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Inhibition of calpain but not caspase activity by spectrin fragments

Abstract

Calpains and caspases are ubiquitous cysteine proteases that are associated with a variety of cellular pathways. Calpains are involved in processes such as long term potentiation, cell motility and apoptosis, and have been shown to cleave non-erythroid (brain) α- and β-spectrin and erythroid β-spectrin. The cleavage of erythroid α-spectrin by calpain has not been reported. Caspases play an important role in the initiation and execution of apoptosis, and have been shown to cleave non-erythroid but not erythroid spectrin. We have studied the effect of spectrin fragments on calpain and caspase activities. The erythroid and non-erythroid spectrin fragments used were from the N-terminal region of α-spectrin, and C-terminal region of β-spectrin, both consisting of regions involved in spectrin tetramer formation. We observed that the all spectrin fragments exhibited a concentration-dependent inhibitory effect on calpain, but not caspase activity. It is clear that additional studies are warranted to determine the physiological significance of calpain inhibition by spectrin fragments. Our findings suggest that calpain activity is modulated by the presence of spectrin partial domains at the tetramerization site. It is not clear whether the inhibitory effect is substrate specific or is a general effect. Further studies of this inhibitory effect may lead to the identification and development of new therapeutic agents specifically for calpains, but not for caspases. Proteins/peptides with a coiled coil helical conformation should be studied for potential inhibitory effects on calpain activity.

Abbreviations

αI-N1:

first 156 amino acid residues of erythroid α-spectrin

αI-N3:

first 368 amino acid residues of erythroid α-spectrin

αII-N1:

first 147 amino acid residues of non-erythroid α-spectrin

αII-N3:

first 359 amino acid residues of non-erythroid α-spectrin

βI-C1:

residues 1898 to 2083 of erythroid β-spectrin

βII-C1:

residues 1906 to 2093 of non-erythroid β-spectrin

Ac-DEVD-pNA:

N-acetyl-Asp-Glu-Val-Asp-p-nitroaniline, a caspase substrate

BSA:

bovine serum albumin

CHAPS:

3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate

DMSO:

dimethyl sulfoxide

DTT:

dithiothreitol

EDTA:

ethylenediamine tetraacetic acid

HEPES:

4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

PBS:

5 mM phosphate buffer at pH 7.4 with 150 mM NaCl

References

  1. Lynch, D.R. and Gleichman, A.J. Picking up the pieces: the roles of functional remnants of calpain-mediated proteolysis. Neuron 53 (2007) 317–319.

    Article  CAS  PubMed  Google Scholar 

  2. Croall, D.E. and Ersfeld, K. The calpains: modular designs and functional diversity. Genome Biol. 8 (2007) 218.

    Article  PubMed  Google Scholar 

  3. Stabach, P.R, Cianci, C.D., Glantz, S.B., Zhang, Z. and Morrow, J.S. Site-directed mutagenesis of alpha II spectrin at codon 1175 modulates its mucalpain susceptibility. Biochemistry 36 (1997) 57–65.

    Article  CAS  PubMed  Google Scholar 

  4. Saez, M.E., Ramirez-Lorca, R., Moron, F.J. and Ruiz, A. The therapeutic potential of the calpain family: new aspects. Drug Discov. Today 11 (2006) 917–923.

    Article  CAS  PubMed  Google Scholar 

  5. Tompa, P., Buzder-Lantos, P., Tantos, A., Farkas, A., Szilagyi, A., Banoczi, Z., Hudecz, F. and Friedrich, P. On the sequential determinants of calpain cleavage. J. Biol. Chem. 279 (2004) 20775–20785.

    Article  CAS  PubMed  Google Scholar 

  6. Moldoveanu, T., Hosfield, C.M., Lim, D., Elce, J.S., Jia, Z. and Davies, P.L. A Ca2+ switch aligns the active site of calpain. Cell 108 (2002) 649–660.

    Article  CAS  PubMed  Google Scholar 

  7. Eto, A., Akita, Y., Saido, T.C., Suzuki, K. and Kawashima, S. The role of calpain-calpastatin system in thyrotropin-releasing hormone-induced selective down-regulation of a protein kinase C isozyme, nPKC, in rat pituitary GH4C1 cells. J. Biol. Chem. 270 (1995) 25115–25120.

    Article  CAS  PubMed  Google Scholar 

  8. Yuen, P.W. and Wang, K.K.W. Calpain inhibitors, novel neuroprotectants and potential anticataractic agents. Drug Future 23 (1998) 741–749.

    Article  CAS  Google Scholar 

  9. Wronski, R., Tompa, P., Hutter-Paier, B., Crailsheim, K., Friedrich, P. and Windisch, M. Inhibitory effect of a brain derived peptide preparation on Ca2+-dependent protease, calpain. J. Neural. Trans. 107 (2000) 145–157.

    Article  CAS  Google Scholar 

  10. Janossy, J., Ubezio, P., Apati, A., Magocsi, M., Tompa, P. and Fridrich, P. Calpain as a multi-site regulator of cell cycle. Biochem. Pharm. 67 (2004) 1513–1521.

    Article  CAS  PubMed  Google Scholar 

  11. Nicholson, D.W. Caspase structure, proteolytic substrates and function during apoptotic cell death. Cell Death Differ. 6 (1999) 1028–1042.

    Article  CAS  PubMed  Google Scholar 

  12. Spira, M.E., Oren, R., Dormann, A., Ilouz, N. and Lev, S. Calcium, protease activation, and cytoskeleton remodeling underlie growth cone formation and neuronal regeneration. Cell. Mol. Neurobiol. 21 (2002) 591–604.

    Article  Google Scholar 

  13. Ai, J., Liu, E., Wang, J., Chen, Y., Yu, J. and Baker, W.J. Calpain inhibitor MDL-28170 reduces the functional and structural deterioration of corpus callosum following fluid percussion injury. J. Neurotrauma 24 (2007) 960–978.

    Article  PubMed  Google Scholar 

  14. Knoblach, S.M., Alroy, D.A., Nikolaeva, M., Cernak, I., Stoica, B.A. and Faden, A.I. Caspase inhibitor z-DEVD-fmk attenuates calpain and necrotic cell death in vitro and after traumatic brain injury. J. Cereb. Blood Flow Metab. 24 (2004) 1119–1132.

    Article  CAS  PubMed  Google Scholar 

  15. Bennett, V. and Baines, A.J. Spectrin and ankyrin-based pathways: metazoan inventions for integrating cells into tissues. Physiol. Rev. 81 (2001) 1353–1392.

    CAS  PubMed  Google Scholar 

  16. Czogalla, A. and Sikorski, A.F. Spectrin and calpain a target and a sniper in the pathology of neuronal cells. Cell. Mol. Life Sci. 62 (2005) 1913–1924.

    Article  CAS  PubMed  Google Scholar 

  17. Glantz, S.B., Cianci, C.D., Iyer, R., Pradhan, D., Wang, K.K. and Morrow, J.S. Sequential degradation of alpha II and beta II spectrin by calpain in glutamate or maitotoxin-stimulated cells. Biochemistry 46 (2007) 502–513.

    Article  CAS  PubMed  Google Scholar 

  18. Lofvenberg, L. and Backman, L. Calpain-induced proteolysis of β-spectrins. FEBS Lett. 443 (1999) 89–92.

    Article  CAS  PubMed  Google Scholar 

  19. Meary, F., Metral, S., Ferreira, C., Eladari, D., Colin, Y., Lecomte, M.C. and Nicolas, G. A mutant alpha II-spectrin designed to resist calpain and caspase cleavage questions the functional importance of this process in vivo. J. Biol. Chem. 282 (2007) 14226–14237.

    Article  CAS  PubMed  Google Scholar 

  20. Wang, K.K.W., Posmantur, R., Nath, R., McGinnis, K., Whitton, M., Talanian, R.V., Glantz, S.B. and Morrow J.S. Simultaneous degradation of αII- and βII-spectrin by caspase 3 (CPP32) in apoptotic cells. J. Biol. Chem. 273 (1998) 22490–22497.

    Article  CAS  PubMed  Google Scholar 

  21. Pineda, J.A., Lewis, S.B., Valadka, A.B., Papa, L., Hannay, H.J., Heaton, S.C., Demery, J.A., Liu, M.C., Aikam, J.M., Akle, V., Brophy, G.M., Tepas, J.J., Wang, K.K., Robertson, C.S. and Hayes, R.L. Clinical significance of alpha II-spectrin breakdown products in cerebrospinal fluid after severe traumatic brain injury. J. Neurotrauma 24 (2007) 354–366.

    Article  PubMed  Google Scholar 

  22. Park, S., Caffrey, M.S., Johnson, M.E. and Fung L.W.-M. Solution structural studies on human erythrocyte alpha-spectrin tetramerization site. J. Biol. Chem. 278 (2003) 21837–21844.

    Article  CAS  PubMed  Google Scholar 

  23. Antoniou, C., Lam, V.Q. and Fung, L.W.-M. Conformational changes at the tetramerization site of erythroid α-spectrin upon binding α-spectrin: a spin label EPR study. Biochemistry 47 (2008) 10765–10772.

    Article  CAS  PubMed  Google Scholar 

  24. Li, Q. and Fung, L.W.-M. Structural and dynamic study of the tetramerization region of non-erythroid α-spectrin: A frayed helix revealed by site-directed spin labeling electron paramagnetic resonance. Biochemistry 48 (2009) 206–215.

    Article  CAS  PubMed  Google Scholar 

  25. Mehboob, S., Song, Y., Witek, M., Long, F., Santarsiero, B., Johnson, M.E. and Fung, L.W.-M. Crystal structure of the non-erythroid α-spectrin tetramerization site reveals differences between erythroid and non-erythroid spectrin tetramer formation. J. Biol. Chem. (2010) doi:10.1074/jbc.M109.080028, in press.

  26. Rackoff, J., Yang, Q. and DePetrillo, P.B. Inhibition of rat PC12 cell calpain activity by glutathione, oxidized glutathione and nitric oxide. Neurosci. Lett. 311 (2001) 129–132.

    Article  CAS  PubMed  Google Scholar 

  27. Antoniou, C. and Fung, L.W.-M. Potential artifacts in using a GST-fusion protein purification system and spin labeling EPR to study protein protein interactions. Anal. Biochem. 376 (2008) 160–162.

    Article  CAS  PubMed  Google Scholar 

  28. Thornberry, N.A. and Lazebnik, Y. Caspases: enemies within. Science 281 (1998) 1312–1316.

    Article  CAS  PubMed  Google Scholar 

  29. Kiss. R., Kovacs, D., Tompa, P. and Perczel, A. Local structural preferences of calpastatin, the intrinsically unstructured protein inhibitor of calpain. Biochemistry 47 (2008) 6936–6945.

    Article  CAS  PubMed  Google Scholar 

  30. Mucsi, Z., Hudecz, F., Hollsi, M., Tompa, P. and Friedrich, P. Binding-induced folding transitions in calpastatin subdomains A and C. Protein Sci. 12 (2003) 2327–2336.

    Article  CAS  PubMed  Google Scholar 

  31. Rutledge, T.W. and Whiteheart, S.W. SNAP-23 is a target for calpain cleavage in activated platelets. J. Biol. Chem. 277 (2002) 37009–37015.

    Article  CAS  PubMed  Google Scholar 

  32. Mehboob, S., Luo, B.H., Fu, W., Johnson, M.E. and Fung, L.W.-M. Conformational studies of the tetramerization site of human erythroid spectrin by cysteine-scanning spin-labeling EPR methods. Biochemistry 44 (2005) 15898–15905.

    Article  CAS  PubMed  Google Scholar 

  33. Mehboob, S., Luo, B.H., Patel, B.M. and Fung, L. W.-M. αβ-spectrin coiled coil association at the tetramerization site. Biochemistry 40 (2001) 12457–12464.

    Article  CAS  PubMed  Google Scholar 

  34. Bennett, V. The spectrin-actin junction of erythrocyte membrane skeletons. Biochim. Biophys. Acta 107 (1989) 107–121.

    Google Scholar 

  35. Serizawa, S., Miyamichi, K., Nakatani, H., Suzuki, M., Saito, M., Yoshihara, Y. and Sakano, H. Negative feedback regulation ensures the one receptor-one olfactory neuron rule in mouse. Science 302 (2003) 2088–2094.

    Article  CAS  PubMed  Google Scholar 

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Correspondence to Leslie W.-M. Fung.

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Rolius, R., Antoniou, C., Nazarova, L.A. et al. Inhibition of calpain but not caspase activity by spectrin fragments. Cell Mol Biol Lett 15, 395–405 (2010). https://doi.org/10.2478/s11658-010-0015-3

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  • DOI: https://doi.org/10.2478/s11658-010-0015-3

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