Human iron regulatory protein 2 is easily cleaved in its specific domain: consequences for the haem binding properties of the protein.
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.
Dycke
Camille
C
CEA, DSV, IRTSV, Laboratoire de Chimie et Biologie des Métaux, 17 rue des Martyrs, Grenoble F-38054, France.
Bougault
Catherine
C
Gaillard
Jacques
J
Andrieu
Jean-Pierre
JP
Pantopoulos
Kostas
K
Moulis
Jean-Marc
JM
D004578:Electron Spin Resonance Spectroscopy; D006418:Heme / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D035942:Iron Regulatory Protein 2 / Q000737:chemistry / Q000378:metabolism*; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding; D013056:Spectrophotometry, Ultraviolet
D006418:Heme; D035942:Iron Regulatory Protein 2