Mining of Caspase-7 Substrates Using a Degradomic Approach

  • Jang, Mi (Translational Research Center, Korea Research Institute of Bioscience and Biotechnology) ;
  • Park, Byoung Chul (Translational Research Center, Korea Research Institute of Bioscience and Biotechnology) ;
  • Kang, Sunghyun (Translational Research Center, Korea Research Institute of Bioscience and Biotechnology) ;
  • Lee, Do Hee (Translational Research Center, Korea Research Institute of Bioscience and Biotechnology) ;
  • Cho, Sayeon (College of Pharmacy, Chung-Ang University) ;
  • Lee, Sang-Chul (Translational Research Center, Korea Research Institute of Bioscience and Biotechnology) ;
  • Bae, Kwang-Hee (Translational Research Center, Korea Research Institute of Bioscience and Biotechnology) ;
  • Park, Sung-Goo (Translational Research Center, Korea Research Institute of Bioscience and Biotechnology)
  • Received : 2007.11.23
  • Accepted : 2008.04.07
  • Published : 2008.08.31

Abstract

Caspases play critical roles in the execution of apoptosis. Caspase-3 and caspase-7 are closely related in sequence as well as in substrate specificity. The two caspases have overlapping substrate specificities with special preference for the DEVD motif. However, they are targeted to different subcellular locations during apoptosis, implying the existence of substrates specific for one or other caspase. To identify new caspase-7 substrates, we digested cell lysates obtained from the caspase-3-deficient MCF-7 cell line with purified recombinant caspase-7, and analyzed spots that disappeared or decreased by 2-DE (we refer to this as the caspase-7 degradome). Several proteins with various cellular functions underwent caspase-7-dependent proteolysis. The substrates of capase-7 identified by the degradomic approach were rather different from those of caspase-3 (Proteomics, 4, 3429-3435, 2004). Among the candidate substrates, we confirmed that Valosin-containing protein (VCP) was cleaved by both capspase-7 and caspase-3 in vitro and during apoptosis. Cleavage occurred at both $DELD^{307}$ and $DELD^{580}$. The degradomic study yielded several candidate caspase-7 substrates and their further analysis should provide valuables clues to the functions of caspase-7 during apoptosis.

Keywords

Acknowledgement

Supported by : Korea Science and Engineering Foundation

References

  1. Ameisen, J.C. (2002). On the origin, evolution, and nature of programmed cell death: a timeline of four billion years. Cell Death Differ. 9, 367-393. https://doi.org/10.1038/sj.cdd.4400950
  2. Byun, Y., Chen, F., Chang, R., Trivedi, M., Green, K.J., and Cryns, V.L. (2001). Caspase cleavage of vimentin disrupts intermediate filaments and promotes apoptosis. Cell Death Differ. 8, 443-450. https://doi.org/10.1038/sj.cdd.4400840
  3. Dai, R.M., Chen, E., Longo, D.L., Gorbea, C.M., and Li, C.C. (1998). Involvement of valosin-containing protein, an ATPase Copurified with IkappaBalpha and 26 S proteasome, in ubiquitin-proteasome-mediated degradation of IkBa. J. BioI. Chem. 273, 3562-3573. https://doi.org/10.1074/jbc.273.6.3562
  4. Denault, J.B., and Salvesen, G.S. (2002). Caspases: keys in the ignrtion of cell death. Chem. Rev. 102, 4489-4500. https://doi.org/10.1021/cr010183n
  5. Fischer, U., Janicke, R.U., and Schulze-Osthoff, K. (2003). Many cuts to ruin: a comprehensive update of caspase substrates. Cell Death Differ. 10, 76-100. https://doi.org/10.1038/sj.cdd.4401160
  6. Hetzer, M., Meyer, H.H., Walther, T.C., Bilbao-Cortes, D., Warren, G., and Mattai, I.W. (2001). Distinct AAA-ATPase p97 com-plexes function in discrete steps of nuclear assembly. Nat. Cell BioI. 3, 1086-1091. https://doi.org/10.1038/ncb1201-1086
  7. Jang, M., Park, B.C., Lee, A.Y., Na, K.S., Kang, S., Bae, K.-H., Myung, P.K., Chung, B.C., Cho, S., Lee, D.H., et al. (2007). Caspase-7 mediated cleavage of proteasome subunits during apoptosis. Biochem. Biophys. Res. Commun. 363, 388-394. https://doi.org/10.1016/j.bbrc.2007.08.183
  8. Joo, W.A., Lee, D.Y., and Kim, C.W. (2003) Deveklpment of an effective sample preparation method for the proteome analysis of oody fluids using 2-D gel electrophoresis. Biosci. Biotechnol. Biochem. 67, 1574-1577. https://doi.org/10.1271/bbb.67.1574
  9. Lee, AY., Park, B.C., Jang, M., Cho, S., Lee, D.H., Lee, S.C., Myung, P.K., and Park, S.G. (2004). Identification of caspase-3 degradome by two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization-time of flight analysis. Proteomics 4, 3429-3436. https://doi.org/10.1002/pmic.200400979
  10. Lee, AY., Lee, Y., Park, Y.K., Bae, K.-H., Cho, S., Lee, D.H., Park, B.C., Kang, S., and Park, S.G. (2008). HS-1-associated protein X-1 is cleaved by caspase-3 during apoptosis. Mol. Cells 25, 86-90.
  11. Lopez-Otin, C., and Overall, C.M. (2002). Protease degradomics: a new challenge for proteomics. Nat. Rev. Mol. Cell Biol. 3, 509-519. https://doi.org/10.1038/nrm858
  12. Na, K.S., Park, B.C., Jang, M., Cho, S., Lee, D.H., Kang, S., Lee, CK, Bae, K.-H., and Park, S.G. (2007). Protein disulfide isom-erase is cleaved by caspase-3 and -7 during apoptosis. Mol. Cells 24, 261-267.
  13. Nicholson, D.W. (1999). Caspase structure, proteolytic substrates, and function during apoptotic cell death. Cell Death Differ. 6, 1028-1042. https://doi.org/10.1038/sj.cdd.4400598
  14. Rabinovich, E., Kerem, A, Frohlich, K.U., Diamant, N., and Bar-Nun, S. (2002). AM-ATPase p97/Cdc48p, a cytosolic chaper-one required for endoplasmic reticulum-associated protein deg-radation. Mol. Cell. Biol. 22, 626-634. https://doi.org/10.1128/MCB.22.2.626-634.2002
  15. Rouiller, I., DeLaBarre, B., May, AP., Weis, W.I., Brunger, AT., Milligan, R.A., and Wilson-Kubalek, E.M. (2002). Conformational changes of the multifunction p97 AM ATPase during its AT-Pase cycle. Nat. Struct. Biol. 9, 950-957. https://doi.org/10.1038/nsb872
  16. Stennicke, H.R., and Salvesen, G.S. (1999). Caspases: preparation and characterization. Methods 17,313-319. https://doi.org/10.1006/meth.1999.0745
  17. Stroh, C., and Schulze-Osthoff, K. (1998). Death by a thousand cuts: an ever increasing list of caspase substrates. Cell Death Differ. 5, 997-1000. https://doi.org/10.1038/sj.cdd.4400451
  18. Thornberry, N.A., and Lazebnik, Y. (1998). Caspases: enemies within. Science 281, 1312-1316. https://doi.org/10.1126/science.281.5381.1312
  19. 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., et al. (1997). A combinatorial approach de-fines specificities of members of the caspase family and granzyme B. Functional relationships established for key mediators of apoptosis. J. Biol. Chem. 272, 17907-17911. https://doi.org/10.1074/jbc.272.29.17907
  20. Vilas, G.L., Corvi, M.M., Plummer, G.J., Seime, AM., Lambkin, G.R., and Berthiaume, L.G. (2006). Posttranslational myristoyla-tion of caspase-activated p21-activated protein kinase 2 (PAK2) potentiates late apoptotic events. Proc. Natl. Acad. Sci. USA 103,6542-6547.
  21. Zhang, X., Shaw, A, Bates, P.A., Newman, R.H., Gowen, B., Or-lova, E., Gorman, M.A., Kondo, H., Dokurno, P., Lally, J., et al. (2000). Structure of the AM ATPase p97. Mol. Cell 6, 1473-1484. https://doi.org/10.1016/S1097-2765(00)00143-X