Cobalt Inhibits the Interaction between Hypoxia-inducible
Factor-
and von Hippel-Lindau Protein by Direct Binding to
Hypoxia-inducible Factor-
*
Yong
Yuan,
George
Hilliard,
Tsuneo
Ferguson, and
David E.
Millhorn
From the Genome Research Institute, University of Cincinnati,
Cincinnati, Ohio 45267-0505
Received for publication, January 15, 2003, and in revised form, February 21, 2003
 |
ABSTRACT |
The hypoxia-inducible factor (HIF) activates the
expression of genes that contain a hypoxia response element. The
-subunits of the HIF transcription factors are degraded by
proteasomal pathways during normoxia but are stabilized under hypoxic
conditions. The von Hippel-Lindau protein (pVHL) mediates the
ubiquitination and rapid degradation of HIF-
(including HIF-1
and
HIF-2
). Post-translational hydroxylation of a proline residue in the
oxygen-dependent degradation (ODD) domain of HIF-
is
required for the interaction between HIF and VHL. It has previously
been established that cobalt mimics hypoxia and causes accumulation of
HIF-1
and HIF-2
. However, little is known about the mechanism by
which this occurs. In an earlier study, we demonstrated that
cobalt binds directly to the ODD domain of HIF-2
. Here we provide
the first evidence that cobalt inhibits pVHL binding to HIF-
even
when HIF-
is hydroxylated. Deletion of 17 amino acids within the ODD
domain of HIF-2
that are required for pVHL binding prevented the
binding of cobalt and stabilized HIF-2
during normoxia. These
findings show that cobalt mimics hypoxia, at least in part, by
occupying the VHL-binding domain of HIF-
and thereby preventing the
degradation of HIF-
.
 |
INTRODUCTION |
Hypoxia is a critical stimulus in many physiological and
disease states (1). Cells respond to hypoxia by regulating the expression of a number of genes, including erythropoietin, vascular endothelial growth factor, and various glycolytic enzymes (2-5). This
regulation is mediated in part by transcription factors of the
hypoxia-inducible factor
(HIF)1 family (6). HIF-1
and HIF-2
are basic helix-loop-helix Per-Arnt-Sim (PAS)
domain proteins (7) that form a heterodimer with the aryl hydrocarbon
nuclear receptor translocator protein. Previous studies have shown that
HIF-1
protein accumulates rapidly during hypoxia without a
significant increase in HIF-1
mRNA levels (8). HIF-2
, which
is also known as endothelial PAS domain protein-1, shares close
sequence and structural homology with HIF-1
(9). Like HIF-1
, the
levels of HIF-2
protein are low during normoxia and accumulate when
cells are exposed to hypoxia, proteasomal inhibitors, transition metals
(e.g. cobalt), iron chelators, or reducing agents (10).
During normoxia, the HIF-
(HIF-1
and HIF-2
are referred to
here simply as HIF-
, except where noted otherwise) proteins are
continuously degraded by ubiquitin- and proteasome-dependent pathway. Detailed studies of HIF-
proteins revealed a 200-amino acid sequence, called the
oxygen-dependent degradation domain (ODD) that is
responsible for its degradation in the presence of oxygen (11, 12). The
von Hippel-Lindau (pVHL) protein, a tumor suppressor protein, mediates
the ubiquitination and degradation of HIF-
by binding to the ODD
domain under conditions of normoxia (13, 14). Recent findings revealed
that pVHL-mediated degradation requires hydroxylation of specific
proline residues within the ODD (15-18). The hydroxylation of these
proline residues may be critical for regulating the HIF levels and,
therefore, transcription of downstream hypoxia-responsive genes.
It has been well documented that cobalt, a transition metal, mimics
hypoxia by causing the stabilization of HIF-
. However, the
biochemical mechanism by which cobalt stabilizes HIF-
remains unknown. A recent model suggested that the hydroxylation of HIF-
is
mediated by a group of HIF-specific hydroxylases and that cobalt can
inactivate the enzymes by occupying an iron-binding site on the proline
hydroxylases (18). We previously reported that HIF-2
binds cobalt
in vitro and that the cobalt-binding site overlaps with the
pVHL-binding site on HIF-2
(19). Here we show that cobalt inhibits
the interaction between pVHL and hydroxylated HIF-
and that cobalt
inhibits the hydroxylation of a key proline residue within the ODD
domain of HIF-2
. This is the first report that cobalt stabilizes
cellular HIF-2
by occupying the VHL-binding domain.
 |
MATERIALS AND METHODS |
Cell Culture and Reagents--
Rat pheochromocytoma (PC12) cells
and CHO cells were obtained from the American Type Culture Collection
(Manassas, VA) and renal clear carcinoma RCC/VHL+ cells
were provided by Dr. Czyzyk-Krzeska (University of Cincinnati). All cells were cultured in Dulbecco's modified Eagle's
medium/Ham's F-12 (Invitrogen) supplemented with 20 mM
HEPES, pH 7.4, 10% fetal bovine serum (Invitrogen), penicillin (100 unit/ml), and streptomycin (100 µg/ml). The medium for
RCC/VHL+ cells was supplemented with 200 µg/ml G418.
Prior to experimentation, cells were grown to ~85% confluence in
100-mm tissue culture dishes (Corning, Inc., Corning, NY).
CoCl2 was from Sigma. Reagents used for hemagglutinin (HA)
immunoprecipitation (anti-HA antibody and protein G/A plus-coupled
agarose) were from Santa Cruz Biotechnology (Santa Cruz, CA). The
HIF-2
antibody was from Novus Biologicals (Littleton, CO).
Plasmids--
The HIF-1
expression vector
(pcDNA3-HIF-1
) was a gift from Dr. Steven L. McKnight
(University of Texas Southwestern, Dallas, TX). The HA-tagged pVHL
plasmid pRC/CMV-HA-VHL was originally from Dr. William G. Kaelin, Jr.
(Harvard Medical School, Boston, MA). HIF-2
expression plasmids were
constructed as described previously (19). pTriEx-HIF-2
(48-688),
expresses amino acids 48-688 of HIF-2
, which includes the wild type
ODD domain. pTriEx-HIF-2
(48-523, 539-688) expresses HIF-2
with
a deletion from amino acid 523 to 539, which forms the core of the ODD
domain and the pVHL-binding site.
Western Blots--
HIF-2
Western blots were performed as
described by Conrad et al. (20).
Binding Assays--
HIF-VHL binding assays were performed as
described by Cockman et al (21). Briefly, 35S-labeled
HIF-
and pVHL were generated in reticulocyte lysates (Promega,
Madison, WI), as described. Proteins were then mixed in binding
buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl,
0.5% Igepal) in the presence or absence of transition metals at
4 °C for 1 h. Samples were then subjected to
immunoprecipitation with an anti-HA antibody and analyzed by SDS-PAGE
followed by autoradiography.
In Vitro Degradation Assay--
35S-labeled proteins
(1 µl) were added to the following reaction: 30 µl of
RCC/VHL+ cell cytoplasmic extract, 6 µl of untreated
rabbit reticulocyte lysate (Promega), 25 µg of ubiquitin (Sigma), 5 µl of 10×ATP-regenerating system (20 mM Tris, pH
7.5, 10 mM ATP, 10 mM magnesium acetate, 300 mM creatine phosphate, 0.5 mg/ml creatine phosphokinase), and water for a final volume of 50 µl. Each reaction was incubated at
30 °C, then stopped by the addition of SDS-PAGE loading buffer at
the indicated times. The labeled proteins were analyzed by SDS-PAGE
followed by autoradiography.
HIF-2
in Vitro Cobalt Binding Experiment--
50 µl of
HisBind Resin (Novagen) was charged with 1 ml of 50 mM
CoCl2. The charged resin was then washed and suspended in binding buffer (20 mM Tris-HCl, pH 7.4, and 500 mM NaCl). Cell extracts were prepared by snap-freezing
cells in cell lysis buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.5% TritonX-100). Cell extracts containing 500 µg of protein were mixed with resin and incubated at 4 °C for 15 min and then washed with 1 ml of binding buffer. The proteins were
washed and eluted with binding buffer containing 60 mM
imidazole, followed by elution with 1 M imidazole. The
remaining uneluted proteins were solubilized with SDS-PAGE sample
buffer, and the resulting samples were subjected to Western blotting
for HIF-2
.
Mass Spectrometry--
Matrix-assisted laser
desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry
analysis was performed with a Bruker Biflex III equipped with reflector
and delayed extraction. Sample preparation and MALDI analysis were
modified according to Lehmann et al. (22). Briefly, equal
volumes of peptides (0.1 mM), matrix (35 mM ATT
in 10 mM Tris, pH 9.0), and metal chloride solution (1.0 mM) were mixed and spotted onto the MALDI target. The
extraction and reflector settings were 19 kV and 20 kV, respectively,
and laser attenuation was set to 54%. The resulting spectra were
summed over 300 shots. The HIF peptide was synthesized by
Bio-Synthesis, Inc. (Lewisville, TX). The sequence of the HIF peptide
is shown in Fig. 3A. The control peptide
(adrenocorticotropic hormone fragment 18-39, ACTH), and the matrix
6-aza-2 thiothymine (ATT) were from Sigma.
 |
RESULTS |
The oxygen-dependent proteolytic destruction of
HIF-
is mediated by the pVHL, which is an E3 ubiquitin ligase (13).
The binding of pVHL to HIF-
is regulated through the
hydroxylation of a proline residue (HIF-1
Pro-564 and HIF-2
Pro-531) in the ODD domain of HIF-
(14, 15). The
hydroxylation of this proline requires active cell extracts, iron, and
oxygen (18). Fig. 1, A and
B shows that HIF-1
and -2
translated in
vitro using reticulocyte lysates in the presence of excess iron
(100 µM FeCl2) enhanced pVHL binding
activity. Iron chelators, such as desferrioxamine (DFO) or dipyridyl,
and the transition metal cobalt chloride stabilize HIF-
during
normoxia. Experiments were designed to determine whether these
hypoxia-mimicking reagents stabilize HIF-
by inhibiting iron-mediated proline hydroxylation of HIF-
. Unhydroxylated HIF-2
was translated in vitro in the absence of excess iron. The
hydroxylation reaction was performed in cell extracts from
RCC/VHL+ cells. This cell line was not selected for any
special reason; in fact, extracts from most mammalian cell lines can
provide the activity required for this hydroxylation reaction.
Supplementary iron (10 µM FeCl2)
substantially increased pVHL binding to HIF-2
, as detected by the
pVHL binding assay, which indicates enhanced hydroxylation of HIF-2
(Fig. 1C). When cobalt (100 µM
CoCl2) or iron chelators (100 µM DFO or
dipyridyl) were present, the inhibition of iron-mediated hydroxylation
of HIF-2
resulted in decreased binding of HIF-2
to HA-VHL (Fig.
1C). Thus, both cobalt and iron chelators inhibit the
iron-mediated hydroxylation of HIF-2
.

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Fig. 1.
Cobalt inhibits iron-mediated hydroxylation
of HIF-2 . Iron supplementation
facilitates the interaction between pVHL and HIF- .
35S-labeled HIF-1 (A) and HIF-2
(B) were generated in reticulocyte lysates in the presence
or absence of Fe2+ and then mixed with HA-VHL in a binding
reaction as described under "Materials and Methods." The
interactions were detected by anti-HA immunoprecipitation and analyzed
by SDS-PAGE followed by autoradiography. C, cobalt and iron
chelators inhibit iron-mediated hydroxylation of HIF-2 as assayed by
the interaction between HIF- and VHL. HIF-2 was produced in
rabbit reticulocyte lysate as described. Hydroxylation of HIF-2 was
performed in extracts from RCC/VHL+ cells in the presence
or absence of iron (10 µM) alone or with either cobalt
(100 µM) or the iron chelators desferrioxamine (DFO, 100 µM) or dipyridyl (100 µM). Reactions were
incubated at 30 °C for 60 min. HIF-VHL binding reactions were
performed in binding buffer by mixing modified HIF-2 and HA-VHL as
described under "Materials and Methods." The interactions were
detected by anti-HA immunoprecipitation and analyzed by SDS-PAGE
followed by autoradiography.
|
|
We next examined the effects of iron chelators and cobalt on the
HIF-VHL interaction. HIF-1
and -2
were translated in reticulocyte lysate in the presence of iron (100 µM
FeCl2), which leads to proline hydroxylation of HIF-
(15). The translation products and HA-VHL were added to the binding
buffer. In some cases, cobalt (100 µM CoCl2)
or the iron chelator (DFO 100 µM) was also added to the
binding buffer. Results from these experiments showed that the addition
of cobalt (100 µM) greatly reduced the interaction between hydroxylated HIF-
and pVHL, whereas DFO had little effect (Fig. 2, A and B).
We next performed a series of experiments to determine the optimal
concentration of cobalt required to inhibit the HIF-VHL interaction. To
do this, we used in vitro translation to generate
[35S]methionine-labeled and hydroxylated HIF-1
and
-2
in the presence of iron (100 µM FeCl2).
We then added various concentrations of CoCl2 to the
binding reactions. We found that cobalt inhibited the interaction
between HIF-
and pVHL in a concentration-dependent manner and that the maximal inhibition occurred at 100 µM
CoCl2 (Fig. 2, C and D). Western blot
analysis revealed that the same concentration of cobalt was required to
stabilize HIF-2
in PC12 cells (Fig. 2D, lower
panel). These findings show that the level of cobalt required to
inhibit the HIF-VHL interaction in vitro is similar to the
concentration needed to stabilize HIF-2
within PC12 cells. These
results suggest that iron chelators stabilize HIF by inhibiting
iron-mediated hydroxylation of the proline residue of HIF. In contrast,
cobalt not only inhibits hydroxylation but also directly inhibits the
HIF-VHL interaction.

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Fig. 2.
Cobalt directly inhibits pVHL and
HIF- interaction. 35S-labeled
and hydroxylated HIF-1 (A) and HIF-2 (B)
were generated in reticulocyte lysates in the presence of
Fe2+ (100 µM) and then mixed with pVHL-HA in
a binding reaction in the presence of either cobalt (100 µM) or DFO (100 µM), as indicated. The
interactions were detected by anti-HA immunoprecipitation and analyzed
by SDS-PAGE followed by autoradiography. Labeled and hydroxylated
HIF-1 (C) and HIF-2 (D) were generated in
reticulocyte lysates in the presence of Fe2+ (100 µM). HIF- proteins were then mixed with HA-VHL in
binding buffer. The binding buffer included different concentrations of
CoCl2 as indicated. The interactions were detected by
anti-HA immunoprecipitation and analyzed by SDS-PAGE followed by
autoradiography. D, lower blot, PC12 cells were
treated with increasing concentrations of cobalt chloride as indicated
for 4 h; HIF-2 protein levels were detected by Western
blotting.
|
|
Experiments were next performed to determine the mechanism by
which cobalt inhibits the HIF-VHL interaction. We previously reported
(19) that HIF-2
binds to cobalt in vitro and that cobalt
and VHL bind to the same site within the ODD. The 17-amino acid
cobalt-binding region contains three glutamic acid residues (Glu) and
three aspartic acid residues (Asp) (Fig.
3A). The carboxyl side groups
of these acidic amino acids provide possible sites for cobalt binding.
When HIF-2
was mutated by removing this 17-amino acid fragment (Fig.
3A), the resulting ODD
mutant had a prolonged
half-life, as determined by in vitro degradation experiments
(Fig. 3B). Moreover, the pVHL binding activity was abolished
in the ODD
HIF-2
protein (Fig. 3C). The
ODD
mutant HIF-2
also lacked cobalt binding activity
(Fig. 3D). These data strongly suggest that cobalt competes
with pVHL for binding to HIF-
. To obtain direct evidence that cobalt
binds to HIF-
, we next used MALDI-TOF to analyze the interaction of cobalt and a synthetic peptide that consisted of the deleted amino acid
sequence shown in Fig. 3A (amino acids 523-539 of
HIF-2
). We found that this peptide binds directly to cobalt, whereas
a control peptide (ACTH) that also contained five acidic amino acids did not bind to cobalt (Fig. 4). This
confirms that cobalt binds directly to a specific amino acid sequence
within the ODD of HIF-
.

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Fig. 3.
Cobalt inhibits the interaction between
HIF- and VHL by occupying a conserved region
of the ODD domain. A, schematic illustration of the
HIF-2 constructs used in the experiment. ODD+ HIF-2
(upper construct) has an intact ODD domain, whereas
ODD HIF-2 (lower construct) has a 17-amino
acid deletion (523-539), which corresponds to the pVHL binding region
of HIF-2 . B, in vitro degradation of the
ODD+ and ODD HIF-2 proteins. Labeled
proteins were produced in reticulocyte lysates. Degradation reactions
were performed as described under "Materials and Methods",
and reactions were stopped at the indicated times. C,
ODD HIF-2 lacks pVHL binding activity. HIF-2
proteins were produced by reticulocyte lysates in the presence of iron
(100 µM FeCl2) and then mixed with HA-VHL in
the binding reaction. Interactions were detected by anti-HA
immunoprecipitation and analyzed by SDS-PAGE followed by
autoradiography. D, ODD HIF-2 lacked the
ability to bind to cobalt in vitro. Plasmids pTriEx-HIF-2
(48-688) and pTriEx-HIF-2 (48-523, 539-688) were transfected into
CHO cells; cells were then treated with 10 µM CbzLLL, a
proteasomal inhibitor. The resulting cell extracts were then used in
cobalt binding assays. Cobalt binding reactions were performed as
described under "Materials and Methods." In each experiment,
lane 1 is loading, lane 2 is run through,
lane 3 is 60 mM imidazole elution, lane
4 is 1 M imidazole elution, and lane 5 is
SDS-PAGE sample buffer elution. The arrow indicates the
amount of HIF-2 that remained bound to the resin after 1 M imidazole washing.
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Fig. 4.
MALDI-TOF analysis of HIF and ACTH peptides
and their interactions with cobalt. A, the calculated
monoisotopic mass of HIF peptide alone is 2349.1086. The
monoisotope peaks for HIF [M+H]+ of 2350.702 m/z and contaminating sodium
[M+Na]+ and potassium [M+K]+ adduct peaks
of 2372.762 and 2388.662 m/z, respectively, are
labeled. B, the calculated monoisotopic mass of ACTH peptide
alone is 2464.191 Dalton. ACTH was used as a calibration and control
peptide; its [M+H]+ is shown. C, HIF peptide
plus cobalt at a molar ratio of 1:10. There are four labeled
monoisotopic peaks: [M+H]+ at 2350.392 m/z, sodium [M+Na]+ and potassium
[M+K]+ adducts at 2372.305 and 2388.369 m/z, respectively, and HIF peptide binding cobalt
[M H+Co2+]+ at 2407.388 m/z. D, ACTH peptide plus cobalt at a
molar ratio of 1:10. The ACTH [M+H]+ peak remained
unchanged in the presence of cobalt. The mass spectra were externally
calibrated. (A.I., absolute intensity).
|
|
 |
DISCUSSION |
The most recent model for oxygen sensing suggests that
iron-mediated hydroxylation occurs via a group of HIF-specific proline hydroxylases (Fig. 5A) (18).
It was suggested that these hydroxylases have an iron binding-center
and that iron is critical for its enzymatic activity. Epstein et
al. (18) further proposed that iron chelators can remove iron from
the iron-binding center of the enzyme and that the iron can
be replaced by cobalt at this site, which inactivates the hydroxylase
activity. This model is consistent with the observations that cobalt
and iron chelators inhibit hydroxylation of HIF. The present study
demonstrates, for the first time, that cobalt also stabilizes HIF-
proteins by binding directly to the ODD and that cobalt inhibits both
hydroxylation and the interaction between hydroxylated HIF-
and pVHL
(Fig. 5B). This conclusion is supported by our MALDI data,
which revealed that cobalt binds directly to a synthetic HIF peptide
with a non-hydroxylated proline residue. In addition, our binding
results show that cobalt inhibits the HIF-VHL interaction even after
the proline residue becomes hydroxylated. Thus, cobalt can bind to HIF
regardless of the hydroxylation state of the proline residue within the
ODD.

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Fig. 5.
Proposed model for oxygen sensing.
A, the current model for oxygen sensing. The HIF-specific
hydroxylase is thought to be an iron-binding protein. In the presence
of oxygen, it catalyzes the hydroxylation of proline 564 within HIF.
Hydroxylated HIF is then ubiquitinated by pVHL. Ubiquitinated HIF is
degraded by proteasomal mechanism. Cobalt inhibits the hydroxylation of
HIF by binding to the iron-binding domain of HIF hydroxylase.
B, revised model based on our new in vitro data.
Hydroxylation of HIF- is mediated by a HIF-specific hydroxylase.
Cobalt binds to the iron center of this enzyme to inactive the
hydroxylase activity. Even if a portion of HIF- becomes
hydroxylated, cobalt can also bind directly to the hydroxylated
proteins to prevent the interaction between HIF- and pVHL, thereby
preventing the degradation of HIF- .
|
|
DFO and cobalt appear to stabilize HIF-
via different mechanisms.
Iron chelation effectively stabilizes HIF-
only when DFO is added to
the translation reaction. The addition of DFO to modified HIF-
in
the binding reaction did not prevent the association of HIF-
with
VHL. Therefore, DFO most likely inhibits hydroxylation of HIF-
by
chelating the iron required for activity of the HIF-specific proline
hydroxylases. In contrast, cobalt prevents VHL binding when added at
either the translation or the binding step. When cobalt was added
during in vitro translation of HIF-
, the final concentration of cobalt in the HIF-VHL binding reaction was 0.5 µM. This is far below the concentrations of cobalt
required to inhibit binding of hydroxylated HIF-
to VHL (50-100
µM). Thus, it is likely that cobalt interferes with
multiple steps of the HIF-
degradation process (see Fig.
5B). When added to the in vitro translation
reaction, it is likely that cobalt prevents hydroxylation of HIF-
by
binding to either HIF-specific proline hydroxylases or HIF-
itself.
When cobalt was added to the binding reaction, it could still bind to
hydroxylated HIF-
to prevent the interaction between HIF and VHL.
The present studies were carried out in vitro and do not
necessarily reflect how cobalt functions within cells. One possible model to explain how cobalt could stabilize HIF-
in cells is illustrated in Fig. 5B. Cobalt may occupy the iron center of
a HIF-specific proline hydroxylase, thereby inactivating the enzyme. Even if a subset of HIF-
did undergo hydroxylation, hydroxylated HIF-
could still bind to cobalt. Cobalt could thereby prevent the
interaction between pVHL and hydroxylated HIF-
, which would prevent
subsequent ubiquitination and degradation of HIF-
. Clearly, further
studies will be needed to test this and other hypotheses and to
characterize the effects of cobalt in vivo.
Because the HIF-
transcription factor plays a critical role in the
cellular response to changes in oxygen levels, it is not surprising
that cells have evolved multiple mechanisms to regulate its activity.
Three residues have been reported to be hydroxylated under normoxic
conditions, including Pro-402, Pro-564, and Asn-803 (15-17, 23).
HIF-
may become oxidized (hydroxylated) at multiple sites under
normoxic conditions to regulate its stability and activity. We have
demonstrated that cobalt inhibits hydroxylation and pVHL binding of
HIF-
at the Pro-564 site. In future studies, it will be important to
determine whether cobalt similarly regulates HIF-
at the other two
hydroxylation sites.
 |
ACKNOWLEDGEMENTS |
We thank G. Doerman for preparation of
figures and Abby Newland for MALDI-TOF assay.
 |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grants HL33831 and HL59945 and by the United States Army.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Genome
Science, Genome Research Inst., University of Cincinnati, 231 Albert
Sabin Way, P. O. Box 670505, Cincinnati, OH 45267-0505. Tel.:
513-558-5473; Fax: 513-558-5422; E-mail: david.millhorn@uc.edu.
Published, JBC Papers in Press, February 26, 2003, DOI 10.1074/jbc.M300463200
 |
ABBREVIATIONS |
The abbreviations used are:
HIF, hypoxia-inducible factor;
ODD, oxygen-dependent
degradation;
pVHL, von Hippel-Lindau protein;
CHO, Chinese
hamster ovary;
HA, hemagglutinin;
MALDI-TOF, matrix-assisted laser
desorption/ionization time-of-flight;
E3, ubiquitin-protein isopeptide
ligase;
DFO, desferrioxamine;
ACTH, adrenocorticotropic hormone
fragment.
 |
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