Identification of a region on hypoxia-inducible-factor prolyl 4-hydroxylases that determines their specificity for the oxygen degradation domains
Résumé
Hypoxia inducible transcription factors (HIFs) are essential for the induction of an adaptive gene expression program under low oxygen tension. The activity of these transcription factors is mainly determined by the stability of HIFα subunit, which is regulated, in an oxygen-dependent manner, by a family of three prolyl-4-hydroxylases (EGLN1-3). HIFα contains two independent Oxygen Degradation Domains (NODD and CODD) that, upon hydroxylation by EGLNs, target HIFα 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 this work we show that while EGLN1 and 2 exerted their function upon any of these ODDs to regulate HIF1α protein levels and activity in vivo, EGLN3 only acted on 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 chimerical constructs generated by domain swapping between EGLN1 and 3. 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 236-52) involved in substrate discrimination. Importantly, mapping of these sequences on EGLN1 tertiary structure indicates that substrate specificity is determined by a region relatively remote from the catalytic site.
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