title: Biochemical characterization of human HIF hydroxylases using HIF protein substrates that contain all three hydroxylation sites.
abstract: The HIF (hypoxia-inducible factor) plays a central regulatory role in oxygen homoeostasis. HIF proteins are regulated by three Fe(II)- and α-KG (α-ketoglutarate)-dependent prolyl hydroxylase enzymes [PHD (prolyl hydroxylase domain) isoenzymes 1-3 or PHD1, PHD2 and PHD3] and one asparaginyl hydroxylase [FIH (factor inhibiting HIF)]. The prolyl hydroxylases control the abundance of HIF through oxygen-dependent hydroxylation of specific proline residues in HIF proteins, triggering subsequent ubiquitination and proteasomal degradation. FIH inhibits the HIF transcription activation through asparagine hydroxylation. Understanding the precise roles and regulation of these four Fe(II)- and α-KG-dependent hydroxylases is of great importance. In the present paper, we report the biochemical characterization of the first HIF protein substrates that contain the CODDD (C-terminal oxygen-dependent degradation domain), the NODDD (N-terminal oxygen-dependent degradation domain) and the CAD (C-terminal transactivation domain). Using LC-MS/MS (liquid chromatography-tandem MS) detection, we show that all three PHD isoenzymes have a strong preference for hydroxylation of the CODDD proline residue over the NODDD proline residue and the preference is observed for both HIF1α and HIF2α protein substrates. In addition, steady-state kinetic analyses show differential substrate selectivity for HIF and α-KG in reference to the three PHD isoforms and FIH.
authors.affiliation: Department of Cancer Research, GlaxoSmithKline, Collegeville, PA 19426, USA.
authors.forename: Melissa B
authors.initials: MB
authors.lastname: Pappalardi
authors.forename: Dean E
authors.initials: DE
authors.lastname: McNulty
authors.forename: John D
authors.initials: JD
authors.lastname: Martin
authors.forename: Kelly E
authors.initials: KE
authors.lastname: Fisher
authors.forename: Yong
authors.initials: Y
authors.lastname: Jiang
authors.forename: Matthew C
authors.initials: MC
authors.lastname: Burns
authors.forename: Huizhen
authors.initials: H
authors.lastname: Zhao
authors.forename: Thau
authors.initials: T
authors.lastname: Ho
authors.forename: Sharon
authors.initials: S
authors.lastname: Sweitzer
authors.forename: Benjamin
authors.initials: B
authors.lastname: Schwartz
authors.forename: Roland S
authors.initials: RS
authors.lastname: Annan
authors.forename: Robert A
authors.initials: RA
authors.lastname: Copeland
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Tummino
authors.forename: Lusong
authors.initials: L
authors.lastname: Luo
mesh_terms: D051792:Basic Helix-Loop-Helix Proteins / Q000737:chemistry* / Q000378:metabolism*; D001665:Binding Sites; D006801:Humans; D006900:Hydroxylation; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000737:chemistry* / Q000378:metabolism*; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism; D011393:Procollagen-Proline Dioxygenase / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D000099162:Endothelial PAS Domain-Containing Protein 1
chemical_list: D051792:Basic Helix-Loop-Helix Proteins; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D007527:Isoenzymes; D011393:Procollagen-Proline Dioxygenase; D000099162:Endothelial PAS Domain-Containing Protein 1