Fas-Induced Caspase Denitrosylation
10.1126/science.284.5414.651
Crossref journal-article
American Association for the Advancement of Science (AAAS)
Science (221)
Abstract

Only a few intracellular S-nitrosylated proteins have been identified, and it is unknown if protein S-nitrosylation/denitrosylation is a component of signal transduction cascades. Caspase-3 zymogens were found to be S-nitrosylated on their catalytic-site cysteine in unstimulated human cell lines and denitrosylated upon activation of the Fas apoptotic pathway. Decreased caspase-3 S-nitrosylation was associated with an increase in intracellular caspase activity. Fas therefore activates caspase-3 not only by inducing the cleavage of the caspase zymogen to its active subunits, but also by stimulating the denitrosylation of its active-site thiol. Protein S-nitrosylation/denitrosylation can thus serve as a regulatory process in signal transduction pathways.

Bibliography

Mannick, J. B., Hausladen, A., Liu, L., Hess, D. T., Zeng, M., Miao, Q. X., Kane, L. S., Gow, A. J., & Stamler, J. S. (1999). Fas-Induced Caspase Denitrosylation. Science, 284(5414), 651–654.

Authors 9
  1. Joan B. Mannick (first)
  2. Alfred Hausladen (additional)
  3. Limin Liu (additional)
  4. Douglas T. Hess (additional)
  5. Ming Zeng (additional)
  6. Qian X. Miao (additional)
  7. Laurie S. Kane (additional)
  8. Andrew J. Gow (additional)
  9. Jonathan S. Stamler (additional)
References 45 Referenced 631
  1. Mannick J. B., Asano K., Izumi K., Kieff E., Stamler J. S., Cell 79, 1137 (1994); (10.1016/0092-8674(94)90005-1) / Cell by Mannick J. B. (1994)
  2. Genaro A. M., Hortelano S., Alvarez A., Martinez C., Bosca L., J. Clin. Invest. 95, 1884 (1995); (10.1172/JCI117869) / J. Clin. Invest. by Genaro A. M. (1995)
  3. Chun S. Y., Eisenhauer K. M., Kubo M., Hsueh A. J. W., Endocrinology 136, 3120 (1995); (10.1210/endo.136.7.7540548) / Endocrinology by Chun S. Y. (1995)
  4. Beauvais F., Laurence M., Dubertret L., FEBS Lett. 361, 229 (1995); (10.1016/0014-5793(95)00188-F) / FEBS Lett. by Beauvais F. (1995)
  5. ; J. B. Mannick X. Q. Miao J. S. Stamler. J. Biol. Chem. 272 24124 (1997); (10.1074/jbc.272.39.24125)
  6. Estevez A., et al., J. Neurosci. 18, 3708 (1998); (10.1523/JNEUROSCI.18-10-03708.1998) / J. Neurosci. by Estevez A. (1998)
  7. Dimmeler S., Haendeler J., Sause A., Zeiher A. M., Cell Growth Differ. 9, 415 (1998). / Cell Growth Differ. by Dimmeler S. (1998)
  8. Dimmeler S., Haendeler J., Nehls M., Zeiher A., J. Exp. Med. 185, 601 (1997); (10.1084/jem.185.4.601) / J. Exp. Med. by Dimmeler S. (1997)
  9. Rossig L., et al., J. Biol. Chem. 274, 6823 (1999). (10.1074/jbc.274.11.6823) / J. Biol. Chem. by Rossig L. (1999)
  10. Kim Y. M., Talanian R. V., Billiar T. R., J. Biol. Chem. 272, 31138 (1997); (10.1074/jbc.272.49.31138) / J. Biol. Chem. by Kim Y. M. (1997)
  11. Geneviva G. D., et al., Am. J. Physiol. 275, L717 (1998). / Am. J. Physiol. by Geneviva G. D. (1998)
  12. 10.1038/41237
  13. Li J., Billiar T. R., Talanian R. V., Kim Y. M., Biochem. Biophys. Res. Commun. 240, 419 (1997); (10.1006/bbrc.1997.7672) / Biochem. Biophys. Res. Commun. by Li J. (1997)
  14. Mohr S., Zech B., Lapetina E. G., Brune B., ibid. 238, 387 (1997); / ibid. by Mohr S. (1997)
  15. ; T. Ogura M. Tatemichi H. Esumi ibid. 236 365 (1997); L. Tenneti D. M. D'Emilia (10.1006/bbrc.1997.6948)
  16. Lipton S. A., Neurosci. Lett. 236, 139 (1997). (10.1016/S0304-3940(97)00780-5) / Neurosci. Lett. by Lipton S. A. (1997)
  17. BJAB and 10C9 are human Epstein-Barr virus–negative Burkitt's lymphoma cell lines. Jurkat H9 and CEM are human T leukemia cell lines. Cells were grown in RPMI medium (Gibco-BRL) supplemented with 10% fetal bovine serum (FBS) (Gemini) 2 mM glutamine (Cellgro) 100 U/ml penicillin and 100 μg/ml streptomycin (Gibco-BRL). For immunoprecipitations 0.7 × 10 8 to 1.2 × 10 8 cells were lysed in 1 ml of NP-40 buffer [150 mM NaCl 1.0% NP-40 50 mM tris (pH 8.0)] containing protease inhibitors and were precleared with protein A–Sepharose beads that had been incubated with normal rabbit serum. Cellular proteins were immunoprecipitated in the dark with 5 μg of an anti–caspase-3 IgG2a monoclonal antibody (Transduction Labs) 20 μl of an anti–caspase-3 rabbit polyclonal antiserum (Pharmingen) 5 μg of a control IgG2a antibody (Sigma) or control normal rabbit serum. The antibody/antigen complexes were isolated with protein A–Sepharose beads (Pharmacia) and then washed five times in high-salt buffer [500 mM NaCl 1% NP-40 50 mM tris (pH 8) 100 μM EDTA and protease inhibitors]. Antigen/antibody complexes were removed from the protein A–Sepharose beads by three 10-min incubations in 70 μl of 5 M MgCl 2 or 100 mM glycine (pH 3) at 4°C before NO measurements. To minimize the possibility of S-nitrosylation subsequent to immunoprecipitation we raised the buffer pH to 5.5 in selected experiments and cleansed the solutions of contaminant nitrite by heating in sealed vessels for 2 hours at 95°C at pH of 2.5 to 3.0. Any residual nitrite in buffers did not correlate with amounts of SNO in samples ( n = 55 R 2 = 0.03). In addition we adapted a methodology designed to exclude the possibility of artifactual S-nitrosylation by blocking free thiols in caspase-3 immunoprecipitates with 1 mM N -ethylmaleimide (NEM) included in wash buffers preceeding elution. Prior exposure to NEM completely blocked NO donor (pH 8)– or nitrite (0.5 N HCl)–mediated S-nitrosylation of recombinant caspase-3 or procaspase-3. Although NEM modestly reduced the NO signal derived from immunoprecipitated caspase-3 (∼25% n = 7) it also reduced by a similar amount the NO signal from highly pure SNO–caspase-3 [0.5 M NaCl 1% NP-40 50 mM tris (pH 8) 0.1 mM EDTA] that had been synthesized in vitro. Thus this approach demonstrated that caspase-3 was S-nitrosylated intracellularly. For protein immunoblot analysis whole-cell lysates or immunoprecipitated proteins were separated on 7% (NOS) or 12% (caspase-3) polyacrylamide gels transferred to nitrocellulose and incubated with 250 ng/ml of anti–caspase-3 monoclonal antibody anti-nNOS monoclonal antibody or anti-iNOS polyclonal antibody (Transduction Labs) or a 1:1 000 dilution of anti–caspase-3 rabbit polyclonal antibody (Pharmingen) followed by a 1:1000 dilution of secondary horseradish peroxidase antibody (Amersham) and then were developed by ECL (Amersham).
  18. Boulakia C. A., et al., Oncogene 12, 529 (1996). / Oncogene by Boulakia C. A. (1996)
  19. Photolysis-chemiluminesecence (assay for SNO) was performed as described [J. S. Stamler and M. Feelisch in Methods in Nitric Oxide Research J. S. Stamler and M. Feelisch Eds. (Wiley Chichester UK 1996) pp. 521–539]. Samples (200 μl) were injected directly into the photolysis unit. The chemical reduction–chemiluminescence assay for nitrite was performed according to the manufacturer (Sievers) and for SNO as described (16) with the following modifications: Immunoprecipitated proteins and cell extracts were assayed both with and without excess HgCl 2 in a paired radical-purged system otherwise according to manufacturer's instructions (NO analyzer Sievers 280); standard curves were derived for both nitrite and S-nitrosoglutathione in the presence and absence of HgCl 2 ; data were normalized to baseline and integrated using Mathcad 7 Professional (MathSoft). SNO was calculated by subtracting the nitrite in samples without HgCl 2 from the nitrite in samples with HgCl 2 and by converting the nitrite difference to SNO from the standard curves of S-nitrosoglutathione (GSNO) ± HgCl 2 . The photolysis and chemical reduction assays are linear to 1 pmol for SNO and nitrite respectively and can differentiate 1 pmol from zero (deionized water). S-Nitrosylated recombinant caspase-3 standards were detected by both methods; the standard curves for SNO–caspase-3 and GSNO were superimposeable in the photolysis assay. The chemical reduction method has not been extensively validated for SNO and thus was only used qualitatively to verify results from photolysis.
  20. J. B. Mannick A. Hausladen L. Liu A. Gow J. S. Stamler data not shown.
  21. Srinivasan A., et al., J. Biol. Chem. 273, 4523 (1998). (10.1074/jbc.273.8.4523) / J. Biol. Chem. by Srinivasan A. (1998)
  22. 10.1016/S0092-8674(00)80434-1
  23. We grew 9 × 10 7 MCF-7 cells to 75% confluence in T150 flasks containing Dulbecco's modified Eagle's medium supplemented with 10% FBS (Gemini) 2 mM glutamine (Cellgro) 100 U/ml penicillin and 100 μg/ml streptomycin (Gibco-BRL). The cells were then either transiently transfected with 120 μg of plasmids—engineered to express either wild-type or mutant procaspase-3 in which cysteine-163 was mutated to alanine (9) using lipofectAMINE per the manufacturer's protocol (Life Technologies)—or further treated with G418 to select for stable clones. The coding sequence of each expression construct was sequenced in its entirety before use. The cells were lysed in NP-40 buffer (48 hours following transient transfection) and caspase-3 immunoprecipitations were done as above. The level of wild-type and mutant caspase-3 in the lysates and immunoprecipitates was determined by protein immunoblot or silver stain analysis.
  24. Fas was cross-linked on the surface of cells with 50 ng/ml of anti-Fas IgM clone CH-11 (Upstate Biotech) for 5 min to 2 hours at 37°C. The cells were then washed at 4°C with phosphate-buffered saline (PBS) and immunoprecipitations were done at selected intervals (6).
  25. Cells were grown for 2 24 or 48 hours in the presence or absence of the NO synthase inhibitor L-NMA (5 mM versus 1 mM L -arginine in the medium) (Calbiochem). The cells were then washed and immunoprecipitations were done (6).
  26. We grew 1 × 10 7 10C9 or Jurkat cells for 24 to 48 hours in the presence or absence of L-NMA as described above. Fas agonist antibody (100 ng/ml clone CH-11 Upstate Biotech) and S-nitrosopenicillamine (500 μM) were then added to the appropriate cultures for 50 to 75 min after which the cells were washed with ice-cold PBS and resuspended in 140 μl of buffer A [100 mM Hepes (pH 7.4) 140 mM NaCl 0.5 mM phenylmethylsulfonyl fluoride 5 μg/ml aprotinin and 10 μg/ml leupeptin]. The cells were then lysed with three cycles of freezing and thawing and the crude cytosol was obtained by centrifugation at 12 000 g for 20 min at 4°C. We mixed 50 to 200 μg of cytosolic protein with 400 μM Ac-DEVD-pNA (Quality Biochemicals) in 150 μl of buffer B [100 mM Hepes (pH 7.4) 20% glycerol and protease inhibitors] and incubated it at 37°C. The caspase-3–like activity was calculated by measuring the increased absorbence at 405 nm every 10 min. The reaction mixture without cell lysate or substrate was used as a control.
  27. Stamler J. S., et al., Proc. Natl. Acad. Sci. U.S.A. 89, 8087 (1992); (10.1073/pnas.89.17.8087) / Proc. Natl. Acad. Sci. U.S.A. by Stamler J. S. (1992)
  28. Molina y Vedia L., et al., J. Biol. Chem. 267, 24929 (1992); (10.1016/S0021-9258(19)73985-4) / J. Biol. Chem. by Molina y Vedia L. (1992)
  29. Stamler J. S., et al., Proc. Natl. Acad. Sci. U.S.A. 89, 444 (1992) ; (10.1073/pnas.89.1.444) / Proc. Natl. Acad. Sci. U.S.A. by Stamler J. S. (1992)
  30. 10.1038/364626a0
  31. Gopalakrishna R., Chen Z. H., Gundimeda U., J. Biol. Chem. 268, 27180 (1993); (10.1016/S0021-9258(19)74235-5) / J. Biol. Chem. by Gopalakrishna R. (1993)
  32. Lander H. M., Ogiste J. S., Pearce S. F., Levi R., Novogrodsky A., ibid. 270, 7017 (1995); / ibid. by Lander H. M. (1995)
  33. 10.1016/0092-8674(94)90269-0
  34. Hausladen A., Privalle C. T., Keng T., DeAngelo J., Stamler J. S., ibid. 86, 719 (1996); / ibid. by Hausladen A. (1996)
  35. Nikitovic D., Holmgren A., Spyrou G., Biochem. Biophys. Res. Commun. 242, 109 (1998); (10.1006/bbrc.1997.7930) / Biochem. Biophys. Res. Commun. by Nikitovic D. (1998)
  36. So H. S., et al., ibid. 247, 809 (1998) ; / ibid. by So H. S. (1998)
  37. 10.1126/science.279.5348.234
  38. 10.1073/pnas.95.10.5773
  39. Li Z., et al., Neuron 20, 1039 (1998); (10.1016/S0896-6273(00)80484-5) / Neuron by Li Z. (1998)
  40. ; G. M. Buga.
  41. Wei L. H., Bauer P. M., Fukuto J. M., Ignarro L. J., Am. J. Physiol. 275, R1256 (1998) ; / Am. J. Physiol. by Wei L. H. (1998)
  42. Clementi E., Brown G. C., Feelisch M., Moncada S., Proc. Natl. Acad. Sci. U.S.A. 95, 7631 (1998); (10.1073/pnas.95.13.7631) / Proc. Natl. Acad. Sci. U.S.A. by Clementi E. (1998)
  43. Saura M., et al., Immunity 10, 21 (1999). (10.1016/S1074-7613(00)80003-5) / Immunity by Saura M. (1999)
  44. Ruiz F., Corrales F. J., Miqueo C., Mato J. M., Hepatology 28, 1051 (1998). (10.1002/hep.510280420) / Hepatology by Ruiz F. (1998)
  45. We thank E. Braunwald V. Dzau and R. Finberg for support and E. Alnemri and G. Salvesen for reagents. Supported by GM57601-01 from the National Institute of General Medicine and a Leukemia Society of America translational research award (J.B.M.) the Amyotrophic Lateral Sclerosis Association (A.G.) and HL52529 and HL59130 from the National Heart Lung and Blood Institute (J.S.S.).
Dates
Type When
Created 23 years, 1 month ago (July 27, 2002, 5:40 a.m.)
Deposited 1 year, 7 months ago (Jan. 13, 2024, 4:22 a.m.)
Indexed 1 week, 2 days ago (Aug. 20, 2025, 9:18 a.m.)
Issued 26 years, 4 months ago (April 23, 1999)
Published 26 years, 4 months ago (April 23, 1999)
Published Print 26 years, 4 months ago (April 23, 1999)
Funders 0

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@article{Mannick_1999, title={Fas-Induced Caspase Denitrosylation}, volume={284}, ISSN={1095-9203}, url={http://dx.doi.org/10.1126/science.284.5414.651}, DOI={10.1126/science.284.5414.651}, number={5414}, journal={Science}, publisher={American Association for the Advancement of Science (AAAS)}, author={Mannick, Joan B. and Hausladen, Alfred and Liu, Limin and Hess, Douglas T. and Zeng, Ming and Miao, Qian X. and Kane, Laurie S. and Gow, Andrew J. and Stamler, Jonathan S.}, year={1999}, month=apr, pages={651–654} }