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Originally published In Press as doi:10.1074/jbc.M500901200 on February 4, 2005

J. Biol. Chem., Vol. 280, Issue 14, 14145-14153, April 8, 2005
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{alpha},{beta}-Unsaturated Ketone Is a Core Moiety of Natural Ligands for Covalent Binding to Peroxisome Proliferator-activated Receptor {gamma}*

Takuma Shiraki{ddagger}§, Narutoshi Kamiya§, Sayaka Shiki{ddagger}, Takashi S. Kodama||**, Akira Kakizuka{ddagger}{ddagger}, and Hisato Jingami{ddagger}§§

From the Departments of {ddagger}Molecular Biology, Computational Biology, and ||Structural Biology, Biomolecular Engineering Research Institute, 6-2-3 Furuedai, Suita-City, Osaka 565-0874, **Japan Biological Information Research Institute Center, Japan Biological Informatics Consortium, 2-41-6 Aomi, Koto-Ku, Tokyo 135-0064, {ddagger}{ddagger}Laboratory of Functional Biology, Graduate School of Biostudies, Kyoto University, Sakyo-Ku, Kyoto 606-8501, and Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Corporation, Kawaguchi 332-0012, Japan

Received for publication, January 25, 2005 , and in revised form, February 4, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) functions in various biological processes, including macrophage and adipocyte differentiation. Several natural lipid metabolites have been shown to activate PPAR{gamma}. Here, we report that some PPAR{gamma} ligands, including 15-deoxy-{Delta}12,14-prostaglandin J2, covalently bind to a cysteine residue in the PPAR{gamma} ligand binding pocket through a Michael addition reaction by an {alpha},{beta}-unsaturated ketone. Using rhodamine-maleimide as well as mass spectroscopy, we showed that the binding of these ligands is covalent and irreversible. Consistently, mutation at the cysteine residue abolished abilities of these ligands to activate PPAR{gamma}, but not of BRL49653 a non-covalent synthetic agonist, indicating that covalent binding of the {alpha},{beta}-unsaturated ketone in the natural ligands was required for their transcriptional activities. Screening of lipid metabolites containing the {alpha},{beta}-unsaturated ketone revealed that several other oxidized metabolites of hydroxyeicosatetraenoic acid, hydroxyeicosadecaenoic acid, and prostaglandins can also function as novel covalent ligands for PPAR{gamma}. We propose that PPAR{gamma} senses oxidation of fatty acids by recognizing such an {alpha},{beta}-unsaturated ketone as a common moiety.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Peroxisome proliferator-activated receptor {gamma} (PPAR{gamma})1 plays important roles for lipid homeostasis, glucose metabolism, and macrophage functions (1–3). The discovery that thiazolidinedione, an anti-diabetic drug, is a potent and selective ligand for PPAR{gamma} (4) accelerated the search for natural PPAR{gamma} ligands. Several lipid metabolites, including phospholipids, polyunsaturated fatty acids, and oxidized fatty acids, have been shown to activate PPAR{gamma} (5–8).

Many structural studies of the PPAR{gamma} ligand-binding domain (LBD) with or without synthetic ligands (9–14) have contributed to better understanding of the binding selectivity and activation mechanism of the synthetic PPAR{gamma} ligands. In the PPAR{gamma} ligand binding pocket, a large hydrophobic region makes contact with ligands, and a hydrogen bond network between PPAR{gamma} and ligands stabilizes helix 12, which may promote binding of coactivator proteins (2). However, there is no structural report for the binding mode of the natural PPAR{gamma} ligands. In the case of PPAR{delta}, its crystal structure complexed with a fatty acid has been reported (15). The ligand, eicosapentaenoic acid, was attached to its binding pocket in two distinct conformations in the crystal. Meanwhile, it has been reported that a natural PPAR{gamma} ligand, 15-deoxy-{Delta}12,14-prostaglandin J2 (15d-PGJ2), has the potential to bind covalently to some proteins, such as H-Ras (16), NF-{kappa}B (17, 18), I{kappa}B kinase (19), and AP-1 (20) via a cysteine residue. In addition, it is reported that the synthetic PPAR{gamma} ligands GW9662, T0070907, and L-764406 (21–23) have been shown to react with the cysteine residue of the PPAR{gamma} LBD. These lines of evidence led to us investigate the binding mode of 15d-PGJ2 to the PPAR{gamma} LBD.

While we built a model structure of 15d-PGJ2-PPAR{gamma} complex, we became aware of the possibility that 15d-PGJ2 covalently binds to the PPAR{gamma} LBD. Here, we have shown that an {alpha},{beta}-unsaturated ketone is a core moiety of endogenous PPAR{gamma} ligands. The ligands containing the {alpha},{beta}-unsaturated ketone can covalently bind to a cysteine residue in PPAR{gamma} LBD through a Michael addition, and the covalent binding is required for PPAR{gamma} activation by these ligands. According to this core moiety, we have identified oxidized eicosanoids, including 5-oxo-eicosatetraenoic acid (ETE), 15-oxoETE, 15-oxo-eicosadecaenoic acid (EDE), 15-keto-prostaglandin E2, 15-keto-prostaglandin E1 (15-keto-PGE1), 15-keto-prostaglandin F2{alpha} (15-keto-PGF2), and 15-keto-prostaglandin F1{alpha} (15-keto-PGF1), as novel endogenous PPAR{gamma} ligands. We analyzed the structure-activity relationship of these oxidized prostaglandins in PPAR{gamma} activation. We also discuss the biological connection between the ligand synthesis and PPAR{gamma} function.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals—PPAR{gamma} ligands and eicosanoids were purchased from Cayman Chemical Co. or Alexis Corporation (Lausen, Switzerland). Rhodamine-maleimide was purchased from Molecular Probes, Inc. Reagents for animal cell culture were obtained from Invitrogen. All other chemicals were purchased from Sigma or WAKO (Osaka, Japan).

Plasmids—For bacterial expression of the PPAR{gamma} LBD, a PPAR{gamma} fragment containing amino acid residues 195–477 was amplified by PCR using oligonucleotides (5'-GA CAT ATG GCG GAG ATC TCC AGT and 5'-G CGG ATC CTA GTA CAA GTC CTT G; underlines indicate introduced NdeI and BamHI sites, respectively) and subcloned into NdeI/BamHI-digested pET28a (Invitrogen). After plasmid DNA was recovered from a colony, the sequence was confirmed.

To obtain the C285A mutant, pCMX-GAL4-hPPAR{gamma}1 was amplified by Pyrobest DNA polymerase (TAKARA Bio. Inc.) using oligonucleotides (5'-ATC TTT CAG GGC GCT CAG TTT CGC TCC and 5'-GGA GCG AAA CTG AGC GCC CTG AAA GAT; underlines indicate mutated codon). The template DNA was removed by digestion with DpnI, and the amplified DNA was directly transformed into Escherichia coli DH5{alpha}. After plasmid DNA was recovered from a colony, the sequence was confirmed. The Y475F mutant was obtained by the same procedure using oligonucleotides (5'-CTG CAG GAG ATC TTC AAG GAC TTG TAC and 5'-GTA CAA GTC CTT GAA GAT CTC CTG CAG; underlines indicate mutated codon).

Protein Purification—The human PPAR{gamma} LBD was expressed in BL21 (DE3) and purified by nickel affinity chromatography as described previously (14). After removal of a polyhistidine tag by thrombin digestion, the PPAR{gamma} LBD was further purified by gel filtration using Superdex200 (Amersham Biosciences). We omitted the reducing agents from all the buffers.

TOF Mass Spectrometry Analysis—After the PPAR{gamma} LBD protein was incubated with the ligands, the protein solution was mixed with 33% acetonitolyl saturated with sinopinic acid at a 1:1 ratio. Then, 2 µl of the sample was spotted onto the grid and dried completely under vacuum. A MALDI-TOF mass spectrometry spectrum was obtained with a Voyager Elite (PerSeptive Biosystems, Framingham, MA).

Rhodamine-Maleimide Assay—The purified PPAR{gamma} LBD (0.1 µM) was mixed with various ligands at the indicated concentrations in a buffer containing 20 mM Tris-HCl, pH 7.4, 150 mM NaCl. The mixture was incubated at room temperature for 30 min. After SDS was added to the reaction mixture to a final concentration of 0.5%, rhodamine-maleimide and tris-(2-carboxyethyl)phosphine hydrochloride were added to the mixture to 0.2 and 1 mM final concentrations, respectively. After incubation at room temperature for 30 min, the samples were treated with SDS-PAGE sample buffer containing {beta}-mercaptoethanol and were separated by SDS-PAGE. Fluorescent signals derived from rhodamine were visualized by a FM-BIO II (Hitachi).

Calculation of Binding Constant—We calculated the kon values of irreversible binding of ligands as follows.


Here, NR and NR-ligand indicate a nuclear receptor and a ligand-conjugated nuclear receptor, respectively. The reaction speed at certain time (t) is shown as Equation 1.

(Eq. 1)
If a is not equal to b, Equation 1 can be converted to Equation 2.

(Eq. 2)
If a is equal to b, Equation 1 can be converted to Equation 3.

(Eq. 3)
We obtained the kon value after nonlinear least square fitting of the data using Equation 2 and Equation 3 by the Newton method.

We calculated the Kd value of competitors as follows.


Here, NR:competitor indicates a reversible complex between a competitor and a nuclear receptor. The reaction equilibrium constant is shown as Equation 4.

(Eq. 4)
The reaction speed of the covalent binding at certain time (t) is shown as Equation 5.

(Eq. 5)
We obtained the Ka value after nonlinear square fitting of the data with the values generated by a differential equation modified from Equations 4 and 5. Kd was obtained as 1/Ka.

Measurement of UV Spectra—Samples were mixed at indicated concentrations and incubated for 20 min at room temperature. UV spectra between 250 and 400 nm were recorded using a Beckman DU640 spectrophotometer. For graphical presentation, UV spectrum of PPAR{gamma} alone was subtracted from that of the ligand-PPAR{gamma} mixture.

Cell Culture and Luciferase Assay—Cell culture, transient transfection, and luciferase assay were described previously (24). In this study, we measured the fluorescence of enhanced yellow fluorescent protein as an internal control of transfection efficiency, using a Fusion {alpha} (PerkinElmer, Inc.).

Model Building—The 15d-PGJ2-PPAR{gamma} covalently bound complex was built using the crystal structure of the PPAR{gamma}-BRL49653-coactivator peptide complex (Protein Data Bank accession code 1FM6 [PDB] ) (12) as the template. The force field and programs used for the potential-energy calculations of the PPAR{gamma} and the coactivator peptide were described in a previous report (25). One of the conformations of 15d-PGJ2, which was randomly generated with a molecular dynamics simulation at 400 K in vacuo, was introduced into the ligand binding pocket of PPAR{gamma} by visual manipulation. Here, the carboxyl group and the cyclopentenone ring in 15d-PGJ2 were placed in the vicinity of the BRL49653head group and the sulfur atom in Cys-285, respectively. All of the Asp, Glu, Arg, Lys, and His residues and the carboxyl group in 15d-PGJ2 were treated as the charged forms. Force field parameters for 15d-PGJ2 covalently bound to Cys-285 were taken from those of the analogous fragments in the AMBER general force field (www.amber.scripps.edu/) for the organic molecule data base. Its partial charges were obtained from charge fitting with the RESP program (26) (www.amber.scripps.edu/Questions/resp.html) after geometry optimization with the Gaussian 98 program (27) using the Hartree-Fock/6–31G* basis set. All of the energy minimization and molecular dynamics calculations were carried out in vacuo. The potential energy of the complex was minimized by using the conjugated gradient method via the following steps. First, the geometry of 15d-PGJ2-conjugated Cys-285 in PPAR{gamma} was allowed to be flexible while the position of the other part was restrained with a harmonic potential, and then all of the complex was allowed to be flexible without restraint. After the energy minimization procedures, a 100-ps heating from 0 to 300 K and a 500-ps equilibration were done. Subsequently, a 500-ps sampling simulation was executed. Here, all of the main-chain atoms of PPAR{gamma} and the coactivator peptide were restrained by applying a harmonic potential to the minimized conformation, and the time step of the molecular dynamics simulation was 1 fs. Finally, the closest structure to the average of the sampled ones was chosen as the complex model.

Reverse Transcription-PCR—THP-1 cells were maintained in RPMI1640 with 10% fetal calf serum at 37 °C. 2 x 106 cells were seeded in 6-cm dishes and treated with ligands for 10 h. Cells were collected by centrifugation, and total RNA was purified from cells by TRIzol reagent (Invitrogen) according to the manufacturer's instructions. Quality of isolated RNA was determined by electrophoresis. Single-stranded cDNA was synthesized from 1 µg of total RNA using reverse transcriptase (Promega) with an oligo(dT)15 primer. PCR primers were designed according to the uniSTS data base; detail is available upon request. PCR products were separated by PAGE and stained with SYBR Gold (Molecular Probes). Images of the gels were obtained by the FM-BIO II (Hitachi).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Model Building of the 15d-PGJ2-PPAR{gamma} Complex—As the first step toward understanding the binding mechanism of the natural PPAR{gamma} ligands, we built a model structure of the 15d-PGJ2-PPAR{gamma} complex according to the superimposed structures of the PPAR{gamma} LBD complexed with several synthetic ligands (Protein Data Bank accession codes 1KNU [PDB] , 1FM6, 1I7I, 4PRG). In the superimposed structures, the polar part of the ligands tended to localize near helix 12 of PPAR{gamma} and the non-polar part near helix 3 (Fig. 1A). To fit 15d-PGJ2 to the binding site of BRL49653 we considered four possible orientations of 15d-PGJ2 in the PPAR{gamma} ligand binding pocket (Fig. 1B). In the models, the cyclopentenone ring (Fig. 1B, a and b) or the carboxyl group (Fig. 1B, c and d) of 15d-PGJ2 could be assigned as a polar moiety. In the latter case, we noticed that a cysteine residue (Cys-285) of PPAR{gamma} located at close proximity to the electrophilic carbon(s) of an {alpha},{beta}-unsaturated ketone in 15d-PGJ2 (Fig. 1B, c and d). The PPAR{gamma} ligand binding pocket was large enough to accommodate 15d-PGJ2 (Fig. 1C).



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  		FIG. 1.
Orientation of ligands in PPAR{gamma} LBD. A, structure of the PPAR{gamma} LBD with BRL49653(Protein Data Bank accession code 1FM6 [PDB] , chain D). Overall structure of the PPAR{gamma} LBD with BRL49653showed that the ligand fit its polar part (red) near helix 12 and its non-polar part (gray) near helix 3 (left panel). Close-up view of the ligand binding cavity with BRL49653(from 1FM6) and YPA (from 1KNU) showed the similarity of the binding mode (right panel). The ligand binding cavity is shown as mesh generated from the data of 1FM6. B, possible orientation of 15d-PGJ2 in the PPAR{gamma} ligand binding pocket. Either the cyclopentenone ring (a and b) or the carboxyl group (c and d) of 15d-PGJ2 was positioned at the polar region of the PPAR{gamma} ligand binding pocket shown in panel A, right. In each case, another two alternative orientations were possible. When the carboxyl group of 15d-PGJ2 was positioned at the polar region of the PPAR{gamma} ligand binding pocket (c and d), {beta}-carbon (blue) of {alpha},{beta}-unsaturated ketone was near the cysteine residue (Cys-285, shown in green) of PPAR{gamma}. C, molecular surface of the PPAR{gamma} LBD and the ligand binding pocket with 15d-PGJ2 in the same orientation as in panel A. A cutaway rendition of the surface is shown on the left. Close-up view of the ligand binding cavity with 15d-PGJ2 is shown on the right. The position of Cys-285 is indicated as a green tube model.

 
Covalent Binding of 15d-PGJ2 to PPAR{gamma} LBD—The model building described above raised the possibility that 15d-PGJ2 is bound to PPAR{gamma} covalently. To test this possibility, we performed a mass spectroscopy experiment. Generally, macromolecules such as proteins are denatured during MALDI-TOF mass spectroscopy analyses, and so small molecules, such as ligands, are detached from the proteins. Actually, with or without the synthetic ligand BRL49653 the PPAR{gamma} LBD showed the same molecular weight as that calculated from the amino acid composition (MW = 34, 534) (Fig. 2A). In contrast, in the presence of 15d-PGJ2, the PPAR{gamma} LBD exhibited a higher molecular weight, which was close to the molecular weight of the PPAR{gamma} LBD plus 15d-PGJ2 (calculated MW = 34, 850) (Fig. 2A). This result indicated that 15d-PGJ2 was bound to PPAR{gamma} even after the PPAR{gamma} was denatured and suggested that 15d-PGJ2 covalently bound to PPAR{gamma}.



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  		FIG. 2.
Covalent binding of 15d-PGJ2 to the PPAR{gamma} LBD. A, mass spectroscopy analysis of PPAR{gamma} LBD with or without ligands. After incubating the PPAR{gamma} LBD with either Me2SO, BRL49653 or 15d-PGJ2, the molecular weight of the ligand-PPAR{gamma} complex was investigated by MALDI-TOF mass spectroscopy. B, schematic representation of the method used to analyze the covalent binding of ligands to the cysteine residue in the PPAR{gamma} LBD. After incubating the PPAR{gamma} LBD with ligands, proteins were denatured by adding SDS. Free cysteine residues were then detected by rhodamine-maleimide. C, concentration-dependent modification of the cysteine residue in the PPAR{gamma} LBD by 15d-PGJ2. D, concentration-dependent competition of 15d-PGJ2 binding to PPAR{gamma} with other ligands. Various concentrations of competitor ligands were simultaneously added to the PPAR{gamma} LBD with 5 µM 15d-PGJ2, and the cysteine modification by 15d-PGJ2 was investigated by rhodamine-maleimide.

 
To confirm that 15d-PGJ2 modifies the cysteine residue in the PPAR{gamma} LBD, we took advantage of rhodamine-maleimide to label free cysteine residues in proteins. Fortunately, there is only one cysteine residue in the PPAR{gamma} LBD. In these experiments, we denatured the protein by adding 0.5% SDS after ligand-protein complex formation, to facilitate rhodamine-maleimide access to the cysteine residue in the PPAR{gamma} LBD (Fig. 2B). The amount of the rhodamine-maleimide-labeled PPAR{gamma} LBD decreased by incubating with 15d-PGJ2 in a concentration-dependent manner (Fig. 2C), indicating that 15d-PGJ2 covalently bound to the cysteine residue of PPAR{gamma}. Based on the irreversible binding reaction model, we calculated kon value of 15d-PGJ2 as 47,165.3 M-1 min-1. We next added the known PPAR{gamma} ligands to the reaction mixture simultaneously with 15d-PGJ2 (Fig. 2D). Excess amount of the competitors can occupy the ligand binding pocket, and so 15d-PGJ2 cannot reach to the cysteine residue. Then addition of 0.5% SDS removes the non-covalently bound competitors, with the result that the cysteine residue in the denatured PPAR{gamma} LBD can be labeled by rhodamine-maleimide. This result indicates that the non-covalent ligands and 15d-PGJ2 share their binding site. Using the kon value of 15d-PGJ2, the Kd values of BRL49653 MCC-555, and 13(S)-HODE were calculated to be 129 nM, 577 nM, and 199 µM, respectively.

Rhodamine-maleimide assay revealed that 15d-PGJ2 specifically modified the cysteine residue of the PPAR{gamma} LBD. Concomitantly, 15d-PGJ2 itself must be modified by binding to the PPAR{gamma} LBD. Then, we measured the UV spectrum of 15d-PGJ2 in the absence or presence of the PPAR{gamma} LBD (Fig. 3A). In the absence of the PPAR{gamma} LBD, 15d-PGJ2 showed peak absorbance at 320 nm (Fig. 3A, a, thin line). After incubating with the PPAR{gamma} LBD, the resultant peak absorbance of 15d-PGJ2 shifted to 295 nm (Fig. 3A, a, thick line). This blue shift of the peak absorbance was also observed even when 15d-PGJ2 was incubated in a free cysteine solution (Fig. 3A, b). However, the resultant peak absorbance was 310 nm, which was different from the PPAR{gamma}-induced peak absorbance but rather similar to that of a 15d-PGJ2 analogue, CAY10410 which lacks the cyclopentenone ring (Fig. 3A, c, thick line). The results suggested that the cysteine residue of the PPAR{gamma} LBD targeted an electrophilic carbon within 15d-PGJ2 that is different from that of free cysteine targets. Considering the similarity of the peak absorbance, the free cysteine may target C9 of 15d-PGJ2 (Fig. 3B), whereas the cysteine residue in the PPAR{gamma} LBD may target C13 of 15d-PGJ2 (Fig. 3B). The amplitude of the UV spectrum of CAY10410was reduced by binding to PPAR{gamma} (Fig. 3A, c), whose peak absorbance was probably shifted to a shorter wavelength than that we had measured. The result indicated that the cysteine residue in the PPAR{gamma} LBD may also target C13 of CAY10410(Fig. 3B) as well as 15d-PGJ2.



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  		FIG. 3.
Change of UV spectrum of 15d-PGJ2 by binding to PPAR{gamma}. A, UV spectra of 15d-PGJ2 (10 µM) before and after incubating with 58 µM PPAR{gamma} indicated the chemical modification in 15d-PGJ2 (a). Blue shift of the peak wavelength of 15d-PGJ2 (10 µM) was also induced by incubating with 2 mM cysteine (b). The resultant peak (thick spectrum) in cysteine-reacted 15d-PGJ2 corresponded to that of a 15d-PGJ2 analogue, CAY10410(c). UV spectra of CAY10410(10 µM) were also altered by incubating with 58 µM PPAR{gamma}. The peak wavelengths of 15d-PGJ2 and CAY10410are indicated as vertical dashed lines. B, explanation of UV spectral rearrangement by the cysteine residue in PPAR{gamma} and the free cysteine. The cysteine residue of the PPAR{gamma} LBD reacts with C13 in 15d-PGJ2 and CAY10410 whereas a free cysteine (Cys) reacts with C9 in 15d-PGJ2.

 
Covalent Binding of Natural Ligands to PPAR{gamma}—PPAR{gamma} is reportedly activated by several natural and synthetic ligands (5–8). For example, Nagy et al. (7) showed that oxidized fatty acids, e.g. 9- and 13-hydroxyoctadecadienoic acid (HODE) and 9- and 13-oxo-octadecadienoic acid (oxoODE) derived from oxidized low density lipoprotein (oxLDL), activated PPAR{gamma} and mediated the cellular effect of oxLDL in macrophages. Then we assessed whether such known PPAR{gamma} ligands possess the ability to bind covalently to PPAR{gamma}. We showed that the molecules (e.g. 15d-PGJ2, CAY10410 9-oxoODE, 13-oxoODE, and T0070907) covalently bound to the cysteine residue in the PPAR{gamma} LBD in the rhodamine-maleimide assay (Fig. 4A). CAY10410also showed weaker blocking activity than 15d-PGJ2. The natural ligands and CAY10410 which covalently bound to the PPAR{gamma} LBD, share the common chemical structure of an {alpha},{beta}-unsaturated ketone (Fig. 4B, boxes). Although 9(S)-HODE and 13(S)-HODE have structures quite similar to those of 9-oxoODE and 13-oxoODE, respectively, they lack an {alpha},{beta}-unsaturated ketone and did not covalently bind to the PPAR{gamma} LBD, indicating that the {alpha},{beta}-unsaturated ketone was a crucial structure for the covalent binding. It is reported that synthetic ligands GW9662, T0070907, and L-764406 covalently bind to the cysteine residue in the PPAR{gamma} LBD (21–23). In this assay, we were able to confirm the cysteine modification by T0070907 (Fig. 4A), which has been shown to react with the cysteine residue of the PPAR{gamma} LBD through a nucleophilic aromatic substitution of the chlorine in the structure (21). Because the cysteine residue of the PPAR{gamma} LBD targets the {beta}-carbon in the {alpha},{beta}-unsaturated ketone, as shown in Fig. 3, the different mode of action was evident between the 15d-PGJ2-represented natural ligands and these synthetic ligands.



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  		FIG. 4.
Naturally occurring ligands covalently bind to the PPAR{gamma} LBD. A, modification of the cysteine residue in the PPAR{gamma} LBD by ligand binding. B, natural ligands that can bind covalently to PPAR{gamma} have a common chemical structure, an {alpha},{beta}-unsaturated ketone (indicated as a box). An electrophilic carbon in the {alpha},{beta}-unsaturated ketone is indicated by an arrow. C, dose-dependent modification of PPAR{gamma} by ligands. The PPAR{gamma} LBD was incubated with various concentrations of 15d-PGJ2 13-oxoODE or 9-oxoODE, and the covalent binding was detected by using rhodamine-maleimide. Quantifications of the band intensities are plotted.

 
Although the binding mode by 15d-PGJ2, 9-oxoODE and 13-oxoODE was achieved by the same chemical reaction, they showed different IC50 to block the rhodamine-maleimide to label the free cysteine residue in the PPAR{gamma} LBD (Fig. 4C). We calculated the kon values of 15d-PGJ2, 13-oxoODE, and 9-oxoODE as 47,165.3, 8,073.7, and 1,535.3 M-1 min-1, respectively.

Requirement of the Covalent Binding for PPAR{gamma} Activation—We evaluated the relationship between the binding modes of the ligands and their activities in a cell-based transcription assay (Fig. 5A). BRL49653was the most potent activator of PPAR{gamma} among the ligands we tested (Fig. 5A, left panel). CAY10410showed transcriptional activity similar to that of 15d-PGJ2 (Fig. 5A, middle panel). On the other hand, when we compared the activities of structurally related molecules, e.g. 13(S)-HODE versus 13-oxoODE and 9(S)-HODE versus 9-oxoODE (Fig. 5A, right panel), the ligands that covalently bound to PPAR{gamma} always showed significantly higher activities than the analogous ligands lacking an {alpha},{beta}-unsaturated ketone. This result is consistent with a previous report that oxo metabolites showed greater activities and affinities than HODE did (7). These results suggested that the covalent binding to the receptor helped the ligands to activate PPAR{gamma}.



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  		FIG. 5.
Requirement of covalent binding for natural ligand-dependent PPAR{gamma} activation. A, dose-dependent activation of transcription by PPAR{gamma} by various ligands in COS-7 cells transfected with the GAL4DBD-PPAR{gamma} LBD, UASG-luciferase, and yellow fluorescent protein. Activation of transcription was detected by luciferase activity, normalized to yellow fluorescent protein fluorescence as an internal control (RLU, relative light unit). B, mutations of the cysteine residue abolish the activity of natural ligands, but not the BRL49653activity. The relative activities compared with that of no ligand in each mutant and the wild type are shown. C, inhibition of 15d-PGJ2- and BRL49653dependent activities by a PPAR{gamma} antagonist. Agonists were applied at the time of 0 h. An antagonist, GW9662, was added 2 h after agonist application, indicated by an arrow. Percent of inhibition by the antagonist compared with the agonist-induced activities is shown.

 
We made three PPAR{gamma} mutants with the cysteine exchanged to alanine (C285A), valine (C285V), or serine (C285S). All of the mutants were still activated by BRL49653at levels equivalent to that of the wild type PPAR{gamma}, whereas they totally lost responsiveness to 15d-PGJ2 and 13-oxoODE (Fig. 5B). Thus, these putative natural ligands indeed functioned as ligands, via novel covalent binding to the cysteine residue in the ligand binding pocket, to activate transcription.

Next, we analyzed the effect of an antagonist on activation by both synthetic and natural ligands for PPAR{gamma}. Two hours after agonist addition, the PPAR{gamma}-specific antagonist GW9662 was applied to the cells expressing GAL4-PPAR{gamma} protein and a luciferase gene as a reporter (Fig. 5C). Both 15d-PGJ2- and BRL49653dependent activities of PPAR{gamma} decreased with the addition of GW9662. However, inhibition of 15d-PGJ2-dependent activity of PPAR{gamma} by GW9662 was more moderate than that of BRL49653dependent activity. Rapid inhibition of BRL49653dependent activity might be due to the displacement of the ligand by the antagonist. On the other hand, resistance of 15d-PGJ2 against the antagonist indicated that 15d-PGJ2 binding to PPAR{gamma} in the cells was irreversible. We considered that slower and more moderate inhibition of 15d-PGJ2-dependent activity was not achieved by the displacement of the ligand but by the competitive binding between 15d-PGJ2 and GW9662 to newly synthesized PPAR{gamma}.

Screening of Novel PPAR{gamma} Ligands—Based on the common structure, {alpha},{beta}-unsaturated ketone in naturally occurring ligands, we looked for other lipid metabolites containing an {alpha},{beta}-unsaturated ketone. Screening the chemical library databases, namiki.db, sumisho_specs.db, and Acd2D_rcg.db, we obtained six candidates for PPAR{gamma} ligands. Two of them were 9- and 13-oxoODE. Three of them, 5-oxoETE, 15-oxoETE, and 15-oxoeicosadecaenoic acid, were identified as PPAR{gamma} ligands (Fig. 6A). Luciferase assay clearly proved that these oxidized eicosanoids, but not their precursors, activated PPAR{gamma} (Fig. 6A). Furthermore, these oxidized eicosanoids covalently bound to the cysteine residue in the PPAR{gamma} LBD as determined by rhodamine-maleimide assay (Fig. 6B).



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  		FIG. 6.
Identification of novel PPAR{gamma} ligands based on a common structural feature. A, activation of PPAR{gamma} by oxidized eicosanoids. The PPAR{gamma} activity was measured in COS-7 cells, using luciferase as a reporter gene. The {alpha},{beta}-unsaturated ketones are indicated as boxes. B, covalent binding of oxidized eicosanoids to the cysteine residue in the PPAR{gamma} LBD. After incubating the PPAR{gamma} LBD with various ligands, the free cysteine in the PPAR{gamma} LBD was detected with rhodamine-maleimide.

 
In addition to a lipoxygenase-dependent pathway where the above metabolites are involved, we investigated the prostaglandin metabolites containing an {alpha},{beta}-unsaturated ketone in a cyclooxygenase-dependent pathway (Fig. 7, A and B). Using cell-based luciferase assay, we proved that oxidized prostaglandin metabolites 15-keto-PGE1, 15-keto-prostaglandin E2, 15-keto-PGF1, and 15-keto-prostaglandin F2{alpha} activated PPAR{gamma} (Fig. 7C, lanes 4, 5, 7, and 8, respectively). Structural relatives, PGE2 and PGF2, did not activate PPAR{gamma} (Fig. 7C, lanes 3 and 6), suggesting that the {alpha},{beta}-unsaturated ketone in the ligands was required for PPAR{gamma} activation through the Michael addition to the cysteine residue in the PPAR{gamma}. The activation by these oxidized prostaglandins was totally abolished by simultaneous application of PPAR{gamma} antagonist T0070907 (Fig. 7D).



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  		FIG. 7.
Identification of oxidized prostaglandin metabolites as PPAR{gamma} ligands. A, synthetic and degradation pathways of lipoxygenase- and cyclooxygease-dependent eicosanoids. Precursors are boxed. Oxidized metabolites are shown in bold. Oxidized prostaglandins investigated in this experiment are underlined. LA, linoleic acid; EDA, eicosadienoic acid; AA, arachidonic acid; DGLA, dihomo-{gamma}-linolenic acid. B, chemical structures of oxidized prostaglandins and their precursors used in this experiment. {alpha},{beta}-Unsaturated ketones are indicated by boxes. C, activities of oxidized prostaglandins in PPAR{gamma}-dependent transcription. Indicated ligands were added to the cells expressing GAL4-PPAR{gamma} at a concentration of 100 µM. 10 µM BRL49653were also shown as a positive control (lane 2). Data are represented as mean ± S.D. D, effect of the PPAR{gamma} antagonist on oxidized prostaglandin-dependent activation of PPAR{gamma}. 15d-PGJ2 (10 µM), 15-keto-PGE1 (10 µM), and 15-keto-PGF1 (50 µM) were added to the cells with or without 1 µM T0070907. Data are represented as mean ± S.D.

 
Concentration Dependence of PPAR{gamma} Activation—Concentration dependence of PPAR{gamma} activation by oxidized prostaglandins revealed that 15-keto-PGF1 and 15-keto-PGE1 showed higher EC50 and maximal activation than 15d-PGJ2 did (Fig. 8A). The order of EC50 of oxidized prostaglandins was 15d-PGJ2, 15-keto-PGE1, and 15-keto-PGF1. BRL49653showed both low EC50 and high maximal activation. This phenomenon indicated that EC50 and maximal activation were independently regulated by each ligand. These oxidized prostaglandins also blocked the maleimide labeling of the PPAR{gamma} LBD in a concentration-dependent manner (Fig. 8B), indicating that they bound to the cysteine residue in the PPAR{gamma} covalently. The order of the affinities was 15d-PGJ2, 15-keto-PGE1, and 15-keto-PGF1, which was correlated with that of EC50.



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  		FIG. 8.
Structure-activity relationship of PPAR{gamma} ligands. A, concentration dependence of oxidized prostaglandin-dependent activation of PPAR{gamma}. B, covalent binding of oxidized prostaglandins to the cysteine residue in the PPAR{gamma} LBD. Concentrations of each ligand were 0.5, 1, 5, 10, 50, and 100 µM from left to right. No ligand is shown as minus. Stars represent reactive carbons. C, superimposition of the model structure of the 15d-PGJ2-PPAR{gamma} complex to the crystal structure of the BRL49653PPAR{gamma} complex. Residues localized within 5 Å of each ligand are shown. The BRL49653 and 15d-PGJ2-PPAR{gamma} complexes are shown in pink and blue, respectively. Residues bound to both ligands are colored black, and those bound to either BRL49653(green) or 15d-PGJ2 (yellow) are shown in red and blue, respectively. D, schematic representation of interactions between 15-ketoPGF1 and PPAR{gamma}. Hydrogen bonds are indicated as blue lines. Atoms of oxidized prostaglandins at the positions indicated as a, b, and c are summarized (inset). E, requirement of the cysteine residue in oxidized prostaglandin-dependent activation of PPAR{gamma}. 15d-PGJ2 (10 µM), 15-keto-PGE1 (10 µM), and 15-keto-PGF1 (50 µM) were added to the cells expressing either wild type (WT) or C285A or Y473F mutant. Abbreviations are BRL, BRL49653 J2, 15d-PGJ2; E1, 15-keto-PGE1; F1, 15-keto-PGF1. Data are represented as mean ± S.D.

 
Structural Models of the Natural Ligand-PPAR{gamma} Complexes—To investigate whether covalent binding of 15d-PGJ2 to the PPAR{gamma} LBD is structurally achievable, we built a structural model of the 15d-PGJ2-PPAR{gamma} complex, based upon the crystal structure of the PPAR{gamma}/retinoid X receptor {alpha} heterodimer complexed with BRL49653and a coactivator peptide (Protein Data Bank accession code 1FM6 [PDB] ) (12). The obtained structure was superimposed onto the PPAR{gamma}-BRL49653 complex, suggesting some similarities and differences between the BRL49653 and 15d-PGJ2-PPAR{gamma} complexes (Fig. 8C). The hydrophobic tail of 15d-PGJ2, which BRL49653lacked, extended toward the tip of helix 11 (L452) and was stabilized through hydrophobic interactions. This binding mode was different from that observed in the eicosapentaenoic acid-PPAR{delta} complex, where the hydrophobic tail of eicosapentaenoic acid extended toward helix 2 or helix 5, rather than helix 11 (15).

To analyze what causes the difference in the maximal activation, we also built model structures of the PPAR{gamma} LBD covalently bound by 15-keto-PGE1 and 15-keto-PGF1. Because these oxidized prostaglandins have very similar structures but very different chemical and physical natures, we took advantage of molecular dynamics simulation to get the model structures instead of homology modeling. Carboxyl groups of these ligands made a hydrogen bond with Tyr-473 (Fig. 8D), which resides in helix 12 and is a key ligand-recognizing residue (12–14). Hydrophobic clusters assisting the ligand binding were also conserved (Fig. 8C and data not shown). In contrast, we observed two differences in hydrogen bonds between the main-chain carbonyl group of Leu-340 and a hydroxyl group of the ligands (Fig. 8D, a) and between the sulfur atom of the cysteine residue and another hydroxyl group (Fig. 8D, b). The former was observed only in the 15-keto-PGF1-PPAR{gamma} complex, and the latter was observed both in the 15-keto-PGF1- and 15-keto-PGE1-PPAR{gamma} complexes, but not in the 15d-PGJ2-PPAR{gamma} complex. These structural differences may produce more stable binding of 15-keto-PGF1 or 15-keto-PGE1 than 15d-PGJ2 and exert the receptor to have higher maximal activity, namely, efficacy.

We observed that all three ligands required the cysteine residue (Fig. 8E, gray bars). According to the structural model described above, we made an additional PPAR{gamma} mutant (Y473F) that showed no response to any ligands (Fig. 8E), supporting our model structure.

Regulation of PPAR{gamma} Target Genes by Oxidized Prostaglandins—Finally, we determined whether oxidized prostaglandins activate PPAR{gamma} target genes in THP-1 cells, which endogenously express PPAR{gamma} protein. Adipophilin (ADFP) was upregulated by PPAR{gamma} ligands (Fig. 9A, left panel), and the upregulation was blocked by simultaneous application of PPAR{gamma} antagonist T0070907 (Fig. 9A, right panel), suggesting that ADFP expression was regulated through PPAR{gamma} (28, 29). Among several PPAR{gamma}-regulated genes that we have identified by microarray,2 we successfully observed that oxidized prostaglandins 15-ketoPGE1 and 15-ketoPGF1 as well as 15d-PGJ2 and 13-oxoODE activated PPAR{gamma}-regulated genes, ADFP, interleukin-1 receptor type I, cyclin-dependent kinase inhibitor 1A, and aldehyde oxidase 1 (Fig. 9B). These results suggested that oxidized prostaglandins acted as PPAR{gamma} ligands and regulated the expression of PPAR{gamma} target genes in vivo.



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  		FIG. 9.
Oxidized prostaglandin-mediated transcriptional regulation through PPAR{gamma} in THP-1 cells. A, reverse transcription-PCR of ADFP gene. ADFP gene was up-regulated by 10 µM PPAR{gamma} ligands BRL49653 15d-PGJ2, and 13-oxoODE (left). The up-regulation was blocked by simultaneous application of PPAR{gamma} antagonist T0070907 (right). B, reverse transcription-PCR of PPAR{gamma} target genes. Newly identified ligands 15-ketoPGE1 (from left to right, 0.3, 3, and 10 µM) and 15-ketoPGF1 (1.66, 16.6, and 50 µM), as well as 15d-PGJ2 (0.3, 3, and 10 µM) and 13-oxoODE (0.3, 3, and 10 µM), up-regulated PPAR{gamma} target genes in a dose-dependent manner. Cdc-like kinase (CLK1) was used as negative control. IL1R1, interleukin-1 receptor type I; AOX1, aldehyde oxidase 1.

 


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
By this study, the currently proposed natural PPAR{gamma} ligands were shown for the first time to share a common structure, the {alpha},{beta}-unsaturated ketone, that enables them to bind to PPAR{gamma} covalently and to exhibit their activities. Although we do not necessarily exclude the possibility that as yet unrevealed high affinity ligands with novel moieties still exist, our studies provide several advances toward answering the enigmas in PPAR{gamma} functions.

First, one can easily imagine that the covalent binding of the ligands to the receptor not only acts as a rigid switch but also enables the receptors to accumulate as active forms, allowing such ligands with low concentrations to function in vivo. Unknown factor(s) may facilitate this covalent binding.

Second, our studies provide clear evidence that the structural moieties within a single molecule are used differently in PPAR{gamma}-dependent and -independent functions. In the case of 15d-PGJ2, the electrophilic carbon (C9) within a cyclopentenone ring has been considered to react with the cysteine residue in NF-{kappa}B and other proteins (17, 30). On the other hand, we consider that the carbon at position 13 reacts with the sulfur atom of the cysteine residue in PPAR{gamma}, according to results from UV absorbance measurements and the activity of CAY10410lacking the cyclopentenone ring (Figs. 2A, 3A, and 5A). Furthermore, the carboxyl group of 15d-PGJ2 is required for the formation of a hydrogen bond with Tyr-473 in helix 12 of PPAR{gamma}. The 15d-PGJ2 biotinylated at the carboxyl group reportedly failed to pull down PPAR{gamma} from cells, whereas it still bound to AP-1 and other proteins (20, 30). Thus, the covalent binding of 15d-PGJ2 to the PPAR{gamma} LBD is different from that in PPAR{gamma}-independent actions.

Third, our finding that several oxidized eicosanoids act as PPAR{gamma} ligands fits well with the expected PPAR{gamma} functions: PPAR{gamma} may function under oxidative conditions and/or inflammation. For example, glucose consumption is related to mitochondrial respiration, which generates free radical. In atherosclerosis, macrophages play important roles in which oxidized low density lipoprotein accumulates as a major source of oxidized lipid (7). Under inflammatory conditions, macrophages express not only cyclooxygease-2 and 12/15-lipoxygenase, to produce prostaglandins and leukotrienes, respectively (31, 32), but also nitric-oxide synthase to generate nitric oxide, another potent oxidative agent (33). In addition, expressions of prostaglandin transporter and 15-hydroxyprostaglandin dehydrogenase, both of which are involved in prostaglandin metabolism to form 15-keto-protstaglandins (34, 35), were modulated during lipopolysaccharide-induced fever (36). Recently, it has been shown that oxidative stress stimulates inflammatory cells to produce 5-oxoETE from 5(S)-hydroxy-6,8,11,14-eicosatetraenoic acid (37). These reports suggest that the production of oxidized eicosanoid metabolites is physiologically regulated under certain conditions, such as inflammation. Thus, PPAR{gamma} may sense such oxidized fatty acids as signaling molecules and function as an inflammation modulator.

On the other hand, the physiological relevance of the lipid metabolites, especially 15d-PGJ2, has long been debated (38). Further studies will be needed to determine whether they really act as endogenous ligands on PPAR{gamma}.


   FOOTNOTES
 
* This work was supported by a research grant endorsed by the New Energy and Industrial Technology Development Organization. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ Both authors contributed equally to this work. Back

§§ To whom correspondence should be addressed. Tel.: 81-6-6872-8214; Fax: 81-6-6872-8210; E-mail: jingami{at}beri.or.jp.

1 The abbreviations used are: PPAR{gamma}, peroxisome proliferator-activated receptor {gamma}; LBD, ligand-binding domain; 15d-PGJ2, 15-deoxy-{Delta}12,14-prostaglandin J2; ETE, eicosatetraenoic acid; 15-keto-PGE1, 15-keto-prostaglandin E1; 15-keto-PGF1, 15-keto-prostaglandin F1{alpha}; HODE, hydroxyoctadecadienoic acid; ODE, octadecadienoic acid; ADFP, adipophilin; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight. Back

2 T. Shiraki and H. Jingami, unpublished data. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Kosuke Morikawa for discussion and Dr. Takuji Oyama for figure preparation.



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