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A Northwestern blotting approach for studying iron regulatory element-binding proteins

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Abstract

At least two proteins binding to iron regulatory elements (IRE) in mRNA are known, designated as iron regulatory proteins (IRP) 1 and 2. Their binding activity is widely studied by electrophoretic mobility shift assays (EMSA), which resolves one or two bands depending on the species. We used Northwestern blotting to resolve this EMSA complex into four components, and identified two other IRE-binding peptides present in HepG2 cell extracts. We designate these six peptide bands A to F on Northwestern blots, ranging in apparent molecular weight from 111 to 37 kDa. Band C is lost when cells are preloaded with iron or when leupeptin (but not several other protease inhibitors) is included in the extraction buffer. Band E is also lost with leupeptin but increases with iron loading. Binding of all bands is sensitive to iron in vitro. Two-dimensional electrophoresis reveals additional processing, especially indicating charge variants of band C. Northwestern bands A and B both react with an antibody to IRP-1 on parallel Western blots. We conclude that cellular processing can produce multiple IRE-binding species that may be involved in a more complex regulation of iron metabolism than generally appreciated. The Northwestern approach should facilitate studies of processing and binding requirements of proteins and peptides that recognize the IRE sequence. (Mol Cell Biochem 268: 67–74, 2005)

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References

  1. Kennedy M, Mende-Mueller L, Blondin G, Beinert H: Purification and characterization of cytosolic aconitase from beef liver and its relationship to the iron-responsive element binding protein. Proc Natl Acad Sci USA 89: 11730–11734, 1992

    Google Scholar 

  2. Menotti E, Henderson BR, Kühn LC: Translational regulation of mRNAs with distinct IRE sequences by iron regulatory proteins 1 and 2. J Biol Chem 273: 1821–1824, 1998

    Article  Google Scholar 

  3. Eisenstein RS: Iron regulatory proteins and the molecular control of mammalian iron metabolism. Annu Rev Nutr 20: 627–662, 2000

    Article  Google Scholar 

  4. Rouault TA, Stout CD, Kaptain S, Harford JB, Klausner RD: Structural relationship between an iron-regulated RNA-binding protein (IRE-BP) and aconitase: Functional implications. Cell 64: 881–883, 1991

    Article  Google Scholar 

  5. Philpott CC, Klausner RD, Rouault TA: The bifunctional iron-responsive element binding protein/cytosolic aconitase: The role of ative-site residues in ligand binding and regulation. Proc Nat Acad Sci USA 91: 7321–7325, 1994

    Google Scholar 

  6. Phillips JD, Kinikini DV, Yu Y, Guo B, Leibold EA: Differential regulation of IRP1 and IRP2 by nitric oxide in rat hepatoma cells. Blood 87: 2983–2992, 1996

    Google Scholar 

  7. Iwai K, Drake SK, Wehr NB, Weissman AM, LaVaute T, Minato N, Klausner RD, Levine RL, Rouault TA: Iron-dependent oxidation, ubiquitination, and degradation of iron regulatory protein 2: Implications for degradation of oxidized proteins. Proc Natl Acad Sci USA 95: 4924–4928, 1998

    Article  Google Scholar 

  8. Yamanaka K, Ishikawa H, Megumi Y, Tokunaga F, Kanie M, Rouault TA, Morishima I, Minato N, Ishimori K, Iwai K: Identification of the ubiquitin-protein ligase that recognizes oxidized IRP2. Nature Cell Biol 5: 336–340, 2003

    Article  Google Scholar 

  9. Hanson ES, Foot LM, Leibold EA: Hypoxia post-translationally activates iron-regulatory protein 2. J Biol Chem 274: 5047–5052, 1999

    Article  Google Scholar 

  10. Martins EA, Robalinho RL, Meneghini R: Oxidative stress induces activation of a cytosolic protein responsible for control of iron uptake. Arch Biochem Biophys 316: 128–134, 1995

    Article  Google Scholar 

  11. Pantopoulos K, Hentze MW: Rapid responses to oxidative stress mediated by iron regulatory protein. EMBO J 14: 2917–2924, 1995

    Google Scholar 

  12. Schalinske KL, Eisenstein RS: Phosphorylation and activation of both iron regulatory proteins 1 and 2 in HL-60 cells. J Biol Chem 271: 7168–7176, 1996

    Article  Google Scholar 

  13. Leibold EA, Munro HN: Cytoplasmic protein binds in vitro to a highly conserved sequence in the 5′ untranslated region of ferritin heavy- and light-subunit mRNAs. Proc Nat Acad Sci USA 85: 2171–2175, 1988

    Google Scholar 

  14. Roy CN, Blemings KP, Deck KM, Davies PS, Anderson EL, Eisenstein RS, Enns CA: Increased IRP1 and IRP2 RNA binding activity accompanies a reduction of the labile iron pool in HFE-expressing cells. J Cell Physiol 190: 218–226, 2002

    Article  Google Scholar 

  15. Liu Z, Yang D, Qiu Z, Lim K-T, Chong P, Gillam S: Identification of domains in Rubella virus genomic RNA and capsid protein necessary for specific interaction. J Virol 70: 2184–2190, 1996

    Google Scholar 

  16. Desvoyes B, Scholthof K-BG: RNA: protein interactions associated with satellites of pancium mosaic virus. FEBS Lett 485: 25–28, 2000

    Article  Google Scholar 

  17. Sugiura R, Kita A, Shimizu Y, Shuntoh H, Slo SO, Kuno T: Feedback regulation of MAPK signalling by an RNA-binding protein. Nature 424: 961–965, 2003

    Article  Google Scholar 

  18. Deschenes-Furry J, Bélanger G, Perrone-Bizzozero N, Jasmin BJ: Post-transcriptional regulation of acetylcholinesterase mRNAs in nerve growth factor-treated PC12 cells by the RNA-binding protein HuD. J Biol Chem 278: 5710–5717, 2003

    Article  Google Scholar 

  19. Müllner EW, Neupert B, Kühn LC: A specific mRNA binding factor regulates the iron-dependent stability of cytoplasmic transferrin receptor mRNA. Cell 58: 373–382, 1989

    Article  Google Scholar 

  20. Gray NK, Quick S, Goossen B, Constable A, Hirling H, Kühn LC, Hentze MW: Recombinant iron-regulatory factor functions as an iron-responsive-element-binding protein, a translational repressor and an aconitase—A functional assay for translational repression and direct demonstration of the iron switch. Eur J Biochem 218: 657–667, 1993

    Google Scholar 

  21. Guo B, Phillips JD, Yu Y, Leibold EA: Iron regulates the intracellular degradation of iron regulatory protein 2 by the proteasome. J Biol Chem 270: 21645–21651, 1995

    Article  Google Scholar 

  22. Flanagan PR, Hajdu A, Adams PC: Iron-responsive element-binding protein in hemochromatosis liver and intestine. Hepatology 22: 828–832, 1995

    Article  Google Scholar 

  23. Haile DJ, Rouault TA, Tang CK, Chin J, Harford JB, Klausner RD: Reciprocal control of RNA-binding and aconitase in the regulation of the iron-responsive element binding protein: Role of the iron–sulfur cluster. Proc Nat Acad Sci USA 89: 7536–7540, 1992

    Google Scholar 

  24. Popovic Z Templeton DM: Iron accumulation and iron-regulatory protein activity in human hepatoma (HepG2) cells. Mol Cell Biochem 265: 37–45

  25. Samaniego F, Chin J, Iwai K, Rouault TA, Klausner RD: Molecular characterization of a second iron-responsive element binding protein, iron regulatory protein 2. Structure, function, and post-translational regulation. J Biol Chem 269: 30904–30910, 1994

    Google Scholar 

  26. Campanella A, Levi S, Cairo G, Biasiotto G, Arosio P: Blotting analysis of native IRP1: A novel approach to distinguish the different forms of IRP1 in cells and tissues. Biochemistry 43: 195–204, 2004

    Google Scholar 

  27. Guo B, Brown FM, Phillips JD, Yu Y, Leibold EA: Characterization and expression of iron regulatory protein 2 (IRP2). Presence of multiple IRP2 transcripts regulated by intracellular iron levels. J Biol Chem 270: 16529–16535, 1995

    Article  Google Scholar 

  28. Lin E, Graziano J, Freyer G: Regulation of the 75-kDa subunit of mitochondrial compex I by iron. J Biol Chem 276: 27685–27692, 2001

    Article  Google Scholar 

  29. Eisenstein RS, Tuazon PT, Schalinske KL, Anderson SA, Traugh JA: Iron-responsive element-binding protein. Phosphorylation by protein kinase C. J Biol Chem 268: 27363–27370, 1993

    Google Scholar 

  30. Basilion JP, Rouault TA, Massinople CM, klausner RD, Burgess WH: The iron-responsive element-binding protein: Localization of the RNA-binding site to the aconitase active site cleft. Proc Natl Acad Sci USA 91: 574–578, 1994

    Google Scholar 

  31. Beinert H, Kennedy MC, Stout CD: Aconitase as iron–sulfur protein, enzyme, and iron-regulatory protein. Chem Rev 96: 2335–2373, 1996

    Article  Google Scholar 

  32. Hirling H, Henderson BR, Kühn LC: Mutational analysis of the [4Fe–4S]-cluster converting iron regulatory factor from its RNA-binding form to cytoplasmic aconitase. EMBO J 13: 453–461, 1994

    Google Scholar 

  33. Neupert B, Menotti E, Kühn LC: A novel method to identify nucleic acid binding sites in proteins by scanning mutagenesis: Application to iron regulatory protein. Nucleic Acids Res 23: 2579–2583, 1995

    Google Scholar 

  34. Kaldy P, Menotti E, Moret R, Kühn LC: Identification of RNA-binding surfaces in iron regulatory protein-1. EMBO J 18: 6073–6083, 1999

    Article  Google Scholar 

  35. Swenson GR, Walden WE: Localization of an RNA binding elelment of the iron responsive element binding protein within a preoteolytic fragment containing iron coordination ligands. Nucleic Acids Res 22: 2627–2633, 1994

    Google Scholar 

  36. Lill R, Kispal G: Maturation of cellular Fe-S proteins: An essential function of mitochondria. Trends Biochem Sci 25: 352–356, 2000

    Article  CAS  PubMed  Google Scholar 

  37. Tong W-H, Rouault T: Distinct iron–sulfur cluster assembly complexes exist in the cytosol and mitochondria of human cells. EMBO J 19: 5692–5700, 2000

    Google Scholar 

  38. Basilion JP, Kennedy MC, Beinert H, Massinople CM, Klausner RD, Rouault TA: Overexpression of iron-responsive element-binding protein and its analytical characterization as the RNA-binding form, devoid of an iron–sulfur cluster. Arch Biochem Biophys 311: 517–522, 1994

    Google Scholar 

  39. Kühn LC: Regulation of mRNA translation and stability in iron metabolism: Is there a redox switch?, In: C. Gitler, A. Danon, (eds). Cellular Implications of Redox Signaling. Imperial College Press, London, 2003, pp 327–360

    Google Scholar 

  40. Konijn AM, Glickstein H, Vaisman B, Meyron-Holtz EG, Slotki IN, Cabantchik ZI: The cellular labile iron pool and intracellular ferritin in K562 cells. Blood 94: 2128–2134, 1999

    Google Scholar 

  41. Meyron-Holtz EG, Vaisman B, Cabantchik ZI, Fibach E, Rouault TA, Hershko C, Konijn AM: Regulation of intracellular iron metabolism in human erythroid precursors by internalized extracellular ferritin. Blood 94: 3205–3211, 1999

    Google Scholar 

  42. Schalinske KL, Anderson SA, Tuazon PT, Chen OS, Kennedy MC, Eisenstein RS: The iron–sulfur cluster of iron regulatory protein 1 modulates the accessibility of RNA binding and phosphorylation sites. Biochemistry 36: 3950–3958, 1997

    Google Scholar 

  43. Rogers JT, Randall JD, Cahill CM, Eder PS, Huang XD, Gunshin H, Leiter L, McPhee J, Sarang SS, Utsuki T, Greig NH, Lahiri DK, Tanzi RE, Bush AI, Giordano T, Gullans SR: An iron-responsive element type II in the 5′-untranslated region of the Alzheimer’s amyloid precursor protein transcript. J Biol Chem 277: 45518–45528, 2002

    Google Scholar 

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  1. Laboratory Medicine and Pathobiology, University of Toronto, 1 King’s College Circle, Toronto, Canada

    Zvezdana Popovic & Douglas M. Templeton

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  1. Zvezdana Popovic
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  2. Douglas M. Templeton
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Correspondence to Douglas M. Templeton.

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Popovic, Z., Templeton, D.M. A Northwestern blotting approach for studying iron regulatory element-binding proteins. Mol Cell Biochem 268, 67–74 (2005). https://doi.org/10.1007/s11010-005-3167-0

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