REVIEW ARTICLE Oxygen-dependent regulation of hypoxia-inducible factors by prolyl and asparaginyl hydroxylation David Lando 1 , Jeffrey J. Gorman 2, *, Murray L. Whitelaw 1 and Daniel J. Peet 1 1 Department of Molecular BioSciences (Biochemistry) and the Centre for Molecular Genetics of Development, University of Adelaide, Australia; 2 CSIRO Health Sciences and Nutrition, Parkville, Victoria, Australia To sustain life mammals have an absolute and continual requirement for oxygen, which is necessary to produce energy for normal cell survival and growth. Hence, main- taining oxygen homeostasis is a critical requirement and mammals have evolved a wide range of cellular and phy- siological responses to adapt to changes in oxygen avail- ability. In the past few years it has become evident that the transcriptional protein complex hypoxia-inducible factor (HIF) is a key regulator of these processes. In this review we will focus on the way oxygen availability regulates HIF proteins and in particular we will discuss the way oxygen- dependent hydroxylation of specific amino acid residues has been demonstrated to regulate HIF function at the level of both protein stability and transcriptional potency. Keywords: oxygen sensing; hypoxia; hydroxylation; transcriptional regulation; hypoxia-inducible factor (HIF). Introduction The development of complex cardiovascular, respiratory and hemopoietic systems in mammals provides a means to efficiently capture and deliver oxygen (O 2 )fromthe environment to every cell of the body. While a sufficient supply of oxygen is essential for energy production, too much oxygen in the form of free radicals (i.e. superoxide, OH – ) can be detrimental [1]. Therefore to maximize oxygen use, as well as at the same time minimize the impact of oxygen free radicals, cells have developed mechanisms to maintain oxygen concentrations within a narrow physiological range. To achieve this mammals regulate oxygen consumption and levels by a combination of both cellular and systemic processes. For example, when oxygen is limiting (hypoxia) individual cells decrease oxidative phos- phorylation and rely on glycolysis as the primary means of ATP production. To facilitate this switch to glycolysis cells up-regulate the expression of a select set of genes, such as those encoding glycolytic enzymes and glucose transporters [2]. Other hypoxic responses monitor global oxygen levels and effect system wide changes in tissue oxygen availability. For instance, the hypoxic induction of the hormone erythropoietin (Epo) by the kidney stimulates red blood cell production to increase the oxygen carrying capacity of the blood [2]. Tissues and cells experiencing reduced oxygen supply, like those associated with wound healing, increase the levels of the angiogenic cytokine vascular endothelial growth factor (VEGF). VEGF then acts on endothelial cells to stimulate the proliferation of new blood vessels, which in turn help maintain an adequate supply of oxygen [3]. However, in many disease states such as cancer, stroke and heart attack these same oxygen delivery systems can become misregulated and hypoxia becomes a major component of the pathophysiology of these diseases [4]. For many years the Epo system was used to study the molecular mechanisms associated with the induction of hypoxia responsive genes and from these investigations the hypoxia-inducible factor (HIF) was identified as a key transcriptional hypoxic regulator of Epo [5,6]. Subsequent research has now found that a large number of other hypoxia-inducible genes (Fig. 1) are also induced by HIF under hypoxic conditions, revealing that HIF functions as a master transcriptional regulator of the adaptive response to hypoxia [7–53]. Hypoxia-inducible factor The HIF transcriptional complex is a heterodimer consist- ing of one of three alpha subunits (HIF-1a,HIF-2a or HIF-3a) and a beta subunit called ARNT [6,54–57]. Correspondence to D. J. Peet, Department of Molecular BioSciences (Biochemistry) and the Centre for Molecular Genetics of Development, University of Adelaide, Adelaide, South Australia, 5005 Australia. Fax: + 61 8 8303 4348, E-mail: daniel.peet@adelaide.edu.au Abbreviations: ARNT, aryl hydrocarbon nuclear translocator; bHLH, basic helix-loop-helix; CAD, carboxy-terminal transactivation domain; CBP, CREB binding protein; CH, cysteine-histidine; CO, carbon monoxide; CREB, cyclic AMP-response element binding protein; DMOG, dimethyloxalylglycine; Dsfx, desferrioxamine; Epo, erythropoietin; FIH-1, factor inhibiting HIF-1; HIF, hypoxia- inducible factor; HPH, HIF prolyl-4-hydroxylase; MAPK, mitogen activated protein kinase; NAD, amino-terminal transactivation domain; NO, nitric oxide; ODD, oxygen-dependent degradation domain; PAS, Per-ARNT-Sim; PHD, prolyl hydroxylase domain-containing protein; RLL, arginine-dileucine; VEGF, vascular endothelial growth factor; VHL, von Hippel-Lindau. *Present address: Institute for Molecular Bioscience, University of Queensland, St Lucia, Queensland, 4067, Australia. (Received 15 October 2002, revised 13 December 2002, accepted 3 January 2003) Eur. J. Biochem. 270, 781–790 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03445.x Both the alpha and ARNT subunits belong to the basic helix-loop-helix (bHLH)/Per-ARNT-Sim (PAS) family of transcription factors. The bHLH domain contains the basic DNA binding region and HLH primary dimerization inter- face. The adjacent PAS domain is comprised of approxi- mately 300 amino acids and is subdivided into two semi conserved repeat regions designated PAS A and PAS B. The role of the PAS domain is to mediate protein–protein interactions and act as a second dimerization interface in conjunction with the HLH motif [58]. ARNT is a general partner protein and is known to heterodimerize with a number of other bHLH-PAS proteins to form transcrip- tionally active complexes [59]. Biochemical comparison of the HIF-1a and HIF-2a subunits have revealed that these proteins share very similar biochemical properties (i.e. dimerize with ARNT to recognize the same DNA recogni- tion sequence) but, surprisingly, each subunit controls quite distinct biological functions during embryo development (i.e. HIF-1a for vascularization, HIF-2a for catecholamine production; for a comprehensive review see [60,61]). Regulation of HIF proteins by hypoxia One of the major challenges facing the HIF research field has been to understand the molecular mechanism by which cells are able to sense oxygen levels and transduce the physiological signal of reduced oxygen levels to HIF. It has been reported that oxygen levels can affect the protein stability, subcellular localization, DNA binding capacity and transcriptional potency of the HIFa subunits, whereas the ARNT subunit is constitutively expressed and its activity not affected by hypoxia (reviewed in [60,61]). While the HIFa subunits may be subject to numerous levels of regulation by oxygen, it has been the recent analysis of HIF- 1a and HIF-2a protein stability and transactivation potency that have provided the greatest insights into oxygen sensing and regulation. HIF protein stability Initial biochemical analysis of HIF-1a revealed that this protein was subject to rapid turnover and degradation at normoxia, whereas hypoxia blocked degradation leading to the accumulation of the HIF-1a protein [62,63]. Treatment with proteasomal inhibitors and mutation of the ubiquitin activating enzyme E1 revealed that HIF-1a was being degraded by the ubiquitin proteasome pathway under normoxic conditions [64]. Subsequent studies mapped the instability region of HIF-1a to a domain of approximately 200 amino acids located carboxy-terminal to the PAS domain [65]. This region was subsequently called the oxygen-dependent degradation domain (ODD) and removal of the entire ODD rendered HIF-1a stable at normoxia. Likewise, analysis of HIF-2a revealed that it was also subject to proteasomal degradation at normoxia [66] via a similar ODD like region [67]. A hallmark of von Hippel-Lindau (VHL) disease is the high degree of vascularization, which is due to the Fig. 1. Hypoxia-inducible factor (HIF) target genes and their roles in oxygen homeostasis. Hypoxia activates the HIF complex which binds to hypoxia response elements (HREs) containing the core recognition sequence 5¢-RCGTG found in numerous genes involved in a variety of cellular and system wide responses to low oxygen stress. 782 D. Lando et al. (Eur. J. Biochem. 270) Ó FEBS 2003 constitutive expression of a large number of hypoxia inducible genes such as VEGF (reviewed in [68]). Because VEGF and other hypoxia inducible genes are known HIF targets, these observations raised the question of whether VHL disease and HIF were somehow related. Using various cell lines deficient in VHL, Maxwell and coworkers demon- strated that VHL –/– cells expressed increased levels of endogenous HIF-1a and HIF-2a protein [69]. Moreover, the protein levels of HIF-1a and HIF-2a could not be further induced by hypoxia in VHL –/– cells. Reintroduction of VHL back into these VHL deficient cell lines resulted in a reduction of normoxic endogenous HIF-1a and HIF-2a protein that could now be induced by hypoxia [69]. Further analysis demonstrated that VHL could physically interact with the HIFa subunits via the ODD [69], and the VHL complex functioned as an ubiquitin ligase capable of ubiquitylating the HIFa subunits at normoxia and targeting them for destruc- tion by the proteasome [70–73]. Along with hypoxia, iron chelating agents such as desferrioxamine (Dsfx) were also able to block VHL interaction, suggesting a requirement for iron in the normoxic degradation of HIFa subunits [69]. HIF transactivation Deletion analysis of HIF-1a protein revealed that HIF-1a contained two transactivation regions, termed the amino- terminal transactivation domain (NAD) and the carboxy- terminal transactivation domain (CAD) [74,75]. Functional analysis revealed that the activity of both the NAD and CAD were enhanced by hypoxia treatment. Since the NAD overlaps with the ODD its increase in transcriptional activity at hypoxia was largely attributed to increased protein stability [75]. However, the increase in transcrip- tional activity of the CAD was not attributed to changes in protein level [75]. Instead, hypoxia was suggested to promote the recruitment of transcriptional coactivator proteins such as CBP/p300 [76–78], steroid receptor coac- tivator-1 (SRC-1), and transcription intermediary factor 2 (TIF2) [77] to the CAD region. Likewise, analysis of HIF- 2a revealed that it also contained two transactivation domains with similar organization [67,79] and, as with HIF- 1a,theCADofHIF-2a was also inducible by hypoxia. While the transactivation capability of HIF-3a is poorly characterized, sequence alignment suggests that HIF-3a lacks an analogous inducible CAD region [57]. Therefore, the transactivation potency of HIF-1a and HIF-2a CADs is negatively regulated by oxygen independ- ent of protein stability, revealing that along with the ODD there exists a second oxygen sensing region near the carboxy- terminus. Moreover, like the ODD the CAD is also sensitive to iron antagonists (i.e. cobalt chloride, Dsfx) suggesting that the mechanism of regulation of both domains involves a common iron dependent process [74,75,79]. Regulation of HIFa subunits by oxygen-dependent prolyl and asparaginyl hydroxylation A variety of oxygen sensors have been described for prokaryotes and yeast [80]; however, for many years the nature of the cellular oxygen sensor in higher organisms remained elusive. A number of interesting models have been proposed to explain how mammalian cells could sense oxygen, including those that involve the hemoprotein, NADPH oxidoreductase, members of the mitochondrial electron transport chain [81], or oxygen-regulated potassium channels [82]. Disappointingly, however, when investigated in more detail none of these models could clearly demon- strate how HIF activation was being universally regulated. To better understand the mechanism of oxygen sensing and signal transduction, considerable effort over the last few years has focused on deciphering the biochemical param- eters by which the ODD and CAD were being regulated by low oxygen levels. By employing both protein interaction and ubiquitylation assays the major VHL binding region was narrowed down to a 20 amino acid stretch within the ODD of both HIF-1a and HIF-2a [70,71,83]. Treatment with hypoxia was able to induce the dissociation of VHL from HIF-1a,suggesting that some cellular activity in normoxic cells maybe respon- sible for promoting VHL association [83]. In support of this hypothesis, a synthetic peptide comprising of the minimal VHL binding motif of HIF-1a was unable to interact with VHL unless pretreated with normoxic cell extracts [84–86]. Likewise, similar biochemical experiments with the CAD demonstrated that a cellular activity was targeting the CAD for repression at normoxia, and hypoxia blocked this cellular activity, thereby promoting the recruitment of coactivator proteins such as CBP/p300 [78]. Then, in an elegant set of experiments, a number of groups concurrently demonstrated that the cellular activity responsible for targeting HIFa for degradation was the enzymatic hydroxylation of a specific proline residue within the ODD. Hydroxylation of this proline residue was shown to promote high affinity binding of VHL protein [84–86]. Subsequently, a second proline hydroxylation site was identified within the ODD and was also shown to promote VHL binding in a hydroxylation dependent manner [87]. Surprisingly, in a similar but distinct mechanism, an enzymatic hydroxylase activity was also found to specifically modify the CAD at normoxia to block p300 binding [88,89]. However, in contrast to the ODD, the hydroxylation activity targeting the CAD was found to occur on an asparagine residue [88]. While the CAD contains a number of proline residues no evidence of hydroxylation of these proline sites has ever been found (D. Lando, J. J. Gorman, M. L. Whitelaw & D. J. Peet, unpublished observations). Hydroxyproline and hydroxyasparagine therefore control HIFa activity by regulating protein–protein interactions; hydroxyproline provides a docking site for VHL binding while hydroxyasparagine prevents binding of the coacti- vator p300. Finally, the long sought after links between oxygen availability and iron in the regulation of HIFa protein stability and transactivation potential were realized when it was demonstrated that hypoxia and iron chelators could block hydroxylation of both the proline and aspara- gine residues, regulating the association of VHL with the ODD [84–87] and p300 with the CAD [88], respectively. HIF prolyl and asparaginyl hydroxylases Prior to the discovery of the HIF hydroxylases, the best characterized prolyl and asparaginyl hydroxylases were those that modify proline residues in collagen [90] and Ó FEBS 2003 Mechanisms of oxygen sensing (Eur. J. Biochem. 270) 783 asparagine or aspartic acid residues in epidermal growth factor (EGF)-like domains [91]. The structures of the catalytic domains of some of these iron dependent hydroxylases have been solved and reveal a conserved double-stranded b-helix enzymatic core commonly referred to as a jellyroll. Within this enzymatic core is a critical 2-His1-carboxylate motif (His-X-Asp/Glu…His), respon- sible for coordinating the binding of the iron atom [92]. By using a combination of protein database mining, and genetic and biochemical assays, three novel HIF prolyl hydroxylase enzymes (designated prolyl hydroxylase do- main-containing proteins (PHDs) 1, 2 and 3 [93], or HIF prolyl hydroxylases (HPHs) 3, 2 and 1, respectively [94]), and one HIF asparaginyl hydroxylase enzyme called factor-inhibiting HIF-1 (FIH-1) [88,95] were identified and shown to hydroxylate the key proline and asparagine residues in HIFa. The enzymatic reactions carried out by the PHD/HPHs and FIH-1 revealed that the hydroxyla- tion reaction requires oxygen (in the form of dioxygen O 2 ), iron (Fe 2+ ) and the cofactor 2-oxoglutarate. The hydroxy- lation reaction is inherently dependent on ambient oxygen because the oxygen atom used to form the proline and asparagine hydroxyl groups is derived directly from molecular oxygen [95,96]. The cofactor 2-oxoglutarate is required because it undergoes a decarboxylation reaction, consuming the remaining oxygen atom to form succinate and CO 2 (Fig. 2). Therefore, the rapid turnover and transcriptional silen- cing of the HIFa protein subunits involves oxygen-depend- ent prolyl and asparaginyl hydroxylation by the PHD/ HPHs and FIH-1 proteins, respectively. These modifica- tions then serve as signals for either VHL binding and polyubiqutylation which targets the HIFa subunits for proteasomal degradation, or blocking coactivator proteins such as p300 from binding the CAD (Fig. 3). The import- ance of the PHD/HPH-HIFa-VHL pathway in the oxygen response is further confirmed by the finding that compo- nents of this pathway are functionally conserved in Caenorhabditis elegans [93] and Drosophila [94,97]. Cur- rently FIH-1 homologues have been predicted to exist in C. elegans and Drosophila [98] but their functionality awaits further confirmation. Oxygen sensing By conducting reactions in a controlled oxygen environment it has been demonstrated that the activity of the PHD/ HPHs are sensitive to graded oxygen levels [93]. Moreover, Fig. 2. General reaction scheme for oxygen-dependent hydroxylation by PHD/HPH and FIH-1 hydroxylases. The hydroxylation of target substrates requires dioxygen (O 2 ), iron [Fe(II)] and the cofactor 2-oxoglutarate. During catalysis the substrate accepts one oxygen atom while 2-oxoglutarate undergoes a decarboxylation reaction consuming the remaining oxygen atom to form succinate and CO 2 . Fig. 3. Regulation of hypoxia inducible factors (HIF) by oxygen-dependent hydroxylation. In oxygenated conditions (normoxia) the asparaginyl and HIF prolyl hydroxylases (FIH-1 and PHD/HPH) hydroxylate (OH) HIFa on specific asparagine (Asn) and proline (Pro) residues, blocking transactivation and targeting HIFa for destruction by ubiquitin proteasome pathway, respectively. Hypoxia and iron antagonists block both PHD/HPH and FIH-1 activity, then HIFa escapes destruction and recruits coactivators (CBP/p300) to induce hypoxia target genes. Oxygen- dependent degradation domain (ODD), carboxy-terminal activation domain (CAD), von Hippel Lindau protein (VHL), cobalt chloride (Co), desferrioxamine (Dfrx), 2¢-2-dipyridyl (DP). 784 D. Lando et al. (Eur. J. Biochem. 270) Ó FEBS 2003 the decreased hydroxylation seems to mirror the progressive cellular increase in HIFa protein levels observed when cells are subjected to similar oxygen gradients [99]. Therefore, it has been speculated that the PHD/HPHs represent primary oxygen sensors. Likewise, it has been suggested, but not yet demonstrated, that FIH-1 catalytic activity is also sensitive to oxygen gradients [95]. Evidence so far suggests that while the PHD/HPHs may act more as gross regulators of HIF activity, FIH-1 may play a more subtle role. For example, when HIFa subunits are fully stabilized, as observed in VHL-deficient cells [69], then FIH-1 activity is saturated by the large excess of HIF protein, rendering the CAD active at normoxia. However, it is likely that FIH-1 plays a crucial role in regulating the transcriptional activity of relatively smaller, yet physiologically relevant, amounts of stabilized HIFa, such as that produced in response to growth factors. Interestingly, reporter assays have demonstrated that overexpression of FIH-1 is still able to partially inhibit CAD activity under hypoxic conditions [98,100]. As hydroxyla- tion of the CAD under hypoxic conditions is essentially absent [88] this inhibition is unlikely to be mediated by hydroxylation. It may be due to direct competition for p300 binding to the CAD, the recruitment of other factors such as histone deacetylases or VHL [98], or may well be an artefact of overexpression. Structural implications Synthetic peptides composed of the minimal VHL binding motifofHIF-1a chemically synthesized with a 4-hydroxy- proline at the critical proline position were shown to bind VHL protein [84–86]. Subsequent structural analysis dem- onstrated that the tight binding of VHL to the hydroxylated peptide was due to the 4-hydroxyproline residue forming critical hydrogen bonds with residues in VHL [101,102]. Taken together, these observations suggest that VHL can specifically recognize a 4-hydroxyproline. Recently the solution structures of the CH1 domains of p300 or CBP bound to the CAD of HIF-1a were solved [103,104]. Analysis of the bound complex revealed that the CAD remains relatively extended, wrapping itself around the globular structure of the CH1 domain in a hand grasp or vice like manner. The critical asparagine residue (Asn803) in the CAD of HIF-1a is found buried deep within the molecular interface and nearly 45% of its surface is concealed in the interface. Asparagine 803 forms two side chain hydrogen bonds with aspartic acid residues in the CAD (Asp799) and the CH1 domain that help to stabilize the complex. Analysis of the asparagine and aspartic acid hydroxylation products in the EGF like domains of other hydroxylated proteins has revealed that the hydroxyl group is attached to the b carbon in the erythro isoform [105]. If the asparagine in the CAD is also hydroxylated on the b-carbon, either erythro or threo isoforms are predicted to destabilize the p300/CBP- HIFa complex formation [103,104]. Apart from hydroxylating the asparagine on the b carbon, FIH-1 could also potentially hydroxylate the asparagine on the side chain amide nitrogen to form a hydroxyamic acid. However, it has been recently reported that the asparagine 803 in HIF-1a is indeed hydroxylated on the b-carbon. Surprisingly, this hydroxylation is in the threo isoform [106], unlike the previously characterized EGF-like domain asparaginyl hydroxylases, which hydroxylate exclusively in the erythro position [105]. Also, unlike other asparaginyl hydroxylase enzymes, which can hydroxylate both asparagine and aspartic acid residues, the FIH-1 enzyme was shown to have a clear preference for asparagine in the CAD of HIF-1a [95]. If the asparagine in the CAD wassubstitutedwithanasparticacidresidue,FIH-1 hydroxylase activity for the aspartic acid residue was only 7% of that obtained with asparagine. This clear difference in amino acid specificity and the production of threo rather than erythro isomers suggests that FIH-1 belongs to a new subfamily of 2-oxoglutarate-dependent asparaginyl hydroxylases. Substrate specificity The three PHD/HPH enzymes have been shown to hydroxylate specific proline residues within the context of two strongly conserved LXXLAP* motifs (P* indicates hydroxy proline acceptor) within the ODD [87,93]. While in-vitro substrate analysis has revealed that the three PHD/ HPHs have differing hydroxylating activity towards the proline residue, they also unfortunately report conflicting evidence showing different enzymes as having highest activity (i.e. PHD-2/HPH-2 [107] vs. PHD-3/HPH-1 [94]). Nevertheless, it will now be interesting to determine which PHD/HPH enzymes are the main regulator of HIFa hydroxylation in the cell under physiological conditions. Expression analysis of the three PHD/HPHs in HeLa cells has revealed that all three mRNAs are expressed at normoxia with PHD-1/HPH-3 exhibiting the greatest expression [93]. Interestingly, the expression of PHD-2/ HPH-2 and PHD-3/HPH-1, but not PHD-1/HPH-3 are induced by hypoxia [93], suggesting a possible role for these inducible enzymes in a negative feedback pathway respon- sible for enhanced degradation of HIFa after re-oxygen- ation. Intriguingly, interaction assays have shown that FIH-1 interacts with the CAD of HIF-1a in a region that does not contain the hydroxylated asparagine residue [98]. This asparagine residue is actually located approximately 20–30 residues carboxy-terminal to the putative FIH-1 binding region. This suggests that for FIH-1 to efficiently hydroxy- late the asparagine residue in HIF-1a it may need to bind to aregioninHIF-1a adjacent to the asparagine motif. To support this notion it has been demonstrated that binding of p300 is not enhanced by the hydroxylase inhibitor dime- thyloxalylglycine (DMOG), or iron antagonists, when the putative FIH-1 binding region is removed [89]. A region spanning the FIH-1 binding site in HIF-1a contains an arginine-dileucine (RLL) motif that has previously been shown to be critical for the normal silencing of the HIF-1a CAD at normoxia [79]. An analogous RLL motif-contain- ing region also operates in a similar silencing fashion in HIF-2a [79], suggesting that this region of both HIF-1a and HIF-2a may contain important elements for targeting FIH-1. The finding that FIH-1 must bind to HIF-1a in a region away from the critical asparagine residue for efficient hydroxylation may help explain the long known phenom- enon that uncoupling the HIF-1a CAD containing residues 786–826 from the adjacent inhibitory domain results in a highly active CAD under normoxic conditions [74,75] that Ó FEBS 2003 Mechanisms of oxygen sensing (Eur. J. Biochem. 270) 785 binds strongly to CBP/p300 irrespective of hypoxia treat- ment [78]. As well as interacting with the CAD region FIH-1 has also been shown to interact with VHL via its b domain [98]. Initially this interaction was thought to be important for the repressive activity of FIH-1 on CAD function [98]; however, a more recent analysis in VHL null cells has shown that VHL is not critical for FIH-1 repressive activity [89]. It is possible that FIH-1 and VHL complexes may operate in additional oxygen regulated processes that affect the transcriptional response of other pathways. Interestingly, both FIH-1 and VHL have been shown to interact with chromatin modifying histone deacetylase (HDAC) enzymes, which are known to play an important role in gene repression [98]. Together these observations raise the possibility that FIH-1 may have multiple roles other than just regulating the CAD of HIF-1a and HIF-2a. Other mechanisms of activation The induction of HIF activity by well established agents such as hypoxia, cobaltous ions and iron chelators can be easily explained by the finding that the PHD/HPHs and FIH-1 are members of the 2-oxoglutarate-dependent family of hydroxylase enzymes that utilize iron and oxygen to modify their target amino acid residues. However, it has also been reported that HIF activity is influenced by particular gas molecules (i.e. NO, CO), reactive oxygen species (i.e. H 2 O 2 ), and phosphorylation events (i.e. p38 MAPK), although the understanding of how these proces- ses may influence PHD/HPHs and FIH-1 function and HIF activity is less clear (reviewed in [7]). NO is a known analogue of dioxygen and analysis of the non heme iron (Fe 2+ ) dependent isopenicillin N synthase enzyme, a closely related oxygenase to the 2-oxoglutarate family, has dem- onstrated that NO can bind to the iron centre of this enzyme [108]. Because NO has only one available oxygen atom for use in catalysis and 2-oxoglutarate-dependent dioxygenases normally require two oxygen atoms for completing the hydroxylation of their substrates (Fig. 2), the binding of NO to the catalytic core of PHD/HPHs and FIH-1 may block enzymatic activity, explaining the reported positive effects of NO on HIF activity [109]. The hydroxylation of substrates by the collagen prolyl-4-hydroxylase has been shown to be inhibited by the artificial generation of radicals at the enzyme active site [110]. Thus, the effects of reactive oxygen species on HIF stability [64] and transactivation [62] may relate to the altering of the redox balance of the cell, which then may affect the catalytic activity of PHD/HPHs and FIH-1. Finally, it is possible that these other reported agents that regulate HIF activity may target components of the VHL ubiquitin ligase or CBP/p300 coactivator complex, or even the PHD/HPHs and FIH-1 enzymes directly. Therapeutic benefits Hypoxia constitutes a major component of many disease states and can have both a proliferative (cancer) or damaging affect (stroke, heart attack) on disease pathogene- sis [4]. Therefore, it has been suggested that inducing HIF activity may be beneficial for stroke and heart attack victims as this would help promote vascularization of damaged tissue. Conversely, blocking HIF activity may be advant- ageous in inhibiting cancer progression as this would help starve growing tumours of oxygen and nutrient supply. Coupled with previous studies that have provided Ôproof of principleÕ that targeting HIF stability [111] and transactiva- tion [78] can enhance oxygen delivery and inhibit cancer progression, respectively, it is reasoned that HIF is an attractive target for pharmaceutical manipulation. With the discoveries that HIF stability and transcriptional activity are controlled by two distinct modifications (prolyl and aspar- aginyl hydroxylation) the development of small molecule drugs to selectively target HIF to differentially modulate its activity should be possible. For instance, while it has been demonstrated that the biological activity of FIH-1 requires 2-oxoglutarate [95,100], an unusual feature of FIH-1 is that it lacks an arginine or lysine residue located on the eighth b strand of the enzymatic core. These conserved residues have previously been demonstrated to be involved in binding 5-carboxylate of 2-oxoglutarate in many other 2-oxoglutarate dependent enzymes [92]. Because these 2-oxoglutarate binding residues are conserved in the PHD/HPHs [93,94], it provides further evidence that FIH- 1 represents a new structural submember of the 2-oxoglu- tarate dependent enzyme family, and raises the possibility that selective agonists and antagonists for PHD/HPHs and FIH-1 can be developed. Furthermore, a preliminary study has found that certain well-established inhibitors of the collagen prolyl hydroxylase enzymes do not inhibit PHD/ HPH activity, suggesting that it may be possible to design pharmacological inhibitors that can selectively target the HIF prolyl hydroxylases [112]. Global oxygen sensing by protein hydroxylation? Apart from the HIF pathway and HIFa subunits, the regulation and activity of a large number of other cellular processes and proteins have also been demonstrated to be influenced by oxygen availability. For example, chronic hypoxia is known to extend the replicative life span of certain cell types such as vascular smooth muscle cells [113]. A recent study attributed this increase in long-term proliferation to enhanced hypoxic phosphorylation of the telomerase cata- lytic component TERT [114]. Apart from the putative hypoxia regulated kinase that phosphorylates TERT, the activity of a number of other protein kinases have also been shown to be regulated by hypoxia. These include p44/ p42MAPK [115], p38 MAPK [116,117] and diacylglycerol kinase [118]. Likewise, the stability of certain messenger RNAs, such as VEGF [119,120], are also known to be increased under hypoxic stress, while the splicing of specific alternative mRNA transcripts has recently been shown to be influenced by low oxygen tension [121]. While the mechanism by which low oxygen stress controls these other processes is unknown it will now be of great interest to determine whether the PHD/HPHs or FIH-1 are involved, or if additional oxygen sensing proteins exist that utilize hydroxylation to modify their target substrates. The use of pharmacological inhibitors such as DMOG should now allow quick and easy analysis of the contribution of post-translational hydroxy- lation in other oxygen sensitive processes. 786 D. Lando et al. (Eur. J. Biochem. 270) Ó FEBS 2003 Conclusions To date three prolyl and one asparaginyl hydroxylase enzymes have been discovered that can target different domains of the HIFa subunits, affecting distinct steps in the induction of the HIF complex. 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(2002) Inhibitory PAS domain protein (IPAS) Is a hypoxia-inducible splicing variant of the hypoxia-inducible factor-3a locus. J. Biol. Chem. 277, 32405–32408. 790 D. Lando et al. (Eur. J. Biochem. 270) Ó FEBS 2003 . REVIEW ARTICLE Oxygen-dependent regulation of hypoxia-inducible factors by prolyl and asparaginyl hydroxylation David Lando 1 , Jeffrey J. Gorman 2, *,. mechanism of regulation of both domains involves a common iron dependent process [74,75,79]. Regulation of HIFa subunits by oxygen-dependent prolyl and asparaginyl hydroxylation A