Detection of intracellular iron by its regulatory effect.
Intracellular iron regulates gene expression by inhibiting the interaction of iron regulatory proteins (IRPs) with RNA motifs called iron-responsive elements (IREs). To assay this interaction in living cells we have developed two fluorescent IRE-based reporters that rapidly, reversibly, and specifically respond to changes in cellular iron status as well as signaling that modifies IRP activity. The reporters were also sufficiently sensitive to distinguish apo- from holotransferrin in the medium, to detect the effect of modifiers of the transferrin pathway such as HFE, and to detect the donation or chelation of iron by siderophores bound to the lipocalin neutrophil gelatinase-associated lipocalin (Ngal). In addition, alternative configurations of the IRE motif either enhanced or repressed fluorescence, permitting a ratio analysis of the iron-dependent response. These characteristics make it possible to visualize iron-IRP-IRE interactions in vivo.
Li
Jau-Yi
JY
College of Physicians and Surgeons, Columbia Univ., 630 W 168th St., New York, NY 10032, USA.
Ram
Gita
G
Gast
Katherine
K
Chen
Xia
X
Barasch
Kimberly
K
Mori
Kiyoshi
K
Schmidt-Ott
Kai
K
Wang
Jianjun
J
Kuo
Hung-Chieh
HC
Savage-Dunn
Cathy
C
Garrick
Michael D
MD
Barasch
Jonathan
J
D000209:Acute-Phase Proteins / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics; D002461:Cell Line, Transformed; D003593:Cytoplasm / Q000378:metabolism; D005434:Flow Cytometry; D005456:Fluorescent Dyes / Q000378:metabolism; D015870:Gene Expression; D017930:Genes, Reporter / Q000502:physiology*; D006801:Humans; D007501:Iron / Q000378:metabolism / Q000494:pharmacology*; D035941:Iron Regulatory Protein 1 / Q000235:genetics / Q000378:metabolism*; D035942:Iron Regulatory Protein 2 / Q000235:genetics / Q000378:metabolism*; D000071068:Lipocalin-2; D054834:Lipocalins; D008164:Luminescent Proteins / Q000235:genetics; D015513:Oncogene Proteins / Q000378:metabolism; D014176:Protein Biosynthesis; D011518:Proto-Oncogene Proteins; D011990:Receptors, Transferrin / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*
D000209:Acute-Phase Proteins; D001426:Bacterial Proteins; D005456:Fluorescent Dyes; C489506:LCN2 protein, human; D000071068:Lipocalin-2; D054834:Lipocalins; D008164:Luminescent Proteins; D015513:Oncogene Proteins; D011518:Proto-Oncogene Proteins; D011990:Receptors, Transferrin; C054562:yellow fluorescent protein, Bacteria; D007501:Iron; D035941:Iron Regulatory Protein 1; D035942:Iron Regulatory Protein 2