Abstract
Hydroxylation of two conserved prolyl residues in the N- and C-terminal oxygen-dependent degradation domains (NODD and CODD) of the alpha-subunit of hypoxia-inducible factor (HIF) signals for its degradation via the ubiquitin-proteasome pathway. In human cells, three prolyl hydroxylases (PHDs 1-3) belonging to the Fe(II) and 2-oxoglutarate (2OG)-dependent oxygenase family catalyze prolyl hydroxylation with differing selectivity for CODD and NODD. Sequence analysis of the catalytic domains of the PHDs in the light of crystal structures for PHD2, and results for other 2OG oxygenases, suggested that either the C-terminal region or a loop linking two beta-strands (beta2 and beta3 in human PHD2) are important in determining substrate selectivity. Mutation analyses on PHD2 revealed that the beta2beta3 loop is a major determinant in conferring selectivity for CODD over NODD peptides. A chimeric PHD in which the beta2beta3 loop of PHD2 was replaced with that of PHD3 displayed an almost complete selectivity for CODD (in competition experiments), as observed for wild-type PHD3. CODD was observed to bind much more tightly to this chimeric protein than the wild type PHD2 catalytic domain.
Highlights
Lindau ubiquitin ligase complex and subsequent degradation by the proteasome [4, 5]
hypoxia-inducible factor (HIF)-1␣ is hydroxylated in its C-terminal transactivation domain (C-TAD) at Asn-803, in a reaction catalyzed by another iron and 2OG-dependent oxygenase, Factor Inhibiting HIF (FIH) [11,12,13]
Competition experiments under limiting substrate conditions indicated that in the presence of CODD, the truncation variant was slightly better than PHD2181–426 at catalyzing NODD hydroxylation
Summary
Materials—HIF-1␣ peptide substrates were obtained from Peptide Protein Research Ltd., Fareham, UK. The NODD peptide sequence used was DALTLLAPAAGDTIISLDF (one-letter amino acid abbreviations), and the CODD peptide sequence DLDLEMLAPYIPMDDDFQL. The C-terminally truncated PHD2181–402 was produced using site-directed mutagenesis (Stratagene) (for primers see Supplemental Table S1). A deletion variant (PHD2⌬loop) was produced whereby residues 238 –250 (the 23 loop) were removed by site-directed mutagenesis (Stratagene) (Supplemental Table S1). PHD2/1A forward and reverse primers (Supplemental Table S1) were designed and used to amplify the sequence encoding for PHD2181–243. PHD2/1B forward and reverse primers (Supplemental Table S1) were used to amplify the sequence encoding for PHD2251–426. A set of primers (Supplemental Table S1) was designed to mutate the DNA sequence encoding PHD2241–251 to the sequence encoding PHD362–73 by six rounds of site-directed mutagenesis (Stratagene). Binding experiments were performed over a range of concentrations and kinetic data were calculated using the BIAevaluation software (GE Healthcare)
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