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Nitric Oxide Inhibits Ornithine Decarboxylase by S-Nitrosylation
Biochemical and Biophysical Research Communications 262, 355–358 (1999) Article ID bbrc.1999.1210, available online at http://www.idealibrary.com on
Nitric Oxide Inhibits Ornithine Decarboxylase by S-Nitrosylation 1 Philip M. Bauer,* Jon M. Fukuto,* Georgette M. Buga,* Anthony E. Pegg,† and Louis J. Ignarro* ,2 *Department of Molecular and Medical Pharmacology, University of California–Los Angeles School of Medicine, Los Angeles, California 90095-1735; and †Department of Cellular and Molecular Physiology, Milton S. Hershey Medical Center, Hershey, Pennsylvania 17033
Received July 22, 1999
Ornithine decarboxylase (ODC) is the initial enzyme in the polyamine synthetic pathway, and polyamines are required for cell proliferation. We have shown previously that nitric oxide (NO) inhibits ODC activity in Caco-2 cells and in crude cell lysate preparations. In this study we examined the mechanism by which NO inhibits the activity of purified ODC. NO, in the form of S-nitrosocysteine (CysNO), S-nitrosoglutathione (GSNO), or 1,1-diethyl-2-hydroxy-2-nitroso-hydrazine (DEA/NO), inhibited enzyme activity in a concentration-dependent manner. CysNO (1 mM) inhibited ODC activity by approximately 90% and 3 mM GSNO by more than 70%. DEA/NO was less potent, inhibiting enzyme activity by 70% at a concentration of 30 mM. Inhibition of enzyme activity by CysNO, GSNO, or DEA/NO was reversible by addition of dithiothreitol or glutathione. Cuprous ion (Cu (I)) also reversed the inhibitory effect of these NO donor agents. The data presented here support the hypothesis that NO inhibits ODC activity via S-nitrosylation of a critical cysteine residue(s) on ODC. © 1999 Academic Press
Ornithine decarboxylase (ODC) is the initial enzyme in the polyamine synthetic pathway. It catalyzes the conversion of L-ornithine to putrescine and CO 2. Putrescine is then converted into the polyamines, spermidine and spermine. Polyamines are essential for cell 1
This work was supported by N.I.H. Grants HL35014 (L.J.I.), HL40922 (L.J.I.), HL58433 (L.J.I.), and CA18138 (A.E.P.). 2 To whom correspondence should be addressed: Department of Molecular and Medical Pharmacology, UCLA School of Medicine, 23-120 CHS, Box 951735, Los Angeles, CA 90095-1735. Fax: (310) 206-0589. E-mail: [email protected]. Abbreviations used: CysNO, S-nitroso-cysteine; DEA/NO, 1,1-diethyl-2-hydroxy-2-nitroso-hydrazine; DETAPAC, diethylenetriaminepentaacetic acid; DTT, dithiothreitol; GSH, glutathione; GSNO, Snitroso-glutathione; NO, nitric oxide; ODC, ornithine decarboxylase.
proliferation and have been shown to stimulate DNA synthesis and to increase the transcription of growthrelated genes (1, 2). In addition, aberrant polyamine metabolism may play an important role in tumor development (3, 4). Nitric oxide (NO) is a diffusible, short-lived radical that is produced in low levels by two constitutive NO synthases (nNOS and eNOS), and in much greater levels by the inducible NO synthase (iNOS). The activation of NO synthase and the consequent increase in NO production leads to the activation of guanylate cyclase and accumulation of cyclic GMP. Increases in NOS activity and addition of exogenous NO have been widely recognized to result in inhibition of cell proliferation (5–9). Additional studies have suggested that both increased NOS activity and exogenously added NO cause cytostasis (10 –14). The mechanisms by which NOS activity or added NO inhibit cell proliferation are not, however, fully understood. This is due, in part, to the fact that in these studies, cyclic GMP did not account for the cytostatic effect of NO in many cell systems. We have previously shown that NO donor agents inhibit cell proliferation of a colon carcinoma cell line (Caco-2) by a cyclic GMP-independent mechanism (9). The cytostatic effect was reversed by the addition of exogenous putrescine, spermine, and spermidine, but not by ornithine, suggesting that ODC activity was inhibited by NO. In addition, NO donor agents were shown to inhibit ODC activity in in vitro preparations of cell lysates (9, 15, 16). ODC contains, in its active site, two cysteine residues (C70 and C360) (17). C360 is required for enzymatic activity, as mutating C360 to alanine reduces enzyme activity by 98% (18, 19). It is known that NO can react with and modify thiols (20 –27). Accordingly, the objective of this study was to test the hypothesis that NO inhibits ODC activity by S-nitrosylation of a cysteine residue(s) on ODC.
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MATERIALS AND METHODS Chemicals and solutions. All chemicals and reagents were purchased from Sigma Chemical unless otherwise noted. L-[1- 14C]ornithine was purchased from Dupont NEN. Ecolite scintillation cocktail was purchased from ICN. S-Nitrosocysteine (CysNO) (21), S-nitrosoglutathione (GSNO) (28), 1,1-diethyl-2-hydroxy-2-nitrosohydrazine (DEA/NO) (29) and the Cu (I)-acetonitrile complex (30) were synthesized as previously described. Preparation of pHIS-ODC. Using pGEM-ODC as a template (19), PCR was performed replacing the initial ATG codon with an in-frame BamH 1 site (GGATCC) replacing the initial methionine of ODC with a glycine. The PCR product was then ligated into the pQE-30 vector (Qiagen), which contains a 6 3 His tag at the amino terminus of the protein-coding region. The resulting plasmid codes for an ODC protein with the following amino terminus: MRGSHHHHHHGS. Expression and purification of His-ODC. The pHIS-ODC plasmid was transformed into XL1-Blue E. Coli (Stratagene) by electroporation. Cells were grown overnight in a 10 ml culture of LB broth supplemented with 50 mg/ml ampicillin. The 10 ml culture was then added to 490 ml of pre-warmed LB1amp and incubated with shaking at 37°C until reaching a density of OD 600 of .0.5. Protein expression was induced by the addition of 300 mM IPTG and the cells were allowed to grow for 4 hours. The cells were centrifuged at 4000 3 g for 10 min at 4°C. The cell pellet was resuspended in 20 ml of buffer A (20 mM Tris-HCl, pH 8.0; 400 mM NaCl; 5 mM imidazole) and sonicated for 3 min (10 sec on, 10 sec off). The sonicated cells were centrifuged for 1 h at 15,000 3 g. The supernatant was diluted to 40 ml with buffer A and loaded onto a 1 ml Talon metal chelate column (Invitrogen). The column was washed with 10 bed volumes of buffer A, followed by 20 bed volumes of buffer B (20 mM Tris-HCl, pH 8.0; 400 mM NaCl; 10 mM imidazole). The protein was eluted into 1 ml fractions with buffer C (20 mM Tris-HCl, pH 8.0; 400 mM NaCl; 200 mM imidazole). The fractions were supplemented with 2.5 mM dithiothreitol (DTT) to stabilize the ODC activity (31). Aliquots of each fraction were run on a 10% SDS-PAGE gel and fractions containing purified ODC were combined. The imidazole was removed using a PD-10 gel filtration column (Pharmacia) pre-equilibrated with ODC assay buffer (25 mM Tris-HCl, pH 7.5; 2.5 mM DTT; 0.1 mM EDTA). The protein was further concentrated using a C-10 centricon microconcentrator (Amicon). When assayed under standard conditions, the His-tagged proteins have activity comparable to non-His-tagged ODC (32). Ornithine decarboxylase assay. ODC activity was determined by monitoring the formation of [ 14C] CO 2 from L-[1- 14C]ornithine (33). Purified ODC was gel filtered on a G-25 sephadex column to remove DTT and preincubated at 37°C for 15 min with test agents. Assays were conducted for 30 minutes at 37°C in 50 mM Tris-HCl, 0.1 mM diethylenetriaminepentaacetic acid (DETAPAC), pH 7.5, containing 25 ng of enzyme, 0.4 mM ornithine (labeled with 0.1 mCi L-[114 C]ornithine; 55 Ci/mmol), 40 mM pyridoxal 59-phosphate and 50 mg/ml bovine serum albumin in the absence or presence of 2.5 mM DTT, 5mM GSH or 20 mM Cu (I) in a final volume of 250 mL. Reactions were conducted in sealed tubes containing filter paper saturated with 10% KOH. Reactions were initiated by addition of enzyme and terminated by addition of 6 N HCl. Following addition of HCl, tubes were kept at 37°C for 60 min and filters were removed and placed in 5 ml of scintillation cocktail and kept in the dark overnight to extinguish chemiluminescene. The samples were then counted in a liquid scintillation spectrometer. Appropriate controls were run for all assays containing DTT, GSH, or Cu (I) as they affected control enzyme activity. GSH increased control activity by ;20%, DTT increased control activity by ;100%, and Cu (I) decreased control activity by ;40%. All samples containing DTT, GSH or Cu (I) were compared to controls treated with the same compound. Samples treated with NO donor agents alone were compared to untreated enzyme assayed in the absence of DTT, GSH or Cu (I).
FIG. 1. Concentration-dependent inhibition of purified recombinant ODC activity by Hg 21 and its reversibility by thiols. DTT was removed from the enzyme by gel filtration through a sephadex G-25 column. ODC was preincubated with various concentrations of HgCl 2 at 37°C for 15 min. The enzyme was then added to the assay buffer containing 0.4 mM L-[1- 14C] ornithine and 40 mM pyridoxal 59phosphate in the absence or presence of 2.5 mM DTT or 5 mM GSH. ODC activity was measured after a 30-min incubation at 37°C as described in Materials and Methods. Data represent the mean 6 SE of duplicate determinations from 3 separate experiments.
RESULTS A reduced thiol group in the active site of ODC is essential for its catalytic activity. Therefore, we studied the effect of HgCl 2, a sulfhydryl reacting agent, on ODC activity. HgCl 2 produced a concentrationdependent inhibition of ODC activity (Fig. 1). Inhibition of enzyme activity was completely reversed by the thiol reducing agent DTT (2.5 mM) or the cysteinecontaining tripeptide glutathione (GSH; 5 mM). In order to determine the effect of NO on ODC the NO donor agents CysNO, GSNO and DEA/NO were employed. The enzyme was preincubated at 37°C for 15 min with each of the NO donors and then assayed for activity for 30 min. All three NO donor agents produced a concentration-dependent inhibition of ODC activity, although with differing potencies (Fig. 2A). CysNO was the most potent, inhibiting ODC activity by ;90% at 1 mM. GSNO inhibited ODC activity by ;70% at 3 mM, while at least 30 mM DEA/NO was required to observe a similar effect. Decomposed DEA/ NO, CysNO and GSNO had no appreciable effect on ODC activity. Addition of DTT or GSH to the assay buffer partially reversed the inhibition of ODC activity by all three NO donor agents (Fig. 2B), which is consistent with a mechanism involving S-nitrosylation of a cysteine residue(s) on ODC. Cu (I) is known to release NO from S-nitrosothiols (34, 35). Therefore, as another means of determining whether ODC is inhibited by S-nitrosylation, we tested if Cu (I) could reverse the inhibition of ODC activity caused by
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CysNO, GSNO, and DEA/NO. Since Cu (I) is unstable and normally disproportionates into Cu (0) and Cu (II) we used a Cu (I)-acetonitrile complex, which is stable until added to aqueous solution. The reactions were performed in the absence of the metal chelator DETAPAC. Addition of 20 mM Cu (I) to the enzyme reaction mixture completely reversed the inhibition of ODC activity caused by all three NO donor agents (Fig. 2C). The addition of acetonitrile to the enzyme mixture had no appreciable effect on enzyme activity. DISCUSSION
FIG. 2. The inhibitory effects of NO donor agents on purified ODC activity and its reversibility by thiols and copper. DTT was separated from the enzyme by gel filtration through a sephadex G-25 column in all experiments. (A) Concentration dependent inhibition of ODC activity by CysNO, GSNO and DEA/NO. ODC was preincubated with various concentrations of CysNO, GSNO or DEA/NO at 37°C for 15 min. The enzyme was then added to the assay buffer containing 0.4 mM ornithine (labeled with 0.1 mCi L-[1- 14C]ornithine; 55 Ci/mmol) and 40 mM pyridoxal 59-phosphate. ODC activity was measured after a 30-min incubation at 37°C as described in Materials and Methods. (B) Reversal of the inhibitory effects of NO by DTT or GSH. ODC was preincubated with 0.5 mM CysNO, 3 mM GSNO, or 30 mM DEA/NO. Enzyme activity was assayed as in A. The assay buffer was prepared in the presence or absence of 2.5 mM DTT or 5 mM GSH. (C) Reversal of the inhibitory effect of NO donor
The present data suggest that DEA/NO, CysNO and GSNO inhibit ODC activity by S-nitrosylation. Hg 21 inhibited enzyme activity in a concentration dependent manner, and inhibition was reversed by addition of either DTT or GSH. This implies that modulation of a thiol group in ODC inhibits catalytic activity, and that reversal of this modification restores catalytic activity. One mM CysNO inhibited ODC activity by approximately 90% and 3 mM GSNO by more than 70%. DEA/NO was less potent, inhibiting enzyme activity by 70% at a concentration of 30 mM. Both DTT and GSH reversed the inhibition of ODC activity caused by all three NO donor agents, which is consistent with a mechanism involving modification of a cysteine residue(s) on ODC. In addition, Cu (I), which is known to release NO from S-nitrosothiols (34, 35), completely reversed the inhibition of ODC activity caused by CysNO, GSNO or DEA/NO. The differences in potencies and chemical properties between DEA/NO and the S-nitrosothiol NO donor agents, CysNO and GSNO, suggest different chemical mechanisms of S-nitrosothiol formation. DEA/NO has a half-life of 2 min and its action can be attributed solely to NO and its subsequent chemistry. In addition, DEA/NO releases 2 molecules of NO for every DEA/NO molecule. Considering the conditions of the assay, it is most likely that S-nitrosylation of ODC by DEA/NO occurs through the formation of N 2O 3, the product of the reaction between NO and O 2. Although N 2O 3 is a potent nitrating agent, the reaction between NO and O 2 is second order with respect to NO and therefore the rate of the reaction is quite slow at low NO concentrations. This is consistent with the lower potency of DEA/NO as an inhibitor of ODC activity. The chemistry of CysNO and GSNO is different from that of DEA/NO. Under the appropriate conditions,
agents by Cu (I). ODC was preincubated with 0.5 mM CysNO, 3 mM GSNO, or 30 mM DEA/NO. 20 mM Cu (I)-acetonitrile was added to the assay buffer immediately after adding enzyme. Enzyme activity was then assayed as in A. *Significantly different from values for NO donor agent alone (p , 0.001). Data represent the mean 6 SE of duplicate determinations from 3 separate experiments.
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S-nitrosothiols can release NO (35). However, S-nitrosothiols also react with other thiols which results in either the release of nitroxyl (HNO) (36), or participation in S-transnitrosation reactions (37). Generation of HNO and its subsequent attack on protein thiol residues would likely lead to irreversible modification of the thiol residue (36). S-Transnitrosation has been suggested to be the predominant mechanism accounting for the biological effects of GSNO (38), which may lead to the S-nitrosylation of thiol containing proteins (39). S-Transnitrosation also occurs in vivo between S-nitroso-albumin and low-molecular weight thiols, and there is evidence that S-transnitrosation reactions are involved in the biological actions of NO (37, 40). Furthermore, the rate of S-transnitrosation reactions is rapid, which is consistent with the relatively high potency of CysNO and GSNO as inhibitors of ODC activity. Therefore, the potency of CysNO and GSNO, along with the evidence for S-nitrosylation of a cysteine residue(s) on ODC, suggest that CysNO and GSNO act by S-transnitrosation. The present study reveals that NO inhibits mammalian ODC activity by S-nitrosylation of a critical thiol residue(s), which is likely to be the C360 residue at the active site of ODC. This may be at least one cyclic GMP-independent mechanism by which NO inhibits cell proliferation, and explains why the cytostatic effect of NO can be reversed by the presence of excess putrescine, spermidine or spermine, but not ornithine (9). In view of the occurrence of S-nitrosothiols in vivo (41) and the fairly high potency of CysNO and GSNO as ODC inhibitors, S-nitrosothiols may represent a physiological mechanism of regulating ODC activity and, therefore, cell proliferation. REFERENCES 1. Ginty, D. D., Osborne, D. L., and Seidel, E. R. (1989) Amer. J. Physiol. 257, G145–G150. 2. Celano, P., Baylin, S. B., and Casero, R. A., Jr. (1989) J. Biol. Chem. 264, 8922– 8927. 3. Pegg, A. E., Shantz, L. M., and Coleman, C. S. (1995) J. Cellular Biochem. Supp. 22, 132–138. 4. McCann, P. P., and Pegg, A. E. (1992) Pharmacol. Ther. 54, 195–215. 5. Blachier, F., Robert, V., Selamnia, M., Mayeur, C., and Duee, P. H. (1996) FEBS Lett. 396, 315–318. 6. Cornwell, T. L., Arnold, E., Boerth, N. J., and Lincoln, T. M. (1994) Amer. J. Physiol. 267, C1405–C1413. 7. Garg, U. C., and Hassid, A. (1989) Amer. J. Physiol. 257, F60 –F66. 8. Granger, D. L., Hibbs, J. B., Jr., Perfect, J. R., and Durack, D. T. (1990) J. Clin. Invest. 85, 264 –273. 9. Buga, G. M., Wei, L. H., Bauer, P. M., Fukuto, J. M., and Ignarro, L. J. (1998) Amer. J. Physiol. 275, R1256 –R1264. 10. Lepoivre, M., Flaman, J. M., Bobe´, P., Lemaire, G., and Henry, Y. (1994) J. Biol. Chem. 269, 21891–21897. 11. Nunokawa, Y., and Tanaka, S. (1992) Biochem. Biophys. Res. Commun. 188, 409 – 415.
12. Punjabi, C. J., Laskin, D. L., Heck, D. E., and Laskin, J. D. (1992) J. Immunol. 149, 2179 –2184. 13. Scott-Burden, T., Schini, V. B., Elizondo, E., Junquero, D. C., and Vanhoutte, P. M. (1992) Circ. Res. 71, 1088 –1100. 14. Stein, C. S., Fabry, Z., Murphy, S., and Hart, M. N. (1995) Mol. Immunol. 32, 965–973. 15. Blachier, F., Briand, D., Selamnia, M., Robert, V., Guihot, G., and Mayeur, C. (1998) Biochem. Pharmacol. 55, 1235–1239. 16. Satriano, J., Ishizuka, S., Archer, D. C., Blantz, R. C., and Kelly, C. J. (1999) Amer. J. Physiol. 276, C892–C899. 17. Kern, A. D., Oliveira, M. A., Coffino, P., and Hackert, M. L. (1999) Structure 7, 567–581. 18. Coleman, C. S., Stanley, B. A., and Pegg, A. E. (1993) J. Biol. Chem. 268, 24572–24579. 19. Lu, L., Stanley, B. A., and Pegg, A. E. (1991) Biochem. J. 277, 671– 675. 20. Wink, D. A., Nims, R. W., Darbyshire, J. F., Christodoulou, D., Hanbauer, I., Cox, G. W., Laval, F., Laval, J., Cook, J. A., Krishna, M. C., DeGraff, W. G., and Mitchell, J. B. (1994) Chemical Res. Toxicol. 7, 519 –525. 21. Stamler, J. S., Simon, D. I., Osborne, J. A., Mullins, M. E., Jaraki, O., Michel, T., Singel, D. J., and Loscalzo, J. (1992) Proc. Nat. Acad. Sci. USA 89, 444 – 448. 22. Stamler, J. S. (1995) Curr. Top. Microbio. Immunol. 196, 19 –36. 23. Li, J., Billiar, T. R., Talanian, R. V., and Kim, Y. M. (1997) Biochem. Biophys. Res. Commun. 240, 419 – 424. 24. Mohr, S., Zech, B., Lapetina, E. G., and Bru¨ne, B. (1997) Biochem. Biophys. Res. Commun. 238, 387–391. 25. Hu, H., Chiamvimonvat, N., Yamagishi, T., and Marban, E. (1997) Circ. Res. 81, 742–752. 26. Upchurch, G. R., Jr., Welch, G. N., and Loscalzo, J. (1995) Adv. Pharm. 34, 343–349. 27. Gow, A. J., Buerk, D. G., and Ischiropoulos, H. (1997) J. Biol. Chem. 272, 2841–2845. 28. Hart, T. W. (1985) Tet. Lett. 26, 2013–2016. 29. Drago, R. S., and Paulik, F. E. (1960) J. Am. Chem. Soc. 82, 96 –98. 30. Hemmerich, P., and Sigwart, C. (1963) Experentia 19, 488 – 489. 31. Pegg, A. E., and Williams-Ashman, H. G. (1981) in Polyamines in Biological Medicine (Morris, D. R., and Marton, L. J., Eds.), pp. 3– 42, Dekker, New York. 32. Coleman, C. S., Stanley, B. A., Viswanath, R., and Pegg, A. E. (1994) J. Biol. Chem. 269, 3155–3158. 33. Coleman, C. S., and Pegg, A. E. (1998) Methods Mol. Bio. 79, 41– 44. 34. Al-Sa’doni, H. H., Megson, I. L., Bisland, S., Butler, A. R., and Flitney, F. W. (1997) Brit. J. Pharmacol. 121, 1047–1050. 35. Singh, R. J., Hogg, N., Joseph, J., and Kalyanaraman, B. (1996) J. Biol. Chem. 271, 18596 –18603. 36. Wong, P. S., Hyun, J., Fukuto, J. M., Shirota, F. N., DeMaster, E. G., Shoeman, D. W., and Nagasawa, H. T. (1998) Biochem. 37, 5362–5371. 37. Liu, Z., Rudd, M. A., Freedman, J. E., and Loscalzo, J. (1998) J. Pharm. Exp. Ther. 284, 526 –534. 38. Park, J. W., Billman, G. E., and Means, G. E. (1993) Biochem. Mol. Bio. Int. 30, 885– 891. 39. Mohr, S., Stamler, J. S., and Bru¨ne, B. (1994) FEBS Lett. 348, 223–227. 40. Scharfstein, J. S., Keaney, J. F., Jr., Slivka, A., Welch, G. N., Vita, J. A., Stamler, J. S., and Loscalzo, J. (1994) J. Clin. Invest. 94, 1432–1439. 41. Stamler, J. S., Jaraki, O., Osbourne, J., Simon, D. I., Keaney, J., Vita, J., Singel, D., Valeri, C. R., and Loscalzo, J. (1992) Proc. Nat. Acad. Sci. USA 89, 7674 –7677.
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Nitric Oxide Inhibits Ornithine Decarboxylase by S-Nitrosylation 1 Philip M. Bauer,* Jon M. Fukuto,* Georgette M. Buga,* Anthony E. Pegg,† and Louis J. Ignarro* ,2 *Department of Molecular and Medical Pharmacology, University of California–Los Angeles School of Medicine, Los Angeles, California 90095-1735; and †Department of Cellular and Molecular Physiology, Milton S. Hershey Medical Center, Hershey, Pennsylvania 17033
Received July 22, 1999
Ornithine decarboxylase (ODC) is the initial enzyme in the polyamine synthetic pathway, and polyamines are required for cell proliferation. We have shown previously that nitric oxide (NO) inhibits ODC activity in Caco-2 cells and in crude cell lysate preparations. In this study we examined the mechanism by which NO inhibits the activity of purified ODC. NO, in the form of S-nitrosocysteine (CysNO), S-nitrosoglutathione (GSNO), or 1,1-diethyl-2-hydroxy-2-nitroso-hydrazine (DEA/NO), inhibited enzyme activity in a concentration-dependent manner. CysNO (1 mM) inhibited ODC activity by approximately 90% and 3 mM GSNO by more than 70%. DEA/NO was less potent, inhibiting enzyme activity by 70% at a concentration of 30 mM. Inhibition of enzyme activity by CysNO, GSNO, or DEA/NO was reversible by addition of dithiothreitol or glutathione. Cuprous ion (Cu (I)) also reversed the inhibitory effect of these NO donor agents. The data presented here support the hypothesis that NO inhibits ODC activity via S-nitrosylation of a critical cysteine residue(s) on ODC. © 1999 Academic Press
Ornithine decarboxylase (ODC) is the initial enzyme in the polyamine synthetic pathway. It catalyzes the conversion of L-ornithine to putrescine and CO 2. Putrescine is then converted into the polyamines, spermidine and spermine. Polyamines are essential for cell 1
This work was supported by N.I.H. Grants HL35014 (L.J.I.), HL40922 (L.J.I.), HL58433 (L.J.I.), and CA18138 (A.E.P.). 2 To whom correspondence should be addressed: Department of Molecular and Medical Pharmacology, UCLA School of Medicine, 23-120 CHS, Box 951735, Los Angeles, CA 90095-1735. Fax: (310) 206-0589. E-mail: [email protected]. Abbreviations used: CysNO, S-nitroso-cysteine; DEA/NO, 1,1-diethyl-2-hydroxy-2-nitroso-hydrazine; DETAPAC, diethylenetriaminepentaacetic acid; DTT, dithiothreitol; GSH, glutathione; GSNO, Snitroso-glutathione; NO, nitric oxide; ODC, ornithine decarboxylase.
proliferation and have been shown to stimulate DNA synthesis and to increase the transcription of growthrelated genes (1, 2). In addition, aberrant polyamine metabolism may play an important role in tumor development (3, 4). Nitric oxide (NO) is a diffusible, short-lived radical that is produced in low levels by two constitutive NO synthases (nNOS and eNOS), and in much greater levels by the inducible NO synthase (iNOS). The activation of NO synthase and the consequent increase in NO production leads to the activation of guanylate cyclase and accumulation of cyclic GMP. Increases in NOS activity and addition of exogenous NO have been widely recognized to result in inhibition of cell proliferation (5–9). Additional studies have suggested that both increased NOS activity and exogenously added NO cause cytostasis (10 –14). The mechanisms by which NOS activity or added NO inhibit cell proliferation are not, however, fully understood. This is due, in part, to the fact that in these studies, cyclic GMP did not account for the cytostatic effect of NO in many cell systems. We have previously shown that NO donor agents inhibit cell proliferation of a colon carcinoma cell line (Caco-2) by a cyclic GMP-independent mechanism (9). The cytostatic effect was reversed by the addition of exogenous putrescine, spermine, and spermidine, but not by ornithine, suggesting that ODC activity was inhibited by NO. In addition, NO donor agents were shown to inhibit ODC activity in in vitro preparations of cell lysates (9, 15, 16). ODC contains, in its active site, two cysteine residues (C70 and C360) (17). C360 is required for enzymatic activity, as mutating C360 to alanine reduces enzyme activity by 98% (18, 19). It is known that NO can react with and modify thiols (20 –27). Accordingly, the objective of this study was to test the hypothesis that NO inhibits ODC activity by S-nitrosylation of a cysteine residue(s) on ODC.
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0006-291X/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
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MATERIALS AND METHODS Chemicals and solutions. All chemicals and reagents were purchased from Sigma Chemical unless otherwise noted. L-[1- 14C]ornithine was purchased from Dupont NEN. Ecolite scintillation cocktail was purchased from ICN. S-Nitrosocysteine (CysNO) (21), S-nitrosoglutathione (GSNO) (28), 1,1-diethyl-2-hydroxy-2-nitrosohydrazine (DEA/NO) (29) and the Cu (I)-acetonitrile complex (30) were synthesized as previously described. Preparation of pHIS-ODC. Using pGEM-ODC as a template (19), PCR was performed replacing the initial ATG codon with an in-frame BamH 1 site (GGATCC) replacing the initial methionine of ODC with a glycine. The PCR product was then ligated into the pQE-30 vector (Qiagen), which contains a 6 3 His tag at the amino terminus of the protein-coding region. The resulting plasmid codes for an ODC protein with the following amino terminus: MRGSHHHHHHGS. Expression and purification of His-ODC. The pHIS-ODC plasmid was transformed into XL1-Blue E. Coli (Stratagene) by electroporation. Cells were grown overnight in a 10 ml culture of LB broth supplemented with 50 mg/ml ampicillin. The 10 ml culture was then added to 490 ml of pre-warmed LB1amp and incubated with shaking at 37°C until reaching a density of OD 600 of .0.5. Protein expression was induced by the addition of 300 mM IPTG and the cells were allowed to grow for 4 hours. The cells were centrifuged at 4000 3 g for 10 min at 4°C. The cell pellet was resuspended in 20 ml of buffer A (20 mM Tris-HCl, pH 8.0; 400 mM NaCl; 5 mM imidazole) and sonicated for 3 min (10 sec on, 10 sec off). The sonicated cells were centrifuged for 1 h at 15,000 3 g. The supernatant was diluted to 40 ml with buffer A and loaded onto a 1 ml Talon metal chelate column (Invitrogen). The column was washed with 10 bed volumes of buffer A, followed by 20 bed volumes of buffer B (20 mM Tris-HCl, pH 8.0; 400 mM NaCl; 10 mM imidazole). The protein was eluted into 1 ml fractions with buffer C (20 mM Tris-HCl, pH 8.0; 400 mM NaCl; 200 mM imidazole). The fractions were supplemented with 2.5 mM dithiothreitol (DTT) to stabilize the ODC activity (31). Aliquots of each fraction were run on a 10% SDS-PAGE gel and fractions containing purified ODC were combined. The imidazole was removed using a PD-10 gel filtration column (Pharmacia) pre-equilibrated with ODC assay buffer (25 mM Tris-HCl, pH 7.5; 2.5 mM DTT; 0.1 mM EDTA). The protein was further concentrated using a C-10 centricon microconcentrator (Amicon). When assayed under standard conditions, the His-tagged proteins have activity comparable to non-His-tagged ODC (32). Ornithine decarboxylase assay. ODC activity was determined by monitoring the formation of [ 14C] CO 2 from L-[1- 14C]ornithine (33). Purified ODC was gel filtered on a G-25 sephadex column to remove DTT and preincubated at 37°C for 15 min with test agents. Assays were conducted for 30 minutes at 37°C in 50 mM Tris-HCl, 0.1 mM diethylenetriaminepentaacetic acid (DETAPAC), pH 7.5, containing 25 ng of enzyme, 0.4 mM ornithine (labeled with 0.1 mCi L-[114 C]ornithine; 55 Ci/mmol), 40 mM pyridoxal 59-phosphate and 50 mg/ml bovine serum albumin in the absence or presence of 2.5 mM DTT, 5mM GSH or 20 mM Cu (I) in a final volume of 250 mL. Reactions were conducted in sealed tubes containing filter paper saturated with 10% KOH. Reactions were initiated by addition of enzyme and terminated by addition of 6 N HCl. Following addition of HCl, tubes were kept at 37°C for 60 min and filters were removed and placed in 5 ml of scintillation cocktail and kept in the dark overnight to extinguish chemiluminescene. The samples were then counted in a liquid scintillation spectrometer. Appropriate controls were run for all assays containing DTT, GSH, or Cu (I) as they affected control enzyme activity. GSH increased control activity by ;20%, DTT increased control activity by ;100%, and Cu (I) decreased control activity by ;40%. All samples containing DTT, GSH or Cu (I) were compared to controls treated with the same compound. Samples treated with NO donor agents alone were compared to untreated enzyme assayed in the absence of DTT, GSH or Cu (I).
FIG. 1. Concentration-dependent inhibition of purified recombinant ODC activity by Hg 21 and its reversibility by thiols. DTT was removed from the enzyme by gel filtration through a sephadex G-25 column. ODC was preincubated with various concentrations of HgCl 2 at 37°C for 15 min. The enzyme was then added to the assay buffer containing 0.4 mM L-[1- 14C] ornithine and 40 mM pyridoxal 59phosphate in the absence or presence of 2.5 mM DTT or 5 mM GSH. ODC activity was measured after a 30-min incubation at 37°C as described in Materials and Methods. Data represent the mean 6 SE of duplicate determinations from 3 separate experiments.
RESULTS A reduced thiol group in the active site of ODC is essential for its catalytic activity. Therefore, we studied the effect of HgCl 2, a sulfhydryl reacting agent, on ODC activity. HgCl 2 produced a concentrationdependent inhibition of ODC activity (Fig. 1). Inhibition of enzyme activity was completely reversed by the thiol reducing agent DTT (2.5 mM) or the cysteinecontaining tripeptide glutathione (GSH; 5 mM). In order to determine the effect of NO on ODC the NO donor agents CysNO, GSNO and DEA/NO were employed. The enzyme was preincubated at 37°C for 15 min with each of the NO donors and then assayed for activity for 30 min. All three NO donor agents produced a concentration-dependent inhibition of ODC activity, although with differing potencies (Fig. 2A). CysNO was the most potent, inhibiting ODC activity by ;90% at 1 mM. GSNO inhibited ODC activity by ;70% at 3 mM, while at least 30 mM DEA/NO was required to observe a similar effect. Decomposed DEA/ NO, CysNO and GSNO had no appreciable effect on ODC activity. Addition of DTT or GSH to the assay buffer partially reversed the inhibition of ODC activity by all three NO donor agents (Fig. 2B), which is consistent with a mechanism involving S-nitrosylation of a cysteine residue(s) on ODC. Cu (I) is known to release NO from S-nitrosothiols (34, 35). Therefore, as another means of determining whether ODC is inhibited by S-nitrosylation, we tested if Cu (I) could reverse the inhibition of ODC activity caused by
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CysNO, GSNO, and DEA/NO. Since Cu (I) is unstable and normally disproportionates into Cu (0) and Cu (II) we used a Cu (I)-acetonitrile complex, which is stable until added to aqueous solution. The reactions were performed in the absence of the metal chelator DETAPAC. Addition of 20 mM Cu (I) to the enzyme reaction mixture completely reversed the inhibition of ODC activity caused by all three NO donor agents (Fig. 2C). The addition of acetonitrile to the enzyme mixture had no appreciable effect on enzyme activity. DISCUSSION
FIG. 2. The inhibitory effects of NO donor agents on purified ODC activity and its reversibility by thiols and copper. DTT was separated from the enzyme by gel filtration through a sephadex G-25 column in all experiments. (A) Concentration dependent inhibition of ODC activity by CysNO, GSNO and DEA/NO. ODC was preincubated with various concentrations of CysNO, GSNO or DEA/NO at 37°C for 15 min. The enzyme was then added to the assay buffer containing 0.4 mM ornithine (labeled with 0.1 mCi L-[1- 14C]ornithine; 55 Ci/mmol) and 40 mM pyridoxal 59-phosphate. ODC activity was measured after a 30-min incubation at 37°C as described in Materials and Methods. (B) Reversal of the inhibitory effects of NO by DTT or GSH. ODC was preincubated with 0.5 mM CysNO, 3 mM GSNO, or 30 mM DEA/NO. Enzyme activity was assayed as in A. The assay buffer was prepared in the presence or absence of 2.5 mM DTT or 5 mM GSH. (C) Reversal of the inhibitory effect of NO donor
The present data suggest that DEA/NO, CysNO and GSNO inhibit ODC activity by S-nitrosylation. Hg 21 inhibited enzyme activity in a concentration dependent manner, and inhibition was reversed by addition of either DTT or GSH. This implies that modulation of a thiol group in ODC inhibits catalytic activity, and that reversal of this modification restores catalytic activity. One mM CysNO inhibited ODC activity by approximately 90% and 3 mM GSNO by more than 70%. DEA/NO was less potent, inhibiting enzyme activity by 70% at a concentration of 30 mM. Both DTT and GSH reversed the inhibition of ODC activity caused by all three NO donor agents, which is consistent with a mechanism involving modification of a cysteine residue(s) on ODC. In addition, Cu (I), which is known to release NO from S-nitrosothiols (34, 35), completely reversed the inhibition of ODC activity caused by CysNO, GSNO or DEA/NO. The differences in potencies and chemical properties between DEA/NO and the S-nitrosothiol NO donor agents, CysNO and GSNO, suggest different chemical mechanisms of S-nitrosothiol formation. DEA/NO has a half-life of 2 min and its action can be attributed solely to NO and its subsequent chemistry. In addition, DEA/NO releases 2 molecules of NO for every DEA/NO molecule. Considering the conditions of the assay, it is most likely that S-nitrosylation of ODC by DEA/NO occurs through the formation of N 2O 3, the product of the reaction between NO and O 2. Although N 2O 3 is a potent nitrating agent, the reaction between NO and O 2 is second order with respect to NO and therefore the rate of the reaction is quite slow at low NO concentrations. This is consistent with the lower potency of DEA/NO as an inhibitor of ODC activity. The chemistry of CysNO and GSNO is different from that of DEA/NO. Under the appropriate conditions,
agents by Cu (I). ODC was preincubated with 0.5 mM CysNO, 3 mM GSNO, or 30 mM DEA/NO. 20 mM Cu (I)-acetonitrile was added to the assay buffer immediately after adding enzyme. Enzyme activity was then assayed as in A. *Significantly different from values for NO donor agent alone (p , 0.001). Data represent the mean 6 SE of duplicate determinations from 3 separate experiments.
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S-nitrosothiols can release NO (35). However, S-nitrosothiols also react with other thiols which results in either the release of nitroxyl (HNO) (36), or participation in S-transnitrosation reactions (37). Generation of HNO and its subsequent attack on protein thiol residues would likely lead to irreversible modification of the thiol residue (36). S-Transnitrosation has been suggested to be the predominant mechanism accounting for the biological effects of GSNO (38), which may lead to the S-nitrosylation of thiol containing proteins (39). S-Transnitrosation also occurs in vivo between S-nitroso-albumin and low-molecular weight thiols, and there is evidence that S-transnitrosation reactions are involved in the biological actions of NO (37, 40). Furthermore, the rate of S-transnitrosation reactions is rapid, which is consistent with the relatively high potency of CysNO and GSNO as inhibitors of ODC activity. Therefore, the potency of CysNO and GSNO, along with the evidence for S-nitrosylation of a cysteine residue(s) on ODC, suggest that CysNO and GSNO act by S-transnitrosation. The present study reveals that NO inhibits mammalian ODC activity by S-nitrosylation of a critical thiol residue(s), which is likely to be the C360 residue at the active site of ODC. This may be at least one cyclic GMP-independent mechanism by which NO inhibits cell proliferation, and explains why the cytostatic effect of NO can be reversed by the presence of excess putrescine, spermidine or spermine, but not ornithine (9). In view of the occurrence of S-nitrosothiols in vivo (41) and the fairly high potency of CysNO and GSNO as ODC inhibitors, S-nitrosothiols may represent a physiological mechanism of regulating ODC activity and, therefore, cell proliferation. REFERENCES 1. Ginty, D. D., Osborne, D. L., and Seidel, E. R. (1989) Amer. J. Physiol. 257, G145–G150. 2. Celano, P., Baylin, S. B., and Casero, R. A., Jr. (1989) J. Biol. Chem. 264, 8922– 8927. 3. Pegg, A. E., Shantz, L. M., and Coleman, C. S. (1995) J. Cellular Biochem. Supp. 22, 132–138. 4. McCann, P. P., and Pegg, A. E. (1992) Pharmacol. Ther. 54, 195–215. 5. Blachier, F., Robert, V., Selamnia, M., Mayeur, C., and Duee, P. H. (1996) FEBS Lett. 396, 315–318. 6. Cornwell, T. L., Arnold, E., Boerth, N. J., and Lincoln, T. M. (1994) Amer. J. Physiol. 267, C1405–C1413. 7. Garg, U. C., and Hassid, A. (1989) Amer. J. Physiol. 257, F60 –F66. 8. Granger, D. L., Hibbs, J. B., Jr., Perfect, J. R., and Durack, D. T. (1990) J. Clin. Invest. 85, 264 –273. 9. Buga, G. M., Wei, L. H., Bauer, P. M., Fukuto, J. M., and Ignarro, L. J. (1998) Amer. J. Physiol. 275, R1256 –R1264. 10. Lepoivre, M., Flaman, J. M., Bobe´, P., Lemaire, G., and Henry, Y. (1994) J. Biol. Chem. 269, 21891–21897. 11. Nunokawa, Y., and Tanaka, S. (1992) Biochem. Biophys. Res. Commun. 188, 409 – 415.
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