Kinetic Rationale for Selectivity toward N- and C-terminal Oxygen-dependent Degradation Domain Substrates Mediated by a Loop Region of Hypoxia-Inducible Factor Prolyl Hydroxylases

Abstract

Hydroxylation of two conserved prolyl residues in the N-and C-terminal oxygen-dependent degradation domains (NODD and CODD) of the ␣-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 ␤-strands (␤2 and ␤3 in human PHD2) are important in determining substrate selectivity. Mutation analyses on PHD2 revealed that the ␤2␤3 loop is a major determinant in conferring selectivity for CODD over NODD peptides. A chimeric PHD in which the ␤2␤3 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.The heterodimeric transcription factor Hypoxia-Inducible Factor (HIF) 2 plays a central role in the response of metazoans to hypoxia (1, 2). Levels of the HIF-␤ subunit are independent of oxygen (3), but under conditions of normoxia, the HIF-␣ subunit is hydroxylated in its oxygen-dependent degradation domain (ODDD), enabling recognition by the von HippelLindau (pVHL) ubiquitin ligase complex and subsequent degradation by the proteasome (4, 5). Under hypoxic conditions, hydroxylation of the HIF-␣ subunit is reduced and it dimerizes with HIF-␤, resulting in the increased expression of hypoxically regulated target genes such as erythropoietin and vascular endothelial growth factor, which enable adaptation to low oxygen concentrations (see reviews in Refs. 6, 7). Hydroxylation of the three HIF-␣ isoforms is catalyzed by prolyl hydroxylases (in humans, PHDs 1-3) (8 -10), belonging to the family of iron(II) and 2-oxoglutarate (2OG)-dependent oxygenases, and occurs at two specific proline residues in the HIF-1␣ ODDD, Pro-402 (N-terminal oxygen-dependent degradation domain, NODD) and Pro-564 (C-terminal oxygen-dependent degradation domain, CODD). HIF-1␣ is also 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) (11-13). Hydroxylation of Asn-803 prevents binding of HIF to the transcriptional co-activator p300/ CBP in an oxygen-dependent fashion.PHD1 is localized to the nucleus, PHD2 and FIH are commonly found in the cytoplasm, and PHD3 is distributed in both the nucleus and cytoplasm (14). One study on relative activities of recombinant human PHDs, produced by rabbit reticulocyte in vitro transcription and translation, on HIF-1␣, 2␣, and 3␣ has given an order of activity of PHD2 ϭ 3 Ͼ 1 (15) although a different study proposes that PHD2 has the greatest ac...

Genetic causes of erythrocytosis and the oxygen-sensing pathway

“…However, the domains are not identical, and there is evidence that there are determinants that convey differences in specificity between the three isoforms. For example, a loop in PHD2 that is distinct from that in PHD3 is responsible for the ability of PHD2 to hydroxylate both the primary and secondary sites of hydroxylation in HIF-1α 96,97 . PHD3, in contrast, displays a marked preference for the primary site of hydroxylation.…”

Isolated erythrocytosis: study of 67 patients and identification of three novel germ-line mutations in the prolyl hydroxylase domain protein 2 (PHD2) gene

“…K423E substitution, harbored by Patient 3, causes the loss of a positive charged side chain with the acquisition of a complementary charged glutamate, three coding residues from the end of the enzyme. Lys423 is an evolutionary conserved residue in ortholog forms of PHD2 (Mus musculus and Rattus norvegicus, and results for other 2OG oxygenases, 23,24 suggested that removal of the C-terminus of the enzyme actually promotes uncoupled turnover of 2OG. However, due to the lack of the experimental solution of the C-terminal PHD2 structure, 15 it is not possible to anticipate the K423E mutation effects on protein stability and function.…”

Oxygenases for oxygen sensing

“…Recent kinetic studies also indicate that the preferred substrates for the PHDs are significantly longer than the relatively short peptides (<20-40 residues) used in some analyses [45,47]. Coupled with the structural analyses, it seems likely that the substrate specificity of the PHDs, in part, is determined by regions relatively remote from the iron center, with biochemical assays suggesting the involvement of a substrate binding "lid" [48,49].…”