Protective effects of 15-deoxy-Delta12,14-prostaglandin J2 against glutamate-induced cell death in primary cortical neuron cultures: induction of adaptive response and enhancement of cell tolerance primarily through up-regulation of cellular glutathione

There is increasing evidence to suggest that reactive oxygen species, including a variety of lipid oxidation products and other physiologically existing oxidative stimuli, can induce an adaptive response and enhance cell tolerance. In the present study, by using cultured cortical neurons, we investigated the effect of electrophilic lipids, such as 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)) and 4-hydroxy-2-nonenal (4-HNE) against the cell death induced by H(2)O(2) and glutamate. Pre-treatment with both 15d-PGJ(2) and 4-HNE at sublethal concentrations resulted in a significant protective effect against oxidative stress, and 15d-PGJ(2), in particular, exhibited a complete protective effect against glutamate-induced neuronal cell death. Pre-treatment with 15d-PGJ(2) increased the intracellular glutathione (GSH) as well as the gene expression of glutamate-cysteine ligase (GCL), the rate-limiting enzyme of GSH synthesis. 15d-PGJ(2) protected cells from glutamate-induced GSH depletion, while the inhibition of cellular GSH synthesis by buthionine sulfoximine abolished the adaptive response induced by 15d-PGJ(2). These findings indicate that at low levels, 15d-PGJ(2) acts as a potent survival mediator against glutamate-induced insults via the induction of an adaptive response primarily through the up-regulation of the intracellular GSH synthesis.     

Identification of actin as a 15-deoxy-Delta12,14-prostaglandin J2 target in neuroblastoma cells: mass spectrometric, computational, and functional approaches to investigate the effect on cytoskeletal derangement

A proteomic approach was used to identify 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2) protein targets in human neuroblastoma SH-SY5Y cells. By using biotinylated 15d-PGJ2, beta-actin was found as the major adducted protein; at least 12 proteins were also identified as minor biotin-positive spots, falling in different functional classes, including glycolytic enzymes (enolase and lactate dehydrogenase), redox enzymes (biliverdin reductase), and a eukaryotic regulatory protein (14-3-3gamma). 15d-PGJ2 induced marked morphological changes in the actin filament network and in particular promoted F-actin depolymerization as confirmed by Western blot analysis. By using a mass spectrometric approach, we found that 15d-PGJ2 reacts with isolated G-actin in a 1:1 stoichiometric ratio and selectively binds the Cys374 site through a Michael adduction mechanism. Computational studies showed that the covalent binding of 15d-PGJ2 induces a significant unfolding of actin structure and in particular that 15d-PGJ2 distorts the actin subdomains 2 and 4, which define the nucleotide binding sites impeding the nucleotide exchange. The functional effect of 15d-PGJ2 on G-actin was studied by polymerization measurement: in the presence of 15d-PGJ2, a lower amount of F-actin forms, as followed by the increase in pyrenyl-actin fluorescence intensity, as the major effect of increasing 15d-PGJ2 concentrations occurs on the maximum extent of actin polymerization, whereas it is negligible on the initial rate of reaction. In summary, the results here reported give an insight into the role of 15d-PGJ2 as a cytotoxic compound in neuronal cell dysfunction. Actin is the main protein cellular target of 15d-PGJ2, which specifically binds through a Michael adduction to Cys374, leading to a protein conformational change that can explain the disruption of the actin cytoskeleton, F-actin depolymerization, and impairment of G-actin polymerization.     

Accumulation of 15-deoxy-delta(12,14)-prostaglandin J2 adduct formation with Keap1 over time: effects on potency for intracellular antioxidant defence induction

The COX (cyclo-oxygenase) pathway generates the reactive lipid electrophile 15d-PGJ2 (15-deoxy-Delta(12,14)-prostaglandin J2), which forms covalent protein adducts that modulate cell signalling pathways. It has been shown that this regulates important biological responses, including protection against oxidative stress, and supports the proposal that 15d-PGJ2 has pharmacological potential. Protective pathways activated by 15d-PGJ2 include those controlling the synthesis of the intracellular antioxidants GSH and the enzyme HO-1 (haem oxygenase-1). The induction of the synthesis of these intracellular antioxidants is, in large part, regulated by covalent modification of Keap1 (Kelchlike erythroid cell-derived protein with 'capn'collar homologyassociated protein 1) by the lipid and the subsequent activation of the EpRE (electrophile-response element). For the first time, we show that the potency of 15d-PGJ2 as a signalling molecule in endothelial cells is significantly enhanced by the accumulation of the covalent adduct with 15d-PGJ2 and endogenous Keap1 over the time of exposure to the prostaglandin. The consequence of this finding is that signalling initiated by electrophilic lipids differs from agonists that do not form covalent adducts with proteins because the constant generation of very lowconcentrations of 15d-PGJ2 can lead to induction of GSH or HO-1. In the course of these studies we also found that a substantial amount (97-99%) of exogenously added 15d-PGJ2 is inactivated in the medium and does not enter the cells to initiate cell signalling. In summary, we propose that the accumulation of covalent adduct formation with signalling proteins provides a mechanism through which endogenous intracellular formation of electrophilic lipids from COX can exert an anti-inflammatory effect in vivo.     

Myosin and actin expression and oxidation in aging muscle.

While the age-related loss in muscle mass partially explains the decline in strength, other yet undefined mechanisms contribute. This study investigates whether changes in myosin-actin stoichiometry and oxidative modification could help explain the decrement in muscle strength with aging. Protein expression and oxidation were evaluated in myosin and actin isolated from the soleus and semimembranosus muscles from young adult, old, and very old Fischer 344 rats. In the soleus muscle, actin and myosin content did not change with aging. In the semimembranosus, actin content was stable, but myosin exhibited decreased content in muscles from very old rats, resulting in a decrease in the myosin-to-actin ratio. 3-Nitrotyrosine and 4-hydroxy-2-nonenal were used as markers of protein oxidative damage. Although myosin and actin are modified with 3-nitrotyrosine and 4-hydroxy-2-nonenal, the extent of chemical modification does not increase with age. The results suggest that the decline in force production with age is not due to the accumulation of these two specific markers of protein oxidation on the myofibrillar proteins. Additionally, age-dependent changes in myofibrillar stoichiometry do not contribute to the decline in force production in the soleus, but may play a role in the semimembranosus with advanced age.     

Reactions of 4-hydroxynonenal with proteins and cellular targets

Peroxidative degradation of lipids yields the aldehyde 4-hydroxy-2-nonenal (4HNE) as a major product. The lipid aldehyde is an electrophile, and reactivity of 4HNE toward protein nucleophiles (i.e., Cys, His, and Lys) has been characterized. Through the use of purified enzymes and isolated cells, various pathways for biotransformation of the lipid aldehyde have been identified and include enzyme-mediated oxidation, reduction, and glutathione conjugation. Uncontrolled oxidative stress can yield excessive lipid peroxidation and 4HNE generation, however, and overwhelm these cellular defenses. Indeed, in vitro and in vivo production of 4HNE in response to pro-oxidant exposure has been demonstrated using antibodies to protein adducts of the lipid aldehyde. Recent evidence suggests a role for protein modification by 4HNE in the pathogenesis of several diseases (e.g., alcohol-induced liver disease); however, the precise mechanism(s) is currently unknown but likely results from adduction of proteins involved in cellular homeostasis or biological signaling.     

Proteomic identification of protein targets for 15-deoxy-Δ(12,14)-prostaglandin J2 in neuronal plasma membrane

15-deoxy-Δ(12,14)-prostaglandin J(2) (15d-PGJ(2)) is one of factors contributed to the neurotoxicity of amyloid β (Aβ), a causative protein of Alzheimer's disease. Type 2 receptor for prostaglandin D(2) (DP2) and peroxysome-proliferator activated receptorγ (PPARγ) are identified as the membrane receptor and the nuclear receptor for 15d-PGJ(2), respectively. Previously, we reported that the cytotoxicity of 15d-PGJ(2) was independent of DP2 and PPARγ, and suggested that 15d-PGJ(2) induced apoptosis through the novel specific binding sites of 15d-PGJ(2) different from DP2 and PPARγ. To relate the cytotoxicity of 15d-PGJ(2) to amyloidoses, we performed binding assay [(3)H]15d-PGJ(2) and specified targets for 15d-PGJ(2) associated with cytotoxicity. In the various cell lines, there was a close correlation between the susceptibilities to 15d-PGJ(2) and fibrillar Aβ. Specific binding sites of [(3)H]15d-PGJ(2) were detected in rat cortical neurons and human bronchial smooth muscle cells. When the binding assay was performed in subcellular fractions of neurons, the specific binding sites of [(3)H]15d-PGJ(2) were detected in plasma membrane, nuclear and cytosol, but not in microsome. A proteomic approach was used to identify protein targets for 15d-PGJ(2) in the plasma membrane. By using biotinylated 15d-PGJ(2), eleven proteins were identified as biotin-positive spots and classified into three different functional proteins: glycolytic enzymes (Enolase2, pyruvate kinase M1 (PKM1) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH)), molecular chaperones (heat shock protein 8 and T-complex protein 1 subunit α), cytoskeletal proteins (Actin β, F-actin-capping protein, Tubulin β and Internexin α). GAPDH, PKM1 and Tubulin β are Aβ-interacting proteins. Thus, the present study suggested that 15d-PGJ(2) plays an important role in amyloidoses not only in the central nervous system but also in the peripheral tissues.     

Differential selectivity of protein modification by the cyclopentenone prostaglandins PGA1 and 15-deoxy-Delta12,14-PGJ2: role of glutathione

Cyclopentenone prostaglandins (cyPG) with antiinflammatory and antiproliferative properties have been envisaged as leads for the development of therapeutic agents. Because cyPG effects are mediated in part by the formation of covalent adducts with critical signaling proteins, it is important to assess the specificity of this interaction. By using biotinylated derivatives of 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)-B) and PGA(1) (PGA(1)-B) we herein provide novel evidence for the differential selectivity of protein modification by distinct cyPG. The marked quantitative and qualitative differences in the binding of 15d-PGJ(2)-B and PGA(1)-B to cellular proteins were related to a differential reactivity in the presence of glutathione (GSH), both in vitro and in intact cells. Therefore GSH levels may influence not only the intensity but also the specificity of cyPG action.     

Site-specific proteomic analysis of lipoxidation adducts in cardiac mitochondria reveals chemical diversity of 2-alkenal adduction

The modification of proteins by lipid peroxidation products has been linked to numerous diseases and age-related disorders. Here we report on the identification of endogenous protein targets of electrophilic 2-alkenals in cardiac mitochondria. An aldehyde/keto-specific chemical labeling and affinity strategy in combination with LC-MS/MS resulted in 39 unique lipoxidation sites on 27 proteins. Several of the target sites were modified by a variety of 2-alkenal products including acrolein, β-hydroxyacrolein, crotonaldehyde, 4-hydroxy-2-hexenal, 4-hydroxy-2-nonenal and 4-oxo-2-nonenal. Many of the adduction sites are implicated in the catalytic function of key mitochondrial enzymes suggesting potential impact on pathways and overall mitochondrial function.     

Interaction of electrophilic lipid oxidation products with mitochondria in endothelial cells and formation of reactive oxygen species

Electrophilic lipids, such as 4-hydroxynonenal (HNE), and the cyclopentenones 15-deoxy-Delta12,14 -prostaglandin J2 (15d-PGJ2) and 15-J2-isoprostane induce both reactive oxygen species (ROS) formation and cellular antioxidant defenses, such as heme oxygenase-1 (HO-1) and glutathione (GSH). When we compared the ability of these distinct electrophiles to stimulate GSH and HO-1 production, the cyclopentenone electrophiles were somewhat more potent than HNE. Over the concentration range required to observe equivalent induction of GSH, dichlorofluorescein fluorescence was used to determine both the location and amounts of electrophilic lipid-dependent ROS formation in endothelial cells. The origin of the ROS on exposure to these compounds was largely mitochondrial. To investigate the possibility that the increased ROS formation was due to mitochondrial localization of the lipids, we prepared a novel fluorescently labeled form of the electrophilic lipid 15d-PGJ2. The lipid demonstrated strong colocalization with the mitochondria, an effect which was not observed by using a fluorescently labeled nonelectrophilic lipid. The role of mitochondria was confirmed by using cells deficient in functional mitochondria. On the basis of these data, we propose that ROS formation in endothelial cells is due to the direct interaction of these lipids with the organelle.     

Proteome Analysis Identified the PPARγ Ligand 15d-PGJ2 as a Novel Drug Inhibiting Melanoma Progression and Interfering with Tumor-Stroma Interaction

Peroxisome proliferator-activated receptors (PPARs) have been originally thought to be restricted to lipid metabolism or glucose homeostasis. Recently, evidence is growing that PPARγ ligands have inhibitory effects on tumor growth. To shed light on the potential therapeutic effects on melanoma we tested a panel of PPAR agonists on their ability to block tumor proliferation in vitro. Whereas ciglitazone, troglitazone and WY14643 showed moderate effects on proliferation, 15d-PGJ2 displayed profound anti-tumor activity on four different melanoma cell lines tested. Additionally, 15d-PGJ2 inhibited proliferation of tumor-associated fibroblasts and tube formation of endothelial cells. 15d-PGJ2 induced the tumor suppressor gene p21, a G₂/M arrest and inhibited tumor cell migration. Shot gun proteome analysis in addition to 2D-gel electrophoresis and immunoprecipitation of A375 melanoma cells suggested that 15d-PGJ2 might exert its effects via modification and/or downregulation of Hsp-90 (heat shock protein 90) and several chaperones. Applying the recently established CPL/MUW database with a panel of defined classification signatures, we demonstrated a regulation of proteins involved in metastasis, transport or protein synthesis including paxillin, angio-associated migratory cell protein or matrix metalloproteinase-2 as confirmed by zymography. Our data revealed for the first time a profound effect of the single compound 15d-PGJ2 on melanoma cells in addition to the tumor-associated microenvironment suggesting synergistic therapeutic efficiency.     

An endogenous electrophile that modulates the regulatory mechanism of protein turnover: inhibitory effects of 15-deoxy-Delta 12,14-prostaglandin J2 on proteasome

Prostaglandin D(2) (PGD(2)), a major cyclooxygenase product in a variety of tissues and cells, readily undergoes dehydration to yield electrophilic PGs, such as 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)). We have previously shown that 15d-PGJ(2) potently induces apoptosis of SH-SY5Y human neuroblastoma cells via accumulation of the tumor suppressor gene product p53. In the study presented here, we investigated the molecular mechanisms involved in the 15d-PGJ(2)-induced accumulation of p53. It was observed that 15d-PGJ(2) potently induced p53 protein expression but scarcely induced p53 gene expression. In addition, exposure of the cells to 15d-PGJ(2) resulted in an accumulation of ubiquitinated proteins and in a significant inhibition of proteasome activities, suggesting that 15d-PGJ(2) acted on the ubiquitin-proteasome pathway, a regulatory mechanism of p53 turnover. The effects of 15d-PGJ(2) on the protein turnover were attributed to its electrophilic feature, based on the observations that (i) the reduction of the double bond in the cyclopentenone ring of 15d-PGJ(2) virtually abolished the effects on protein turnover, (ii) overexpression of an endogenous redox regulator, thioredoxin 1, significantly retarded the inhibition of proteasome activities and accumulations of p53 and ubiquitinated proteins induced by 15d-PGJ(2), and (iii) treatment of SH-SY5Y cells with biotinylated 15d-PGJ(2) indeed resulted in the formation of a 15d-PGJ(2)-proteasome conjugate. These data suggest that the modulation of proteasome activity may be involved in the mechanism responsible for the accumulation of p53 and subsequent induction of apoptotic cell death induced by 15d-PGJ(2).     

Membrane-mediated amyloidogenesis and the promotion of oxidative lipid damage by amyloid beta proteins

Evidence of oxidative stress and the accumulation of fibrillar amyloid beta proteins (Abeta) in senile plaques throughout the cerebral cortex are consistent features in the pathology of Alzheimer disease. To define a mechanistic link between these two processes, various aspects of the relationship between oxidative lipid membrane damage and amyloidogenesis were characterized by chemical and physical techniques. Earlier studies of this relationship demonstrated that oxidatively damaged synthetic lipid membranes promoted amyloidogenesis. The studies reported herein specify that 4-hydroxy-2-nonenal (HNE) is produced in both synthetic lipids and human brain lipid extracts by oxidative lipid damage and that it can account for accelerated amyloidogenesis. Abeta promotes the copper-mediated generation of HNE from polyunsaturated lipids, and in turn, HNE covalently modifies the histidine side chains of Abeta. HNE-modified Abeta have an increased affinity for lipid membranes and an increased tendency to aggregate into amyloid fibrils. Thus, the prooxidant activity of Abeta leads to its own covalent modification and to accelerated amyloidogenesis. These results illustrate how lipid membranes may be involved in templating the pathological misfolding of Abeta, and they suggest a possible chemical mechanism linking oxidative stress with amyloid formation.     

The covalent modification of spectrin in red cell membranes by the lipid peroxidation product 4-hydroxy-2-nonenal

Spectrin strengthens the red cell membrane through its direct association with membrane lipids and through protein-protein interactions. Spectrin loss reduces the membrane stability and results in various types of hereditary spherocytosis. However, less is known about acquired spectrin damage. Here, we showed that α- and β-spectrin in human red cells are the primary targets of the lipid peroxidation product 4-hydroxy-2-nonenal (HNE) by immunoblotting and mass spectrometry analyses. The level of HNE adducts in spectrin (particularly α-spectrin) and several other membrane proteins was increased following the HNE treatment of red cell membrane ghosts prepared in the absence of MgATP. In contrast, ghost preparation in the presence of MgATP reduced HNE adduct formation, with preferential β-spectrin modification and increased cross-linking of the HNE-modified spectrins. Exposure of intact red cells to HNE resulted in selective HNE-spectrin adduct formation with a similar preponderance of HNE-β-spectrin modifications. These findings indicate that HNE adduction occurs preferentially in spectrin at the interface between the skeletal proteins and lipid bilayer in red cells and suggest that HNE-spectrin adduct aggregation results in the extrusion of damaged spectrin and membrane lipids under physiological and disease conditions.     

A 4-oxo-2(E)-nonenal-derived glutathione adduct from 15-lipoxygenase-1-mediated oxidation of cytosolic and esterified arachidonic acid

15(S)-Hydroperoxy-[5Z,8Z,11Z,13E]-eicosatetraenoic acid (15(S)-HpETE) undergoes homolytic decomposition to bifunctional electrophiles such as 4-oxo-2(E)-nonenal. 4-Oxo-2(E)-nonenal reacts with glutathione to form a thiadiazabicyclo-4-oxo-2(E)-nonenal–glutathione adduct (TOG). Therefore, this endogenous glutathione adduct can serve as a specific biomarker of lipid hydroperoxide-mediated 4-oxo-2(E)-nonenal formation. A monocyte/macrophage cell line was generated to constitutively express human 15-lipoxygenase-1. In these cells, TOG was formed from 15(S)-HpETE-derived 4-oxo-2(E)-nonenal in a nonlinear dose-dependent manner upon arachidonic acid treatment. The lipoxygenase inhibitor cinnamyl-3,4-dihydroxy-α-cyanocinnamate abolished arachidonic acid-mediated TOG formation. The calcium ionophore A23187 was also used to induce the formation of 15(S)-HpETE from esterified arachidonic acid present in the membrane lipids. In the 15-lipoxygenase-1-expressing cells, the calcium ionophore A23187 significantly increased TOG levels compared with mock-transfected cells. This was due to the 15-lipoxygenase-mediated formation of 15(S)-HpETE in the forms of free fatty acid and esterified lipids, which was subsequently converted to 4-oxo-2(E)-nonenal. The increase in TOG formation was again abrogated by pretreatment with cinnamyl-3,4-dihydroxy-α-cyanocinnamate. Only 8.7% 15(S)-HETE (both the free fatty acid and its esterified form in the cell membrane) was formed after ionophore A23187 stimulation compared with that formed after the addition of arachidonic acid. In contrast, the TOG levels after treatment with ionophore A23187 or arachidonic acid were comparable. Thus, it is likely that esterified 15(S)-HpETE underwent homolytic decomposition to 4-oxo-2(E)-nonenal more efficiently than the free 15(S)-HpETE that was formed in the cytosol.     

Role of mitogen-activated protein kinases in 4-hydroxy-2-nonenal-induced actin remodeling and barrier function in endothelial cells

In vivo and in vitro studies indicate that 4-hydroxy-2-nonenal (4-HNE), generated by cellular lipid peroxidation or after oxidative stress, affects endothelial permeability and vascular tone. However, the mechanism(s) of 4-HNE-induced endothelial barrier function is not well defined. Here we provide evidence for the first time on the involvement of mitogen-activated protein kinases (MAPKs) in 4-HNE-mediated actin stress fiber formation and barrier function in lung endothelial cells. Treatment of bovine lung microvascular endothelial cells with hydrogen peroxide (H(2)O(2)), as a model oxidant, resulted in accumulation of 4-HNE as evidenced by the formation of 4-HNE-Michael protein adducts. Exposure of cells to 4-HNE, in a dose- and time-dependent manner, decreased endothelial cell permeability measured as transendothelial electrical resistance. The 4-HNE-induced permeability changes were not because of cytotoxicity or endothelial cell apoptosis, which occurred after prolonged treatment and at higher concentrations of 4-HNE. 4-HNE-induced changes in transendothelial electrical resistance were calcium independent, as 4-HNE did not alter intracellular free calcium levels as compared with H(2)O(2) or diperoxovanadate. Stimulation of quiescent cells with 4-HNE (1-100 microm) resulted in phosphorylation of ERK1/2, JNK, and p38 MAPKs, and actin cytoskeleton remodeling. Furthermore, pretreatment of bovine lung microvascular endothelial cells with PD 98059 (25 microm), an inhibitor of MEK1/2, or SP 600125 (25 microm), an inhibitor of JNK, or SB 202190 (25 microm), an inhibitor of p38 MAPK, partially attenuated 4-HNE-mediated barrier function and cytoskeletal remodeling. These results suggest that the activation of ERK, JNK, and p38 MAP kinases is involved in 4-HNE-mediated actin remodeling and endothelial barrier function.     

Thioredoxin as a molecular target of cyclopentenone prostaglandins

Prostaglandin (PG) D2, a major cyclooxygenase product in a variety of tissues and cells, readily undergoes dehydration to yield the bioactive cyclopentenone-type PGs of the J2 series, such as 15-deoxy-Delta12,14-PGJ2 (15d-PGJ2). We have shown previously that 15d-PGJ2 is a potent electrophile that causes intracellular oxidative stress and redox alteration in human neuroblastoma SH-SY5Y cells. In the present study, based on the observation that the electrophilic center of 15d-PGJ2 was involved in the pro-oxidant effect, we investigated the role of thioredoxin 1 (Trx), an endogenous redox regulator, against 15d-PGJ2-induced oxidative cell injury. It was observed that the 15d-PGJ2-induced oxidative stress was significantly suppressed by the Trx overexpression. In addition, the treatment of SH-SY5Y cells with biotinylated 15d-PGJ2 resulted in the formation of a 15d-PGJ2-Trx adduct, indicating that 15d-PGJ2 directly modified the endogenous Trx in the cells. To further examine the mechanism of the 15d-PGJ2 modification of Trx, human recombinant Trx treated with 15d-PGJ2 was analyzed by mass spectrometry. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis of the 15d-PGJ2-treated human recombinant Trx demonstrated the addition of one molecule of 15d-PGJ2 per protein molecule. Moreover, the electrospray ionization-liquid chromatography/mass spectrometry/mass spectrometry analysis identified two cysteine residues, Cys-35 and Cys-69, as the targets of 15d-PGJ2. These residues may represent the direct sensors of the electrophilic PGs that induce the intracellular redox alteration and neuronal cell death.     

Modification and activation of Ras proteins by electrophilic prostanoids with different structure are site-selective

Cyclopentenone prostanoids (cyP) arise as important modulators of inflammation and cell proliferation. Although their physiological significance has not been fully elucidated, their potent biological effects have spurred their study as leads for the development of therapeutic agents. A key determinant of cyP action is their ability to bind to thiol groups in proteins or in glutathione through Michael addition. Even though several protein targets for cyP addition have been identified, little is known about the structural determinants from the protein or the cyP that drive this modification. The results herein presented provide the first evidence that cyP with different structures target distinct thiol sites in a protein molecule, namely, H-Ras. Whereas 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2) and Delta12-PGJ2 preferentially target the C-terminal region containing cysteines 181 and 184, PGA1 and 8-iso-PGA1 bind mainly to cysteine 118, located in the GTP-binding motif. The biological counterparts of this specificity are the site-selective modification and activation of H-Ras in cells and the differential interaction of cyP with H, N, and K-Ras proteins. Cysteine 184 is unique to H-Ras, whereas cysteine 118 is present in the three Ras homologues. Consistent with this, PGA1 binds to and activates H-, N-, and K-Ras, thus differing from the preferential interaction of 15d-PGJ2 with H-Ras. These results put forward the possibility of influencing the selectivity of cyP-protein addition by modifying cyP structure. Furthermore, they may open new avenues for the development of cyP-based drugs.