Demonstration and characterization of the iron regulatory protein in human brain.
Iron regulatory proteins (IRPs) are cytoplasmic RNA binding proteins that regulate expression of ferritin, erythroid 5-aminolevulinic acid synthase, and transferrin receptor through interaction with conserved RNA stem-loop structures called iron-responsive elements (IREs). Two IRPs (IRP1 and IRP2) have been reported. In the present study we provide evidence for and initial characterization of the IRPs in human brain. Two RNA-protein complexes were obtained by RNA band shift assay on cytoplasmic extracts from human brain. Competition studies indicate that the formations of the RNA-protein complexes are specific to the IRE structure. UV crosslinking of brain cytoplasmic extracts with ferritin IRE RNA transcripts revealed a single RNA-protein complex with a molecular mass of 110 kDa. A single band at 100 kDa was obtained with IRP1 antiserum on western blot analysis of brain cytoplasmic extracts, and a supershift in the RNA-protein complexes was observed with an IRP1 antiserum. Two cDNA clones were isolated from a human brain cDNA library with IRP1 cDNA probes, and both of these cDNA probes recognized a single mRNA species (4.0 kb) from human astrocytoma cells. Purified human brain IRP protein has a molecular mass of approximately 100 kDa and is capable of forming two RNA-protein complexes with ferritin IRE RNA and reacts strongly with IRP1 antiserum. These data indicate that IRP1 is predominant in the adult human brain and, in this tissue, is capable of forming a double IRE/IRP complex. This latter observation suggests the brain IRP undergoes posttranslational modification, the result of which may influence the stability of the IRE/IRP complex.
Hu
J
J
Department of Neuroscience and Anatomy, Penn State University College of Medicine, M. S. Hershey Medical Center, Hershey, Pennsylvania 17033, USA.
Connor
J R
JR
D000328:Adult; D001254:Astrocytoma / Q000378:metabolism; D001667:Binding, Competitive; D001921:Brain / Q000378:metabolism*; D006801:Humans; D007158:Immunologic Techniques; D007501:Iron / Q000378:metabolism; D035941:Iron Regulatory Protein 1; D035942:Iron Regulatory Protein 2; D035925:Iron-Regulatory Proteins; D007506:Iron-Sulfur Proteins / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D051154:Wnt2 Protein
D035925:Iron-Regulatory Proteins; D007506:Iron-Sulfur Proteins; D011518:Proto-Oncogene Proteins; D016601:RNA-Binding Proteins; D051154:Wnt2 Protein; D007501:Iron; D035941:Iron Regulatory Protein 1; D035942:Iron Regulatory Protein 2