Characterization of an oxygen/redox-dependent degradation domain of hypoxia-inducible factor alpha (HIF-alpha) proteins

Hypoxia-inducible factors are heterodimeric DNA-binding complexes that cont rol the hypoxia responses of several genes and regulate the adaptive respon ses to the lack of oxygen. The complex is composed of two b-HLH protein sub units, HIF-1 beta (ARNT), that is constitutively expressed, and a HIF-alpha subunit, that is present only in hypoxic cells. HIF-alpha proteins are con tinuously synthesized, but are rapidly degraded by the ubiquitin-proteasome system under oxic conditions. Hypoxia, transition metals, iron chelators, and several antioxidants stabilize the HIF-alpha proteins, allowing the for mation of the transcriptionally active HIF complex. However, the sequences and mechanisms involved in the regulated degradation of the alpha protein s ubunits are poorly understood. Analysis of the available cloned sequences o f human and mouse members of the HIF-alpha family of proteins revealed an a rea of about 15 amino acids with strong sequence conservation between all t he members. This area corresponds to the region encompassing amino acids 55 7-571 of the hHIF-1 alpha subunit. Fragments of HIF-1 alpha and HIF-3 alpha proteins containing this conserved sequence were able to confer hypoxia re gulation when expressed as fusion proteins in Hep-3B cells. Regulation was observed with all the known hypoxia "mimics," including the reducing thiol donor N-mercaptopropionylglycine (NMPG). Selective alanine substitutions of amino acids 561-568 stabilized the protein in normoxic conditions. Further more, transfection with an expression vector containing a fragment of hHIF- 1 alpha comprising amino acids 540-580 enhanced transactivation activity of the full-length hHIF-1 alpha protein. These results suggest that the above -mentioned conserved sequences are likely involved in the hypoxic stabiliza tion of HIF-alpha proteins. The mechanisms and the interacting ubiquitin-li gases involved in the selective degradation process remain unknown. (C) 199 9 Academic Press.