., Huang J., LeBlanc A., Smith D., Rigby M., Shearman M. S. Involvement of caspases in proteolytic cleavage of Alzheimer's amyloid-beta precursor protein and amyloidogenic A beta peptide formation. Cell. 1999 Apr 30;97(3):395–406. doi: 10.1016/s0092-8674(00)80748-5. [DOI] [PubMed] [Google Scholar]
  11. Grøn H., Meldal M., Breddam K. Extensive comparison of the substrate preferences of two subtilisins as determined with peptide substrates which are based on the principle of intramolecular quenching. Biochemistry. 1992 Jul 7;31(26):6011–6018. doi: 10.1021/bi00141a008. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. Margolin N., Raybuck S. A., Wilson K. P., Chen W., Fox T., Gu Y., Livingston D. J. Substrate and inhibitor specificity of interleukin-1 beta-converting enzyme and related caspases. J Biol Chem. 1997 Mar 14;272(11):7223–7228. doi: 10.1074/jbc.272.11.7223. [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. Mehlen P., Rabizadeh S., Snipas S. J., Assa-Munt N., Salvesen G. S., Bredesen D. E. The DCC gene product induces apoptosis by a mechanism requiring receptor proteolysis. Nature. 1998 Oct 22;395(6704):801–804. doi: 10.1038/27441. [DOI] [PubMed] [Google Scholar]
  16. Meldal M., Breddam K. Anthranilamide and nitrotyrosine as a donor-acceptor pair in internally quenched fluorescent substrates for endopeptidases: multicolumn peptide synthesis of enzyme substrates for subtilisin Carlsberg and pepsin. Anal Biochem. 1991 May 15;195(1):141–147. doi: 10.1016/0003-2697(91)90309-h. [DOI] [PubMed] [Google Scholar]
  17. Merritt E. A., Murphy M. E. Raster3D Version 2.0. A program for photorealistic molecular graphics. Acta Crystallogr D Biol Crystallogr. 1994 Nov 1;50(Pt 6):869–873. doi: 10.1107/S0907444994006396. [DOI] [PubMed] [Google Scholar]
  18. Mittl P. R., Di Marco S., Krebs J. F., Bai X., Karanewsky D. S., Priestle J. P., Tomaselli K. J., Grütter M. G. Structure of recombinant human CPP32 in complex with the tetrapeptide acetyl-Asp-Val-Ala-Asp fluoromethyl ketone. J Biol Chem. 1997 Mar 7;272(10):6539–6547. doi: 10.1074/jbc.272.10.6539. [DOI] [PubMed] [Google Scholar]
  19. Nicholson D. W. Caspase structure, proteolytic substrates, and function during apoptotic cell death. Cell Death Differ. 1999 Nov;6(11):1028–1042. doi: 10.1038/sj.cdd.4400598. [DOI] [PubMed] [Google Scholar]
  20. Odake S., Kam C. M., Narasimhan L., Poe M., Blake J. T., Krahenbuhl O., Tschopp J., Powers J. C. Human and murine cytotoxic T lymphocyte serine proteases: subsite mapping with peptide thioester substrates and inhibition of enzyme activity and cytolysis by isocoumarins. Biochemistry. 1991 Feb 26;30(8):2217–2227. doi: 10.1021/bi00222a027. [DOI] [PubMed] [Google Scholar]
  21. Perona J. J., Craik C. S. Structural basis of substrate specificity in the serine proteases. Protein Sci. 1995 Mar;4(3):337–360. doi: 10.1002/pro.5560040301. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  23. Rano T. A., Timkey T., Peterson E. P., Rotonda J., Nicholson D. W., Becker J. W., Chapman K. T., Thornberry N. A. A combinatorial approach for determining protease specificities: application to interleukin-1beta converting enzyme (ICE). Chem Biol. 1997 Feb;4(2):149–155. doi: 10.1016/s1074-5521(97)90258-1. [DOI] [PubMed] [Google Scholar]
  24. Rotonda J., Nicholson D. W., Fazil K. M., Gallant M., Gareau Y., Labelle M., Peterson E. P., Rasper D. M., Ruel R., Vaillancourt J. P. The three-dimensional structure of apopain/CPP32, a key mediator of apoptosis. Nat Struct Biol. 1996 Jul;3(7):619–625. doi: 10.1038/nsb0796-619. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. Schechter I., Berger A. On the size of the active site in proteases. I. Papain. Biochem Biophys Res Commun. 1967 Apr 20;27(2):157–162. doi: 10.1016/s0006-291x(67)80055-x. [DOI] [PubMed] [Google Scholar]
  27. Simon M. M., Hausmann M., Tran T., Ebnet K., Tschopp J., ThaHla R., Müllbacher A. In vitro- and ex vivo-derived cytolytic leukocytes from granzyme A x B double knockout mice are defective in granule-mediated apoptosis but not lysis of target cells. J Exp Med. 1997 Nov 17;186(10):1781–1786. doi: 10.1084/jem.186.10.1781. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Smith M. M., Shi L., Navre M. Rapid identification of highly active and selective substrates for stromelysin and matrilysin using bacteriophage peptide display libraries. J Biol Chem. 1995 Mar 24;270(12):6440–6449. doi: 10.1074/jbc.270.12.6440. [DOI] [PubMed] [Google Scholar]
  29. Stennicke H. R., Birktoft J. J., Breddam K. Characterization of the S1 binding site of the glutamic acid-specific protease from Streptomyces griseus. Protein Sci. 1996 Nov;5(11):2266–2275. doi: 10.1002/pro.5560051113. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Stennicke H. R., Deveraux Q. L., Humke E. W., Reed J. C., Dixit V. M., Salvesen G. S. Caspase-9 can be activated without proteolytic processing. J Biol Chem. 1999 Mar 26;274(13):8359–8362. doi: 10.1074/jbc.274.13.8359. [DOI] [PubMed] [Google Scholar]
  31. Stennicke H. R., Jürgensmeier J. M., Shin H., Deveraux Q., Wolf B. B., Yang X., Zhou Q., Ellerby H. M., Ellerby L. M., Bredesen D. Pro-caspase-3 is a major physiologic target of caspase-8. J Biol Chem. 1998 Oct 16;273(42):27084–27090. doi: 10.1074/jbc.273.42.27084. [DOI] [PubMed] [Google Scholar]
  32. 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]
  33. Stennicke H. R., Salvesen G. S. Caspases: preparation and characterization. Methods. 1999 Apr;17(4):313–319. doi: 10.1006/meth.1999.0745. [DOI] [PubMed] [Google Scholar]
  34. 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]
  35. 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]
  36. 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]
  37. 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]
  38. Turk D., Guncar G., Podobnik M., Turk B. Revised definition of substrate binding sites of papain-like cysteine proteases. Biol Chem. 1998 Feb;379(2):137–147. doi: 10.1515/bchm.1998.379.2.137. [DOI] [PubMed] [Google Scholar]
  39. Walker N. P., Talanian R. V., Brady K. D., Dang L. C., Bump N. J., Ferenz C. R., Franklin S., Ghayur T., Hackett M. C., Hammill L. D. Crystal structure of the cysteine protease interleukin-1 beta-converting enzyme: a (p20/p10)2 homodimer. Cell. 1994 Jul 29;78(2):343–352. doi: 10.1016/0092-8674(94)90303-4. [DOI] [PubMed] [Google Scholar]
  40. Watt W., Koeplinger K. A., Mildner A. M., Heinrikson R. L., Tomasselli A. G., Watenpaugh K. D. The atomic-resolution structure of human caspase-8, a key activator of apoptosis. Structure. 1999 Sep 15;7(9):1135–1143. doi: 10.1016/s0969-2126(99)80180-4. [DOI] [PubMed] [Google Scholar]
  41. Wilson K. P., Black J. A., Thomson J. A., Kim E. E., Griffith J. P., Navia M. A., Murcko M. A., Chambers S. P., Aldape R. A., Raybuck S. A. Structure and mechanism of interleukin-1 beta converting enzyme. Nature. 1994 Jul 28;370(6487):270–275. doi: 10.1038/370270a0. [DOI] [PubMed] [Google Scholar]
  42. Wolf B. B., Green D. R. Suicidal tendencies: apoptotic cell death by caspase family proteinases. J Biol Chem. 1999 Jul 16;274(29):20049–20052. doi: 10.1074/jbc.274.29.20049. [DOI] [PubMed] [Google Scholar]