Hypoxia-inducible factor prolyl-hydroxylase: purification and assays of PHD2.
The adaptation of animals to oxygen availability is mediated by a transcription factor termed hypoxia-inducible factor (HIF). HIF is an alpha (alpha)/beta (beta) heterodimer that binds hypoxia response elements (HREs) of target genes, including some of medicinal importance, such as erythropoietin (EPO) and vascular endothelial growth factor (VEGF). While the concentration of the HIF-beta subunit, a constitutive nuclear protein, does not vary with oxygen availability, the abundance and activity of the HIF-alpha subunits are tightly regulated via oxygen-dependent modification of specific residues. Hydroxylation of prolyl residues (Pro402 and Pro564 in HIF-1alpha) promotes interaction with the von Hippel-Lindau E3 ubiquitin ligase and, consequently, proteolytic destruction by the ubiquitin-proteasome pathway. This prolyl hydroxylation is catalyzed by the prolyl-hydroxylase domain (PHD) containing enzymes for which three isozymes have been identified in humans (1-3). Additionally, asparaginyl hydroxylation (Asn803 in HIF-1alpha) by factor-inhibiting HIF (FIH) ablates interaction of the HIF-alpha subunit with the coactivator p300, providing an alternative mechanism for down-regulation of HIF-dependent genes. Under hypoxic conditions, when oxygen-mediated regulation of the alpha-subunits is curtailed or minimized, dimerization of the alpha- and beta-subunits occurs with subsequent target gene upregulation. Therapeutic activation of HIF signaling has been suggested as a potential treatment for numerous conditions, including ischemia, stroke, heart attack, inflammation, and wounding. One possible route to achieve this is via inhibition of the HIF hydroxylases. This chapter details methods for the purification and assaying of PHD2, the most abundant PHD and the most important in setting steady-state levels of HIF-alpha. Assays are described that measure the activity of PHD2 via direct and indirect means. Furthermore, conditions for the screening of small molecules against PHD2 are described.
Hewitson
Kirsty S
KS
Department of Chemistry, Chemistry Research Laboratory, University of Oxford, Oxford, United Kingdom.
Schofield
Christopher J
CJ
Ratcliffe
Peter J
PJ
D000818:Animals; D002245:Carbon Dioxide / Q000737:chemistry; D002853:Chromatography, Liquid; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D051793:Hypoxia-Inducible Factor 1 / Q000737:chemistry*; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000737:chemistry; D013058:Mass Spectrometry; D010100:Oxygen / Q000737:chemistry; D010101:Oxygen Consumption; D011393:Procollagen-Proline Dioxygenase / Q000032:analysis* / Q000737:chemistry* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D013386:Succinates / Q000032:analysis / Q000737:chemistry
D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D011994:Recombinant Proteins; D013386:Succinates; D002245:Carbon Dioxide; D011393:Procollagen-Proline Dioxygenase; D010100:Oxygen