162 Inflammation & Allergy - Drug Targets, 2013, 12, 162-171
Modulation of GSTP1-1 Oligomerization by Electrophilic Inflammatory Mediators and Reactive Drugs
Francisco J. Sánchez-Gómez§,1, Carlos García Dorado§,1, Pedro Ayuso1,2, José A.G. Agúndez2, María A. Pajares3 and Dolores Pérez-Sala*,1
1Chemical and Physical Biology Department, Centro de Investigaciones Biológicas, Consejo Superior de
Investigaciones Científicas, Ramiro de Maeztu, 9, 28040 Madrid, Spain
2Department of Pharmacology, Universidad de Extremadura, Cáceres, Spain
3Instituto de Investigaciones Biomédicas Alberto Sols (CSIC-UAM), Arturo Duperier 4, 28029 Madrid and Molecular
Hepatology group, IdiPAZ, Pº de la Castellana 261, 28046, Madrid, Spain
Abstract: Glutathione S transferase P1-1 plays a key role in the metabolism of inflammatory mediators and drugs, thus modulating the inflammatory response. Active GSTP1-1 is a homodimer with cysteine residues close to the active site that can undergo oligomerization in response to stress, a process that affects enzyme activity and interactions with signaling and redox-active proteins.
Cyclopentenone prostaglandins (cyPG) are endogenous reactive lipid mediators that participate in the regulation of inflammation and may covalently modify proteins through Michael addition. cyPG with dienone structure, which can bind to vicinal cysteines, induce an irreversible oligomerization of GSTP1-1. Here we have characterized the oligomeric state of GSTP1-1 in Jurkat cells treated with 15-deoxy- 12,14-PGJ2 (15d-PGJ2). 15d-PGJ2 induces both reversible and irreversible GSTP1-1 oligomerization as shown by blue-native 2D electrophoresis. Interestingly, GSTP1-1 dimers were the main species detected by analytical gel filtration chromatography in control cells, whereas only oligomers, compatible with a tetrameric association state, were found in 15d-PGJ2-treated cells.
cyPG-induced GSTP1-1 oligomerization also occurred in cell-free systems. Therefore, we employed this model to assess the effects of endogenous reactive species and drugs. Inflammatory mediators, such as 15d-PGJ2 and 12-PGJ2, and drugs like chlorambucil, phenylarsine oxide or dibromobimane elicited whereas ethacrynic acid hampered GSTP1-1 oligomerization or intra-molecular cross-linking in cell-free systems, yielding GSTP1-1 species specific for each compound.
These observations situate GSTP1-1 at the cross-roads of inflammation and drug action behaving as a target for both inflammatory mediators and reactive drugs, which induce or reciprocally modulate GSTP1-1 oligomerization or conformation.
Glutathione S transferases (GST) are abundant cytosolic enzymes which promote the detoxification of both endogenous and exogenous electrophiles by catalyzing their conjugation with glutathione (GSH). Among GSTs, the isoform glutathione S-transferase pi 1 (GSTP1-1) is expressed at high levels in numerous cell types and has been involved both in tumorigenesis and chemoresistance [1]. GSTP1-1 may be overexpressed in certain tumors and metabolize anti-cancer drugs and endogenous electrophilic compounds. In addition to this enzymatic role, it participates in cellular homeostasis through protein-protein interactions and by non-covalent binding or sequestration of numerous
*Address correspondence to this author at the Department of Chemical and Physical Biology, Centro de Investigaciones Biológicas, C.S.I.C., Ramiro de Maeztu, 9, 28040 Madrid, Spain; Tel: 34918373112; Fax: 34915360432; E-mail: [email protected] §Contributed equally to this work.
substances, for which a “ligandin” role for this enzyme has been proposed [2-4]. GSTP1-1 participates in cellular redox homeostasis by maintaining certain thiol groups in their reduced state and by regulating glutathionylation, and it may contribute to sustain cell viability upon certain insults [5, 6]. Moreover, allelic variants of this enzyme possess different kinetic properties towards certain substrates [4], and associate with different susceptibility to cancer or allergy [7, 8] or ability to reduce lipid peroxidation [9].
The active form of GSTP1-1 is a non-covalent homodimer composed by two 22 kDa monomers. GSTP1-1 possesses several reactive thiol residues and has been shown to be the target for covalent modification by various drugs or toxic compounds, including ethacrynic acid, acrolein, chlorambucil or ibuprofen, which may inactivate the enzyme [10-13]. Indeed, GSTP1-1 has been used as a model to assess the ability of certain compounds to covalently modify cellular proteins [14, 15]. In turn, some reactive drugs, electrophilic stress and exposure to inflammatory mediators with electrophilic nature, like the cyclopentenone
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prostaglandin 15-deoxy- 12,14-PGJ2 (15d-PGJ2), may induce GSTP1-1 levels in a cell type-dependent manner [16].
Cyclopentenone prostaglandins (cyPG) are endogenous lipidic mediators, generated by dehydration of PG, which are involved in the modulation of inflammation. In particular, a role for 15d-PGJ2 in the onset and resolution of inflammation has been reported [17, 18]. cyPG are electrophilic compounds which possess an , -unsaturated carbonyl group in the cyclopentane ring and may form covalent adducts with nucleophilic groups in proteins, mainly thiol groups, through Michael addition [19]. Covalent modification of critical proteins in proinflammatory and antioxidant pathways constitutes the best characterized mechanism for cyPG action [20, 21]. Covalent modification plays also a key role in the activation of transcription factor PPAR by 15d-PGJ2 [22]. Interestingly, cyPG may exert a concentration or context-dependent double-faceted role. Nanomolar or low micromolar concentrations of cyPG elicit or potentiate an inflammatory response in certain cell types [23-25]. In contrast, micromolar concentrations or continued exposure to nanomolar levels may elicit anti-inflammatory responses in most cell types studied as well as in vivo [25-27].
We have recently shown that GSTP1-1 is a target for direct covalent modification by cyPG, which leads to inactivation [6]. Moreover, in cells, cyPG may induce a variety of posttranslational modifications in GSTP1-1, including thiolation as a consequence of oxidative stress [28], and importantly, oligomerization [29]. GSTP1-1 oligomerization bears a particular interest because it has been reported to influence stress signaling cascades, releasing kinases which may lead to the induction of apoptosis [30]. Certain cyPG, including 15d-PGJ2 and 12-PGJ2 possess two electrophilic carbons and have been shown to elicit intra- or inter-molecular protein cross-linking by simultaneously reacting with two vicinal cysteine residues [31]. Notably, dienone cyPG induce GSTP1-1 oligomerization, which is resistant under denaturing conditions [29]. Several agents including xenobiotics, UV irradiation and oxidative stress also elicit GSTP1-1
oligomerization, although in most cases as a reversible process [29, 30]. This implies that several mediators, as well as drugs, may both bind to GST and alter its interaction patterns in cells. However, in spite of its structural and functional importance, the factors governing GST oligomerization are poorly understood.
Here we have undertaken the characterization of GSTP1-1 oligomerization induced by dienone cyPG. Moreover, we have used a cell-free assay to explore the ability of various mediators and drugs (structures shown in Scheme 1) to elicit or modify GSTP1-1 oligomerization, as a means to identify interactions potentially leading to the modulation of cellular GST functions.
METHODS
Reagents
Electrophilic prostaglandins were from Cayman Chemical. Purified GSTP1-1 was from Alpha Diagnostic International. Anti-GSTp monoclonal antibody was from BD Biosciences. HRP-conjugated anti-mouse Igs antibodies were from Dako. The ECL chemiluminiscence reagent was from GE Healthcare.
Cell Culture and Processing
Jurkat cells were cultured in RPMI 1640 (Gibco) supplemented with 10% (v/v) FBS, 100 U/ml penicillin and 100 μg/ml streptomycin. Treatment of intact cells with cyPG was carried out in serum-free medium for 16 h, as previously described [29]. In order to preserve GSTP1-1 oligomerization state, cells were washed with PBS containing 50 mM iodoacetamide [32], before lysis in 20 mM Tris-HCl pH 7.5, 0.1 mM EDTA, 0.1 mM EGTA, 0.1 mM -mercaptoethanol, 0.5% (w/v) SDS, and 50 mM iodoacetamide containing protease inhibitors (2 μg/ml each of trypsin inhibitor, leupeptin and aprotinin, and 1.3 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride), as previously described [29].
Scheme 1. Structures of various compounds used in this study. *, Electrophilic carbons formally susceptible to Michael type additions; #, carbons susceptible of attack by nucleophilic agents; ‡, arsine oxide reacts with sulfides.
15-deoxy-∆12,14-Prostaglandin J2
∆12-Prostaglandin J2
O
COOH
COOH
OOH
AsO
O O
BrBr
N
N
N
ClCl
O
OH
O
OHO
Cl
Cl
O
Dibromobimane
Phenylarsine oxide
Chlorambucil Ethacrynic acid
*
*
*
*
* *
#
‡
#
# #
164 Inflammation & Allergy - Drug Targets, 2013, Vol. 12, No. 3 Sánchez-Gómez et al.
Blue-Native 2D Electrophoresis
Jurkat cells treated with vehicle (DMSO) or 10 μM 15d-PGJ2 were lysed in buffer containing 1% (v/v) Triton X-100 and 40 mM iodoacetamide without reducing agents. Aliquots containing twenty micrograms of protein were mixed with sample buffer (300 mM Tris-HCl pH 6.8, 40% (v/v) glycerol, 0.1% (w/v) Coomassie blue G and 500 mM aminocaproic acid) [33]. Samples were analyzed in the first dimension in 12.5% polyacrylamide gels without SDS using a two-buffer system: the cathode buffer contained 0.002% (w/v) Coomassie blue G, which was absent from the anode buffer. When the dye front reached one third of the gel length, the cathode buffer was substituted by anode buffer. After electrophoresis gel lanes were cut, placed over 12.5% SDS-PAGE gels and overlaid with Laemmli sample buffer containing 100 mM DTT for electrophoresis. Gels from the second dimension were subjected to western blot with anti-GSTP1-1 antibody (1:1000) and secondary antibody (1:2000), as previously described [29].
Subcellular Fractionation
Cells were lysed in 20 mM Tris-HCl pH 7.5, 0.1 mM EDTA, 0.1 mM EGTA, 0.1 mM -mercaptoethanol, 50 mM sodium fluoride, 0.1 mM sodium orthovanadate and 50 mM iodoacetamide with protease inhibitors (2 μg/ml each of trypsin inhibitor, leupeptin and aprotinin, and 1.3 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride) and lysates were subjected to ultracentrifugation at 100,000xg for 1 h at 4oC, as previously described [34], to obtain supernatant (S100) and pellet (P100) fractions.
Analytical Gel Filtration Chromatography
S100 fractions from Jurkat cells treated with vehicle or 10 μM 15d-PGJ2 (100 μl containing a minimum of 50 μg protein) were injected into a Superose 12 10/30 HR gel filtration column (GE Healthcare) connected to an Äkta Purifier system (GE Healthcare). Equilibration and elution were carried out with 10 mM Tris-HCl pH 7.5, 0.1 mM EDTA, 0.1 mM EGTA, 150 mM NaCl and protease inhibitors at a constant flow rate of 0.3 ml/min at room temperature, and 210 μl fractions were collected. Protein detection in column fractions was carried out by dot-blot (100 μl) using anti-GSTp antibody as described for western blots. The protein standards (GE Healthcare and Sigma) used and their elution volumes were as follows: Blue Dextran (2000 kDa), 7.02 ml; tyroglobulin (669 kDa), 9.03 ml; apoferritin (443 kDa), 9.87 ml; -amylase (200 kDa), 11.13 ml; aldolase (158 kDa), 12.18 ml; conalbumin (75 kDa), 13.65 ml; ovalbumin (43 kDa), 14.7 ml; soybean trypsin inhiibitor (21.5 kDa), 15.96 ml; and ATP (551 Da), 17.4 ml. The optimal separation range for this column according to the manufacturer’s brochure is 300 kDa to 1 kDa and the void volume 2000 kDa. The KAV for standards in the separation range was calculated using the following equation:
KAV = (Ve-Vo)/(Vf-Vo)
where Ve is the elution volume of the standard, Vo is the void volume (7.02 ml) and Vf, the final volume (17.4 ml). The calibration curve was obtained by representing KAV against the log of the molecular mass for each standard.
Oligomerization Assays
For in vitro oligomerization assays S100 fractions were obtained as above, except that they were isolated from untreated Jurkat cells lysed in the absence of iodoacetamide. Aliquots of S100 fractions containing 20 μg of protein were mixed with 20 mM Tris-HCl pH 7.0, 45 mM NaCl, 5 mM MgCl2 and 0.1 mM DTT, and incubated with the indicated agents at room temperature for 2 h. For these assays we used cyPG concentrations in the range of those previously found to induce oligomerization of GSTP1-1 in vitro [29]. Other agents were used at a 2-fold molar excess over cyPG. After the incubation, 50 mM iodoacetamide was added to the mixtures, which were left in the dark for 25 min before addition of Laemmli sample buffer with or without -mercaptoethanol for analysis under reducing or non-reducing conditions, respectively.
RESULTS
Characterization of GSTP1-1 Oligomers Induced by
cyPG with Dienone Structure
We have previously observed that treatment of various cell types with cyPG dienones induces GSTP1-1 oligomerization which in some cases may account for nearly 50% of the total cellular GSTP1-1 protein and is associated with enzyme inactivation [29]. As it is shown in Fig. (1A), treatment of Jurkat cells with 15d-PGJ2 induces the oligomerization of GSTP1-1 (Fig. 1A), which is clearly evidenced on SDS-PAGE gels under denaturing conditions. Under non-reducing conditions, several oligomeric species are visible. Some of these species may originate by disulfide bond formation since they are reversed in the presence of -mercaptoethanol. In addition, a band migrating faster than the GSTP1-1 monomer was visible in samples from cells treated with 15d-PGJ2. This band was previously identified as a monomer bearing an intra-molecular disulfide bond, based on its behavior in 2D non-reducing/reducing electrophoresis, where this band runs as a spot with the mobility of the monomer [29]. Notably, samples from cells treated with 15d-PGJ2 analyzed under reducing conditions show a major oligomeric band with an apparent molecular mass of 70 kDa that will be referred to from now on as the 70 kDa oligomeric species.
To get insight into the nature of cyPG-induced GSTP1-1 oligomers, lysates from control and 15d-PGJ2-treated cells were analyzed under native conditions using blue-native electrophoresis, which is known to preserve certain protein-protein interactions (Fig. 1B). Using this technique we observed that in control cells, GSTP1-1 appeared as a homogeneous species with a size compatible with a monomer (approximately 29 kDa, Fig. 1B). Although GSTP1-1 is thought to exist as homodimers in cells, our results indicate that they dissociate under the conditions used for blue-native electrophoresis. In contrast, in cells treated with 15d-PGJ2 a broad band was detected with an apparent molecular weight compatible with the formation of dimers or higher order oligomers. Interestingly, analysis of these complexes in the second dimension showed that they were integrated by GST species that resolved as monomers and oligomeric species. These oligomers were stable under denaturing conditions, thus confirming previous observations.
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Fig. (1). Induction of GSTP1-1 oligomerization by 15d-PGJ2 in Jurkat cells. (A) Analysis of GSTP1-1 species by SDS-PAGE electrophoresis. Jurkat cells were treated with 10 μM 15d-PGJ2 for 16 h, after which, cell lysates were obtained in the presence of 0.5% SDS and 50 mM iodoacetamide and analyzed by SDS-PAGE under non-reducing or reducing conditions and Western blot with anti-GSTP1-1 antibody. (B) Cells treated as in (A) were lysed in the presence of 1% Triton X-100 and 40 mM iodoacetamide and analyzed by blue-native electrophoresis. Arrows mark the position of the species resulting from the dissociation of the oligomers in the second dimension. Results shown are representative of at least three assays with similar results in each case.
Next, we assessed whether GSTP1-1 oligomerization altered its subcellular localization or solubility within cells. For this purpose, cells were lysed under various conditions and lysates were subjected to ultracentrifugation. Under all conditions assayed, all GSTP1-1 species remained fully soluble (Fig. 2). Therefore, we went on to analyze GSTP1-1 oligomers by analytical gel filtration chromatography using S100 fractions from control and 15d-PGJ2-treated cells. The SDS-PAGE analysis of the material injected into the column under reducing conditions is depicted in Fig. (2A, right panels), showing the prominent 70 kDa oligomeric GSTP1-1 band in samples from 15d-PGJ2-treated cells. The elution profiles of GSTP1-1 were then assessed by dot-blot of the corresponding fractions with the specific antibody and are depicted in Fig. (2B). As it can be observed, although GSTP1-1-containing fractions eluted as a single peak in the analysis of S100 fractions from both control and 15d-PGJ2-treated cells, the elution volumes were markedly different in both cases. The elution volume of GSTP1-1-containing fractions in the analysis of S100 fractions from control cells corresponded to that expected for a 43 kDa protein, thus compatible with a dimeric association state (theoretical molecular weight 44 kDa). Remarkably, in the analysis of
S100 fractions from 15d-PGJ2-treated cells, GSTP1-1 eluted as a single peak at volumes corresponding to a 97 kDa protein, that is, with the apparent size expected for a tetramer. This peak may contain a mixture of species, including reversibly and irreversibly associated oligomers, as well as oligomers containing both types of monomer associations. Only the reversible cross-links will dissociate when the sample is analyzed under denaturing SDS-PAGE conditions leading to the complex patterns composed by monomers and oligomers observed in gels.
Study of GSTP1-1 Oligomerization Patterns in Cell-Free
Systems
We have previously shown that dienone cyPG can induce the oligomerization of purified GSTP1-1 [29]. Here, we were interested in exploring the ability of cyPG to induce GSTP1-1 oligomerization in cell-free systems. For this purpose, S100 fractions from Jurkat cells were incubated in vitro with vehicle or 15d-PGJ2 and subsequently analyzed by SDS-PAGE under reducing and non-reducing conditions, followed by western blot and detection of GSTP1-1 (Fig. 3). Analysis of vehicle-treated S100 fractions under non-reducing conditions showed the presence of monomeric GSTP1-1 as the major form, although several faint oligomeric species and a band migrating faster than the GSTP1-1 monomer could be detected in a variable proportion. These oligomeric species and the fast migrating band were not detected under reducing conditions; thus, they probably arise from reversible oxidations taking place during the isolation or incubation of S100 fractions. This is also consistent with the fact that these bands are not detected in samples obtained from intact cells (shown in the left panel for comparison), which are lysed in the presence of iodoacetamide to prevent further oxidation and disulfide exchange (see methods). Analysis of 15d-PGJ2-treated S100 fractions under non-reducing conditions showed a modest increase in the 70 kDa oligomeric species (marked with an arrow in Fig. 3), together with an increase in poorly-defined larger oligomeric species and a decrease in the proportion of the fast migrating band. The fact that cyPG induce an increase in this fast migrating band in intact cells may represent an indirect effect related to the ability of these compounds to induce oxidative stress leading to reversible GSTP1-1 oxidations. 15d-PGJ2-induced oligomers were resistant under reducing conditions. Remarkably, the 70 kDa oligomeric species detected in intact cells and S100 fractions treated with 15d-PGJ2 is also observed in assays performed with the purified protein (Fig. 3, right panel). In view of the ability of 15d-PGJ2 to induce GSTP1-1 oligomerization in isolated S100 fractions we used this system to explore the ability of several compounds to alter GSTP1-1 oligomerization patterns, both under non-reducing and reducing conditions.
12-PGJ2 is a cyPG generated from PGJ2 in the presence
of albumin. This cyPG also possesses dienone structure and reproduces some of the effects of 15d-PGJ2. When analyzed under non-reducing conditions, 12-PGJ2-treated S100 fractions showed a weak increase in the 70 kDa oligomeric band, together with an increase in poorly resolved higher oligomeric species (Fig. 4A). Remarkably, analysis under reducing conditions allowed the detection of a well-defined
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major oligomeric species at 70 kDa. Interestingly, the size of this species coincides with the major oligomer induced by 15d-PGJ2 or 12-PGJ2 in intact cells [29].
The antitumoral drug chlorambucil has been reported to interact with GSTP1-1 [12]. This drug induced a pattern very similar to that elicited by 12-PGJ2, the most salient feature being the formation of an oligomer with an apparent mass of 70 kDa, which was resistant under reducing conditions, thus indicating the generation of protein cross-links (Fig. 4A).
The bifunctional compounds dibromobimane (DBB) and phenylarsine oxide (PAO) induced an increase in the proportion of several GSTP1-1 oligomeric species in S100 fractions, as detected under non-reducing conditions, with the major species showing the apparent molecular weight of
a dimer (Fig. 4B). A variable increase in the intensity of the faster migrating band was also observed. Interestingly, PAO-induced conformational changes were totally reversible. In contrast, DBB-induced oligomers as well as the fast migrating band were resistant under reducing conditions, thus indicating the occurrence of irreversible intra- and inter-molecular cross-links in GSTP1-1 in DBB-treated samples.
Other drugs have been reported to interact with GST, including the anti-inflammatory drug ibuprofen, the anti-tumoral compound etoposide and ethacrynic acid, a diuretic drug and GST inhibitor [13, 35, 36]. Therefore, we explored the effect of these drugs on GSTP1-1 association states, as well as their potential to interfere with cyPG-induced oligomerization. Under the conditions of the assay, ibuprofen and etoposide did not induce appreciable
Fig. (2). Analysis of GSTP1-1 oligomeric species by analytical gel filtration chromatography. (A) Jurkat cells were treated with 15d-PGJ2 as above and lysates were obtained in the absence or presence of detergent, subjected to ultracentrifugation and the indicated fractions analyzed by western blot (sup, supernatant). (B) S100 fractions obtained from control and 15d-PGJ2-cells were subjected to gel filtration chromatography on a Superose 12 10/30 HR column connected to an Äkta Purifier system. The elution profile of GSTP1-1, as assessed by densitometric scanning of the dot-blot of the collected fractions is shown. The elution volumes and the molecular mass (in kDa) of the standards used for calibration are depicted (BD, blue Dextran; Am, -amylase; Al, aldolase; Con, conalbumin; STI, soybean trypsin inhibitor).
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alterations in the GST oligomerization pattern (Fig. 5). Remarkably, incubation with ethacrynic acid reduced the proportion of all oligomers and intra-molecular disulfide-containing species with respect to the control samples. Interestingly, ethacrynic acid also reduced 12-PGJ2-induced oligomerization both under non-reducing and reducing conditions. As it is discussed below, this could be due to conformational alterations induced by covalent or non-covalent binding of this inhibitor. Taken together, these results show that GSTP1-1 oligomerization can be differentially modulated by various drugs and inflammatory mediators.
DISCUSSION
GST enzymes are highly reactive proteins which may interact both with endogenous mediators and drugs. GSTP1-1 modification may inhibit its enzymatic activity and affect its oligomerization, thus influencing its association with cellular proteins important for signaling and for the maintenance of redox status. Our results show that cyPG as well as drugs induce GSTP1-1 reversible and/or irreversible oligomerization resulting in specific patterns (a simplified scheme is presented in Fig. 6). Moreover, certain agents can interfere with cyPG-elicited oligomerization. Therefore, GSTP1-1 modification and oligomerization may integrate signals from inflammation and pharmacological treatments transducing them into redox alterations and/or activation of stress kinases.
cyPG-induced GSTP1-1 oligomerization occurs both in cells and in cell-free systems. Results from gel filtration suggest that the main cyPG-induced oligomer is a tetramer. This association state could be generated by the cross-linking of two dimers. However, the species observed in SDS-PAGE (70 kDa) shows an apparent size more consistent with a trimer. Therefore, the possibility exists that within the tetramer, three of the subunits may be covalently bound, whereas the fourth remains dissociable under denaturing conditions, as schematized in Fig. (6). Alternatively, a compact tetramer, for instance, one containing intra-
Fig. (4). Effect of various agents on GSTP1-1 oligomerization in S100 fractions. S100 fractions treated in the presence of the agents indicated at 12.5 μM were analyzed as above. (A) Effect of 12-PGJ2 and chlorambucil. (B) Effect of dibromobimane (DBB) and phenylarsine oxide (PAO). Results are representative of at least three assays for every condition.
molecular cross-links, could migrate faster in SDS-PAGE gels. Identification of the precise interactions leading to this species requires further studies. We previously observed that
Fig. (3). Effect of 15d-PGJ2 on GSTP1-1 oligomerization state in a cell-free system. S100 fractions obtained from control cells were treated in vitro with vehicle (DMSO) or with 12.5 μM 15d-PGJ2 and analyzed by SDS-PAGE under non-reducing or reducing conditions, as indicated. The position of GSTP1-1 species was detected by western blot. Results are representative of at least three assays for every condition. The position of GSTP1-1 oligomers obtained by treatment of intact cells or of the purified protein is shown for comparison. Arrows mark the position of the 70 kDa oligomeric species.
15d-PGJ2 - + - + - + - +
Non-reducing ReducingIntact cells S100 S100 Purified protein
168 Inflammation & Allergy - Drug Targets, 2013, Vol. 12, No. 3 Sánchez-Gómez et al.
Fig. (5). Effect of various agents on GSTP1-1 oligomerization per se and in the presence of 12-PGJ2. S100 fractions were incubated in the presence of vehicle, ibuprofen, ethacrynic acid or etoposide at 25 μM for 1 h, after which, 12-PGJ2 was added and incubation was continued for 2 h. Incubation mixtures were processed as described in Methods and GSTP1-1 oligomeric species were detected by western blot.
Fig. (6). Scheme summarizing some of the potential interactions between electrophilic inflammatory mediators and reactive drugs with GSTP1-1. GSTP1-1 is present in cellular cytosol as a non-covalent dimer. Several bifunctional drugs as well as inflammatory mediators may induce reversible (in the case of PAO) or irreversible GSTP1-1 cross-linking, giving rise to a putative tetramer, potentially formed by covalent and non-covalent interactions. The interaction with ethacrynic acid blocks this effect possibly shielding residues important for oligomerization.
Cys101 is required for 15d-PGJ2-induced oligomerization in intact cells [29]. Importantly, Cys101 is not present in murine GSTP1-1. Therefore, studies in murine models may miss GSTP1-1 interactions important for the response to oxidative or electrophilic stress taking place in humans. Since the GSTP1-1 monomer presents great flexibility, Cys101 could be involved in interactions with other cysteine residues, even though they appear distant in the crystal structure. For instance, Cys101 has been reported to form an
intra-molecular disulfide bond with Cys47 [37]. Moreover, in the GSTP1-1 dimer, the Cys101 residues from each monomer are within cross-linking distance both by cyPG and DBB [29], and chlorambucil has been reported to induce GSTP1-1 cross-linking potentially involving Cys101 of one monomer and Cys47 of the other [12]. In addition, cyPG could induce protein oligomerization through mechanisms involving conformational alterations (see for [19] review). For certain proteins, such as UCH-L1, modification by 15d-PGJ2 may provoke protein unfolding leading to the formation of aggregates [38]. Thus, the formation of GSTP1-1 oligomers could be also the result of partial unfolding favoring association or protein aggregation.
Other bifunctional agents, including DBB and PAO give rise to the appearance of several GSTP1-1 species in cell-free systems, the reversibility of which is consistent with the chemical nature of the modification by either agent, as we have previously shown [31]. Similarly to cyPG, these agents are able to cross-link cysteine residues which are within a distance of 3 to 6 Å. Interestingly, both DBB and PAO induce intra-molecular cross-links leading to more compact monomers that migrate faster in SDS-PAGE gels, and that in the case of DBB appear to be irreversible.
Interestingly, in intact cells, low concentrations of ethacrynic acid or etoposide may induce reversible GSTP1-1 dimerization through a mechanism dependent on oxidative stress [29, 39]. Nevertheless, these compounds did not induce or even reduced GSTP1-1 oligomerization in cell-free systems. Ethacrynic acid is a known inhibitor of GSTP1-1 which has been shown to form an adduct with Cys47 [10] or with Cys101 in Cys47Ser mutants [36]. Our results show that ethacrynic acid reduces basal and 12-PGJ2-induced oligomerization in S100 fractions. Moreover, ethacrynic acid blocks the binding of biotinylated 15d-PGJ2 to recombinant GSTP1-1, as well as 15d-PGJ2-induced oligomerization (results not shown). Therefore, either covalent or non-covalent binding of ethacrynic acid to GSTP1-1 could block the residues involved in oligomerization, induce conformational changes or steric hindrance precluding access of 12-PGJ2 to those residues. Indeed, according to the crystal structure, non-covalent binding of ethacrynic acid or ethacrynic acid-GSH conjugate to GSTP1-1 would shield Cys101 and Cys47 from access by other compounds [40]. In this context, the presence of the substrate, GSH, is also an important factor that may affect the modification and oligomerization of GSTP1-1 by various agents [12, 29]. Therefore, alterations of the cellular redox status and GSH content may influence GSTP1-1 oligomerization and/or inactivation by various agents.
Importantly, the possibility exists that the GSTP1-1 oligomeric species may contain other protein(s). Several putative candidates could be considered since GSTP1-1 has been reported to associate with various proteins in cells, including JNK and TRAF2, the interaction having important consequences for stress and TNF- -mediated signaling [30, 41]. Association of GSTP1-1 with these kinases reportedly keeps them in an inactive state. Stimuli inducing GSTP1-1 oligomerization release these kinases potentially contributing to the activation of downstream cascades resulting in apoptosis in various cancer cell lines [29, 42]. Nevertheless, the mechanisms involved are not completely understood
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since GSTP1-1 depletion has been reported both to lead to increased JNK constitutive activity [43] and to reduce JNK-dependent apoptosis [44], depending on the experimental system. Therefore, additional interactions must exist which are context-dependent that influence the outcome of GSTP1-1 oligomerization. GSTP1-1 has also been reported to interact with and activate peroxiredoxins 1 and 6 and 1-cysteine peroxiredoxin [9, 45, 46], and to be a substrate for transglutaminase [47]. Nevertheless, the observation that cyPG-treatment of purified GSTP1-1 induces an association state similar to that observed in cells or in S100 fractions indicates that there is no need for the participation of other proteins in oligomerization.
In addition to its role in cancer chemoresistance, GST may play an important role in the modulation of inflammation. In preliminary assays we have observed that overexpression of GSTP1-1 reduces the activity of a peroxisomal proliferator response element (PPRE) reporter, both under basal conditions and upon stimulation with 15d-PGJ2. Although the mechanisms involved have not been elucidated, several possibilities for the effect of GSTP1-1 on PPRE activity could be hypothesized, including a reduction in the formation of endogenous PPAR ligands (i.e. products of lipid peroxidation), their conjugation to GSH, which would facilitate their export, as well as their sequestration. In addition, the fact that 15d-PGJ2 and other electrophilic lipids are able to induce GSTP1-1 expression in certain cell systems [16] could give rise to a feedback regulation of the action of inflammatory mediators. Lastly, the observation that certain drugs can induce GSTP1-1 oligomerization per se, as well as alter the pattern of 12-PGJ2-induced oligomerization, opens new possibilities for understanding the regulation of this complex system.
In the pathophysiological context, it should also be taken into account that there are several allelic variants of GSTP1-1, which differ in their efficiency to catalyze the conjugation of various drugs, including chlorambucil, to GSH [4], to modify the risk of developing severe adverse drug reactions with anticancer drugs [48] and to differentially regulate the ability of peroxiredoxin 6 to detoxify lipid peroxides [9]. Also, the frequent Ile105Val polymorphic variant (SNP id. rs1695) presents substantially higher affinity for certain GST inhibitors with antiproliferative properties [49]. GST polymorphisms may also influence the outcome of inflammatory responses, the capacity to cope with oxidative stress, the development of certain allergic diseases [8] and the risk of developing colorectal cancer [50]. Whether the allelic variants of GSTP1-1 present different oligomerization behavior remains to be explored.
In summary, assessment of GSTP1-1 oligomerization in the different experimental systems used in this study offers complementary information. Whereas intact cells represent the most relevant system, S100 fractions avoid indirect effects arising from the induction of cell toxicity or oxidative stress by the agents employed while preserving potential hetero-oligomerization partners, which are absent in the purified protein preparations. Integration of the information obtained from these various experimental systems suggests that GSTP1-1 behaves as a sensor for both inflammatory agents and reactive drugs, which may act in concert to modulate GSTP1-1 oligomerization and functions.
CONFLICT OF INTEREST
The authors confirm that this article content has no conflicts of interest.
ACKNOWLEDGEMENTS
We are indebted to B. Díez-Dacal for help with preliminary experiments and to M.J. Carrasco for technical assistance. This work was supported by grants from MINECO SAF2009-11642 and SAF2012-36519 to DP-S and BFU2005-00050, BFU2008-00666 and BFU2009-08977 to MAP and from ISCIII, RETIC RD07/0064/0007 and RD12/0013/0008 to DPS and RD12/0013/0002 to JAGA.
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Received: February 19, 2013 Revised: April 6, 2013 Accepted: April 12, 2013
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