Article | Published:

Potent and selective caspase-2 inhibitor prevents MDM-2 cleavage in reversine-treated colon cancer cells

Cell Death & Differentiation (2019) | Download Citation

Abstract

Most caspases can be positioned unambiguously within the regulated cell death networks of apoptosis and pyroptosis, but the role of caspase-2, a highly conserved protease within the family, remains enigmatic. This is mainly due to lack of selective chemical and biochemical tools for the investigation of this protease. In this study, we used our hybrid combinatorial substrate library (HyCoSuL) approach to broadly profile caspase-2 substrate specificity using peptide scanning libraries. This screen uncovered previously unknown caspase-2 peptidyl substrate preferences, which were further used to develop caspase-2 selective fluorogenic substrates and covalent, irreversible AOMK inhibitors. Finally, we used the champion inhibitor (NH-23-C2) in reversine-treated HCT-116 colon cancer cells to selectively block caspase-2 activity and caspase-2-mediated MDM-2 cleavage. In addition, we showed that NH-23-C2 does not block caspase-3 or caspase-8, which makes it a powerful chemical tool to dissect the true role of caspase-2 in various biological setups.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Edited by A. Ashkenazi

References

  1. 1.

    Shalini S, Dorstyn L, Dawar S, Kumar S. Old, new and emerging functions of caspases. Cell Death Differ. 2015;22:526–39.

  2. 2.

    Thornberry NA, Rano TA, Peterson EP, Rasper DM, Timkey T, Garcia-Calvo M, et al. 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;272:17907–11.

  3. 3.

    Shi J, Zhao Y, Wang K, Shi X, Wang Y, Huang H, et al. Cleavage of GSDMD by inflammatory caspases determines pyroptotic cell death. Nature. 2015;526:660–5.

  4. 4.

    Kumar S, Kinoshita M, Noda M, Copeland NG, Jenkins NA. Induction of apoptosis by the mouse Nedd2 gene, which encodes a protein similar to the product of the Caenorhabditis elegans cell death gene ced-3 and the mammalian IL-1 beta-converting enzyme. Genes Dev. 1994;8:1613–26.

  5. 5.

    Wang L, Miura M, Bergeron L, Zhu H, Yuan J. Ich-1, an Ice/ced-3-related gene, encodes both positive and negative regulators of programmed cell death. Cell. 1994;78:739–50.

  6. 6.

    Bouchier-Hayes L, Green DR. Caspase-2: the orphan caspase. Cell Death Differ. 2012;19:51–57.

  7. 7.

    Tiwari M, Sharma LK, Vanegas D, Callaway DA, Bai Y, Lechleiter JD, et al. A nonapoptotic role for CASP2/caspase 2: modulation of autophagy. Autophagy. 2014;10:1054–70.

  8. 8.

    Slee EA, Harte MT, Kluck RM, Wolf BB, Casiano CA, Newmeyer DD, et al. Ordering the cytochrome c-initiated caspase cascade: hierarchical activation of caspases-2, -3, -6, -7, -8, and -10 in a caspase-9-dependent manner. J Cell Biol. 1999;144:281–92.

  9. 9.

    Fava LL, Bock FJ, Geley S, Villunger A. Caspase-2 at a glance. J Cell Sci. 2012;125(Pt 24):5911–5.

  10. 10.

    Krumschnabel G, Sohm B, Bock F, Manzl C, Villunger A. The enigma of caspase-2: the laymen’s view. Cell Death Differ. 2009;16:195–207.

  11. 11.

    Cheung HH, Lynn Kelly N, Liston P, Korneluk RG. Involvement of caspase-2 and caspase-9 in endoplasmic reticulum stress-induced apoptosis: a role for the IAPs. Exp Cell Res. 2006;312:2347–57.

  12. 12.

    Machado MV, Michelotti GA, Jewell ML, Pereira TA, Xie G, Premont RT, et al. Caspase-2 promotes obesity, the metabolic syndrome and nonalcoholic fatty liver disease. Cell Death Dis. 2016;7:e2096.

  13. 13.

    Tu S, McStay GP, Boucher LM, Mak T, Beere HM, Green DR. In situ trapping of activated initiator caspases reveals a role for caspase-2 in heat shock-induced apoptosis. Nat Cell Biol. 2006;8:72–7.

  14. 14.

    Bonzon C, Bouchier-Hayes L, Pagliari LJ, Green DR, Newmeyer DD. Caspase-2-induced apoptosis requires bid cleavage: a physiological role for bid in heat shock-induced death. Mol Biol Cell. 2006;17:2150–7.

  15. 15.

    Manzl C, Peintner L, Krumschnabel G, Bock F, Labi V, Drach M, et al. PIDDosome-independent tumor suppression by Caspase-2. Cell Death Differ. 2012;19:1722–32.

  16. 16.

    Fava LL, Schuler F, Sladky V, Haschka MD, Soratroi C, Eiterer L, et al. The PIDDosome activates p53 in response to supernumerary centrosomes. Genes Dev. 2017;31:34–45.

  17. 17.

    Dorstyn L, Puccini J, Wilson CH, Shalini S, Nicola M, Moore S, et al. Caspase-2 deficiency promotes aberrant DNA-damage response and genetic instability. Cell Death Differ. 2012;19:1288–98.

  18. 18.

    Castedo M, Perfettini JL, Roumier T, Valent A, Raslova H, Yakushijin K, et al. Mitotic catastrophe constitutes a special case of apoptosis whose suppression entails aneuploidy. Oncogene. 2004;23:4362–70.

  19. 19.

    Kumar S. Caspase 2 in apoptosis, the DNA damage response and tumour suppression: enigma no more? Nat Rev Cancer. 2009;9:897–903.

  20. 20.

    Vakifahmetoglu-Norberg H, Zhivotovsky B. The unpredictable caspase-2: what can it do? Trends Cell Biol. 2010;20:150–9.

  21. 21.

    Miles MA, Kitevska-Ilioski T, Hawkins CJ. Old and novel functions of caspase-2. Int Rev Cell Mol Biol. 2017;332:155–212.

  22. 22.

    Ho LH, Taylor R, Dorstyn L, Cakouros D, Bouillet P, Kumar S. A tumor suppressor function for caspase-2. Proc Natl Acad Sci USA. 2009;106:5336–41.

  23. 23.

    Ahmed Z, Kalinski H, Berry M, Almasieh M, Ashush H, Slager N, et al. Ocular neuroprotection by siRNA targeting caspase-2. Cell Death Dis. 2011;2:e173.

  24. 24.

    Jesenberger V, Procyk KJ, Yuan J, Reipert S, Baccarini M. Salmonella-induced caspase-2 activation in macrophages: a novel mechanism in pathogen-mediated apoptosis. J Exp Med. 2000;192:1035–46.

  25. 25.

    Bouchier-Hayes L, Oberst A, McStay GP, Connell S, Tait SW, Dillon CP, et al. Characterization of cytoplasmic caspase-2 activation by induced proximity. Mol Cell. 2009;35:830–40.

  26. 26.

    Zheng TS, Hunot S, Kuida K, Momoi T, Srinivasan A, Nicholson DW, et al. Deficiency in caspase-9 or caspase-3 induces compensatory caspase activation. Nat Med. 2000;6:1241–7.

  27. 27.

    Parrish AB, Freel CD, Kornbluth S. Cellular mechanisms controlling caspase activation and function. Cold Spring Harb Perspect Biol. 2013;5:a008672.

  28. 28.

    Julien O, Zhuang M, Wiita AP, O’Donoghue AJ, Knudsen GM, Craik CS, et al. Quantitative MS-based enzymology of caspases reveals distinct protein substrate specificities, hierarchies, and cellular roles. Proc Natl Acad Sci USA. 2016;113:E2001–10.

  29. 29.

    Wejda M, Impens F, Takahashi N, Van Damme P, Gevaert K, Vandenabeele P. Degradomics reveals that cleavage specificity profiles of caspase-2 and effector caspases are alike. J Biol Chem. 2012;287:33983–95.

  30. 30.

    Talanian RV, Quinlan C, Trautz S, Hackett MC, Mankovich JA, Banach D, et al. Substrate specificities of caspase family proteases. J Biol Chem. 1997;272:9677–82.

  31. 31.

    Benkova B, Lozanov V, Ivanov IP, Mitev V. Evaluation of recombinant caspase specificity by competitive substrates. Anal Biochem. 2009;394:68–74.

  32. 32.

    McStay GP, Salvesen GS, Green DR. Overlapping cleavage motif selectivity of caspases: implications for analysis of apoptotic pathways. Cell Death Differ. 2008;15:322–31.

  33. 33.

    Maillard MC, Brookfield FA, Courtney SM, Eustache FM, Gemkow MJ, Handel RK, et al. Exploiting differences in caspase-2 and -3 S(2) subsites for selectivity: structure-based design, solid-phase synthesis and in vitro activity of novel substrate-based caspase-2 inhibitors. Bioorg Med Chem. 2011;19:5833–51.

  34. 34.

    Poreba M, Szalek A, Kasperkiewicz P, Rut W, Salvesen GS, Drag M. Small molecule active site directed tools for studying human caspases. Chem Rev. 2015;115:12546–629.

  35. 35.

    Poreba M, Groborz K, Navarro M, Snipas SJ, Drag M, Salvesen GS. Caspase selective reagents for diagnosing apoptotic mechanisms. Cell Death Differ. 2018;26:229–44.

  36. 36.

    Poreba M, Kasperkiewicz P, Snipas SJ, Fasci D, Salvesen GS, Drag M. Unnatural amino acids increase sensitivity and provide for the design of highly selective caspase substrates. Cell Death Differ. 2014;21:1482–92.

  37. 37.

    Poreba M, Salvesen GS, Drag M. Synthesis of a HyCoSuL peptide substrate library to dissect protease substrate specificity. Nat Protoc. 2017;12:2189–214.

  38. 38.

    Stennicke HR, Salvesen GS. Caspases: preparation and characterization. Methods. 1999;17:313–9.

  39. 39.

    Poreba M, Szalek A, Kasperkiewicz P, Drag M. Positional scanning substrate combinatorial library (PS-SCL) approach to define caspase substrate specificity. Methods Mol Biol. 2014;1133:41–59.

  40. 40.

    Poreba M, Solberg R, Rut W, Lunde NN, Kasperkiewicz P, Snipas SJ, et al. Counter selection substrate library strategy for developing specific protease substrates and probes. Cell Chem Biol. 2016;23:1023–35.

  41. 41.

    Rut W, Poreba M, Kasperkiewicz P, Snipas SJ, Drag M. Selective substrates and activity-based probes for imaging of the human constitutive 20s proteasome in cells and blood samples. J Med Chem. 2018;61:5222–34.

  42. 42.

    Scaffidi C, Medema JP, Krammer PH, Peter ME. FLICE is predominantly expressed as two functionally active isoforms, caspase-8/a and caspase-8/b. J Biol Chem. 1997;272:26953–8.

  43. 43.

    Tang Y, Wells JA, Arkin MR. Structural and enzymatic insights into caspase-2 protein substrate recognition and catalysis. J Biol Chem. 2011;286:34147–54.

  44. 44.

    Chauvier D, Ankri S, Charriaut-Marlangue C, Casimir R, Jacotot E. Broad-spectrum caspase inhibitors: from myth to reality? Cell Death Differ. 2007;14:387–91.

  45. 45.

    Oliver TG, Meylan E, Chang GP, Xue W, Burke JR, Humpton TJ, et al. Caspase-2-mediated cleavage of Mdm2 creates a p53-induced positive feedback loop. Mol Cell. 2011;43:57–71.

  46. 46.

    Fava LL, Schuler F, Sladky V, Haschka MD, Soratroi C, Eiterer L, et al. The PIDDosome activates p53 in response to supernumerary centrosomes. Genes Dev. 2017;31:34–45.

  47. 47.

    Lopez-Garcia C, Sansregret L, Domingo E, McGranahan N, Hobor S, Birkbak NJ, et al. BCL9L dysfunction impairs caspase-2 expression permitting aneuploidy tolerance in colorectal cancer. Cancer Cell. 2017;31:79–93.

  48. 48.

    LeBlanc H, Lawrence D, Varfolomeev E, Totpal K, Morlan J, Schow P, et al. Tumor-cell resistance to death receptor--induced apoptosis through mutational inactivation of the proapoptotic Bcl-2 homolog Bax. Nat Med. 2002;8:274–81.

  49. 49.

    Kitevska T, Roberts SJ, Pantaki-Eimany D, Boyd SE, Scott FL, Hawkins CJ. Analysis of the minimal specificity of caspase 2 and identification of Ac-VDTTD-AFC as a caspase 2 selective peptide substrate. Biosci Rep. 2014;34:127.

Download references

Acknowledgements

This project received funding from the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement no. 661187 (to MP) and was supported by the National Science Centre grant 2011/03/B/ST5/01048 in Poland (to MD), the “TEAM/2017-4/32” project, which is carried out within the TEAM programme of the Foundation for Polish Science cofinanced by the European Union under the European Regional Development Fund (to MD), and the NIH grant R01 GM099040 to (GSS). Sanford Burnham Prebys NCI Cancer Center Support Grant P30 CA030199.

Author information

Affiliations

  1. NCI Designated Cancer Center, Sanford Burnham Prebys Medical Discovery Institute, 10901 North Torrey Pines Road, La Jolla, CA, 92037, USA

    • Marcin Poreba
    • , Katarzyna Groborz
    • , Scott J. Snipas
    • , Guy S. Salvesen
    •  & Marcin Drag
  2. Department of Bioorganic Chemistry, Faculty of Chemistry, Wroclaw University of Science and Technology, Wyb. Wyspianskiego 27, 50-370, Wroclaw, Poland

    • Marcin Poreba
    • , Wioletta Rut
    • , Katarzyna Groborz
    •  & Marcin Drag

Authors

  1. Search for Marcin Poreba in:

  2. Search for Wioletta Rut in:

  3. Search for Katarzyna Groborz in:

  4. Search for Scott J. Snipas in:

  5. Search for Guy S. Salvesen in:

  6. Search for Marcin Drag in:

Contributions

MP and MD conceived the project; MP, WR, GS, and MD designed and MP, WR, and KG performed the experiments; SJS contributed new reagents and tools. MP drafted the manuscript and all authors edited the manuscript. All authors reviewed the results and approved the final version of the manuscript.

Conflict of interest

The authors declare that they have no conflict of interest.

Corresponding authors

Correspondence to Marcin Poreba or Marcin Drag.

Supplementary information

About this article

Publication history

Received

Revised

Accepted

Published

DOI

https://doi.org/10.1038/s41418-019-0329-2