Iron cannot decrease iron regulatory protein binding activity in cell extracts if they are simultaneously treated with an uncoupler of oxi-dative phosphorylation.. Physiologic concentrat
Trang 1protein-1 complex by ATP binding and hydrolysis
Zvezdana Popovic and Douglas M Templeton
Laboratory Medicine and Pathobiology, University of Toronto, Canada
Mechanisms of post-transcriptional regulation of gene
expression include control of initiation of translation
and regulation of mRNA degradation Among the
best-studied models for these processes is regulation of
proteins involved in iron homeostasis These control
mechanisms involve functional iron-responsive
ele-ments (IREs) in the 5¢-UTRs or 3¢-UTRs of mRNAs
that interact with iron regulatory proteins (IRPs),
depending upon the amount of iron present in the cell
Two IRPs have been identified: IRP-1, which contains
a 4Fe)4S iron–sulfur cluster [1], and IRP-2, which
does not [2,3] IRP-1 has 30% amino acid identity to mitochondrial aconitase [4], a 4Fe)4S enzyme involved
in the tricarboxylic acid cycle IRP-1 is generally believed to interconvert between an enzymatically inac-tive IRE-binding state and a nonbinding form with aconitase activity, the latter requiring an intact 4Fe-4S cluster Thus, the simple model for iron sensing by IRP-1 involves direct association of iron with the iron– sulfur center to form a complete 4Fe)4S cluster
A linkage between cellular iron levels and energy metabolism is suggested by the influence of agents that
Keywords
ATP binding; ATP hydrolysis; energy
metabolism; iron regulatory proteins;
iron-responsive element
Correspondence
D M Templeton, Laboratory Medicine and
Pathobiology, University of Toronto, 1 King’s
College Circle, Toronto, Ontario, M5S 1A8,
Canada
Fax: +1 416 978 5959
Tel: +1 416 978 3972
E-mail: doug.templeton@utoronto.ca
(Received 22 February 2007, revised 29
March 2007, accepted 24 April 2007)
doi:10.1111/j.1742-4658.2007.05843.x
Iron regulatory protein-1 binding to the iron-responsive element of mRNA
is sensitive to iron, oxidative stress, NO, and hypoxia Each of these agents changes the level of intracellular ATP, suggesting a link between iron levels and cellular energy metabolism Furthermore, restoration of iron regula-tory protein-1 aconitase activity after NO removal has been shown to require mitochondrial ATP We demonstrate here that the iron-responsive element-binding activity of iron regulatory protein is ATP-dependent in HepG2 cells Iron cannot decrease iron regulatory protein binding activity
in cell extracts if they are simultaneously treated with an uncoupler of oxi-dative phosphorylation Physiologic concentrations of ATP inhibit iron-responsive element⁄ iron regulatory protein binding in cell extracts and binding of iron-responsive element to recombinant iron regulatory protein-1 ADP has the same effect, in contrast to the nonhydrolyzable analog adenosine 5¢-(b,c-imido)triphosphate, indicating that in order to inhibit iron regulatory protein-1 binding activity, ATP must be hydrolyzed Indeed, recombinant iron regulatory protein-1 binds ATP with a Kd of
86 ± 17 lm in a filter-binding assay, and can be photo-crosslinked to azido-ATP Upon binding, ATP is hydrolyzed The kinetic parameters [Km¼ 5.3 lm, Vmax¼ 3.4 nmolÆmin)1Æ(mg protein))1] are consistent with those of a number of other ATP-hydrolyzing proteins, including the RNA-binding helicases Although the iron-responsive element does not itself hydrolyze ATP, its presence enhances iron regulatory protein-1’s ATPase activity, and ATP hydrolysis results in loss of the complex in gel shift assays
Abbreviations
AMP-PNP, adenosine 5¢-(b,c-imido)triphosphate; ATP-cS, adenosine 5¢-O-(3-thiotriphosphate); CCCP, carbonyl cyanide
m-chlorophenylhydrazone; EMSA, electrophoretic mobility shift assay; IRE, iron-responsive element; IRP, iron regulatory protein.
Trang 2affect ATP levels and regulate IRP-1 In addition to
iron, NO [5], H2O2 [5] and oxidative stress in general
[6] influence the activity of IRP-1 All these agents
decrease the level of intracellular ATP, and
concomit-antly increase IRP-1 binding activity, suggesting
integ-rated regulatory mechanisms In addition, IRP-1 may
be regulated by phosphorylation [7] Generation of
NO results in an increase in IRP-1 activity, and a
decrease in cytosolic aconitase activity [8–12] NO
donors also lead to ATP depletion [13], consistent with
inhibition by NO of a number of enzymes involved in
ATP synthesis through glycolysis [14], electron
trans-port, and the tricarboxylic acid cycle [15] ATP
deple-tion with NO parallels an increase in IRP-1 binding
activity Exposure to H2O2 promotes removal of the
4Fe)4S cluster of IRP-1, again increasing IRE-binding
activity It is proposed that the H2O2-mediated
conver-sion from cytosolic aconitase to an IRE-binding
protein is a result of a signaling pathway rather than
of direct chemical modification of the 4Fe)4S cluster
by H2O2 [9,16,17] Indeed, superoxide, H2O2, and HO
are all capable of damaging components of the
elec-tron transport apparatus, and thus can disrupt
mitoch-ondrial function and limit ATP production [18], while
increasing IRP-1 binding activity Hypoxia also
decrea-ses ATP as cells switch their primary means of energy
production from the tricarboxylic acid cycle to
glyco-lysis [19], and modulates cellular iron homeostasis in
human hepatoma and erythroleukemia cells by
enhan-cing IRP-1 binding capacity [20], although in rodent
cells it has been found to decrease total IRP binding
[21], perhaps due to a higher IRP-2⁄ IRP-1 ratio in the
rodent [20]
The present study was undertaken to examine the
possible interaction of ATP with the IRE–IRP system
We have determined that ATP binds to IRP-1, is
hydrolyzed, and disrupts IRE–IRP-1 binding
Results
Effect of uncoupling oxidative phosphorylation
on IRP binding activity
We treated HepG2 cells with iron (20 lgÆmL)1) for 3 h
in the presence or absence of carbonyl cyanide
m-chlo-rophenylhydrazone (CCCP), and RNA binding was
subsequently examined by electrophoretic mobility
shift assay (EMSA) with human ferritin H-chain IRE
Iron treatment decreased IRP binding activity to 60%
of control values, but simultaneous treatment with iron
and 10 lm CCCP prevented the iron-dependent
inhibition of IRP binding activity (Fig 1A) In
parallel, ATP was measured in samples subjected to
each treatment In the absence of oxidative phosphory-lation, the ATP levels in CCCP-treated cells were about half of those in the control cells Iron treatment did not significantly affect ATP levels in either CCCP-treated or control cells (Fig 1B) Western blots
of IRP-1 demonstrate that the IRP-1 protein level does not change with iron and⁄ or CCCP treatment (Fig 1C) IRP-2 was weakly detected by western blot-ting of extracts from HepG2 cells (data not shown)
Purification of IRP-1 Recombinant His-tagged hIRP-1 purified on an Ni2+ affinity column retained several lower molecular weight bands (Fig 2A), some of which were also detectable
by western blotting with antibody to IRP-1 (Fig 2B), consistent with lower molecular weight fragments in both native and recombinant preparations reported earlier [22] However, the possibility that these peptides could present artefactual ATP binding or ATPase activity prompted us to purify the protein further Recombinant IRP-1 was purified to homogeneity by elution from biotinylated IRE–streptavidin agarose with 1 m KCl (fraction E2, Fig 2) Fraction E2 is essentially pure on an overexposed silver-stained gel (Fig 2A), is positive for IRP-1 on western blot (Fig 2B), and retains IRE-binding properties on EMSA (Fig 2C) Experiments reported below as using affinity-purified recombinant protein were performed
on material purified as fraction E2
ATP modulates IRE–IRP interaction in vitro
To determine whether ATP directly influences IRP– IRE complexation, we incubated HepG2 cell extracts with different ATP concentrations prior to the addi-tion of labeled IRE and subsequent EMSA (Fig 3) Binding activity was substantially decreased by 2.5 mm ATP and was undetectable at 5 mm ATP When puri-fied recombinant IRP-1 was tested for IRE binding in the presence of ATP, the results were similar (Fig 3), suggesting that interaction can occur among IRP-1, IRE and ATP without involvement of cellular proteins lacking IRP activity Inclusion of 2% b-mercaptoetha-nol in the reaction mixture had no effect with either HepG2 extract, Ni2+–nitrolotriacetic acid-purified recombinant protein, or affinity-purified recombinant protein (Fig 3B)
ATP is hydrolyzed to inhibit IRE–IRP binding
To determine the specificity of ATP’s effect on the IRE–IRP interaction, complex formation was studied
Trang 3in the presence of other nucleotide triphosphates CTP,
GTP, and UTP had no significant effect on IRP-1
bind-ing in either HepG2 cell extracts (Fig 4A) or with
recombinant protein (Fig 4B) To determine whether
ATP hydrolysis is required to modulate IRP–IRE
inter-action, we analyzed IRE–IRP complex formation in the
presence of the ATP analogs adenosine
5¢-O-(3-thiotri-phosphate) (ATP-cS) and adenosine
5¢-(b,c-imido)tri-phosphate (AMP-PNP) The imidodi5¢-(b,c-imido)tri-phosphate analog
AMP-PNP, which is nonhydrolyzable between the b
and c phosphorus atoms, had no effect on IRE binding
either in cell extracts or with recombinant protein In
whole cell extracts, the IRE complex was inhibited by
only about 30% by ATP-cS (5 mm) However, ATP-cS
had the same inhibitory effect as 5 mm ATP on the
IRE complex formed with recombinant IRP-1 ATP-cS
is hydrolyzed by some phosphatases and ATPases [23], and the results may reflect a difference between native and recombinant protein Furthermore, 5 mm ADP inhibited the IRP–IRE complex to the same extent as
5 mm ATP, suggesting that ATP is hydrolyzed to ADP
to produce the inhibitory effect Note that after ATP depletion in Fig 1, binding is not abolished by cellular ADP, presumably because [ATP]⁄ [ADP] ratios are nor-mally 50–100 in respiring cells [24]
IRP-1 but not IRE has ATPase activity Having demonstrated that ATP interferes with the IRE–IRP complex, we performed experiments to
Fig 1 Effect of an uncoupler of oxidative phosphorylation on IRP binding activity HepG2 cells growing in complete medium were left untreated (Control) or treated with 20 lgÆmL)1of Fe as ferric ammonium citrate, 10 l M CCCP, or 20 lgÆmL)1of Fe and 10 l M CCCP together, for 3 h (A) EMSA of IRP–IRE binding activity in total cell extracts using the ferritin IRE probe Binding activity without b-mercapto-ethanol treatment was quantified by scanning densitometry of the autoradiograms, and expressed as a percentage of control Values in the histogram are means ± SD from three separate experiments Representative autoradiograms before and after b-mercaptoethanol treatment are also shown Values differing from control by one-way ANOVA followed by Dunnett’s test are indicated; *P < 0.05, **P < 0.01 (B) Meas-urement of cellular ATP levels following treatments as in (A) ATP was measured in whole cell lysates with a luciferin ⁄ luciferase assay kit, and expressed relative to the values in untreated control cells Values are means ± SD from three independent experiments expressed relat-ive to control taken as 100% in each experiment, and statistical differences from control are indicated as in (A) The absolute concentration
in control cells was 2.27 ± 0.79 pmol ATPÆlg)1protein (C) Level of IRP-1 in whole cell extracts following different treatments as in (A) Equal amounts of protein (80 lg per lane) were subjected to western blot analysis with anti-IRP-1 serum The position of IRP-1 just above the 100 kDa molecular mass marker (compare Fig 2) is indicated by the arrow.
Trang 4establish whether IRE or IRP-1 has ATP hydrolytic
activity ATPase activities were measured with a
sensi-tive assay that monitors the production of inorganic
[32P]phosphate from [32P]ATP[cP] by TLC
Recombin-ant IRP-1 (200–800 ng; 250 nm to 1 lm) has ATPase
activity in the presence of 300 lm ATP (Fig 5A,B)
that correlates with the amount of protein in the
reac-tion A reaction with BSA as a protein control had
negligible activity After boiling of IRP-1 for 5 min,
less than 3% activity remained Size exclusion chroma-tography of recombinant IRP-1 could not separate IRE-binding activity from ATPase activity (data not shown) Purified IRE RNA was devoid of ATPase activity (Fig 5C)
The kinetics of ATP hydrolysis were studied with affinity-purified recombinant IRP-1 Release of inor-ganic phosphate was linear with time (Fig 6A) and plateaued with increasing protein at about 7 nmol phos-phateÆmin)1Æmg)1 IRP-1 (Fig 6B) Dependence on [ATP] yielded an apparent Km value of 5.3 ± 1.7 lm with respect to ATP hydrolysis and Vmax ¼ 3.4 ± 1.7 nmolÆmin)1Æmg)1(Fig 6C,D)
ATP hydrolysis can be used for production of energy to do work or to phosphorylate proteins Although we detected free [32P]phosphate on TLC,
we still checked for protein phosphorylation by separation of an ATP hydrolysis mixture containing IRP-1 by SDS⁄ PAGE and subsequent autoradiogra-phy We could not detect any autophosphorylation of IRP-1 (data not shown) In a similar experiment, Eisenstein et al [25] demonstrated that purified rat liver IRP-1 is a substrate for different protein kinases but does not autophosphorylate in the presence of [32P]ATP[cP]
IRP-1 binds ATP More direct proof that ATP binds directly to IRP-1 was sought in a photolabeling experiment Recombin-ant IRP-1 was incubated with 8-azido-[32P]ATP[aP] for 2 min at 4C, and this was followed by UV-induced covalent crosslinking of bound [a-32 P]nucleo-tide The proteins were separated by SDS⁄ PAGE, and
C
Fig 3 ATP inhibits IRP–IRE binding activity IRE binding activity
was measured by EMSA using either cytosolic extract from HepG2
cells or recombinant human IRP-1 after addition of the indicated
concentration of ATP A representative gel is shown in (A), and the
values in the histograms (C) are means ± SD from three
independ-ent experimindepend-ents Values marked *** differ from control ([ATP] ¼ 0)
at P < 0.001 (B) EMSA of HepG2 cell extracts, Ni 2+ –nitrilotriacetic
acid-purified ecombinant IRP-1 (Ni-IRP-1), and affinity-purified IRP-1
(A-IRP-1) without ATP, with 5 m M ATP, or with 5 m M ATP plus 2%
b-mercaptoethanol (ME).
Fig 2 Purification of IRP-1 Recombinant IRP-1 was purified from E coli by Ni 2+ –nitrilotriacetic acid agarose chromatography and IRE affinity chromatography (A) Silver-stained gels of molecular mass markers (lane M; sizes in kDa indicated by arrows), crude E coli lysate (lane L),
200 ng (N2) and 100 ng (N1) of Ni2+–nitrilotriacetic acid agarose-purified IRP-1, and 10 lL aliquots of successive 50 lL elutions from an IRE affinity column with 1 M KCl-containing elution buffer (E1, E2) (B) Western blot analysis with IRP-1 antibody Lanes represent 500 ng of
Ni 2+ –nitrilotriacetic acid agarose-purified IRP-1 (N5), and IRE affinity chromatography flow through (FT), wash (W), and successive 1 M KCl elutions (E1–E4) (C) IRE binding activity by EMSA of 500 ng of Ni2+–nitrilotriacetic acid agarose-purified IRP-1 (N5), and IRE affinity chroma-tography flow through (FT), wash (W), and 1 M KCl elutions (E1, E2).
Trang 5proteins that had bound to 8-azido-[32P]ATP[aP] were
visualized by autoradiography of dried gels Specificity
of binding to IRP-1 was demonstrated by competition
with unlabeled ATP, ATP-cS, and unlabeled
8-azido-ATP, and by lack of competition with the related
pur-ine nucleotide, GTP (Fig 7A,B) Binding of increasing
amounts of 8-azido-[32P]ATP[aP] (Fig 7C) indicated a
Kd> 30 lm To characterize the binding further, a
filter-binding assay was performed with up to 500 lm
[32P]ATP[aP] and gave a Kd¼ 86 ± 17 lm (Fig 7D)
ATPase activity is enhanced in the presence
of IRE Binding to RNA and ATPase activity are characteris-tics of RNA helicases, and RNA binding increases the ATPase activity of these proteins [26] To test whether the ATPase activity of IRP-1 is also enhanced in the presence of IRE, we measured hydrolysis of [32P]ATP[cP] in the presence of 0–400 ng of IRE (Fig 8) Compared to the amount of ATP hydrolyzed with 400 ng of IRP alone, addition of IRE increased the release of inorganic [32P]phosphate by about 50% IRE alone at 400 ng had no hydrolytic activity (Fig 5C)
Discussion
We have demonstrated here that treatment of HepG2 cells with an uncoupler of oxidative phosphorylation depleted them of ATP and prevented suppression of
Fig 4 Requirement for ATP hydrolysis to inhibit IRP–IRE binding
activity EMSA was performed for IRE binding to HepG2 cytosolic
extract (upper panel) or recombinant IRP-1 (lower panel) The first
lane in each panel is without added nucleotide In subsequent
lanes, the indicated nucleotide analog was added prior to protein,
to a final nucleotide concentration of 5 m M Representative
auto-radiograms are shown, and the bars show means ± SD of the
intensities from separate experiments (HepG2 extract, n ¼ 4;
recombinant IRP-1, n ¼ 3), expressed relative to the nucleotide-free
lane, taken as 100% Values marked ** are significantly lower than
the nucleotide-free lane; P < 0.01.
A
B
C
Fig 5 Recombinant IRP-1 has ATPase activity (A) TLC autoradio-gram of [ 32 P]ATP[cP] reaction mixtures containing 300 l M ATP in the presence of increasing amounts of recombinant IRP-1 The position of free inorganic phosphate is indicated by the arrow In the last lane, 800 lg of BSA is included as a nonspecific protein control (B) Combined results (means ± SD) of three repetitions of experiments as in (A), with inorganic phosphate quantitated by scraping and scintillation counting Significant increases in the pres-ence of IRP-1 are indicated; **P < 0.01, ***P < 0.001 (C) TLC autoradiogram of [ 32 P]ATP[cP] reaction mixtures in the presence of increasing amounts of IRE mRNA.
Trang 6IRP activity by iron A similar effect of CCCP-induced
ATP depletion was observed on the reconstitution of
cytosolic and mitochondrial aconitase activities after
removal of NO-generating agents [27] That is, the
high IRE-binding activity of IRP-1 after NO exposure
remained elevated in CCCP-treated cells after cessation
of NO flux Moreover, Bouton et al [27] also observed
that IRP-1 cannot dissociate from IRE if mitochondria
are unable to produce ATP They suggested that
IRP-1–IRE complex dissociation, an obligate step upstream
of 4Fe)4S cluster repair, utilizes an ATP-dependent
mechanism Although this is a plausible explanation
for our results, we have not attempted to rule out
other possibilities For instance, interference with
mito-chondrial function and dissipation of the
mitochond-rial transmembrane potential by CCCP could prevent
reconstitution by interfering with mitochondrial
4Fe)4S cluster synthesis, although Li et al have
dem-onstrated that reconstitution of mammalian cytosolic
aconitase probably involves cytosolic forms of enzymes
of cluster synthesis [28]
Although iron treatment has a dramatic impact on
iron–sulfur proteins and the bioenergetic function of
mitochondria [29], 3 h of iron treatment of our HepG2
cultures did not significantly increase the ATP level
compared to that in control cells (Fig 1) Oexle et al
[30] found a significant increase in ATP after 24 h in
iron-treated, differentiating K562 cells as compared to
control or deferoxamine-treated cells, and aconitase
activity was also increased in iron-treated cells, as
expected Thus, one may speculate that iron regulates
IRP binding activity in part through changes in ATP
concentration (Fig 1) and⁄ or that ATP is required for iron-mediated regulation of IRP-1 binding activity On the other hand, a Kd of 86 lm argues against direct regulation of IRP-1 binding by ATP Total cellular ATP levels are c 5 mm [32], and this would suggest that IRP-1 would be saturated with ATP under normal circumstances, preventing IRE binding Indeed, this might account for the observation, derived from IRP-1 gene-ablated mice, that IRP-2 rather than IRP-1 is responsible for the post-transcriptional regulation of iron homeostasis, and that IRP-1 functions as a cyto-solic aconitase, rather than as an RNA-binding pro-tein, in most cells [33] However, the lower Kmvalues
of many ATPases in the micromolar range suggest that they experience lower local concentrations of ATP, e.g due to compartmentalization Furthermore, at least 50% of cytosolic ADP is protein-bound [34] Thus, whether IRP-1 experiences subsaturating cytosolic ATP concentrations in the cell remains an open ques-tion Certainly, our estimated Km value of 5.3 lm is quite in keeping with the values reported for a number
of other ATP-hydrolyzing proteins, e.g Escherichia coli DnaK (20 lm) [35], Hsc70 (1.4 lm) [36], and F1-ATPase (15 lm) [37] Furthermore, these proteins have
Vmax values in the range 1.1–3.5 nmolÆmin)1Æmg)1, comparable to the value of 3.4 nmolÆmin)1Æmg)1 measured here, supporting a physiologic relevance
of the observed ATP binding
The requirement for more than 1 mm ATP to abol-ish binding in EMSA (Fig 3) is at first sight inconsis-tent with a Kdof 86 lm The Kmand Kdvalues are in reasonable agreement, and it may be that factors other
Fig 6 Kinetics of ATP hydrolysis by
affinity-purified recombinant IRP-1 (A) Time
dependence of ATP hydrolysis IRP-1
(20 ng) was incubated in a final volume of
20 lL with 10 n M [ 32 P]ATP[cP] in the
pres-ence of 10 l M ATP for 1 h, and [ 32
P]phos-phate release was measured by TLC (B)
Dependence of ATP hydrolysis on IRP-1
concentration The indicated amount of IRP
was included in the reaction with conditions
as in (A) (C) A representative thin layer
chromatogram of ATP hydrolysis with 20 ng
of affinity-purified recombinant IRP-1, 10 n M
[32P]ATP[cP] and the indicated amount of
unlabeled ATP for 1 h Bk is the reaction
mixture with no added IRP-1 or cold ATP.
(D) Rate data calculated from (C) Nonlinear
regression (r 2 ¼ 0.67) of the rate data gives
Km¼ 5.3 ± 1.7 l M and Vmax¼ 3.4 ± 0.3
nmolÆmin)1Æmg)1.
Trang 7than binding and hydrolysis, e.g a conformational
change induced by high ATP concentrations, are
necessary for disruption of RNA binding However,
other factors in the EMSA protocol, including changes
in Mg2+ and ATP concentrations during
electropho-retic separation, may influence binding, and we think
this the more likely explanation; direct comparison of
the ATP dependence of EMSA and binding of ATP to
purified protein under optimized conditions may not be
appropriate Nor can the concentration of ATP
neces-sary to disrupt IRP–IRE binding on EMSA be readily
compared to effective concentrations in vivo, where additional interactions may be involved
Disruption of the 4Fe)4S cluster of IRP-1 with 2% b-mercaptoethanol is widely used to uncover total IRP-1 binding activity However, in our experiments addition of 2% b-mercaptoethanol did not reconstitute IRP-1 binding inhibited by ATP, and nor was the pro-tein degraded Similarly, Gonzalez et al [38] reported that IRP binding activity inhibited by interferon-c⁄ lipopolysaccharide-dependent NO production and phorbol ester treatment was not recovered by exposure
of IRP-1 to 2% b-mercaptoethanol, and nor was this due to protein degradation Furthermore, we previously found that b-mercaptoethanol somewhat diminished the binding activity achieved by in vitro treatment with deferoxamine [39] Thus, while b-mercaptoethanol can disrupt the 4Fe)4S cluster and facilitate binding of IRP-1, it may also decrease bind-ing by openbind-ing up the protein to internal disulfide bond formation [40] These data suggest a novel mech-anism for ATP-dependent inhibition of IRP-1 binding activity that cannot be recovered by reductive treat-ment with b-mercaptoethanol
We demonstrate a requirement for ATP hydrolysis
in order to inhibit recombinant IRP-1 binding activity, and both ATP and ADP suppress binding (Fig 3) The mechanism of activation of IRP-1 by oxidative stress might well involve ATP Thus, IRP-1 activation with H2O2requires Mg2+, and is sensitive to treatment with alkaline phosphatase that results in a 90% reduc-tion of ATP levels [41] Furthermore, ATP-cS and GTP-cS inhibited IRP-1 activation by H2O2 signifi-cantly Pantopoulos & Hentze [41] conclude that IRP-1 activation by H2O2 in permeabilized cells appears to require ATP and GTP, indicating an energy dependence of the process and⁄ or the involvement of a phosphorylation–dephosphorylation cycle We found that ATP-cS causes only about 30% inhibition of binding in whole cell extracts but 80% inhibition with recombinant protein, and this difference may arise from different characteristics of native HepG2 protein and recombinant protein, such as the presence of a His-tag on the latter Although ATP-cS is often con-sidered a nonhydrolyzable ATP analog, it undergoes hydrolysis in some circumstances, and is a substrate for RNA-dependent nucleotide hydrolysis by helicases [23] Because hydrolysis of a phosphorothioate group
is actually predicted to be faster than that of the corresponding phosphate on the basis of chemical considerations, it has been suggested that the rate-determining step may be a conformational change that takes place after substrate binding [23] Native and recombinant proteins may have different
D
C
Fig 7 Photolabeling of IRP-1 with 8-azido-ATP (A) Two hundred
nanograms of affinity-purified recombinant IRP-1 was reacted with
5 l M 8-azido-[ 32 P]ATP[aP], and then subjected to gradient
SDS ⁄ PAGE; and the gel was then silver stained (Ag), and
autoradio-graphed ( 32 P) The arrow indicates the position of IRP-1 at 105 kDa.
(B) Autoradiograph after photo-crosslinking of 4 lg portions of
Ni 2+ –nitrilotriacetic acid-purified recombinant IRP-1 with 5 l M
8-azi-do-[32P]ATP[aP] alone in control lanes (C), or in the presence of
competitors: 20 m M ATP (A2), 20 m M GTP (G2), 100 m M ATP
(A10), 100 m M GTP (G10), 20 m M ATP-cS (c), and 20 m M unlabeled
8-azido-ATP (Z) (C) Photo-crosslinking of 4 lg portions of Ni2+
–nitril-otriacetic acid-purified recombinant IRP-1 with increasing
concentra-tions of 8-azido-[ 32 P]ATP[aP] as indicated The reaction mixture was
electrophoresed, silver stained, and autoradiographed (D)
Filter-binding assay of 0.62 lg of IRP-1 incubated with 100 n M
[ 32 P]ATP[aP] and the indicated concentration of unlabeled ATP.
The reaction mixture was collected on nitrocellulose membranes
as described in Experimental procedures The saturation plot
was fitted by nonlinear regression (r 2 ¼ 0.98) to give values of
K d ¼ 86 ± 17 l M and B max ¼ 4.6 ± 0.3, typical of three such
experiments.
Trang 8tional flexibilities Alternatively, lower inhibition by
ATP-cS in cell extracts may be due to the presence of
additional cellular proteins from HepG2 cytosol that
are involved in IRP–IRE complex formation
Further-more, ATP-cS, but not AMP-PNP, inhibited IRP–IRE
binding in extracts from fibroblasts treated with the
analogs in culture [41] Therefore, the 30% inhibition
of binding in our cell extracts still suggests that ATP
should be hydrolyzed in order to interact with the
IRP-1–IRE complex This is confirmed by the lack of
any effect of the nonhydrolyzable imidodiphosphate
analog AMP-PNP
Direct binding of ATP to IRP-1 was demonstrated
by photolabeling with radioactive photosensitive
8-azi-do-[32P]ATP[aP] (Fig 7), a method that has been used
to identify ATP-binding sites on proteins, e.g on
histi-dine permease [42] and chaperonin GroEL [43]
Labe-ling with 8-azido-[32P]ATP[aP], together with evidence
of binding of [32P]ATP[aP] but not [32P]ATP[cP] in the
filter assay (data not shown), is a strong indication of
an ATPase activity of IRP-1 Competition by ATP
and analogs, but not GTP (Fig 7) or CTP (data not
shown), supports specificity of the binding site(s) A
large excess of unlabeled ATP was necessary to
elimin-ate the signal The reason for this is unknown, but it
has been observed previously in similar ATP
photo-labeling experiments [44–47]
A simple model, then, is that IRP-1 is bound
to RNA at lower ATP concentrations, but at higher
concentrations it hydrolyzes ATP and dissociates from
RNA This means that a low local ATP level
main-tains the IRP-1–IRE complex at high iron
concentra-tion, and when the ATP level increases, the complex
dissociates Increased ATP hydrolysis in the presence
of IRE is reminiscent of an important ATPase protein
family) the helicases [48] They unwind duplex RNAs
in concert with the hydrolysis of nucleoside For
example, the RNA-binding helicase eIF4A undergoes cyclic conformational changes upon ATP binding and hydrolysis such that the eIF4A–ADP complex has a greatly decreased affinity for ssRNA [26] Our data do not show whether ATP inhibits the binding of IRP-1
to IRE, or instead facilitates dissociation of the com-plex Enhancement of ATP hydrolysis by IRE in the presence of IRP-1, but absence of hydrolysis in the presence of IRE alone, suggests that ATP may interact with the complex However, the enhancement is not dramatic, and whether ATP inhibits formation or increases dissociation of the IRE–IRP-1 complex can-not be definitively determined from the present data
If formation of the IRP-1–IRE complex depends on iron concentration, but its dissociation depends on ATP concentration, then the expression of IRE-con-taining genes would actually depend on both iron and ATP levels At low ATP levels, transferrin receptor mRNA would be stable, and transferrin receptor on the membrane would continue to take up iron In par-allel, ferritin mRNA (and other 5¢-IRE mRNAs) would still have IRP-1 bound, and ferritin synthesis would be blocked, so iron would not be stored in ferr-itin but relocated to iron-binding proteins, many of them located in mitochondria and involved in energy metabolism The present observations suggest an expanded role for the IRE–IRP system in regulating cellular energy metabolism
Experimental procedures
Cell culture and iron loading HepG2 cells (no HB 8065) were obtained from the Ameri-can Type Culture Collection (Rockville, MD) and grown in a-MEM supplemented with 10% fetal bovine serum, peni-cillin (100 UÆmL)1) and streptomycin (100 lgÆmL)1) Cells
Fig 8 IRE stimulates ATPase activity of
IRP-1 (A) Hydrolysis reaction of
[ 32 P]ATP[cP] The blank contains neither
pro-tein nor RNA Other lanes contain 400 ng of
recombinant IRP-1 and the indicated amount
of IRE RNA Values are means ± SD from
three separate experiments *Significantly
greater than IRP-1 alone (P < 0.05) (B)
Autoradiogram of a thin layer chromatogram
showing the release of [ 32 P]phosphate.
Lanes are as in (A).
Trang 9were plated at a density of 4· 106
cells per 60 mm dish, allowed to attach and grow overnight, and then treated
with iron (20 lgÆmL)1) as ferric ammonium citrate, 10 lm
CCCP, or both After 3 h, cells were harvested and protein
was extracted (see below)
Preparation of cytoplasmic extracts
Monolayers of HepG2 cells were scraped in NaCl⁄ Pi and
centrifuged at 11 000 g for 1 min with an Eppendorf 5415R
centrifuge The pellet was resuspended in extraction buffer
(EB: 10 mm Hepes, pH 7.6, 3 mm MgCl2, 40 mm KCl,
1 mm dithiothreitol, 0.2% Nonidet P-40) After sonication
for 5 s, the suspension was centrifuged for 1 min at
11 000 g with an Eppendorf 5415R centrifuge The
super-natant was recentrifuged under the same conditions for use
in the IRE-binding assay [49] The protein content of the
extracts was determined with a Bradford-based protein
determination kit (Bio-Rad, Mississauga, ON, Canada)
The protein extract was aliquoted and stored at) 80 C
Preparation of RNA transcripts
Transcription was performed in vitro with 1 lg of BamH1
linearized plasmid pSPTfer [50], coding the human ferritin
H-chain IRE (provided by L C Ku¨hn, ISREC, Epalinges,
Switzerland) in the presence of 50 lCi of [32P]CTP[aP]
(800 CiÆmmol)1; ICN, Costa Mesa, CA) and T7 RNA
Polymerase using a Promega in vitro transcription system
(Promega, Madison, WI) Full-length transcripts were
puri-fied on QuickSpin columns (Roche Molecular Biochemicals,
Laval, PQ, Canada) for radiolabeled RNA purification
according to the manufacturer’s procedure
Purification of recombinant IRP-1
Human recombinant IRP-1 was purified from E coli
trans-formed with pT7-his-IRP-1 plasmid (a gift from K
Pantop-oulos, McGill University) as previously described [51]
Bacterial pellets were resuspended in 20 mm Tris⁄ HCl
(pH 8.0), with 250 mm NaCl and 0.5% Nonidet P-40,
fro-zen, thawed, and sonicated The lysates were clarified by
centrifugation (4300 g, Sorvall centrifuge with SS34 rotor)
Ni2+–nitrilotriacetic acid agarose beads (Qiagen,
Mississ-auga, ON, Canada) were washed twice with buffer N
(24 mm Hepes, pH 7.6, with 150 mm potassium acetate,
1.5 mm MgCl2, and 5% glycerol) KCl was added to the
lysate to a final concentration of 0.4 m, and the lysate was
tumbled with Ni2–nitrilotriacetic acid agarose beads for 1 h
at 4C This mixture was poured into a column and
washed sequentially at 4C with buffer N plus 0.4 m KCl,
buffer N alone, and buffer N with 5 mm imidazole IRP-1
was eluted with buffer N containing 50 mm imidazole
Recombinant protein was further purified with the use of
streptavidin-conjugated paramagnetic spheres (Invitrogen Canada Inc., Burlington, ON, Canada) and biotinylated IRE RNA [52] For biotinylation, 100 lL of a transcription reaction mixture was prepared containing 5 lL of 10 mm GTP, UTP and ATP, 2.5 lL of 10 mm CTP, and 2.5 lL of
10 mm biotin-14-CTP (Invitrogen Canada Inc.) according
to the manufacturer’s instruction Biotin-14-CTP is a CTP analog that contains biotin attached to the N4 position via
a 14-atom linker Full-length transcripts were purified on QuickSpin columns and used for affinity chromatography Streptavidin beads (100 lL) were washed two times with
200 lL of binding buffer (20 mm Hepes, pH 7.5, 150 mm KCl, 1 mm phenylmethanesulfonyl fluoride), resuspended in
100 lL of binding buffer, and added to biotinylated IRE (10 lg in 150 lL of binding buffer) Biotinylated IRE was bound to streptavidin beads for 10 min at 4C, and unbound IRE was washed with 100 lL of binding buffer Fifty micrograms of fresh IRP-1 was then added to mag-netic beads in 100 mm KCl in 200 lL of binding buffer, and tumbled for 90 min at 4C Beads were washed three times with 400 lL of washing buffer (20 mm Hepes,
pH 7.5, 150 mm KCl, 2 mm MgCl2, 0.5 mm dithiothreitol, 0.5 mm phenylmethanesulfonyl fluoride, 5% glycerol, and 0.1% Nonidet P-40), and eluted five times with 50 lL of elution buffer (50 mm Hepes, pH 7.5, 0.5 mm EDTA, 1 m KCl, 1 mm phenylmethanesulfonyl fluoride, 5% glycerol)
EMSA RNA–protein interactions were analyzed in cytoplasmic extracts containing 15–30 lg of total protein, or with
400 ng of Ni2+–nitrilotriacetic acid-isolated recombinant IRP-1 or 20 ng of affinity-purified IRP-1 Extracts were mixed with a molar excess of labeled ferritin H-chain IRE (10 ng) in EB buffer without Nonidet P-40 and with or without ATP (final volume 20 lL) In some experiments, various nucleotide analogs were added at 5 mm final con-centration to the EB buffer prior to adding protein and IRE RNAse T1 was added after 10 min of incubation, and
2 lL of 2.5 mgÆmL)1 heparin was added after a further
10 min for an additional 10 min period IRE–IRP com-plexes were resolved in 6% nondenaturing polyacrylamide gels In some experiments, parallel samples were treated with 2% b-mercaptoethanol before addition of the labeled RNA probe to unmask total IRP binding activity
Western blot analysis Proteins were resolved by 8% SDS⁄ PAGE, transferred to nitrocellulose membranes, incubated with antibodies to IRP-1 or IRP-2 (Alpha Diagnostic International, San Anto-nio, TX) (1 : 5000 dilution), and detected using ECL west-ern blot detection reagents (Amersham Bioscience, Baie d’Urfe´, PQ, Canada)
Trang 108-Azido-[32P]nucleotide binding experiments
Recombinant human IRP-1 (0.2–4 lg) was combined with
8-azido-[32P]ATP[aP] (Affinity Labeling Technologies,
Lex-ington, KY; specific activity 10–15 CiÆmmol)1) at a final
concentration of 20 lm and assay buffer (25 mm Tris,
pH 7.6, 100 mm KCl, 5 mm MgCl2and 10% glycerol) in a
total volume of 20 lL according to the manufacturer’s
pro-tocol After 2 min of incubation on ice, the samples were
crosslinked immediately with a 254 nm UV lamp for 2 min
The samples were separated on either a 4–15% gradient or
8% SDS⁄ PAGE gels (Bio-Rad), silver stained, dried, and
exposed to X-ray film
Filter-binding assay
ATP-binding activity was determined by a filter-binding
assay as previously described [53] IRP-1 (0.5–1 lg) was
incubated with 100 nm [32P]ATP[aP] (Perkin Elmer
Can-ada, Woodridge, ON, Canada; 3000 CiÆmmol)1) at room
temperature for 30 min in 20 lL of binding buffer (25 mm
Tris, pH 7.6, 100 mm KCl, 5 mm MgCl2, 1 mm
dithiothrei-tol, and 10% glycerol) containing different concentrations
of unlabeled ATP Nitrocellulose membranes were soaked
briefly in 0.4 m KOH, rinsed thoroughly, and mounted on
a dot blot apparatus The binding reaction was stopped by
addition of 70 lL of ice-cold buffer, and the reaction
mix-ture was loaded on the dot blot appratus in triplicate 30 lL
aliquots, and washed twice with 200 lL of cold binding
buffer Nitrocellulose membranes were exposed to film, and
each spot was cut out and counted by liquid scintillation
ATP hydrolysis and TLC
ATP hydrolysis was determined by measuring the release of
[32P]phosphate from [32P]ATP[cP] (Perkin Elmer Canada;
3000 CiÆmmol)1) [36] IRP-1 was added to 10 nm [32P]ATP
[cP] in 25 mm Tris⁄ HCl (pH 7.6), 100 mm KCl, 5 mm
MgCl2, 10% glycerol and varying concentration of ATP in
a final volume of 20 lL After incubation for 60 min at
room temperature, 2 lL was spotted on polyethyleneimine
cellulose TLC plates (Sigma, St Louis, MO, USA) IRE
(100–800 ng) was analyzed under the same conditions The
spotted samples were resolved using 0.5 m lithium chloride
in 0.5 m formic acid and visualized by autoradiography
Spots containing the released [32P]phosphate were cut and
measured by scintillation counting
ATP measurement
To extract ATP, cells were harvested in cold 0.6 m HClO4
and centrifuged at 9000 g for 1 min with an Eppendorf
5415R centrifuge The pellet was saved for protein
deter-mination by Lowry’s method [54,55] The supernatant was
neutralized with 5 m KOH and 0.4 m imidazole, and centri-fuged again Aliquots of ATP-containing extract were saved
at ) 80 C ATP was measured with a commercial
lucifer-in⁄ luciferase (ENLITEN) kit (Promega) according to the manufacturer’s protocol Briefly, 100 lL of a 1 : 500 diluted sample was added to a microplate, and 50 lL of ENLITEN
rL⁄ L reagent was added immediately before measurement and mixed by repeated pipetting Measurements were taken
in a microplate luminometer (MicroLumat Plus; ED & G Berthold, Gaithersburg, MD, USA) Three measurements were made for each sample and were compared to a stan-dard curve of ATP solutions ranging from 10 to 750 nm
Statistical methods Values from multiple experiments are expressed as mean ± SD Differences between means in multiple com-parisons were analyzed by one-way anova Comcom-parisons of multiple values against a control value were performed using Dunnett’s post hoc test Kinetic data and binding plots were fitted by nonlinear regression using the program prism(GraphPad Software, San Diego, CA)
Acknowledgements
This work was supported by grants from the Heart and Stroke Foundation of Canada (grant T4134) and the Canadian Institutes of Health Research (grant MT11270) We thank Dr Tania Christova for helpful discussions
References
1 Kennedy MC, Mende-Mueller L, Blondin GA & Beinert
H (1992) Purification and characterization of cytosolic aconitase from beef liver and its relationship to the iron-responsive element binding protein Proc Natl Acad Sci USA 89, 11730–11734 (erratum appears in Proc Natl Acad Sci USA 90, 2556)
2 Samaniego F, Chin J, Iwai K, Rouault TA & Klausner
RD (1994) Molecular characterization of a second iron-responsive element binding protein, iron regulatory protein 2 Structure, function, and post-translational regulation J Biol Chem 269, 30904–30910
3 Guo B, Yu Y & Leibold EA (1994) Iron regulates cyto-plasmic levels of a novel iron-responsive element-bind-ing protein without aconitase activity J Biol Chem 269, 24252–24260
4 Rouault TA, Stout CD, Kaptain S, Harford JB & Klausner RD (1991) Structural relationship between an iron-regulated RNA-binding protein (IRE-BP) and aco-nitase: functional implications Cell 64, 881–883
5 Martins EL, Robalinho RL & Meneghini R (1995) Oxi-dative stress induces activation of a cytosolic protein