Microbiology and Biotechnology Letters (한국미생물·생명공학회지)

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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The Specific Binding Mechanism of the Antimicrobial Peptide CopA3 to Caspases

  • Ho Kim (Division of Biohealthcare, College of Health Science, Daejin University)
  • Received : 2023.05.22
  • Accepted : 2023.06.28
  • Published : 2023.09.28
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Abstract

We recently found that the insect-derived antimicrobial peptide CopA3 (LLCIALRKK) directly binds to and inhibits the proteolytic activation of caspases, which play essential roles in apoptotic processes. However, the mechanism of CopA3 binding to caspases remained unknown. Here, using recombinant GST-caspase-3 and -6 proteins, we investigated the mechanism by which CopA3 binds to caspases. We showed that replacement of cysteine in CopA3 with alanine caused a marked loss in its binding activity towards caspase-3 and -6. Exposure to DTT, a reducing agent, also diminished their interaction, suggesting that this cysteine plays an essential role in caspase binding. Experiments using deletion mutants of CopA3 showed that the last N-terminal leucine residue of CopA3 peptide is required for binding of CopA3 to caspases, and that C-terminal lysine and arginine residues also contribute to their interaction. These conclusions are supported by binding experiments employing direct addition of CopA3 deletion mutants to human colonocyte (HT29) extracts containing endogenous caspase-3 and -6 proteins. In summary, binding of CopA3 to caspases is dependent on a cysteine in the intermediate region of the CopA3 peptide and a leucine in the N-terminal region, but that both an arginine and two adjacent lysines in the C-terminal region of CopA3 also contribute. Collectively, these results provide insight into the interaction mechanism and the high selectivity of CopA3 for caspases.

Keywords

Acknowledgement

This work was supported by the Daejin University Research Grants in 2023.

References

  1. Shalini S, Dorstyn L, Dawar S, Kumar S. 2015. Old, new and emerging functions of caspases. Cell Death Differ. 22: 526-539.  https://doi.org/10.1038/cdd.2014.216
  2. Kamada S, Funahashi Y, Tsujimoto Y. 1997. Caspase-4 and caspase-5, members of the ICE/CED-3 family of cysteine proteases, are CrmA-inhibitable proteases. Cell Death Differ. 4: 473-478.  https://doi.org/10.1038/sj.cdd.4400268
  3. Miura M, Zhu H, Rotello R, Hartwieg EA, Yuan J. 1993. Induction of apoptosis in fibroblasts by IL-1 beta-converting enzyme, a mammalian homolog of the C. elegans cell death gene ced-3. Cell 75: 653-660.  https://doi.org/10.1016/0092-8674(93)90486-A
  4. Ray CA, Black RA, Kronheim SR, Greenstreet TA, Sleath PR, Salvesen GS, et al. 1992. Viral inhibition of inflammation: cowpox virus encodes an inhibitor of the interleukin-1 beta converting enzyme. Cell 69: 597-604.  https://doi.org/10.1016/0092-8674(92)90223-Y
  5. Zhou Q, Snipas S, Orth K, Muzio M, Dixit VM, Salvesen GS. 1997. Target protease specificity of the viral serpin CrmA. Analysis of five caspases. J. Biol. Chem. 272: 7797-7800.  https://doi.org/10.1074/jbc.272.12.7797
  6. Ekert PG, Silke J, Vaux DL. 1999. Caspase inhibitors. Cell Death Differ. 6: 1081-1086.  https://doi.org/10.1038/sj.cdd.4400594
  7. Rano TA, Timkey T, Peterson EP, Rotonda J, Nicholson DW, Becker JW, et al. 1997. A combinatorial approach for determining protease specificities: application to interleukin-1beta converting enzyme (ICE). Chem. Biol. 4: 149-155.  https://doi.org/10.1016/S1074-5521(97)90258-1
  8. Mittl PR, Di Marco S, Krebs JF, Bai X, Karanewsky DS, Priestle JP, et al. 1997. Structure of recombinant human CPP32 in complex with the tetrapeptide acetyl-Asp-Val-Ala-Asp fluoromethyl ketone. J. Biol. Chem. 272: 6539-6547.  https://doi.org/10.1074/jbc.272.10.6539
  9. Walker NP, Talanian RV, Brady KD, Dang LC, Bump NJ, Ferenz CR, et al. 1994. Crystal structure of the cysteine protease interleukin-1 beta-converting enzyme: a (p20/p10)2 homodimer. Cell 78: 343-352.  https://doi.org/10.1016/0092-8674(94)90303-4
  10. Kang JK, Hwang JS, Nam HJ, Ahn KJ, Seok H, Kim SK, et al. 2011. The insect peptide coprisin prevents Clostridium difficile-mediated acute inflammation and mucosal damage through selective antimicrobial activity. Antimicrob. Agents Chemother. 55: 4850-4857.  https://doi.org/10.1128/AAC.00177-11
  11. Lee J, Hwang JS, Hwang IS, Cho J, Lee E, Kim Y, et al. 2012. Coprisin-induced antifungal effects in Candida albicans correlate with apoptotic mechanisms. Free Radic. Biol. Med. 52: 2302-2311.  https://doi.org/10.1016/j.freeradbiomed.2012.03.012
  12. Lee J, Lee D, Choi H, Kim HH, Kim H, Hwang JS, et al. 2014. Structure-activity relationships of the intramolecular disulfide bonds in coprisin, a defensin from the dung beetle. BMB Rep. 47: 625-630.  https://doi.org/10.5483/BMBRep.2014.47.11.262
  13. Kim YH, Hwang JS, Yoon IN, Lee JH, Lee J, Park KC, et al. 2021. The insect peptide CopA3 blocks programmed cell death by directly binding caspases and inhibiting their proteolytic activation. Biochem. Biophys. Res. Commun. 547: 82-88.  https://doi.org/10.1016/j.bbrc.2021.01.107
  14. Hwang JS, Lee J, Kim YJ, Bang HS, Yun EY, Kim SR, et al. 2009. Isolation and characterization of a defensin-like peptide (Coprisin) from the Dung Beetle, Copris tripartitus. Int. J. Pept. 2009: 136284. 
  15. Yoon IN, Hwang JS, Lee JH, Kim H. 2019. The antimicrobial peptide CopA3 inhibits Clostridium difficile toxin a-induced viability loss and apoptosis in neural cells. J. Microbiol. Biotechnol. 29: 30-36.  https://doi.org/10.4014/jmb.1809.08065
  16. Kim IW, Lee JH, Kwon YN, Yun EY, Nam SH, Ahn MY, et al. 2013. Anticancer activity of a synthetic peptide derived from harmoniasin, an antibacterial peptide from the ladybug Harmonia axyridis. Int. J. Oncol. 43: 622-628.  https://doi.org/10.3892/ijo.2013.1973
  17. Kim DH, Lee IH, Nam ST, Hong J, Zhang P, Lu LF, et al. 2015. Antimicrobial peptide, lumbricusin, ameliorates motor dysfunction and dopaminergic neurodegeneration in a mouse model of Parkinson's disease. J. Microbiol. Biotechnol. 25: 1640-1647.  https://doi.org/10.4014/jmb.1507.07011
  18. Seo M, Lee JH, Baek M, Kim MA, Ahn MY, Kim SH, et al. 2017. A novel role for earthworm peptide Lumbricusin as a regulator of neuroinflammation. Biochem. Biophys. Res. Commun. 490: 1004-1010.  https://doi.org/10.1016/j.bbrc.2017.06.154
  19. Kim DH, Lee IH, Nam ST, Hong J, Zhang P, Hwang JS, et al. 2014. Neurotropic and neuroprotective activities of the earthworm peptide Lumbricusin. Biochem. Biophys. Res. Commun. 448: 292-297.  https://doi.org/10.1016/j.bbrc.2014.04.105
  20. Kim H, Kokkotou E, Na X, Rhee SH, Moyer MP, Pothoulakis C, et al. 2005. Clostridium difficile toxin A-induced colonocyte apoptosis involves p53-dependent p21(WAF1/CIP1) induction via p38 mitogen-activated protein kinase. Gastroenterology 129: 1875-1888.  https://doi.org/10.1053/j.gastro.2005.09.011
  21. Arbach H, Butler C, McMenimen KA. 2017. Chaperone activity of human small heat shock protein-GST fusion proteins. Cell Stress Chaperones 22: 503-515.  https://doi.org/10.1007/s12192-017-0764-2
  22. Nadkarni DV. 2020. Conjugations to endogenous cysteine residues. Methods Mol. Biol. 2078: 37-49.  https://doi.org/10.1007/978-1-4939-9929-3_3
  23. Martinez-Zapien D, Ruiz FX, Poirson J, Mitschler A, Ramirez J, Forster A, et al. 2015. Structure of the E6/E6AP/p53 complex required for HPV-mediated degradation of p53. Nature 529: 541-545.  https://doi.org/10.1038/nature16481
  24. Hu M, Luo Q, Alitongbieke G, Chong S, Xu C, Xie L, et al. 1996. Celastrol-induced Nur77 interaction with TRAF2 alleviates inflammation by promoting mitochondrial ubiquitination and autophagy. Mol. Cell. 66: 141-153 e146. 
  25. Adamidou T, Arvaniti KO, Glykos NM. 2011. Folding simulations of a nuclear receptor box-containing peptide demonstrate the structural persistence of the LxxLL motif even in the absence of its cognate receptor. J. Phys. Chem B. 122: 106-116. https://doi.org/10.1021/acs.jpcb.7b10292