Prolyl 4-hydroxylase domain (PHD) proteins are oxygen-dependent enzymes that hydroxylate hypoxia-inducible transcription factor (HIF) ␣-subunits, leading to their subsequent ubiquitination and degradation. Paradoxically, the expression of two family members (PHD2 and PHD3) is induced in hypoxic cell culture despite the reduced availability of the oxygen co-substrate, and it has been suggested that they become functionally relevant following re-oxygenation to rapidly terminate the HIF response. Here we show that PHDs are also induced in hypoxic mice in vivo, albeit in a tissue-specific manner. As demonstrated under chronically hypoxic conditions in vitro, PHD2 and PHD3 show a transient maximum but remain upregulated over more than 10 days, suggesting a feedback down-regulation of HIF-1␣ which then levels off at a novel set point. Indeed, hypoxic induction of PHD2 and PHD3 is paralleled by the attenuation of endogenous HIF-1␣. Using an engineered oxygen-sensitive reporter gene in a cellular background lacking endogenous HIF-1␣ and hence inducible PHD expression, we could show that increased exogenous PHD levels can compensate for a wide range of hypoxic conditions. Similar data were obtained in a reconstituted cellfree system in vitro. In summary, these results suggest that due to their high O 2 K m values, PHDs have optimal oxygensensing properties under all physiologically relevant oxygen concentrations; increased PHDs play a functional role even under oxygen-deprived conditions, allowing the HIF system to adapt to a novel oxygen threshold and to respond to another hypoxic insult. Furthermore, such an autoregulatory oxygen-sensing system would explain how a single mechanism works in a wide variety of differently oxygenated tissues.

IntroductionWhen O 2 delivery is impaired, the resulting hypoxia activates homeostatic mechanisms at the systemic and cellular level. 1 This response involves changes in gene expression mediated by hypoxiainducible factor-1 (HIF-1), the master transcription factor of oxygen-regulated genes. HIF-1 is a heterodimeric protein comprising the oxygen-sensitive ␣-subunit (HIF-1␣, or the more cell-typespecifically expressed HIF-2␣) and the oxygen-insensitive ␤-subunit. 2 Oxygen-regulated gene expression involves binding of HIF to cis-regulatory hypoxia response elements (HREs) of HIF target genes such as erythropoietin or vascular endothelial growth factor. 3 The molecular basis for the hypoxia-induced stability and activity of HIF-1␣ and HIF-2␣ is the O 2 -dependent hydroxylation of distinct prolyl residues. [4][5][6] A family of oxygen-, iron-and 2-oxoglutarate-dependent prolyl-4-hydroxylases has been described recently to hydroxylate the oxygen-labile ␣ subunits of HIF-1 and HIF-2. 5,7,8 This family consists of 3 members called prolyl-4-hydroxylase domain (PHD) 1, PHD2, PHD3, or HIF prolyl hydroxylase (HPH) 3, HPH2, and HPH1, respectively. 4,5 Following prolyl-4-hydroxylation of the critical prolyl residues under normoxic conditions, the ubiquitin ligase von Hippel-Lindau tumor suppressor protein (pVHL) recognizes HIF␣ subunits and targets them for rapid ubiquitination and proteasomal degradation. 9 Binding of pVHL strictly requires prior modification of human HIF-1␣ and HIF-2␣ by prolyl-4-hydroxylation at prolines 402 and 564 or prolines 405 or 531, respectively. 10,11 Limited oxygen supply prevents HIF␣ hydroxylation and degradation. 12 In addition to protein stability, oxygen-dependent C-terminal asparagine hydroxylation of HIF␣ by factor-inhibiting HIF (FIH) prevents transcriptional cofactor recruitment, thereby fine-tuning HIF-1 activity after a further decrease in oxygen availability. 13 Most interestingly, in addition to HIF␣, ankyrin repeats present in IB and NF-B family members have recently been described to be hydroxylated by FIH, demonstrating that hydroxylation is not restricted to the HIF signaling pathway. 14 Besides similarities in the hydroxylation reaction in vitro, the 3 PHDs differ in their ability to hydroxylate HIF-1␣ in vivo and in their organ-specific expression pattern. [15][16][17] Moreover, the phenotypes of PHD knock-out mice demonstrate divergent roles of the 3 PHDs during embryonic development. 18 These data indicate that under physiologic conditions, PHD1, PHD2, and PHD3 mediate different, probably even HIF-independent, oxygen-regulated signal transduction pathways.By searching for novel targets of PHD3 using yeast 2-hybrid technology, we identified activating transcription factor-4 (ATF-4) as a novel interaction partner, and we found that PHD3 confers oxygen-dependent ATF-4 protein stability in a pVHL-independent manner. ATF-4-deficient mice are severely anemic during fetal development, apparently because of an impairment in definitive hematopoiesis. 19 In addition, overexpression of A...

Nonnatural residues can endow proteins with desirable properties. Here, replacing a proline residue that has a cis peptide bond in native ribonuclease A with 5,5-dimethyl-l-proline is shown to accelerate protein folding by 6-fold and enhance conformational stability by DeltaTm = 2.8 +/- 0.3 degrees C while having no effect on enzymatic activity. The rational use of this and other prosthetic segments could enable chemotherapeutic proteins to survive longer in vivo or retain activity after oral administration.

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