Mammalian IRPs (iron regulatory proteins), IRP1 and IRP2, are cytosolic RNA-binding proteins that post-transcriptionally control the mRNA of proteins involved in storage, transport, and utilization of iron. In iron-replete cells, IRP2 undergoes degradation by the ubiquitin/proteasome pathway. Binding of haem to a 73aa-Domain (73-amino-acid domain) that is unique in IRP2 has been previously proposed as the initial iron-sensing mechanism. It is shown here that recombinant IRP2 and the 73aa-Domain are sensitive to proteolysis at the same site. NMR results suggest that the isolated 73aa-Domain is not structured. Iron-independent cleavage of IRP2 within the 73aa-Domain also occurs in lung cancer (H1299) cells. Haem interacts with a cysteine residue only in truncated forms of the 73aa-Domain, as shown by a series of complementary physicochemical approaches, including NMR, EPR and UV–visible absorption spectroscopy. In contrast, the cofactor is not ligated by the same residue in the full-length peptide or intact IRP2, although non-specific interaction occurs between these molecular forms and haem. Therefore it is unlikely that the iron-dependent degradation of IRP2 is mediated by haem binding to the intact 73aa-Domain, since the sequence resembling an HRM (haem-regulatory motif) in the 73aa-Domain does not provide an axial ligand of the cofactor unless this domain is cleaved.
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December 2007
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Research Article|
November 28 2007
Human iron regulatory protein 2 is easily cleaved in its specific domain: consequences for the haem binding properties of the protein
Camille Dycke;
Camille Dycke
1
*CEA, DSV, IRTSV, Laboratoire de Chimie et Biologie des Métaux, 17 rue des Martyrs, Grenoble F-38054, France
†LCBM, CNRS, Grenoble, France
‡Université Joseph Fourier, Grenoble, France
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Catherine Bougault;
Catherine Bougault
‡Université Joseph Fourier, Grenoble, France
§Laboratoire de Résonance Magnétique Nucléaire, Institut de Biologie Structurale – Jean-Pierre Ebel, 41 rue Jules Horowitz, F-38027 Grenoble, France
∥IBS, CNRS, Grenoble, France
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Jacques Gaillard;
Jacques Gaillard
¶Département de Recherche Fondamentale sur la Matière Condensée, Service de Chimie Inorganique et Biologique, 17 rue des Martyrs, Grenoble F-38054, France
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Jean-Pierre Andrieu;
Jean-Pierre Andrieu
‡Université Joseph Fourier, Grenoble, France
∥IBS, CNRS, Grenoble, France
**Laboratoire d'Enzymologie Moléculaire, Institut de Biologie Structurale – Jean-Pierre Ebel, 41 rue Jules Horowitz, F-38027 Grenoble, France
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Kostas Pantopoulos;
Kostas Pantopoulos
††Lady Davis Institute for Medical Research, 3999 Côte Ste Catherine, Montréal, QC, Canada H3T 1E2
‡‡Department of Medicine, McGill University, Montréal, QC, Canada
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Jean-Marc Moulis
Jean-Marc Moulis
2
†LCBM, CNRS, Grenoble, France
‡Université Joseph Fourier, Grenoble, France
2To whom correspondence should be addressed (email jean-marc.moulis@cea.fr).
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Publisher: Portland Press Ltd
Received:
July 24 2007
Revision Received:
August 28 2007
Accepted:
August 31 2007
Accepted Manuscript online:
August 31 2007
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© The Authors Journal compilation © 2007 Biochemical Society
2007
Biochem J (2007) 408 (3): 429–439.
Article history
Received:
July 24 2007
Revision Received:
August 28 2007
Accepted:
August 31 2007
Accepted Manuscript online:
August 31 2007
Citation
Camille Dycke, Catherine Bougault, Jacques Gaillard, Jean-Pierre Andrieu, Kostas Pantopoulos, Jean-Marc Moulis; Human iron regulatory protein 2 is easily cleaved in its specific domain: consequences for the haem binding properties of the protein. Biochem J 15 December 2007; 408 (3): 429–439. doi: https://doi.org/10.1042/BJ20070983
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