Catalytic Properties of the Asparaginyl Hydroxylase (FIH) in the Oxygen Sensing Pathway Are Distinct from Those of Its Prolyl 4-Hydroxylases

Abstract

The activity of hypoxia-inducible transcription factor HIF, an ␣␤ heterodimer that has an essential role in adaptation to low oxygen availability, is regulated by two oxygen-dependent hydroxylation events. Hydroxylation of specific proline residues by HIF prolyl 4-hydroxylases targets the HIF-␣ subunit for proteasomal destruction, whereas hydroxylation of an asparagine in the C-terminal transactivation domain prevents its interaction with the transcriptional coactivator p300. The HIF asparaginyl hydroxylase is identical to a previously known factor inhibiting HIF (FIH). We report here that recombinant FIH has unique catalytic and inhibitory properties when compared with those of the HIF prolyl 4-hydroxylases. FIH was found to require particularly long peptide substrates so that omission of only a few residues from the N or C terminus of a 35-residue HIF-1␣ sequence markedly reduced its substrate activity. Hydroxylation of two HIF-2␣ peptides was far less efficient than that of the corresponding HIF-1␣ peptides. The K m of FIH for O 2 was about 40% of its atmospheric concentration, being about one-third of those of the HIF prolyl 4-hydroxylases but 2.5 times that of the type I collagen prolyl 4-hydroxylase. Several 2-oxoglutarate analogs were found to inhibit FIH but with distinctly different potencies from the HIF prolyl 4-hydroxylases. For example, the two most potent HIF prolyl 4-hydroxylase inhibitors among the compounds studied were the least effective ones for FIH. It should therefore be possible to develop specific small molecule inhibitors for the two enzyme classes involved in the hypoxia response.The hypoxia-inducible transcription factor HIF 1 has a major role in the conserved oxygen-sensitive response pathway that is activated in hypoxic cells. HIF-regulated hypoxia-inducible genes are involved in angiogenesis, vascularization, and anaerobic energy production, for instance. HIFs are ␣␤ heterodimers in which the stability of the ␣ subunit is regulated in an oxygen-dependent manner (for reviews, see Refs. 1-4). Hydroxylation of at least one of two proline residues, Pro 402 and Pro 564 , in the oxygen-dependent degradation domain of human HIF-1␣ mediates the interaction of HIF-␣ with the von Hippel Lindau E3 (ubiquitin-protein isopeptide ligase) ubiquitin ligase complex that targets it for rapid proteasomal degradation under normoxic conditions (5-8). This hydroxylation is catalyzed in humans by three novel cytoplasmic and nuclear HIF prolyl 4-hydroxylases (9 -11) that are distinct from the well characterized collagen prolyl 4-hydroxylases, which reside in the lumen of the endoplasmic reticulum (12-16). Under hypoxic conditions, the oxygen-requiring process of hydroxylation is prevented, and HIF-␣ escapes degradation and dimerizes with HIF-␤, the dimer then recognizing a specific element in the promoters of hypoxia-responsive target genes (1-4).Transcriptional activation in an oxygen-dependent manner is another key step that regulates HIF-␣ activity. Hydroxylation of a specific asparagine residue, Asn 80...

The asparaginyl hydroxylase factor-inhibiting HIF is essential for tumor growth through suppression of the p53–p21 axis

“…Within this microenvironment, the relative contribution of the two hydroxylases, which require oxygen for activity, remains to be clarified. The estimated K m values for oxygen for the PHDs and FIH in vitro are quite different: 250 and 90 mM, respectively (Koivunen et al, 2003). Consistent with this work, a recent study showed in in vivo experiments, using the endogenous human HIF-1a protein, that both prolyl hydroxylation was more sensitive to inhibition to hypoxia (Hx) than asparaginyl hydroxylation (Tian et al, 2011).…”

Turn me on: regulating HIF transcriptional activity

“…40 Recognition and hydroxylation of the CAD by FIH-1 have also been reported to require a minimal substrate sequence of approximately 35 residues, including residues 788-822, which essentially spans sites 1 and 2. 29 Hydroxylation is markedly reduced by deletion of only a few residues from either the N-or C-terminus, demonstrating the importance of both site 1 and site 2 in FIH-1 binding and hydroxylation.…”

“…Interestingly, since FIH-1 appears to have a higher affinity for O 2 than the PHDs based on in vitro experiments, 29 it follows that as the severity of hypoxia increases, the PHDs would be inactivated first, while FIH-1 would require more severe hypoxia to lose activity (Figure 4). Indeed, overexpressed FIH-1 can still exert a catalytic effect at 0.2% Figure 3 Normoxic silencing of the CAD by FIH-1-mediated hydroxylation.…”

“…The estimated K m values of FIH-1 and the PHDs for molecular oxygen were first reported to be approximately 90 and 250 mM, respectively. 29,30 Although these values were determined in vitro using relatively short peptides as substrates, they do provide a guide as to the oxygen sensitivity of these enzymes within cells. Given that physiological oxygen concentrations are around 4-40 mM and below the estimated K m , and the enzymes are present within cells in limiting amounts, this implies that the hydroxylases are poised to respond to physiologically relevant changes in intracellular oxygen concentrations, with any decrease in oxygen tension likely to impart a direct reduction in the catalytic rate of the enzymes (reviewed in Schofield and Ratcliffe 31 and Fandrey et al 32 ).…”

“…While the a-helical conformation at site 2 is similar when bound to FIH or CBP/p300, the a-helix at site 1 is only observed when it is bound to CBP/p300 substrates more efficiently hydroxylated by FIH than HIF-2a substrates in vitro. 29 Although there is quite high conservation at the amino-acid level between the HIF-1a and HIF-2a-CADs, hHIF-1a has a conserved alanine at position 804, whereas HIF-2a has a valine in the equivalent position. As stated previously, this residue forms a hydrogen bond required for the correct orientation of the target asparagine into the active site of FIH-1.…”