Cloning and characterization of the rat HIF-1 alpha prolyl-4-hydroxylase-1 gene
- Protein Expr Purif. 2005 Aug;42(2):295-304. doi: 10.1016/j.pep.2005.03.036.
- 1. Systems Biology, Berlex Biosciences, Richmond, CA 94806, USA. [email protected]
Prolyl-4-hydroxylase domain-containing Enzymes (PHDs) mediate the oxygen-dependent regulation of the heterodimeric transcription factor hypoxia-inducible factor-1 (HIF-1). Under normoxic conditions, one of the subunits of HIF-1, HIF-1alpha, is hydroxylated on specific proline residues to target HIF-1alpha for degradation by the ubiquitin-proteasome pathway. Under hypoxic conditions, the hydroxylation by the PHDs is attenuated by lack of the oxygen substrate, allowing HIF-1 to accumulate, translocate to the nucleus, and mediate HIF-mediated gene transcription. In several mammalian species including humans, three PHDs have been identified. We report here the cloning of a full-length rat cDNA that is highly homologous to the human and murine PHD-1 Enzymes and encodes a protein that is 416 Amino acids long. Both cDNA and protein are widely expressed in rat tissues and cell types. We demonstrate that purified and crude baculovirus-expressed rat PHD-1 exhibits HIF-1alpha specific prolyl hydroxylase activity with similar substrate affinities and is comparable to human PHD-1 protein.