From the Department of Biochemistry and Molecular Biology and
Center for Experimental BioInformatics, University of Southern Denmark,
DK-5320 Odense M., Denmark, the Department of Medical Nutrition,
Karolinska Institute, S-14157 Huddinge, Sweden, and the
Institute of Clinical Biochemistry and
§§ Department of Biochemistry and Molecular
Biology, University of Bergen, N-5021 Bergen, Norway
Received for publication, February 5, 2001
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ABSTRACT |
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The peroxisome proliferator-activated receptor
Members of the nuclear receptor superfamily mediate
ligand-dependent transactivation of genes controlling
development, differentiation, and homeostasis in response to
nutritional, metabolic, and hormonal signals (1). The peroxisome
proliferator-activated receptor Activation of nuclear receptor-mediated transcription involves an
agonist-dependent release of co-repressors and recruitment of co-activators. Accumulating evidence obtained by x-ray
crystallography has revealed a significant ligand-dependent
conformational change involving repositioning of the conserved AF-2
helix in the ligand-binding domains of nuclear receptors (8-11). This
ligand-induced conformational change has been demonstrated to be a
determining event governing interactions with co-activators and
co-repressors (see Refs. 12-14; reviewed in Ref. 15). The crystal
structures of the PPAR A large variety of long-chain fatty acids, eicosanoids, and synthetic
compounds have been shown to serve as PPAR Plasmids--
pSG5-mPPAR Ligands--
Linoleic acid was purchased from Sigma-Aldrich.
Wy14643 and 9-cis-retinoic acid were from Biomol.
S-Hexadecyl-CoA was synthesized as described by Rosendal
et al. (39), and tetradecylthioacetic acid (TTA) was
synthesized as described by Berge et al. (40). BRL49653 was
kindly provided by Novo Nordisk A/S.
Sf21 Whole Cell Extracts--
Rat PPAR In Vitro Transcription and Translation--
PPAR Electrophoretic Mobility Shift Assays--
The DNA probe was
prepared by annealing 32-base pair oligonucleotides covering the
PPAR/RXR binding motif in the promoter of the acyl-CoA oxidase (ACO)
gene (32). The probe was labeled by filling in the 3'-recessive ends
with [ Differential Protease Sensitivity Assay--
2-µl aliquots of
the 35S-labeled, in vitro translated protein was
incubated for 20 min at 25 °C in a total volume of 8 µl of binding
buffer with the addition of ligand or vehicle. The final concentrations
in the binding buffer were 22 mM Tris-HCl, pH 8, 75 mM KCl, 5% (v/v) glycerol, and 2 µM
dithioerythritol. Linoleic acid and S-hexadecyl-CoA were
used in final concentrations of 120 µM and 5-15
µM, respectively. Chymotrypsin (Roche Molecular Biochemicals) was dissolved in 50 mM
NH4HCO3. Following incubation, 2 µl of
chymotrypsin was added to final concentrations of 0-120 µg/ml or
0-260 µg/ml. The digestions were carried out at 25 °C for 20 min
and stopped by boiling in Tris-Tricine loading buffer (0.1 M Tris-HCl, pH 6.8, 24% (v/v) glycerol, 8% (w/v) SDS, 0.2 M dithiothreitol, 0.02% (w/v) G-250 Coomassie Brilliant
Blue). The resulting peptide mixtures were separated by SDS-PAGE and visualized using a PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA).
Palmitoyl-CoA-Agarose Bead Assay--
Whole cell extracts of
yeast (BJ2168) expressing mPPAR GST Pull-downs--
GST fusion proteins were captured on
glutathione-Sepharose beads as described (36). 8 µl of
35S-labeled in vitro translated mPPAR Ligand-induced Binding of PPAR PPAR
Recombinant full-length PPAR S-Hexadecyl-CoA Increases the Sensitivity of PPAR S-Hexadecyl-CoA Abolishes Ligand-induced Interaction between SRC-1
and PPAR S-Hexadecyl-CoA Exerts Its Action on PPAR
As shown above, the presence of S-hexadecyl-CoA
differentially altered the ability of PPAR In the present work, we present evidence suggesting that acyl-CoA
esters directly affect the conformation and function of PPAR Long-chain acyl-CoA esters have been estimated to have a van der Waals
volume of not less than 850 Å3 (58). This size would
exclude acyl-CoA esters from the ligand-binding pocket of most nuclear
receptors except for the PPARs with ligand-binding pockets of ~1300
Å3 (16, 17). It was recently reported that docosahexaenoic
acid is a ligand for RXR Biochemical and structural studies have revealed a unifying principle
determining the interaction of nuclear receptors with co-activators and
co-repressors involving an at least partially overlapping binding site
(13-15, 59). The hydrophobic face of helical regions in the receptor
interacting domains of co-activators or co-repressors harboring an
LXXLL core motif or a related CoRNR motif,
respectively, interacts with a hydrophobic pocket formed by helices
3-5 and the AF-2 helix in PPAR From the analysis of the structure of the estrogen receptor bound to
agonists or antagonists, it is evident that subtle distortions in the
placement of the AF-2 helix may have a profound effect on the
interaction with co-activators or co-repressors (10). Our finding that
S-hexadecyl-CoA decreases interaction with SRC-1 and
increases recruitment of NCoR indicates that the bulky CoA head
influences directly or indirectly the positioning of the AF-2 helix.
Thus, the bulky CoA head group of S-hexadecyl-CoA may
prevent the AF-2 helix from folding back, forcing the AF-2 helix to adopt an extended conformation contrasting with the unliganded conformation that allows the AF-2 helix to fold back. The increased sensitivity of PPAR Examination of the crystal structure of PPAR Several genes are transcriptionally regulated by antagonistic
cross-talk between PPAR and HNF-4 (PPAR
) is a ligand-activated transcription factor and a key
regulator of lipid homeostasis. Numerous fatty acids and eicosanoids
serve as ligands and activators for PPAR
. Here we demonstrate that S-hexadecyl-CoA, a nonhydrolyzable palmitoyl-CoA analog,
antagonizes the effects of agonists on PPAR
conformation and
function in vitro. In electrophoretic mobility shift
assays, S-hexadecyl-CoA prevented agonist-induced
binding of the PPAR
-retinoid X receptor
heterodimer to the
acyl-CoA oxidase peroxisome proliferator response element. PPAR
bound specifically to immobilized palmitoyl-CoA and Wy14643, but
not BRL49653, abolished binding. S-Hexadecyl-CoA increased in a dose-dependent and reversible manner the
sensitivity of PPAR
to chymotrypsin digestion, and the
S-hexadecyl-CoA-induced sensitivity required a functional
PPAR
ligand-binding pocket. S-Hexadecyl-CoA prevented
ligand-induced interaction between the co-activator SRC-1 and PPAR
but increased recruitment of the nuclear receptor co-repressor NCoR. In
cells, the concentration of free acyl-CoA esters is kept in the low
nanomolar range due to the buffering effect of high affinity
acyl-CoA-binding proteins, especially the acyl-CoA-binding protein. By
using PPAR
expressed in Sf21 cells for electrophoretic
mobility shift assays, we demonstrate that S-hexadecyl-CoA
was able to increase the mobility of the PPAR
-containing heterodimer
even in the presence of a molar excess of acyl-CoA-binding protein,
mimicking the conditions found in vivo.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(PPAR
,1 NR1C1 (2)) belongs
to the nuclear hormone receptor superfamily (3). Through
heterodimerization with the retinoid X receptors (4) (NR2B1-3) and
binding to DR-1 response elements, PPAR
regulates transcription of
several genes encoding enzymes involved in lipid metabolism (5, 6).
Accordingly, PPAR
is predominantly expressed in tissues with a high
turnover of fatty acids (7).
and PPAR
ligand-binding domains (16, 17)
have revealed an overall folding pattern similar to that observed for
other nuclear receptor ligand-binding domains (8-11). However, the
PPAR ligand-binding pocket is substantially larger than those of other
nuclear receptors, and this may in part explain the observed
promiscuity in terms of ligand binding (16, 17). The interior of the
ligand-binding pocket has been suggested to be solvent-accessible via a
channel between helix 3 and the
-sheet. The entrance is lined by
polar side chains, and its dimension indicates that ligands may enter the cavity without affecting the overall structure of the receptor (16-18). Crystallization of a ternary complex containing the PPAR
ligand-binding domain, the PPAR
agonist BRL49653, and
the nuclear receptor-binding domain of the steroid receptor
co-activator-1 (SRC-1) has revealed that association between liganded
nuclear receptors and co-activators depends on conserved residues in
helix 3 and the AF-2 helix forming a charge clamp and hydrophobic
interactions involving helices 3, 4, and 5 and the AF-2 helix (16).
Although the three-dimensional structure of PPAR
has yet to be
reported, it has been shown that the C terminus of the ligand-binding
domain is essential for the ligand-induced co-activator interaction
(19-22).
ligands and
activators (17, 23-25). Several natural and synthetic PPAR
ligands
are activated to the corresponding CoA esters (26-28), and these have
been demonstrated to accumulate in tissues of treated rats (29, 30).
Generally, the formation of CoA esters has been considered a process
that merely reduces the availability of the activating PPAR
ligands
(31). In the present study, we present evidence that acyl-CoA esters
directly affect PPAR
conformation and function in a manner
indicating that acyl-CoA esters may act as PPAR
antagonists.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
was kindly supplied by
J. D. Tugwood (32). The plasmid encoding SRC-1 (pBKCMV-hSRC1) was
a kind gift from B. W. O'Malley (33).
pGEX-NCoR-(2239-2453) for bacterial expression of GST-NCoR
(amino acids 2239-2453) was kindly provided by M. A. Lazar (34).
Plasmids pTLI-mPPAR
AB, and pTLI-mPPAR
AB/
425-468 were kindly
provided by M. Leid (35). The following constructs have been described
previously: rPPAR
(p7zf-PPARBE) and rRXR
(prRXRT7) (4,
23), pGEX-5X-1-mPPAR
-LBD (amino acids 166-469) (36), and
pCA4-rRXR
(37). The plasmid pCA2-mPPAR
was constructed by
subcloning the murine PPAR
cDNA into the
BamHI/SalI site of pCA2, which is derived from
pCA4 by insertion of the CUP1 promoter cassette into pRS314 (38). The
murine PPAR
fragment was obtained by polymerase chain reaction from
pSG5-mPPAR
using BamHI/SalI-tagged primers.
was expressed
in Spodoptera frugiperda Sf21 insect cells as
previously described (4). The Sf21 whole cell homogenate was
prepared by disrupting 4 × 107 cells in 2.3 ml of
buffer (25 mM KPO4, pH 7.4, 100 mM
KCl, 1 mM EDTA, 2 mM dithioerythritol, 1 mM phenylmethylsulfonyl fluoride, 1 mg/ml leupeptin, 1 mg/ml antipain, 1 mg/ml pepstatin, 0.01 trypsin inhibitory
units/ml aprotinin) with 50 strokes in an all glass Dounce
homogenizer. The ionic strength was increased by slowly adding 1.2 ml
of 1 M KCl, and the homogenate was incubated on ice for 30 min before centrifugation (35,000 rpm, 42 min) in a Beckman Ti70 rotor.
The supernatant was stored in aliquots in liquid nitrogen. The protein
concentration, as determined by spectrophotometry (41), was 3-4
µg/µl.
, RXR
, and
SRC-1 were synthesized in vitro by using the TNT®-coupled
transcription/translation system (Promega) with or without
[35S]cysteine/methionine amino acid mixture (ICN). PPAR
and RXR were synthesized in the presence of 0.5-1.0 µM
zinc acetate.
-32P]dCTP (10 µCi, 3000 Ci/mmol; Amersham
Pharmacia Biotech) using the Klenow fragment of the Escherichia
coli DNA polymerase. Binding reactions were performed in a total
of 25 µl containing Sf21 extract, 2.4 µg of
poly(dI-dC) (Amersham Pharmacia Biotech), 25 mM
Hepes, pH 7.6, 40 mM KCl, 0.1 mM EDTA, 1 mM dithioerythritol, and 10% (v/v) glycerol. The reactions
were preincubated for 20 min on ice, after which 0.5 ng of probe was
added, and the reactions were incubated for 20 min at room temperature.
Free DNA and DNA-protein complexes were resolved by electrophoresis (12 V/cm, 1.5 h at 4 °C) on a 4% (w/v)
polyacrylamide/bisacrylamide gel (30:1) in a buffer containing 50 mM Tris-HCl, pH 8.5, 380 mM glycine, and 2 mM EDTA. Ligand-induced complex formation assays were
performed as described above, except that in vitro
translated PPAR
and RXR
were used instead of Sf21 extract,
the total reaction volume was 20 µl, and reactions contained
130 mM KCl and 5% (v/v) glycerol. Combined
electrophoretic mobility assay (EMSA) and Western blotting was
performed as described previously (42) using a polyclonal affinity-purified anti-rPPAR
antibody (21).
and/or rRXR
were prepared as
previously described (37). Yeast extract (7.5 µl) was incubated at
room temperature for 30 min in 42 mM NaCl, 100 mM Tris-HCl, pH 6.7, 4% (v/v) glycerol, 1 mM
dithioerythritol, 0.2 mM MgCl2, 1 mM phenylmethylsulfonyl fluoride, and
CompleteTM protease inhibitor mixture (Roche Molecular
Biochemicals) with 10 µl of palmitoyl-CoA beads (Sigma-Aldrich) or 10 µl of protein A-beads (Amersham Pharmacia Biotech). BRL49653 was
added to a final concentration of 1 µM, Wy14643 was added
to final concentrations of 100 nM to 10 µM,
and 9-cis-retinoic acid was added to a final concentration
of 10 µM. The beads were washed twice in 150 µl of 40 mM KCl, 100 mM Tris, pH 6.7, 4% (v/v)
glycerol, 1 mM dithioerythritol, and 1 mM
phenylmethylsulfonyl fluoride in the presence of ligand or vehicle.
Bound proteins were eluted by boiling in SDS sample buffer, separated
by SDS-PAGE, and evaluated by immunoblot analysis. The anti-PPAR
antibody was raised against the mouse PPAR
AB domain and
affinity-purified, and anti-RXR
antibody was from Santa Cruz
Biotechnology, Inc. (Santa Cruz, CA). Enhanced chemiluminescence detection was used for visualization.
, or
SRC-1 was incubated in buffer A (50 mM NaCl, 20 mM Tris-HCl, pH 7.9, 0.1% (v/v) Nonidet P-40, 10% (v/v)
glycerol, and 1% (w/v) essentially fatty acid-free milk powder) with
100 µM Wy14643, 30 µM TTA, or vehicle. 10 µl of GST fusion protein on beads was added, and interaction was allowed to proceed for 2 h at 4 °C. The beads were washed three times in 150 µl of buffer A and finally once in 150 µl of buffer A
without milk powder. The bound proteins were eluted by boiling in
SDS-PAGE sample buffer, resolved by electrophoresis on a 10% SDS-polyacrylamide gel, and visualized using the PhosphorImager. Quantitation of the content of discrete bands was done using the ImageQuant version 5.0 software (Molecular Dynamics).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
·RXR
to the ACO PPRE Is
Antagonized by the Nonhydrolyzable Palmitoyl-CoA Analog
S-Hexadecyl-CoA--
It is a well established fact that a large number
of fatty acids, eicosanoids, and hypolipidemic drugs activate
PPAR
-mediated transactivation, and different protocols have been
established to demonstrate that several of these activators are
bona fide PPAR
ligands (17, 23-25). Ligand-induced
complex formation assay provides one such simple and sensitive method
for analyzing the ability of PPAR activators to bind to PPAR and induce
DNA binding of a PPAR·RXR complex to a peroxisome
proliferator-responsive element (PPRE) (24). We have used this well
established assay to examine whether S-hexadecyl-CoA, a
nonhydrolyzable analog of palmitoyl-CoA (39), was able to modulate
ligand-induced binding of the PPAR
·RXR
heterodimer to the ACO
PPRE. The concentration of S-hexadecyl-CoA used in these
experiments was well below the critical micelle concentration (43). As
a PPAR
ligand, we used the fatty acid analog TTA, which has
been shown to efficiently promote PPAR
·RXR
·ACO PPRE complex
formation (24) and to induce PPAR
-mediated transactivation. Fig.
1 demonstrates that the addition of TTA
to limiting amounts of in vitro translated PPAR
and
RXR
, as reported previously (24), induced a considerable increase in
the binding of the PPAR
·RXR
heterodimer to the ACO PPRE probe in an electrophoretic mobility shift assay (EMSA) (Fig. 1,
lanes 2 and 3), whereas no binding of
the RXR
homodimer was observed (lane 8).
Interestingly, the presence of 5 µM
S-hexadecyl-CoA abrogated TTA-induced PPAR
·RXR
·ACO
PPRE complex formation (Fig. 1, lane 4).
Increasing the concentration of TTA restored PPAR
·RXR
·ACO PPRE complex formation in the presence of S-hexadecyl-CoA
(Fig. 1, lanes 5-7), demonstrating that the
inhibitory effect of S-hexadecyl-CoA was reversible and that
competition between fatty acid and acyl-CoA appears to regulate
PPAR
·RXR
·ACO PPRE complex formation.
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Fig. 1.
S-Hexadecyl-CoA reversibly
antagonizes fatty acid-induced
PPAR ·RXR
·PPRE
complex formation. One µl of a mixture of in vitro
transcribed/translated rPPAR
and rRXR
(10:1, v/v) or rRXR
alone was incubated with a 32P-labeled oligonucleotide
containing the PPRE of the rat ACO promoter in the presence of
different concentrations of TTA and S-hexadecyl-CoA as
indicated. The free and bound PPRE were separated by PAGE.
Binds Palmitoyl-CoA--
The ligand-induced complex
formation assay described above indicated that fatty acid and acyl-CoA
might compete for binding to PPAR
or, alternatively, that
simultaneous binding of an acyl-CoA ester and an agonistic ligand to
the PPAR
·RXR
complex antagonized the effect of the PPAR
agonist. Due to the hydrophobic characteristics and micelle forming
capacity of fatty acids and their CoA derivatives, we have been
unsuccessful in performing reliable simple binding competition
experiments between fatty acids and acyl-CoAs. To circumvent these
problems, we reckoned that a possible direct binding of acyl-CoA to
PPAR
might be revealed by specific binding of PPAR
to
palmitoyl-CoA covalently coupled to agarose beads. Since the covalent
bond between the palmitoyl-CoA and the agarose beads joins the amino
group of the CoA moiety with the agarose matrix, this approach,
furthermore, circumvented the inherent problem associated with assays
involving acyl-CoA esters, namely the hydrolysis of the labile
thioester bond. This is particularly important in the context of
competition assays to determine ligand binding to PPARs, since
hydrolysis of the acyl-CoA would generate a fatty acid, which would act
as a regular agonist and thereby give false Kd
values for the binding of acyl-CoA esters to the PPARs. In contrast, in
a bead-based pull-down assay, hydrolysis of the covalently coupled
palmitoyl-CoA would release the free fatty acid, and hence, if PPAR
only interacted with the free fatty acid, no specific retention of
PPAR
would be observed.
and RXR
were expressed in yeast, and
whole cell extracts were prepared as described (37). The extracts were
incubated with palmitoyl-CoA agarose beads or protein A-agarose beads
(control) in the absence or presence of PPAR-selective or RXR-selective
ligands. After washing, bound material was recovered by boiling in SDS
sample buffer, and PPAR
and RXR
were detected by Western
blotting. Fig. 2A shows that PPAR
preferentially was retained on the palmitoyl-CoA-agarose beads
compared with the protein A-agarose beads. The addition of the
PPAR
-selective ligand Wy14643 prevented PPAR
binding to the
palmitoyl-CoA beads in a dose-dependent manner, whereas the
addition of the PPAR
-selective ligand BRL49653 was without effect on
PPAR
binding. In contrast, no specific interaction between RXR
and the palmitoyl-CoA agarose beads was detected irrespective of the presence of PPAR- or RXR-selective ligands (Fig.
2B). We conclude that the established PPAR
ligand Wy14643 and palmitoyl-CoA compete for binding to PPAR
.
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Fig. 2.
Wy14643 and acyl-CoA compete for direct
binding to PPAR . Immunoblot analysis of
proteins retained on palmitoyl-CoA- or protein A-coupled agarose beads
incubated with whole cell extracts of yeast expressing either mPPAR
and rRXR
(A) or rRXR
alone (B). Bound
proteins were recovered by boiling in SDS sample buffer and analyzed by
immunoblotting. For competition, the PPAR
-selective ligand, Wy14643,
the PPAR
-selective ligand, BRL49653, or the RXR ligand,
9-cis-retinoic acid, was added to the binding reactions as
indicated.
to
Chymotrypsin--
Differential protease sensitivity assays have been
widely used to examine the effect of ligand binding on receptor
conformation. We applied this technique to compare the effects of a
known PPAR
ligand, linoleic acid, and S-hexadecyl-CoA on
PPAR
conformation. 35S-Labeled PPAR
or RXR
was
incubated with S-hexadecyl-CoA, linoleic acid, or vehicle
(0.5% (v/v) Me2SO), digested with increasing concentrations of chymotrypsin for 20 min at 25 °C, and the
digestion products were analyzed by SDS-PAGE. Binding of synthetic
agonists to PPAR
has been shown to decrease the sensitivity to
chymotrypsin digestion, resulting in preservation of protease-resistant
fragments (35). In contrast, we found that S-hexadecyl-CoA
in a dose-dependent manner increased the sensitivity of
PPAR
to chymotrypsin, as indicated by the rapid disappearance of the
diagnostic 26-kDa protease-resistant fragment (Fig.
3A). In comparison, incubation with 15 µM S-hexadecyl-CoA did not influence
the sensitivity of RXR
to chymotrypsin, indicating that the effect
of S-hexadecyl-CoA was receptor-dependent (Fig.
3B). To further corroborate the notion that the effects of
S-hexadecyl-CoA on PPAR
conformation were reversible and
did not result from an irreversible denaturing action, the following
experiment was performed. The 35S-labeled PPAR
was
incubated for 20 min with either 10 µM
S-hexadecyl-CoA or water, and then each mixture received
either 120 µM linoleic acid or vehicle (0.5%
Me2SO). Incubation was continued for 20 min, and each of
the four incubations was subjected to digestion with increasing amounts
of chymotrypsin. Fig. 4 shows that
incubation with linoleic acid as expected decreased the sensitivity of
PPAR
to chymotrypsin, whereas incubation with
S-hexadecyl-CoA increased the sensitivity to chymotrypsin.
It is noteworthy that the addition of linoleic acid to PPAR
preincubated with S-hexadecyl-CoA partially restored
resistance to chymotrypsin digestion. Thus, S-hexadecyl-CoA interacted reversibly with PPAR
and appeared to compete with the
agonist linoleic acid for binding to PPAR
. It has previously been
shown that deletion of the putative helices H10-12 of the C-terminal
region in the PPAR
ligand-binding domain abolishes agonist-induced protease protection and transactivation (35). To
examine whether the presence of this region was required for S-hexadecyl-CoA-induced protease sensitivity, the truncated
forms mPPAR
AB and mPPAR
AB/
425 (35) were digested in the
presence of either linoleic acid or S-hexadecyl-CoA.
mPPAR
AB contains the entire ligand-binding domain, whereas the
putative helices 10-12 are deleted in mPPAR
AB/
425 (35). As
shown in Fig. 5, in the presence of
linoleic acid, digestion of mPPAR
AB was decreased, whereas
S-hexadecyl-CoA increased the sensitivity to chymotrypsin. However, neither linoleic acid nor S-hexadecyl-CoA affected
the sensitivity of mPPAR
AB/
425 (Fig. 5). Thus, it
appears that helices 10-12 are required for interaction of
S-hexadecyl-CoA with PPAR
as well as for interaction of
linoleic acid with PPAR
.
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Fig. 3.
S-Hexadecyl-CoA increases the
sensitivity of PPAR to chymotrypsin
digestion. In vitro transcribed/translated
35S-labeled mPPAR
(A) or mRXR
(B) was incubated with increasing concentrations of
S-hexadecyl-CoA or vehicle (water) as indicated for 20 min.
followed by digestion with increasing concentrations of chymotrypsin
for 20 min at 25 °C. The digests were analyzed by SDS-PAGE followed
by PhosphorImaging.
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Fig. 4.
Opposing effects of fatty acid and acyl-CoA
on the sensitivity of PPAR to chymotrypsin
digestion. In vitro transcribed/translated
35S-labeled mPPAR
was preincubated with
either S-hexadecyl-CoA (10 µM) or vehicle for
20 min. Then each preincubation mixture was further incubated for 20 min with either linoleic acid (120 µM) or vehicle. The
mixtures were subjected to limited digestion with chymotrypsin and the
digests were analyzed by SDS-PAGE followed by PhosphorImaging.
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Fig. 5.
The PPAR C-terminal
region is required for both agonist- and acyl-CoA-dependent
alteration of the sensitivity to chymotrypsin digestion. In
vitro transcribed/translated 35S-labeled mPPAR
truncation mutants mPPAR
AB and mPPAR
AB
425 were incubated
in the presence of vehicle, 120 µM linoleic, acid or 10 µM S-hexadecyl-CoA for 20 min and then
subjected to limited digestion with increasing concentrations of
chymotrypsin. The digests were analyzed by SDS-PAGE followed by
PhosphorImaging.
but Enhances Interaction of NCoR with PPAR
--
Nuclear
receptor-mediated transactivation is controlled by a complex interplay
between co-activators and co-repressors. Agonist binding enhances
recruitment of co-activators, whereas the hallmarks of antagonists are
decreased or abolished interaction with co-activators coupled with
induced or enhanced interaction with co-repressors (44-50). The
experiments presented above indicate that S-hexadecyl-CoA has the characteristics of a PPAR
antagonist. To further address this possibility, the effects of S-hexadecyl-CoA on the
interaction between PPAR
and the co-activator SRC-1 or the
co-repressor NCoR were determined using GST pull-down assays. Fig.
6A demonstrates that
S-hexadecyl-CoA abolished agonist-induced recruitment of SRC-1 to PPAR
in a dose-dependent manner and even
decreased interaction below that observed with the unliganded receptor.
In contrast, S-hexadecyl-CoA enhanced almost 3-fold the
interaction between NCoR and PPAR
. Thus, by these
criteria, S-hexadecyl-CoA behaves like a PPAR
antagonist.
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Fig. 6.
S-Hexadecyl-CoA antagonizes
ligand-induced interaction between PPAR
and SRC-1, but enhances
PPAR
-NCoR interaction. A,
bacterially expressed GST-PPAR
(ligand binding domain)
immobilized on glutathione-Sepharose was incubated with in
vitro transcribed/translated 35S-labeled SRC-1 in the
presence of vehicle, Wy14643, or S-hexadecyl-CoA.
B, bacterially expressed GST-mNCoR (amino acids 2239-2453)
immobilized on glutathione-Sepharose was incubated with in
vitro transcribed/translated 35S-labeled mPPAR
in the presence of vehicle, Wy14643, or S-hexadecyl-CoA.
Bound proteins were recovered by boiling in SDS sample buffer,
separated by SDS-PAGE, and quantified by PhosphorImaging.
Even When
Complexed to Its Natural Carrier Protein, the Acyl-CoA-binding
Protein--
In the cell, the concentrations of free acyl-CoA esters
and fatty acids are kept in the low or medium nanomolar range by the buffering action of the acyl-CoA-binding protein (ACBP) and fatty acid-binding proteins, respectively (for a review, see Ref. 51). Thus,
in order to evaluate the possible biological significance of acyl-CoA
esters or for that matter fatty acids in PPAR-mediated signaling, it is
imperative to establish assay conditions that mimic or at least
approach in vivo conditions. These requirements have only
been met partially in one report analyzing the interaction between
fatty acids and PPAR
using a fluorescence-based assay (52). During
our initial EMSA experiments using saturating or nearly saturating
amounts of in vitro translated PPAR
and RXR
, we noted
that the mobility of the PPAR
·RXR
·ACO PPRE complex in the
presence of S-hexadecyl-CoA or TTA-CoA was marginally
increased (data not shown). Subsequently, we discovered that this
effect was much more pronounced in whole cell extracts of Sf21
insect cells expressing recombinant rat PPAR
, suggesting that this
might form the basis for a highly sensitive assay for
S-hexadecyl-CoA interaction with PPAR
. No binding was
observed in extract prepared from mock-infected cells (Fig.
7A). The increase in
electrophoretic mobility was not observed using millimolar
concentrations of the detergent lauryl-sarcosine, underscoring the
specific action of S-hexadecyl-CoA (results not shown).
Sf21 cells contain an RXR analog, in this work referred to as
Sf "RXR" (related to ultraspiracle (NR2B4 (2)) in
Drosophila (53)), with which PPAR
is able to
heterodimerize and subsequently bind to the ACO PPRE (4, 54).
Accordingly, combining in vitro translated PPAR with extract of mock-infected cells allowed efficient binding to the ACO PPRE in
EMSA experiments (results not shown). Using this assay, we next asked
whether a preformed complex between S-hexadecyl-CoA and ACBP
was able to modulate the mobility of the rPPAR
·Sf"RXR"·ACO PPRE complex. Fig. 7B demonstrates that even in the presence
of a molar surplus of ACBP, S-hexadecyl-CoA was able to
increase the mobility of the rPPAR
·Sf"RXR"·ACO PPRE complex.
By combining EMSA with Western blotting (42) we demonstrated that the
shifted band indeed contained PPAR
(Fig. 7C).
Using the established Kd for binding of long-chain
acyl-CoA to ACBP, the concentration of free S-hexadecyl-CoA
in a solution containing 10 µM
S-hexadecyl-CoA and 15 µM ACBP was
calculated as 0.2 nM. Thus, using conditions that mimic the
in vivo conditions with respect to acyl-CoA availability, S-hexadecyl-CoA imparted an increase in the electrophoretic mobility of the rPPAR
·Sf"RXR"·ACO PPRE complex.
View larger version (17K):
[in a new window]
Fig. 7.
The electrophoretic mobility of the
rPPAR ·Sf"RXR"·PPRE complex is
increased by S-hexadecyl-CoA as well as by
S-hexadecyl-CoA complexed with ACBP. Whole cell
extracts of Sf21 cells infected with an expression vector
without (Sf21) or with (Sf21(PPAR
)) the rPPAR
cDNA were incubated with a 32P-labeled oligonucleotide
containing the PPRE of the rat ACO promoter in the presence of vehicle
or S-hexadecyl-CoA (A). B,
S-hexadecyl-CoA was added as a preformed complex with ACBP
as indicated. C, combined EMSA and Western blotting
revealing the shifted position of PPAR
in the presence of
S-hexadecyl-CoA.
to interact with
co-activators and co-repressors. Formally, it was therefore possible
that the increased electrophoretic mobility reflected an altered
molecular mass of the rPPAR
·Sf"RXR"·ACO PPRE complex.
However, a Ferguson analysis (55) revealed that the presence of 10 µM S-hexadecyl-CoA, 10 µM ACBP,
or a 10 µM ACBP plus 10 µM
S-hexadecyl-CoA complex did not alter the molecular mass
of the rPPAR
·Sf"RXR"·ACO PPRE complex (results not shown).
Thus, the increased electrophoretic mobility of the
rPPAR
·Sf"RXR"·ACO PPRE complex in the presence of
S-hexadecyl-CoA is not due to an altered molecular mass of the bound heterodimer but rather reflects an altered conformation or
change in charge of the heterodimer.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
. Using
a variety of in vitro approaches, we show that the nonhydrolyzable acyl-CoA analogue, S-hexadecyl-CoA,
antagonizes ligand-induced formation of a PPAR
·RXR
·ACO
PPRE complex. We were able to demonstrate specific binding of PPAR
to immobilized palmitoyl-CoA, and furthermore, we show that
S-hexadecyl-CoA increases the sensitivity of PPAR
to
chymotrypsin digestion in a manner that depended on the integrity of
the ligand-binding pocket. We show that S-hexadecyl-CoA,
like well established antagonists for other receptors, abolishes
ligand-induced interaction with a co-activator, SRC-1, and conversely
increases recruitment of a co-repressor, NCoR. Importantly, we show
that S-hexadecyl-CoA is able to affect a PPAR
-containing
complex in the presence of a molar excess of the natural cellular
acyl-CoA carrier, ACBP. These observations, taken together with our
recent finding that ACBP and acyl-CoA esters are present in the nuclei
of rat hepatocytes (56), are compatible with the notion that acyl-CoA
esters also in vivo might be involved in the regulation of
PPAR
activity. Our results are furthermore supported by
recent data showing interaction between acyl-CoA esters and PPAR
and
PPAR
in competition binding experiments with the labeled synthetic
dual agonist, KRP-297 (57).
, raising the question of whether acyl-CoA esters might also influence the PPAR
·RXR
heterodimer via
RXR
. However, as mentioned above, the size of the
ligand-binding pocket of RXR
is not compatible with specific binding
of acyl-CoA esters, and accordingly, we detected no alteration in the
sensitivity to chymotrypsin digestion when RXR
was incubated with
S-hexadecyl-CoA, and similarly, we observed no binding of
RXR
to palmitoyl-CoA.
(13-16, 59).
Ligand-dependent positioning of the AF-2 helix and
differences in the regions flanking the LXXLL and
CoRNR motifs are critically involved in the differential interaction of co-activators and co-repressors with liganded and unliganded nuclear receptors, respectively (13, 15, 59). Interestingly,
the crystal structure of PPAR
shows that the AF-2 helix even in the
unliganded receptor may fold back against the body of the receptor,
assuming a conformation similar to the conformation stabilized by
interactions between the polar head group of ligands and the AF-2 helix
(16, 18, 60), and as a consequence, interaction with co-activators and
co-repressors may be less stringently regulated by ligands in the PPAR
subfamily in comparison with other nuclear receptor subfamilies.
to chymotrypsin digestion upon binding of S-hexadecyl-CoA is also indicative of a less compact conformation.
and PPAR
(16, 17)
led to the suggestion that ligands might enter the ligand-binding pocket via a channel between helix 3 and the
-sheet. In addition, the crystal structure of liganded PPAR
and PPAR
revealed
prominent interactions between the polar head group of the different
agonists and the AF-2 helix (16-18). In contrast, co-crystallization
of the partial agonist GW0072 with the ligand-binding domain of PPAR
revealed a mode of binding in which the carboxylic group of GW0072 was
oriented toward the loop region between helices 2' and 3 with no
contacts to the AF-2 helix (61). In this context, it is intriguing that
we observe specific binding of PPAR
to palmitoyl-CoA immobilized via
the CoA head group. If the palmitoyl-CoA entered the ligand-binding pocket via the channel between helix 3 and the
-sheet, this suggests that the orientation of palmitoyl-CoA mimicked that of GW0072. Alternatively, positioning of the palmitoyl-CoA molecule with the acyl
chain in the characteristic tail-down configuration would imply that
the acyl-CoA ligand entered the ligand-binding pocket via the AF-2
side. Interaction of PPAR
with immobilized PPAR
agonists would
clearly be of interest to examine this possibility.
through a shared DNA binding motif
(62-64). It is well established that PPAR
is activated by polyunsaturated fatty acids (23, 24), and recently myristoyl-CoA and
palmitoyl-CoA were reported to bind to HNF-4
and activate HNF-4
-mediated transactivation, whereas
-3 and
-6 polyunsaturated acyl-CoA esters and stearoyl-CoA were
shown to antagonize HNF-4
-mediated transactivation (65). Based on
this finding, it was proposed that the ratio of fatty acids to acyl-CoA
esters and the composition of acyl-CoA esters might regulate cross-talk
between PPAR
and HNF-4
(65). However, it should be noted that
recent data based on molecular modeling of HNF-4
and protease
protection experiments have questioned the role of acyl-CoA esters in
the regulation of HNF-4
activity (58). Thus, it remains to be
established conclusively whether HNF-4
is a target for
acyl-CoA-dependent regulation. If so, our findings add
another level to the interplay between PPAR
and HNF-4
, indicating
that acyl-CoA esters, apart from activating HNF-4
, down-regulate
PPAR
-mediated transactivation via direct binding to PPAR
, thereby
imparting a conformation that reduces co-activator interaction and
enhances recruitment of co-repressors (Fig.
8).
View larger version (26K):
[in a new window]
Fig. 8.
Model of the opposing effects of
transcriptional agonists and acyl-CoA esters on
PPAR . A, ligands and acyl-CoA
compete for binding to PPAR
, resulting in
acyl-CoA-dependent suppression of the ligand-induced
formation of a PPAR
(P)·RXR (R)·PPRE
complex. This phenomenon is observed when the concentrations of PPAR
and RXR
are limiting. B, when the concentrations of
PPAR
and RXR
are high, formation of the PPAR
·RXR
·PPRE
complex is unaffected by ligands. The transcriptional activity of the
DNA-bound PPAR
·RXR
complex is determined by the recruitment of
cofactors. Acyl-CoA antagonizes the ligand-enhanced recruitment of the
co-activator SRC-1 but imparts an increased affinity for the
co-repressor NCoR. Arrows in gray denote DR-1
half-sites with the consensus sequence AGGTCA.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Drs. J. D. Tugwood, B. W. O'Malley, M. A. Lazar, and Dr. M. Leid for the generous gift of plasmids and Novo Nordisk A/S for supplying ligands.
![]() |
FOOTNOTES |
---|
* This work was supported in part by grants from the Danish Biotechnology Program, the Danish Natural Science Research Council, and the Novo Nordisk Foundation.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.
Supported by a grant from the Nordic Research Academy. Present
address: Dept. of Biochemistry and Molecular Biology, University of
Bergen, N-5009 Bergen, Norway.
§ These authors contributed equally to this work.
** Present address: Institut für Genetik, Kernforschungszentrum, D-7602 Karlsruhe, Germany.
¶¶ To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5320 Odense M., Denmark. Tel.: 45 6550 2408; Fax: 45 6550 2467; E-mail: kak@bmb.sdu.dk.
Published, JBC Papers in Press, February 20, 2001, DOI 10.1074/jbc.M101073200
1
The abbreviations and trivial names used
are: PPAR, peroxisome proliferator-activated receptor; ACBP,
acyl-CoA-binding protein; ACO, acyl-CoA oxidase; BRL49653,
(±)-5-([4-[2-methyl-2-(pyridylamino)-ethoxy]phenyl]methyl) 2,4-thiazolidinedione; DR-1, direct repeat separated by one nucleotide; EMSA, electrophoretic mobility shift assay; GST, glutathione
S-transferase; HNF-4, hepatocyte nuclear factor-4
;
NCoR, nuclear receptor co-repressor; PAGE, polyacrylamide gel
electrophoresis; mPPAR, mouse peroxisome proliferator-activated
receptor; rPPAR, rat peroxisome proliferator-activated receptor; RXR,
retinoid X receptor; rRXR, rat RXR; PAGE, polyacrylamide gel
electrophoresis; SRC-1, steroid receptor co-activator-1; TTA, tetradecylthioacetic acid; Wy14643,
4-chloro-6-(2,3-xylidino)-2-pyrimidinylthioacetic acid; PPRE,
peroxisome proliferator-responsive element; Tricine, N-[2-hydroxy- 1,1-bis(hydroxymethyl)ethyl]glycine.
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