HYPOXIA-INDUCIBLE FACTOR 1-ALPHA (HIF-1-ALPHA) IS A NONHEME IRON PROTEIN - IMPLICATIONS FOR OXYGEN SENSING

The hypoxia-inducible factor 1 complex (HIF-1) is involved in the tran scriptional activation of several genes, like erythropoietin and vascu lar endothelial growth factor, that are responsive to the lack of oxyg en. The HIF-1 complex is composed of two b-HLH proteins: HIF-1 beta, t hat is constitutively expressed, and HIF-1 alpha, that is present only in hypoxic cells. The HIF-1 alpha subunit is continuously synthesized and degraded by the ubiquitin-proteasome under oxic conditions. Hypox ia, transition metals, iron chelators, and several antioxidants stabil ize the HIF-1 alpha protein, allowing the formation of the transcripti onally active HIF-1 complex. The mechanisms of oxygen sensing and the pathways leading to HIF-1 alpha stabilization are unclear. Because the involvement of a heme protein oxygen sensor has been postulated, we t ested the heme sensor hypothesis by using a luciferase-expressing cell line (B-l), that is highly responsive to hypoxia, Exposure of B-l cel ls to carbon monoxide and heme synthesis inhibitors failed to show any effect on the hypoxia responsiveness of these cells, suggesting that heme proteins are not involved in hypoxia sensing. Measurement of iron in recombinantly expressed HIF-1 alpha protein revealed that this pro tein binds iron in vivo. Iron binding was localized to a 129-amino aci d peptide between sequences 529 and 658 of the HIF-1 alpha protein. Al though the exact structure of the iron center has not been yet defined , a 2:1 metal/protein molar ratio suggests a di-iron center, probably similar to the one found in hemerythrin. This finding is compatible wi th a model where redox: reaction may occur directly in the iron center of the HIF-1 alpha subunit, affecting its survival in oxic conditions .