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Positional Scanning Substrate Combinatorial Library (PS-SCL) Approach to Define Caspase Substrate Specificity

  • Marcin Poręba
  • Aleksandra Szalek
  • Paulina Kasperkiewicz
  • Marcin Drąg
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1133)

Abstract

Positional scanning substrate combinatorial library (PS-SCL) is a powerful tool for studying substrate specificity of proteolytic enzymes. Here, we describe the protocol for analyzing S4-S2 pockets preferences of caspases using PS-SCL. Additionally, we describe procedures for the identification of optimal substrates sequence after PS-SCL, solid phase synthesis, and purification of selected fluorogenic substrates, as well as their kinetic analysis.

Key words

Substrate specificity Caspase Fluorogenic substrate Combinatorial library Cysteine protease 

Notes

Acknowledgments

This work was supported by the National Science Centre grant 2011/03/B/ST5/01048 and the Foundation for Polish Science in Poland. This work is co-financed by the European Union as part of the European Social Fund.

References

  1. 1.
    Drag M, Salvesen GS (2010) Emerging principles in protease-based drug discovery. Nat Rev Drug Discov 9(9):690–701. doi: 10.1038/nrd3053 PubMedCrossRefPubMedCentralGoogle Scholar
  2. 2.
    Fuentes-Prior P, Salvesen GS (2004) The protein structures that shape caspase activity, specificity, activation and inhibition. Biochem J 384(Pt 2):201–232. doi: 10.1042/BJ20041142 PubMedGoogle Scholar
  3. 3.
    Salvesen GS, Dixit VM (1997) Caspases: intracellular signaling by proteolysis. Cell 91(4):443–446PubMedCrossRefGoogle Scholar
  4. 4.
    Pop C, Salvesen GS (2009) Human caspases: activation, specificity, and regulation. J Biol Chem 284(33):21777–21781. doi: 10.1074/jbc.R800084200, R800084200PubMedCrossRefGoogle Scholar
  5. 5.
    Thornberry NA, Lazebnik Y (1998) Caspases: enemies within. Science 281(5381):1312–1316PubMedCrossRefGoogle Scholar
  6. 6.
    Poreba M, Drag M (2010) Current strategies for probing substrate specificity of proteases. Curr Med Chem 17(33):3968–3995PubMedCrossRefGoogle Scholar
  7. 7.
    Ostresh JM, Winkle JH, Hamashin VT et al (1994) Peptide libraries: determination of relative reaction rates of protected amino acids in competitive couplings. Biopolymers 34(12):1681–1689. doi: 10.1002/bip.360341212 PubMedCrossRefGoogle Scholar
  8. 8.
    Rano TA, Timkey T, Peterson EP et al (1997) A combinatorial approach for determining protease specificities: application to interleukin-1beta converting enzyme (ICE). Chem Biol 4(2):149–155, S1074-5521(97)90258-1 [pii]PubMedCrossRefGoogle Scholar
  9. 9.
    Sleath PR, Hendrickson RC, Kronheim SR et al (1990) Substrate specificity of the protease that processes human interleukin-1 beta. J Biol Chem 265(24):14526–14528PubMedGoogle Scholar
  10. 10.
    Howard AD, Kostura MJ, Thornberry N et al (1991) IL-1-converting enzyme requires aspartic acid residues for processing of the IL-1 beta precursor at two distinct sites and does not cleave 31-kDa IL-1 alpha. J Immunol 147(9):2964–2969PubMedGoogle Scholar
  11. 11.
    Thornberry NA, Bull HG, Calaycay JR et al (1992) A novel heterodimeric cysteine protease is required for interleukin-1 beta processing in monocytes. Nature 356(6372):768–774. doi: 10.1038/356768a0 PubMedCrossRefGoogle Scholar
  12. 12.
    Thornberry NA, Molineaux SM (1995) Interleukin-1 beta converting enzyme: a novel cysteine protease required for IL-1 beta production and implicated in programmed cell death. Protein Sci 4(1):3–12. doi: 10.1002/pro.5560040102 PubMedCrossRefGoogle Scholar
  13. 13.
    Thornberry NA, Rano TA, Peterson EP et al (1997) 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 272(29):17907–17911PubMedCrossRefGoogle Scholar
  14. 14.
    Garcia-Calvo M, Peterson EP, Rasper DM et al (1999) Purification and catalytic properties of human caspase family members. Cell Death Differ 6(4):362–369. doi: 10.1038/sj.cdd.4400497 PubMedCrossRefGoogle Scholar
  15. 15.
    Wachmann K, Pop C, van Raam BJ et al (2010) Activation and specificity of human caspase-10. Biochemistry 49(38):8307–8315. doi: 10.1021/bi100968m PubMedCrossRefPubMedCentralGoogle Scholar
  16. 16.
    Mikolajczyk J, Scott FL, Krajewski S et al (2004) Activation and substrate specificity of caspase-14. Biochemistry 43(32):10560–10569. doi: 10.1021/bi0498048 PubMedCrossRefGoogle Scholar
  17. 17.
    Edwards PD, Mauger RC, Cottrell KM et al (2000) Synthesis and enzymatic evaluation of a P1 arginine aminocoumarin substrate library for trypsin-like serine proteases. Bioorg Med Chem Lett 10(20):2291–2294PubMedCrossRefGoogle Scholar
  18. 18.
    Backes BJ, Harris JL, Leonetti F et al (2000) Synthesis of positional-scanning libraries of fluorogenic peptide substrates to define the extended substrate specificity of plasmin and thrombin. Nat Biotechnol 18(2):187–193. doi: 10.1038/72642 PubMedCrossRefGoogle Scholar
  19. 19.
    Harris JL, Backes BJ, Leonetti F et al (2000) Rapid and general profiling of protease specificity by using combinatorial fluorogenic substrate libraries. Proc Natl Acad Sci U S A 97(14):7754–7759. doi: 10.1073/pnas.140132697, 140132697 [pii]PubMedCrossRefPubMedCentralGoogle Scholar
  20. 20.
    Maly DJ, Leonetti F, Backes BJ et al (2002) Expedient solid-phase synthesis of fluorogenic protease substrates using the 7-amino-4-carbamoylmethylcoumarin (ACC) fluorophore. J Org Chem 67(3):910–915, jo016140o [pii]PubMedCrossRefGoogle Scholar
  21. 21.
    Walters J, Pop C, Scott FL et al (2009) A constitutively active and uninhibitable caspase-3 zymogen efficiently induces apoptosis. Biochem J 424(3):335–345. doi: 10.1042/BJ20090825 PubMedCrossRefPubMedCentralGoogle Scholar
  22. 22.
    Choe Y, Leonetti F, Greenbaum DC et al (2006) Substrate profiling of cysteine proteases using a combinatorial peptide library identifies functionally unique specificities. J Biol Chem 281(18):12824–12832. doi: 10.1074/jbc.M513331200 PubMedCrossRefGoogle Scholar
  23. 23.
    Debela M, Magdolen V, Schechter N et al (2006) Specificity profiling of seven human tissue kallikreins reveals individual subsite preferences. J Biol Chem 281(35):25678–25688. doi: 10.1074/jbc.M602372200, M602372200 [pii]PubMedCrossRefGoogle Scholar
  24. 24.
    Hachmann J, Snipas SJ, van Raam BJ et al (2012) Mechanism and specificity of the human paracaspase MALT1. Biochem J 443(1):287–295. doi: 10.1042/BJ20120035 PubMedCrossRefPubMedCentralGoogle Scholar
  25. 25.
    Drag M, Mikolajczyk J, Bekes M et al (2008) Positional-scanning fluorigenic substrate libraries reveal unexpected specificity determinants of DUBs (deubiquitinating enzymes). Biochem J 415(3):367–375. doi: 10.1042/BJ20080779, BJ20080779 [pii]PubMedCrossRefPubMedCentralGoogle Scholar
  26. 26.
    Garcia-Calvo M, Peterson EP, Leiting B et al (1998) Inhibition of human caspases by peptide-based and macromolecular inhibitors. J Biol Chem 273(49):32608–32613PubMedCrossRefGoogle Scholar
  27. 27.
    Stennicke HR, Salvesen GS (1999) Caspases: preparation and characterization. Methods 17(4):313–319. doi: 10.1006/meth.1999.0745 PubMedCrossRefGoogle Scholar
  28. 28.
    Ekici OD, Li ZZ, Campbell AJ et al (2006) Design, synthesis, and evaluation of aza-peptide Michael acceptors as selective and potent inhibitors of caspases-2, -3, -6, -7, -8, -9, and -10. J Med Chem 49(19):5728–5749. doi: 10.1021/jm0601405 PubMedCrossRefGoogle Scholar
  29. 29.
    Fu J, Yang Y, Zhang XW et al (2010) Discovery of 1H-benzo[d][1,2,3]triazol-1-yl 3,4,5-trimethoxybenzoate as a potential antiproliferative agent by inhibiting histone deacetylase. Bioorg Med Chem 18(24):8457–8462. doi: 10.1016/j.bmc.2010.10.049 PubMedCrossRefGoogle Scholar
  30. 30.
    Carpino LA, Han GY (1972) 9-Fluorenylmethoxycarbonyl amino-protecting group. J Org Chem 37(22):3404–3409CrossRefGoogle Scholar
  31. 31.
    Kaiser E, Colescott RL, Bossinger CD et al (1970) Color test for detection of free terminal amino groups in the solid-phase synthesis of peptides. Anal Biochem 34(2):595–598PubMedCrossRefGoogle Scholar
  32. 32.
    Chang CD, Meienhofer J (1978) Solid-phase peptide synthesis using mild base cleavage of N alpha-fluorenylmethyloxycarbonylamino acids, exemplified by a synthesis of dihydrosomatostatin. Int J Pept Protein Res 11(3):246–249PubMedCrossRefGoogle Scholar
  33. 33.
    Chan WC, White PD (2000) Fmoc solid phase peptide synthesis, the practical approach series. Oxford University Press, New York, pp 1–74Google Scholar
  34. 34.
    Merrifield RB (1963) Solid phase peptide synthesis. I. The synthesis of a tetrapeptide. J Am Chem Soc 85(14):2149–2154CrossRefGoogle Scholar
  35. 35.
    McStay GP, Salvesen GS, Green DR (2008) Overlapping cleavage motif selectivity of caspases: implications for analysis of apoptotic pathways. Cell Death Differ 15(2):322–331. doi: 10.1038/sj.cdd.4402260 PubMedCrossRefGoogle Scholar
  36. 36.
    Stennicke HR, Salvesen GS (1997) Biochemical characteristics of caspases-3, -6, -7, and -8. J Biol Chem 272(41):25719–25723PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Marcin Poręba
    • 1
  • Aleksandra Szalek
    • 1
  • Paulina Kasperkiewicz
    • 1
  • Marcin Drąg
    • 1
  1. 1.Division of Bioorganic Chemistry, Faculty of ChemistryWroclaw University of TechnologyWroclawPoland

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