Amoxicillin Haptenation of α-Enolase is Modulated by Active Site Occupancy and Acetylation.

Allergic reactions to antibiotics are a major concern in the clinic. ß-lactam antibiotics are the class most frequently reported to cause hypersensitivity reactions. One of the mechanisms involved in this outcome is the modification of proteins by covalent binding of the drug (haptenation). Hence, interest in identifying the corresponding serum and cellular protein targets arises. Importantly, haptenation susceptibility and extent can be modulated by the context, including factors affecting protein conformation or the occurrence of other posttranslational modifications. We previously identified the glycolytic enzyme α-enolase as a target for haptenation by amoxicillin, both in cells and in the extracellular milieu. Here, we performed an in vitro study to analyze amoxicillin haptenation of α-enolase using gel-based and activity assays. Moreover, the possible interplay or interference between amoxicillin haptenation and acetylation of α-enolase was studied in 1D- and 2D-gels that showed decreased haptenation and displacement of the haptenation signal to lower pI spots after chemical acetylation of the protein, respectively. In addition, the peptide containing lysine 239 was identified by mass spectrometry as the amoxicillin target sequence on α-enolase, thus suggesting a selective haptenation under our conditions. The putative amoxicillin binding site and the surrounding interactions were investigated using the α-enolase crystal structure and molecular docking. Altogether, the results obtained provide the basis for the design of novel diagnostic tools or approaches in the study of amoxicillin-induced allergic reactions.

A non-randomized prospective study on the diagnostic performance of perineal prostatic biopsy, directed via diffusion nuclear resonance, in patients with suspected prostate cancer and previous negative transrectal prostate biopsy.

BACKGROUND: A definition of the best strategy is necessary to optimize the follow-up of patients with previous negative transrectal guided ultrasound biopsy (TRUS-GB) and the persistence of raised prostate-specific antigen (PSA).The purpose of this study was to evaluate the prostate cancer (PCa) diagnostic rate of targeted transperineal ultrasound guided biopsy (TPUS-GB) with cognitive multiparametric magnetic resonance imaging (mpMRI) registration with concurrent systematic biopsy in patients with previous negative systematic TRUS-GB and persistently elevated PSA levels. MATERIALS AND METHODS: In this prospective study conducted at the University Infanta Sofia Hospital from April 2016 to November 2017, patients with one previous negative systematic TRUS-GB and persistently high PSA levels were referred for mpMRI prostate scans. All patients underwent systematic TPUS-GB and those patients with suspicious findings on mpMRI scans, Pirads 3 and 4-5, underwent a subsequent cognitive guidance mpMRI-TPUS-GB. RESULTS: In total, 71 patients were included in this study. Suspicious findings on mpMRI scans prior to TPUS-GB were found in 50 patients (70.4%). 16 patients were diagnosed with prostate cancer (22.5%), of whom 14 (87.5%) had a mpMRI scan with Pirads 3 or Pirads 4-5. Patients with Pirads 3, 4 or 5 showed negative results in almost all cores taken by concurrent systematic TPUS-GB. CONCLUSIONS: Cognitive mpMRI-TPUS fusion biopsy is a useful tool to diagnose PCa in patients with previous negative prostate biopsy. The samples obtained from the suspicious areas in the mpMRI detect more cases of intermediate and high risk PCa compared to the samples obtained at random or from non-suspicious areas.

Amoxicillin Inactivation by Thiol-Catalyzed Cyclization Reduces Protein Haptenation and Antibacterial Potency.

Serum and cellular proteins are targets for the formation of adducts with the ß-lactam antibiotic amoxicillin. This process could be important for the development of adverse, and in particular, allergic reactions to this antibiotic. In studies exploring protein haptenation by amoxicillin, we observed that reducing agents influenced the extent of amoxicillin-protein adducts formation. Consequently, we show that several thiol-containing compounds, including dithiothreitol, N-acetyl-L-cysteine, and glutathione, perform a nucleophilic attack on the amoxicillin molecule that is followed by an internal rearrangement leading to amoxicillin diketopiperazine, a known amoxicillin metabolite with residual activity. Increased diketopiperazine conversion is also observed with human serum albumin but not with L-cysteine, which mainly forms the amoxicilloyl amide. The effect of thiols is catalytic and can render complete amoxicillin conversion. Interestingly, this process is dependent on the presence of an amino group in the antibiotic lateral chain, as in amoxicillin and ampicillin. Furthermore, it does not occur for other ß-lactam antibiotics, including cefaclor or benzylpenicillin. Biological consequences of thiol-mediated amoxicillin transformation are exemplified by a reduced bacteriostatic action and a lower capacity of thiol-treated amoxicillin to form protein adducts. Finally, modulation of the intracellular redox status through inhibition of glutathione synthesis influenced the extent of amoxicillin adduct formation with cellular proteins. These results open novel perspectives for the understanding of amoxicillin metabolism and actions, including the formation of adducts involved in allergic reactions.

Biotin-Labelled Clavulanic Acid to Identify Proteins Target for Haptenation in Serum: Implications in Allergy Studies.

Clavulanic acid (CLV) and amoxicillin, frequently administered in combination, can be independently involved in allergic reactions. Protein haptenation with ß-lactams is considered necessary to activate the immune system. The aim of this study was to assess the suitability of biotinylated analogues of CLV as probes to study protein haptenation by this ß-lactam. Two synthetic approaches afforded the labeling of CLV through esterification of its carboxylic group with a biotin moiety, via either direct binding (CLV-B) or tetraethylenglycol linker (CLV-TEG-B). The second analogue offered advantages as solubility in aqueous solution and potential lower steric hindrance for both intended interactions, with the protein and with avidin. NMR reactivity studies showed that both CLV and CLV-TEG-B reacts through ß-lactam ring opening by aliphatic amino nitrogen, however with different stability of resulting conjugates. Unlike CLV conjugates, that promoted the decomposition of clavulanate fragment, the conjugates obtained with the CLV-TEG-B remained linked, as a whole structure including biotin, to nucleophile and showed a better stability. This was a desired key feature to allow CLV-TEG-B conjugated protein detection at great sensitivity. We have used biotin detection and mass spectrometry (MS) to detect the haptenation of human serum albumin (HSA) and human serum proteins. MS of conjugates showed that HSA could be modified by CLV-TEG-B. Remarkably, HSA preincubation with CLV excess only reduced moderately the incorporation of CLV-TEG-B, which could be attributed to different protein interferences. The CLV-TEG-B fragment with opened ß-lactam was detected bound to the 404-430HSA peptide of the treated protein. Incubation of human serum with CLV-TEG-B resulted in the haptenation of several proteins that were identified by 2D-electrophoresis and peptide mass fingerprinting as HSA, haptoglobin, and heavy and light chains of immunoglobulins. Taken together, our results show that tagged-CLV keeps some of the CLV features. Moreover, although we observe a different behavior in the conjugate stability and in the site of protein modification, the similar reactivity indicates that it could constitute a valuable tool to identify protein targets for haptenation by CLV with high sensitivity to get insights into the activation of the immune system by CLV and mechanisms involved in ß-lactams allergy.

Asthma and allergic rhinitis associate with the rs2229542 variant that induces a p.Lys90Glu mutation and compromises AKR1B1 protein levels.

Asthma and rhinitis are two of the main clinical manifestations of allergy, in which increased reactive oxygen or electrophilic species can play a pathogenic role. Aldose reductase (AKR1B1) is involved in aldehyde detoxification and redox balance. Recent evidence from animal models points to a role of AKR1B1 in asthma and rhinitis, but its involvement in human allergy has not been addressed. Here, the putative association of allergic rhinitis and asthma with AKR1B1 variants has been explored by analysis of single-strand variants on the AKR1B1 gene sequence in 526 healthy subjects and 515 patients with allergic rhinitis, 366 of whom also had asthma. We found that the rs2229542 variant, introducing the p.Lys90Glu mutation, was significantly more frequent in allergic patients than in healthy subjects. Additionally, in cells transfected with expression vectors carrying the wild-type or the p.Lys90Glu variant of AKR1B1, the mutant consistently attained lower protein levels than the wild-type and showed a compromised thermal stability. Taken together, our results show that the rs2229542 variant associates with asthma and rhinitis, and hampers AKR1B1 protein levels and stability. This unveils a connection between the genetic variability of aldose reductase and allergic processes.

Prospective nonrandomized study of diagnostic accuracy comparing prostate cancer detection by transrectal ultrasound-guided biopsy to magnetic resonance imaging with subsequent MRI-guided biopsy in biopsy-naïve patients.

BACKGROUND: To evaluate the diagnostic efficacy in cancer prostate (PCa) of Multiparametric prostate magnetic resonance imaging (mp-MRI) targeted biopsy compared to standard systematic transrectal ultrasound-guided biopsy (TRUSGB) in biopsy-naïve patients. METHODS: A total of 168 biopsy-naïve men with clinical suspicion of PCa due to elevated PSA levels and/or an abnormal digital rectal examination were consecutively enrolled from July 2011 to July 2014. All patients underwent TRUSGB. Patients with equivocal (Pi-rads 3) or suspicious lesion (Pi-rads 4-5), were additionally biopsied using two cores, by the same operator (cognitive technique). RESULTS: Among the 168 cases, mp-MRI was equivocal for PCa (Pi-rads 3) in 46 subjects (27.4%) and suspicious (Pi-rads 4, 5) in 40 cases (23.8%). Of the 69 patients with PCa, standard TRUSGB showed Gleason ≥7 in 75% of patients with Pirads 3 and 77.8% in cases with Pirads 4-5 on mp-MRI. Among the 40 patients with Pi-rads 4-5 lesion on the MRI, cognitive mp-MRI-guided biopsy (MRCGB) detected a higher number of cases of PCa with a Gleason score equal or superior to 7 (90%) with a higher negative predictive value (97.5%) than cases with Pi-rads 3 lesion or subjects with TRUSGB alone. CONCLUSIONS: mp-MRI followed by selective biopsy seems to be a valuable tool to improve the diagnosis of intermediate and high risk PCa compared to standard TRUSGB.

Adduct Formation and Context Factors in Drug Hypersensitivity: Insight from Proteomic Studies.

Drug hypersensitivity reactions result from the activation of the immune system by drugs or their metabolites. The clinical presentations of drug hypersensitivity can range from relatively mild local manifestations to severe systemic syndromes that can be lifethreatening. As in other allergic reactions, the causes are multifactorial as genetic, metabolic and concomitant factors may influence the occurrence of drug hypersensitivity. Formation of drug protein adducts is considered a key step in drug adverse reactions, and in particular in the immunological recognition in drug hypersensitivity reactions. Nevertheless, noncovalent interactions of drugs with receptors in immune cells or with MHC clefts and/or exposed peptides can also play an important role. In recent years, development of proteomic approaches has allowed the identification and characterization of the protein targets for modification by drugs in vivo and in vitro, the nature of peptides exposed on MHC molecules, the changes in protein levels induced by drug treatment, and the concomitant modifications induced by danger signals, thus providing insight into context factors. Nevertheless, given the complexity and multifactorial nature of drug hypersensitivity reactions, understanding the underlying mechanisms also requires the integration of knowledge from genomic, metabolomic and clinical studies.

Detoxifying Enzymes at the Cross-Roads of Inflammation, Oxidative Stress, and Drug Hypersensitivity: Role of Glutathione Transferase P1-1 and Aldose Reductase.

Phase I and II enzymes are involved in the metabolism of endogenous reactive compounds as well as xenobiotics, including toxicants and drugs. Genotyping studies have established several drug metabolizing enzymes as markers for risk of drug hypersensitivity. However, other candidates are emerging that are involved in drug metabolism but also in the generation of danger or costimulatory signals. Enzymes such as aldo-keto reductases (AKR) and glutathione transferases (GST) metabolize prostaglandins and reactive aldehydes with proinflammatory activity, as well as drugs and/or their reactive metabolites. In addition, their metabolic activity can have important consequences for the cellular redox status, and impacts the inflammatory response as well as the balance of inflammatory mediators, which can modulate epigenetic factors and cooperate or interfere with drug-adduct formation. These enzymes are, in turn, targets for covalent modification and regulation by oxidative stress, inflammatory mediators, and drugs. Therefore, they constitute a platform for a complex set of interactions involving drug metabolism, protein haptenation, modulation of the inflammatory response, and/or generation of danger signals with implications in drug hypersensitivity reactions. Moreover, increasing evidence supports their involvement in allergic processes. Here, we will focus on GSTP1-1 and aldose reductase (AKR1B1) and provide a perspective for their involvement in drug hypersensitivity.

Molecular Interactions and Implications of Aldose Reductase Inhibition by PGA1 and Clinically Used Prostaglandins.

Aldose reductase (AKR1B1) is a critical drug target because of its involvement in diabetic complications, inflammation, and tumorigenesis. However, to date, development of clinically useful inhibitors has been largely unsuccessful. Cyclopentenone prostaglandins (cyPGs) are reactive lipid mediators that bind covalently to proteins and exert anti-inflammatory and antiproliferative effects in numerous settings. By pursuing targets for modification by cyPGs we have found that the cyPG PGA1 binds to and inactivates AKR1B1. A PGA1-AKR1B1 adduct was observed, both by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and by SDS-PAGE using biotinylated PGA1 (PGA1-B). Insight into the molecular interactions between AKR1B1 and PGA1 was advanced by molecular modeling. This anticipated the addition of PGA1 to active site Cys298 and the potential reversibility of the adduct, which was supported experimentally. Indeed, loss of biotin label from the AKR1B1-PGA1-B adduct was favored by glutathione, indicating a retro-Michael reaction, which unveils new implications of cyPG-protein interaction. PGA1 elicited only marginal inhibition of aldehyde reductase (AKR1A1), considered responsible for the severe adverse effects of many AKR1B1 inhibitors. Interestingly, other prostaglandins (PGs) inhibited the enzyme, including non-electrophilic PGE1 and PGE2, currently used in clinical practice. Moreover, both PGA1 and PGE1 reduced the formation of sorbitol in an ex-vivo model of diabetic cataract to an extent comparable to that attained by the known AKR inhibitor epalrestat. Taken together, these results highlight the role of PGs as AKR1B1 inhibitors and the interest in PG-related molecules as leads for the development of novel pharmacological tools.

NOX4-dependent Hydrogen peroxide promotes shear stress-induced SHP2 sulfenylation and eNOS activation.

Laminar shear stress (LSS) triggers signals that ultimately result in atheroprotection and vasodilatation. Early responses are related to the activation of specific signaling cascades. We investigated the participation of redox-mediated modifications and in particular the role of hydrogen peroxide (H2O2) in the sulfenylation of redox-sensitive phosphatases. Exposure of vascular endothelial cells to short periods of LSS (12 dyn/cm(2)) resulted in the generation of superoxide radical anion as detected by the formation of 2-hydroxyethidium by HPLC and its subsequent conversion to H2O2, which was corroborated by the increase in the fluorescence of the specific peroxide sensor HyPer. By using biotinylated dimedone we detected increased total protein sulfenylation in the bovine proteome, which was dependent on NADPH oxidase 4 (NOX4)-mediated generation of peroxide. Mass spectrometry analysis allowed us to identify the phosphatase SHP2 as a protein susceptible to sulfenylation under LSS. Given the dependence of FAK activity on SHP2 function, we explored the role of FAK under LSS conditions. FAK activation and subsequent endothelial NO synthase (eNOS) phosphorylation were promoted by LSS and both processes were dependent on NOX4, as demonstrated in lung endothelial cells isolated from NOX4-null mice. These results support the idea that LSS elicits redox-sensitive signal transduction responses involving NOX4-dependent generation of hydrogen peroxide, SHP2 sulfenylation, and ulterior FAK-mediated eNOS activation.

Protein lipoxidation: Detection strategies and challenges.

Enzymatic and non-enzymatic lipid metabolism can give rise to reactive species that may covalently modify cellular or plasma proteins through a process known as lipoxidation. Under basal conditions, protein lipoxidation can contribute to normal cell homeostasis and participate in signaling or adaptive mechanisms, as exemplified by lipoxidation of Ras proteins or of the cytoskeletal protein vimentin, both of which behave as sensors of electrophilic species. Nevertheless, increased lipoxidation under pathological conditions may lead to deleterious effects on protein structure or aggregation. This can result in impaired degradation and accumulation of abnormally folded proteins contributing to pathophysiology, as may occur in neurodegenerative diseases. Identification of the protein targets of lipoxidation and its functional consequences under pathophysiological situations can unveil the modification patterns associated with the various outcomes, as well as preventive strategies or potential therapeutic targets. Given the wide structural variability of lipid moieties involved in lipoxidation, highly sensitive and specific methods for its detection are required. Derivatization of reactive carbonyl species is instrumental in the detection of adducts retaining carbonyl groups. In addition, use of tagged derivatives of electrophilic lipids enables enrichment of lipoxidized proteins or peptides. Ultimate confirmation of lipoxidation requires high resolution mass spectrometry approaches to unequivocally identify the adduct and the targeted residue. Moreover, rigorous validation of the targets identified and assessment of the functional consequences of these modifications are essential. Here we present an update on methods to approach the complex field of lipoxidation along with validation strategies and functional assays illustrated with well-studied lipoxidation targets.

L-Plastin S-glutathionylation promotes reduced binding to ß-actin and affects neutrophil functions.

Posttranslational modifications (PTMs) of cytoskeleton proteins due to oxidative stress associated with several pathological conditions often lead to alterations in cell function. The current study evaluates the effect of nitric oxide (DETA-NO)-induced oxidative stress-related S-glutathionylation of cytoskeleton proteins in human PMNs. By using in vitro and genetic approaches, we showed that S-glutathionylation of L-plastin (LPL) and ß-actin promotes reduced chemotaxis, polarization, bactericidal activity, and phagocytosis. We identified Cys-206, Cys-283, and Cys-460as S-thiolated residues in the ß-actin-binding domain of LPL, where cys-460 had the maximum score. Site-directed mutagenesis of LPL Cys-460 further confirmed the role in the redox regulation of LPL. S-Thiolation diminished binding as well as the bundling activity of LPL. The presence of S-thiolated LPL was detected in neutrophils from both diabetic patients and db/db mice with impaired PMN functions. Thus, enhanced nitroxidative stress may results in LPL S-glutathionylation leading to impaired chemotaxis, polarization, and bactericidal activity of human PMNs, providing a mechanistic basis for their impaired functions in diabetes mellitus.

Acute hypoxia produces a superoxide burst in cells.

Oxygen is a key molecule for cell metabolism. Eukaryotic cells sense the reduction in oxygen availability (hypoxia) and trigger a series of cellular and systemic responses to adapt to hypoxia, including the optimization of oxygen consumption. Many of these responses are mediated by a genetic program induced by the hypoxia-inducible transcription factors (HIFs), regulated by a family of prolyl hydroxylases (PHD or EGLN) that use oxygen as a substrate producing HIF hydroxylation. In parallel to these oxygen sensors modulating gene expression within hours, acute modulation of protein function in response to hypoxia is known to occur within minutes. Free radicals acting as second messengers, and oxidative posttranslational modifications, have been implied in both groups of responses. Localization and speciation of the paradoxical increase in reactive oxygen species production in hypoxia remain debatable. We have observed that several cell types respond to acute hypoxia with a transient increase in superoxide production for about 10 min, probably originating in the mitochondria. This may explain in part the apparently divergent results found by various groups that have not taken into account the time frame of hypoxic ROS production. We propose that this acute and transient hypoxia-induced superoxide burst may be translated into oxidative signals contributing to hypoxic adaptation and preconditioning.

S-glutathionylation: relevance in diabetes and potential role as a biomarker.

Glutathione is considered the main regulator of redox balance in the cellular milieu due to its capacity for detoxifying deleterious molecules. The oxidative stress induced as a result of a variety of stimuli promotes protein oxidation, usually at cysteine residues, leading to changes in their activity. Mild oxidative stress, which may take place in physiological conditions, induces the reversible oxidation of cysteines to sulfenic acid form, while pathological conditions are associated with higher rates of reactive oxygen species production, inducing the irreversible oxidation of cysteines. Among these, neurodegenerative disorders, cardiovascular diseases and diabetes have been proposed to be pathogenetically linked to this state. In diabetes-associated vascular complications, lower levels of glutathione and increased oxidative stress have been reported. S-glutathionylation has been proposed as a posttranslational modification able to protect proteins from over-oxidizing environments. S-glutathionylation has been identified in proteins involved in diabetic models both in vitro and in vivo. In all of them, S-glutathionylation represents a mechanism that regulates the response to diabetic conditions, and has been described to occur in erythrocytes and neutrophils from diabetic patients. However, additional studies are necessary to discern whether this modification represents a biomarker for the early onset of diabetic vascular complications.

Modulation of GSTP1-1 oligomerization by electrophilic inflammatory mediators and reactive drugs.

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-Δ12,14-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 Δ12-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.

SirT1 regulation of antioxidant genes is dependent on the formation of a FoxO3a/PGC-1α complex.

UNLABELLED: SirT1 is a class III histone deacetylase that has been implicated in metabolic and reactive oxygen species control. In the vasculature it has been shown to decrease endothelial superoxide production, prevent endothelial dysfunction and atherosclerosis. However, the mechanisms that mediate SirT1 antioxidant functions remain to be characterized. The transcription factor FoxO3a and the transcriptional coactivator peroxisome proliferator activated receptor γ-coactivator 1α (PGC-1α) have been shown to induce the expression of antioxidant genes and to be deacetylated by SirT1. AIMS: Here we investigated SirT1 regulation of antioxidant genes and the roles played by FoxO3a and PGC-1α in this regulation. RESULTS: We found that SirT1 regulates the expression of several antioxidant genes in bovine aortic endothelial cells, including Mn superoxide dismutase (MnSOD), catalase, peroxiredoxins 3 and 5 (Prx3, Prx5), thioredoxin 2 (Trx2), thioredoxin reductase 2 (TR2), and uncoupling protein 2 (UCP-2) and can be localized in the regulatory regions of these genes. We also found that knockdown of either FoxO3a or PGC-1α prevented the induction of antioxidant genes by SirT1 over-expression. Furthermore, SirT1 increased the formation of a FoxO3a/PGC-1α complex as determined by co-immunoprecipitation (IP) assays, concomitantly reducing H2O2-dependent FoxO3a and PGC-1α acetylation. Data showing that FoxO3a knockdown increases PGC-1α acetylation levels and vice versa, suggest that SirT1 activity on FoxO3a and PGC-1α may be dependent of the formation of a FoxO3a/PGC-1α complex. INNOVATION: A unifying mechanism for SirT1 activities is suggested. CONCLUSION: We show that SirT1 regulation of antioxidant genes in vascular endothelial cells depends on the formation of a FoxO3a/PGC-1α complex.

15-Deoxy-Δ(12,14)-prostaglandin J2 exerts pro- and anti-inflammatory effects in mesangial cells in a concentration-dependent manner.

Cyclopentenone prostaglandins play a modulatory role in inflammation, in part through their ability to covalently modify key proinflammatory proteins. Using mesangial cells as a cellular model of inflammation we have observed that 15-deoxy-Δ(12,14)-prostaglandin J(2) (15d-PGJ(2)) exerts a biphasic effect on cell activation by cytokines, with nanomolar concentrations eliciting an amplification of nitric oxide (NO) production and iNOS and COX-2 levels, and concentrations of 5 µM and higher inhibiting proinflammatory gene expression. An analog of 15d-PGJ(2) lacking the cyclopentenone structure (9,10-dihydro-15d-PGJ(2)) showed reduced ability to elicit both types of effects, suggesting that the electrophilic nature of 15d-PGJ(2) is important for its biphasic action. Interestingly, the switch from stimulatory to inhibitory actions occurred within a narrow concentration range and correlated with the ability of 15d-PGJ(2) to induce heme oxygenase 1 and γ-GCSm expression. These events are highly dependent on the triggering of the antioxidant response, which is considered as a sensor of thiol group modification. Indeed, the levels of the master regulator of the antioxidant response Nrf2 increased upon treatment with concentrations of 15d-PGJ(2) above 5 µM, an effect that could not be mimicked by 9,10-dihydro-15d-PGJ(2). Thus, an interplay of redox and electrophilic signalling mechanisms can be envisaged by which 15d-PGJ(2), as several other redox mediators, could contribute both to the onset and to the resolution of inflammation in a context or concentration-dependent manner.

Critical role of hydrogen peroxide signaling in the sequential activation of p38 MAPK and eNOS in laminar shear stress.

Laminar shear stress (LSS) is a protective hemodynamic regulator of endothelial function and limits the development of atherosclerosis and other vascular wall diseases related to pathophysiological generation of reactive oxygen species. LSS activates several endothelial signaling responses, including the activation of MAPKs and eNOS. Here, we explored the mechanisms of activation of these key endothelial signaling pathways. Using the cone/plate model we found that LSS (12 dyn/cm(2)) rapidly promotes endothelial intracellular generation of superoxide and hydrogen peroxide (H(2)O(2)). Physiological concentrations of H(2)O(2) (flux of 0.1 nM/min and 15 µM added extracellularly) significantly activated both eNOS and p38 MAPK. Pharmacological inhibition of NADPH oxidases (NOXs) and specific knockdown of NOX4 decreased LSS-induced p38 MAPK activation. Whereas the absence of eNOS did not alter LSS-induced p38 MAPK activation, pharmacological inhibition and knockdown of p38α MAPK blocked H(2)O(2)- and LSS-induced eNOS phosphorylation and reduced (•)NO levels. We propose a model in which LSS promotes the formation of signaling levels of H(2)O(2), which in turn activate p38α MAPK and then stimulate eNOS, leading to increased (•)NO generation and protection of endothelial function.

Cyclopentenone prostaglandins with dienone structure promote cross-linking of the chemoresistance-inducing enzyme glutathione transferase P1-1.

Glutathione transferase P1-1 (GSTP1-1) plays crucial roles in cancer chemoprevention and chemoresistance and is a key target for anticancer drug development. Oxidative stress or inhibitor-induced GSTP1-1 oligomerization leads to the activation of stress cascades and apoptosis in various tumor cells. Therefore, bivalent glutathione transferase (GST) inhibitors with the potential to interact with GST dimers are been sought as pharmacological and/or therapeutic agents. Here we have characterized GSTP1-1 oligomerization in response to various endogenous and exogenous agents. Ethacrynic acid, a classic GSTP1-1 inhibitor, 4-hydroxy-nonenal, hydrogen peroxide, and diamide all induced reversible GSTP1-1 oligomerization in Jurkat leukemia cells through the formation of disulphide bonds involving Cys47 and/or Cys101, as suggested by reducing and nonreducing SDS-polyacrylamide gel electrophoresis analysis of cysteine to serine mutants. Remarkably, the electrophilic prostanoid 15-deoxy-Δ(12,14)-prostaglandin J(2) (15d-PGJ(2)) induced irreversible GSTP1-1 oligomerization, specifically involving Cys101, a residue present in the human but not in the murine enzyme. 15d-PGJ(2)-induced GSTP1-1 cross-linking required the prostaglandin (PG) dienone structure and was associated with sustained c-Jun NH(2)-terminal kinase activation and induction of apoptosis. It is noteworthy that 15d-PGJ(2) elicited GSTP1-1 cross-linking in vitro, a process that could be mimicked by other dienone cyclopentenone PG, such as Δ(12)-PGJ(2), and by the bifunctional thiol reagent dibromobimane, suggesting that cyclopentenone PG may be directly involved in oligomer formation. Remarkably, Δ(12)-PGJ(2)-induced oligomeric species were clearly observed by electron microscopy showing dimensions compatible with GSTP1-1 tetramers. These results provide the first direct visualization of GSTP1-1 oligomeric species. Moreover, they offer novel strategies for the modulation of GSTP1-1 cellular functions, which could be exploited to overcome its role in cancer chemoresistance.