Tài liệu Báo cáo khoa học: Oxygen-dependent regulation of hypoxia-inducible factors by prolyl and asparaginyl hydroxylation pdf

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:
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. The numerous enzymes and
their various targets may have evolved to help manipulate
the magnitude of the HIF transcriptional response by
providing a variable mechanism to gradually alter the
activity of the HIFa subunits in response to subtle changes
in oxygen levels. Furthermore, other studies have suggested
that the nuclear accumulation of HIFa subunits may also be
oxygen regulated [76], and it will now be interesting to
establish if this or other components of HIF regulation are
also influenced by the above or other hydroxylation
mediated events.
Acknowledgements
D. J. P. is the W. Bruce Hall Cancer Research Fellow supported by the
Cancer Council of South Australia, and this work was also supported
by the National Heart Foundation and National Health and Medical
Research Council of Australia.
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