Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 2000 Jun 1;348(Pt 2):315–320.

Oxalomalate, a competitive inhibitor of aconitase, modulates the RNA-binding activity of iron-regulatory proteins.

M Festa 1, A Colonna 1, C Pietropaolo 1, A Ruffo 1
PMCID: PMC1221068  PMID: 10816424

Abstract

We investigated the effect of oxalomalate (OMA, alpha-hydroxy-beta-oxalosuccinic acid), a competitive inhibitor of aconitase, on the RNA-binding activity of the iron-regulatory proteins (IRP1 and IRP2) that control the post-transcriptional expression of various proteins involved in iron metabolism. The RNA-binding activity of IRP was evaluated by electrophoretic mobility-shift assay of cell lysates from 3T3-L1 mouse fibroblasts, SH-SY5Y human cells and mouse livers incubated in vitro with OMA, with and without 2-mercaptoethanol (2-ME). Analogous experiments were performed in vivo by prolonged incubation (72 h) of 3T3-L1 cells with OMA, and by injecting young mice with equimolar concentrations of oxaloacetate and glyoxylate, which are the precursors of OMA synthesis. OMA remarkably decreased the binding activity of IRP1 and, when present, of IRP2, in all samples analysed. In addition, the recovery of IRP1 by 2-ME in the presence of OMA was constantly lower versus control values. These findings suggest that the severe decrease in IRP1 RNA-binding activity depends on: (i) linking of OMA to the active site of aconitase, which prevents the switch to IRP1 and explains resistance to the reducing agents, and (ii) possible interaction of OMA with some functional amino acid residues in IRP that are responsible for binding to the specific mRNA sequences involved in the regulation of iron metabolism.

Full Text

The Full Text of this article is available as a PDF (226.1 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Adinolfi A., Guarriera-Bobyleva V., Olezza S., Ruffo A. Inhibition by oxalomalate of rat liver mitochondrial and extramitochondrial aconitate hydratase. Biochem J. 1971 Nov;125(2):557–562. doi: 10.1042/bj1250557. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Adinolfi A., Moratti R., Olezza S., Ruffo A. Control of the citric acid cycle by glyoxylate. The mechanism of inhibition of oxoglutarate dehydrogenase, isocitrate dehydrogenase and aconitate hydratase. Biochem J. 1969 Sep;114(3):513–518. doi: 10.1042/bj1140513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Adinolfi A., Speranza M. L., Guarriera-Bobyleva V., Ruffo A. Citrate metabolism in liver of rats treated with glyoxylate and oxaloacetate. Ital J Biochem. 1973 Mar-Jun;22(2):92–104. [PubMed] [Google Scholar]
  4. Eisenstein R. S., Blemings K. P. Iron regulatory proteins, iron responsive elements and iron homeostasis. J Nutr. 1998 Dec;128(12):2295–2298. doi: 10.1093/jn/128.12.2295. [DOI] [PubMed] [Google Scholar]
  5. Guo B., Brown F. M., Phillips J. D., Yu Y., Leibold E. A. Characterization and expression of iron regulatory protein 2 (IRP2). Presence of multiple IRP2 transcripts regulated by intracellular iron levels. J Biol Chem. 1995 Jul 14;270(28):16529–16535. doi: 10.1074/jbc.270.28.16529. [DOI] [PubMed] [Google Scholar]
  6. Guo B., Phillips J. D., Yu Y., Leibold E. A. Iron regulates the intracellular degradation of iron regulatory protein 2 by the proteasome. J Biol Chem. 1995 Sep 15;270(37):21645–21651. doi: 10.1074/jbc.270.37.21645. [DOI] [PubMed] [Google Scholar]
  7. Guo B., Yu Y., Leibold E. A. Iron regulates cytoplasmic levels of a novel iron-responsive element-binding protein without aconitase activity. J Biol Chem. 1994 Sep 30;269(39):24252–24260. [PubMed] [Google Scholar]
  8. Haile D. J., Rouault T. A., Harford J. B., Kennedy M. C., Blondin G. A., Beinert H., Klausner R. D. Cellular regulation of the iron-responsive element binding protein: disassembly of the cubane iron-sulfur cluster results in high-affinity RNA binding. Proc Natl Acad Sci U S A. 1992 Dec 15;89(24):11735–11739. doi: 10.1073/pnas.89.24.11735. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Henderson B. R., Kühn L. C. Differential modulation of the RNA-binding proteins IRP-1 and IRP-2 in response to iron. IRP-2 inactivation requires translation of another protein. J Biol Chem. 1995 Sep 1;270(35):20509–20515. doi: 10.1074/jbc.270.35.20509. [DOI] [PubMed] [Google Scholar]
  10. Hentze M. W., Kühn L. C. Molecular control of vertebrate iron metabolism: mRNA-based regulatory circuits operated by iron, nitric oxide, and oxidative stress. Proc Natl Acad Sci U S A. 1996 Aug 6;93(16):8175–8182. doi: 10.1073/pnas.93.16.8175. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hentze M. W., Rouault T. A., Harford J. B., Klausner R. D. Oxidation-reduction and the molecular mechanism of a regulatory RNA-protein interaction. Science. 1989 Apr 21;244(4902):357–359. doi: 10.1126/science.2711187. [DOI] [PubMed] [Google Scholar]
  12. Hirling H., Henderson B. R., Kühn L. C. Mutational analysis of the [4Fe-4S]-cluster converting iron regulatory factor from its RNA-binding form to cytoplasmic aconitase. EMBO J. 1994 Jan 15;13(2):453–461. doi: 10.1002/j.1460-2075.1994.tb06280.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Iwai K., Klausner R. D., Rouault T. A. Requirements for iron-regulated degradation of the RNA binding protein, iron regulatory protein 2. EMBO J. 1995 Nov 1;14(21):5350–5357. doi: 10.1002/j.1460-2075.1995.tb00219.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kennedy M. C., Emptage M. H., Dreyer J. L., Beinert H. The role of iron in the activation-inactivation of aconitase. J Biol Chem. 1983 Sep 25;258(18):11098–11105. [PubMed] [Google Scholar]
  15. Klausner R. D., Rouault T. A., Harford J. B. Regulating the fate of mRNA: the control of cellular iron metabolism. Cell. 1993 Jan 15;72(1):19–28. doi: 10.1016/0092-8674(93)90046-s. [DOI] [PubMed] [Google Scholar]
  16. Melefors O., Hentze M. W. Iron regulatory factor--the conductor of cellular iron regulation. Blood Rev. 1993 Dec;7(4):251–258. doi: 10.1016/0268-960x(93)90012-s. [DOI] [PubMed] [Google Scholar]
  17. Paraskeva E., Hentze M. W. Iron-sulphur clusters as genetic regulatory switches: the bifunctional iron regulatory protein-1. FEBS Lett. 1996 Jun 24;389(1):40–43. doi: 10.1016/0014-5793(96)00574-1. [DOI] [PubMed] [Google Scholar]
  18. Philpott C. C., Haile D., Rouault T. A., Klausner R. D. Modification of a free Fe-S cluster cysteine residue in the active iron-responsive element-binding protein prevents RNA binding. J Biol Chem. 1993 Aug 25;268(24):17655–17658. [PubMed] [Google Scholar]
  19. Philpott C. C., Klausner R. D., Rouault T. A. The bifunctional iron-responsive element binding protein/cytosolic aconitase: the role of active-site residues in ligand binding and regulation. Proc Natl Acad Sci U S A. 1994 Jul 19;91(15):7321–7325. doi: 10.1073/pnas.91.15.7321. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. RUFFO A., ADINOLFI A., BUDILLON G., CAPOBIANCO G. Control of the citric acid cycle by glyoxylate. 2. Mechanism of the inhibition of respiration in liver and kidney particles. Biochem J. 1962 Dec;85:593–600. doi: 10.1042/bj0850593. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. RUFFO A., ROMANO M., ADINOLFI A. Inhibition of aconitase by glyoxylate plus oxaloacetate. Biochem J. 1959 Aug;72:613–618. doi: 10.1042/bj0720613. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. RUFFO A., TESTA E., ADINOLFIA, PELIZZA G. Control of the etric acid cycle by glyoxylate. I. A new inhibitor of aconitase formed by the condensation of glyoxylate with oxaloacetate. Biochem J. 1962 Dec;85:588–593. doi: 10.1042/bj0850588. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Recalcati S., Conte D., Cairo G. Preferential activation of iron regulatory protein-2 in cell lines as a result of higher sensitivity to iron. Eur J Biochem. 1999 Jan;259(1-2):304–309. doi: 10.1046/j.1432-1327.1999.00038.x. [DOI] [PubMed] [Google Scholar]
  24. Rouault T. A., Klausner R. D. Iron-sulfur clusters as biosensors of oxidants and iron. Trends Biochem Sci. 1996 May;21(5):174–177. [PubMed] [Google Scholar]
  25. Rouault T. A., Stout C. D., Kaptain S., Harford J. B., Klausner R. D. Structural relationship between an iron-regulated RNA-binding protein (IRE-BP) and aconitase: functional implications. Cell. 1991 Mar 8;64(5):881–883. doi: 10.1016/0092-8674(91)90312-m. [DOI] [PubMed] [Google Scholar]
  26. Ruffo A., Testa E., Adinolfi A., Pelizza G., Moratti R. Control of the citric acid cycle by glyoxylate. Mechanism of the inhibition by oxalomalate and gamma-hydroxy-alpha-oxoglutarate. Biochem J. 1967 Apr;103(1):19–23. doi: 10.1042/bj1030019. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Theil E. C. The iron responsive element (IRE) family of mRNA regulators. Regulation of iron transport and uptake compared in animals, plants, and microorganisms. Met Ions Biol Syst. 1998;35:403–434. [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES