Identification of a region on hypoxia-inducible-factor prolyl 4-hydroxylases that determines their specificity for the oxygen degradation domains

Universidad Autónoma de Madrid, Madrid, Madrid, Spain
Biochemical Journal (Impact Factor: 4.4). 01/2008; 408(2):231-40. DOI: 10.1042/BJ20071052
Source: PubMed


HIFs [hypoxia-inducible (transcription) factors] are essential for the induction of an adaptive gene expression programme under low oxygen partial pressure. The activity of these transcription factors is mainly determined by the stability of the HIFalpha subunit, which is regulated, in an oxygen-dependent manner, by a family of three prolyl 4-hydroxylases [EGLN1-EGLN3 (EGL nine homologues 1-3)]. HIFalpha contains two, N- and C-terminal, independent ODDs (oxygen-dependent degradation domains), namely NODD and CODD, that, upon hydroxylation by the EGLNs, target HIFalpha for proteasomal degradation. In vitro studies indicate that each EGLN shows a differential preference for ODDs, However, the sequence determinants for such specificity are unknown. In the present study we showed that whereas EGLN1 and EGLN2 acted upon any of these ODDs to regulate HIF1alpha protein levels and activity in vivo, EGLN3 only acted on the CODD. With the aim of identifying the region within EGLNs responsible for their differential substrate preference, we investigated the activity and binding pattern of different EGLN deletions and chimaeric constructs generated by domain swapping between EGLN1 and EGLN3. These studies revealed a region of 97 residues that was sufficient to confer the characteristic substrate binding observed for each EGLN. Within this region, we identified the minimal sequence (EGLN1 residues 236-252) involved in substrate discrimination. Importantly, mapping of these sequences on the EGLN1 tertiary structure indicates that substrate specificity is determined by a region relatively remote from the catalytic site.

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    • "S247 is located in the variable region (variable with respect to PHD1, PHD2 and PHD3 sequences) between strands β2 and β3 (referred to as ‘β2β3 loop’) in the published crystal structures of PHD2 (McDonough et al. 2006, Chowdhury et al. 2009). Subsequent biophysical analyses have shown that this region is flexible and involved in determining the prolyl hydroxylation site selectivity of the PHDs (Villar et al. 2007, Flashman et al. 2008, Chowdhury et al. 2009). However, a point mutation at this position (Ser289Lys) that could be produced in the soluble form was as active as wild-type PHD2 (in studies using recombinant tPHD2) with respect to hydroxylation of the peptide fragments of the N- and C-terminal oxygen-dependent degradation domains of HIF-1α when these were tested either separately or simultaneously in competition revealing that (at least) under standard incubation conditions, mutations at Ser289 are unlikely to affect absolute activity or selectivity with respect to the hydroxylation sites on HIF-α (data not shown). "
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    • "Hydroxylation of Pro564 and/or 402 residues in HIF-1a is a prerequisite for its interaction with the von Hippel-Lindau (VHL) protein yielding a complex that provides HIF ubiquitinylation and subsequent proteasomal degradation (Kaelin, 2005). Hydroxylation of Pro564 occurs prior to Pro402 (Chan et al., 2005), though some experiments contradict this finding (Villar et al., 2007). Hydroxylation of HIF-1a Asn803 blocks its interaction with transcriptional proactivator p300 (Lando et al., 2002). "
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