Electrophilic Lipid Research Articles

Senescent cells inhibit mouse myoblast differentiation via the SASP-lipid 15d-PGJ2 mediated modification and control of HRas

Senescent cells are characterized by multiple features such as increased expression of senescence-associated β-galactosidase activity (SA β-gal) and cell cycle inhibitors such as p21 or p16. They accumulate with tissue damage and dysregulate tissue homeostasis. In the context of skeletal muscle, it is known that agents used for chemotherapy such as Doxorubicin (Doxo) cause buildup of senescent cells, leading to the inhibition of tissue regeneration. Senescent cells influence the neighboring cells via numerous secreted factors which form the senescence-associated secreted phenotype (SASP). Lipids are emerging as a key component of SASP that can control tissue homeostasis. Arachidonic acid-derived lipids have been shown to accumulate within senescent cells, specifically 15d-PGJ2, which is an electrophilic lipid produced by the non-enzymatic dehydration of the prostaglandin PGD2. This study shows that 15d-PGJ2 is also released by Doxo-induced senescent cells as an SASP factor. Treatment of skeletal muscle myoblasts with the conditioned medium from these senescent cells inhibits myoblast fusion during differentiation. Inhibition of L-PTGDS, the enzyme that synthesizes PGD2, diminishes the release of 15d-PGJ2 by senescent cells and restores muscle differentiation. We further show that this lipid post-translationally modifies Cys184 of HRas in C2C12 mouse skeletal myoblasts, causing a reduction in the localization of HRas to the Golgi, increased HRas binding to Ras Binding Domain (RBD) of RAF Kinase (RAF-RBD), and activation of cellular Mitogen Activated Protein (MAP) kinase–Extracellular Signal Regulated Kinase (Erk) signaling (but not the Akt signaling). Mutating C184 of HRas prevents the ability of 15d-PGJ2 to inhibit the differentiation of muscle cells and control the activity of HRas. This work shows that 15d-PGJ2 released from senescent cells could be targeted to restore muscle homeostasis after chemotherapy.

Senescent cells inhibit mouse myoblast differentiation via the SASP-lipid 15d-PGJ2 mediated modification and control of HRas.

Senescent cells are characterized by multiple features such as increased expression of senescence-associated β-galactosidase activity (SA β-gal) and cell cycle inhibitors such as p21 or p16. They accumulate with tissue damage and dysregulate tissue homeostasis. In the context of skeletal muscle, it is known that agents used for chemotherapy such as Doxorubicin (Doxo) cause buildup of senescent cells, leading to the inhibition of tissue regeneration. Senescent cells influence the neighboring cells via numerous secreted factors which form the senescence-associated secreted phenotype (SASP). Lipids are emerging as a key component of SASP that can control tissue homeostasis. Arachidonic acid-derived lipids have been shown to accumulate within senescent cells, specifically 15d-PGJ2, which is an electrophilic lipid produced by the non-enzymatic dehydration of the prostaglandin PGD2. This study shows that 15d-PGJ2 is also released by Doxo-induced senescent cells as an SASP factor. Treatment of skeletal muscle myoblasts with the conditioned medium from these senescent cells inhibits myoblast fusion during differentiation. Inhibition of L-PTGDS, the enzyme that synthesizes PGD2, diminishes the release of 15d-PGJ2 by senescent cells and restores muscle differentiation. We further show that this lipid post-translationally modifies Cys184 of HRas in C2C12 mouse skeletal myoblasts, causing a reduction in the localization of HRas to the Golgi, increased HRas binding to Ras Binding Domain (RBD) of RAF Kinase (RAF-RBD), and activation of cellular Mitogen Activated Protein (MAP) kinase-Extracellular Signal Regulated Kinase (Erk) signaling (but not the Akt signaling). Mutating C184 of HRas prevents the ability of 15d-PGJ2 to inhibit the differentiation of muscle cells and control the activity of HRas. This work shows that 15d-PGJ2 released from senescent cells could be targeted to restore muscle homeostasis after chemotherapy.

Plasmalogen oxidation induces the generation of excited molecules and electrophilic lipid species.

Plasmalogens are glycerophospholipids with a vinyl ether linkage at the sn-1 position of the glycerol backbone. Despite being suggested as antioxidants due to the high reactivity of their vinyl ether groups with reactive oxygen species, our study reveals the generation of subsequent reactive oxygen and electrophilic lipid species from oxidized plasmalogen intermediates. By conducting a comprehensive analysis of the oxidation products by liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS), we demonstrate that singlet molecular oxygen [O2 (1Δg)] reacts with the vinyl ether bond, producing hydroperoxyacetal as a major primary product (97%) together with minor quantities of dioxetane (3%). Furthermore, we show that these primary oxidized intermediates are capable of further generating reactive species including excited triplet carbonyls and O2 (1Δg) as well as electrophilic phospholipid and fatty aldehyde species as secondary reaction products. The generation of excited triplet carbonyls from dioxetane thermal decomposition was confirmed by light emission measurements in the visible region using dibromoanthracene as a triplet enhancer. Moreover, O2 (1Δg) generation from dioxetane and hydroperoxyacetal was evidenced by detection of near-infrared light emission at 1,270 nm and chemical trapping experiments. Additionally, we have thoroughly characterized alpha-beta unsaturated phospholipid and fatty aldehydes by LC-HRMS analysis using two probes that specifically react with aldehydes and alpha-beta unsaturated carbonyls. Overall, our findings demonstrate the generation of excited molecules and electrophilic lipid species from oxidized plasmalogen species unveiling the potential prooxidant nature of plasmalogen-oxidized products.

Abstract 1823 Plasmalogen derived products of reactive chlorinating species targeting: A family of bioactive electrophilic lipids

Abstract 1823 Plasmalogen derived products of reactive chlorinating species targeting: A family of bioactive electrophilic lipids

Fluorescent probe to quantify lipid-derived electrophiles in edible oils.

In the presence of molecular oxygen, edible oils can be oxidized to form a multitude of α,β-unsaturated carbonyl products collectively called 'lipid-derived electrophiles'. These molecules affect the taste of fat-containing foods but also act as electrophiles by covalently binding to protein amines/thiols and DNA nucleotides. The chemical modification of proteins by lipid-derived electrophiles appears to play an important role in human health, but the quantification of this diverse class of compounds remains a challenge. In this study, we describe a method capable of measuring the relative content of α,β-unsaturated carbonyls in food containing edible oils by using a "turn-on" fluorescent probe. The detection of electrophiles is based on a pre-fluorescent probe, 7-mercapto-4-methyl-coumarin (C-SH). The fluorescence of C-SH increases after nucleophilic addition to electrophilic lipid oxidation products. Since different lipid-derived electrophiles will react at a different rate with our fluorescent probe, we expressed the probe's response against a standard electrophile: trans-2-nonenal. In this assay, electrophiles which react more quickly will have a more dominant weight in the measurements carried out. Using this analytical technique, we can compare electrophilic content in French fries from several restaurants, and find they have lower amounts of lipid-derived electrophiles versus frozen fries baked at home. We also demonstrate that potato chips sealed in a reduced oxygen atmosphere will have a low 'electrophilic content' that increases over time, whereas chips in oxygen-permeable packaging initially have a higher 'electrophilic content' that does not increase as much over time. The relative ease of fluorescence measurements using microplate readers coupled with a simple oil extraction protocol should allow this method to quantify 'electrophilic content' in several food sources.

Utility of expression of 4-hydroxynonenal tested by immunohistochemistry for cervical cancer.

Cervical cancer (CC) is a leading cause of mortality in women around the world, with the highest incidence rate still being in developing countries. The most common aetiological factor is infection with high-risk human papilloma virus viral strains. Oxidative stress through generation of reactive oxygen species leads to lipid peroxidation and DNA damage. Studies show that reactive lipid electrophiles such as 4-hydroxynonenal (4-HNE) produced in the process play an important role in cancer signalling pathways and are a good biomarker for oxidative stress. We aim to investigate the prognostic role of 4-HNE as a biomarker for oxidative stress in patients in early and advanced stages of CC measured by immunohistochemistry. This is a retrospective study of 69 patients treated at our Department of Oncogynaecology. Paraffin embedded tumour tissues were immunohistochemically tested for the levels of expression of 4-HNE. The results for H-score, Allred score, and combined score were investigated for association with tumour size, lymph node status, andInternational Federation of Gynaecology and Obstetrics stage. 4-hydroxynonenal showed higher expression in more advanced stages of CC and in cases with involved lymph nodes. Tumour size was not associated with the levels of 4-HNE. To best of our knowledge, this is the first study to use immunohistochemistry to examine the expression of 4-HNE as a prognostic factor in CC. The 3 score systems showed similar results. The pattern of 4-HNE histological appearance is dependent on the histological origin of cancer and is not universal.

Digestive interaction between dietary nitrite and dairy products generates novel nitrated linolenic acid products

Nitrated fatty acids are important anti-inflammatory and protective lipids formed in the gastric compartment, with conjugated linoleic acid (rumenic acid, RA, 9Z,11E-18:2) being the primary substrate for lipid nitration. The recently reported identification of nitrated rumelenic acid (NO2-RLA) in human urine has led to hypothesize that rumelenic acid (RLA, 9Z,11E,15Z-18:3) from dairy fat is responsible for the formation of NO2-RLA. To evaluate the source and mechanism of NO2-RLA formation, 15N labeled standards of NO2-RLA were synthesized and characterized. Afterward, milk fat with different RA and RLA levels was administered to mice in the presence of nitrite, and the appearance of nitrated fatty acids in plasma and urine followed. We confirmed the formation of NO2-RLA and defined the main metabolites in plasma, urine, and tissues. In conclusion, RLA obtained from dairy products is the main substrate for forming this novel electrophilic lipid reported to be present in human urine.

Differential Contributions of Distinct Free Radical Peroxidation Mechanisms to the Induction of Ferroptosis.

Ferroptosis is a form of regulated cell death driven by lipid peroxidation of polyunsaturated fatty acids (PUFAs). Lipid peroxidation can propagate through either the hydrogen-atom transfer (HAT) or peroxyl radical addition (PRA) mechanism. However, the contribution of the PRA mechanism to the induction of ferroptosis has not been studied. In this study, we aim to elucidate the relationship between the reactivity and mechanisms of lipid peroxidation and ferroptosis induction. We found that while some peroxidation-reactive lipids, such as 7-dehydrocholesterol, vitamins D3 and A, and coenzyme Q10, suppress ferroptosis, both nonconjugated and conjugated PUFAs enhanced cell death induced by RSL3, a ferroptosis inducer. Importantly, we found that conjugated PUFAs, including conjugated linolenic acid (CLA 18:3) and conjugated linoleic acid (CLA 18:2), can induce or potentiate ferroptosis much more potently than nonconjugated PUFAs. We next sought to elucidate the mechanism underlying the different ferroptosis-inducing potency of conjugated and nonconjugated PUFAs. Lipidomics revealed that conjugated and nonconjugated PUFAs are incorporated into distinct cellular lipid species. The different peroxidation mechanisms predict the formation of higher levels of reactive electrophilic aldehydes from conjugated PUFAs than nonconjugated PUFAs, which was confirmed by aldehyde-trapping and mass spectrometry. RNA sequencing revealed that protein processing in the endoplasmic reticulum and proteasome are among the most significantly upregulated pathways in cells treated with CLA 18:3, suggesting increased ER stress and activation of unfolded protein response. These results suggest that protein damage by lipid electrophiles is a key step in ferroptosis.

Reversibility and Low Commitment to Forward Catalysis in the Conjugation of Lipid Alkenals by Glutathione Transferase A4-4.

High concentrations of electrophilic lipid alkenals formed during oxidative stress are implicated in cytotoxicity and disease. However, low concentrations of alkenals are required to induce antioxidative stress responses. An established clearance pathway for lipid alkenals includes conjugation to glutathione (GSH) via Michael addition, which is catalyzed mainly by glutathione transferase isoform A4 (GSTA4-4). Based on the ability of GSTs to catalyze hydrolysis or retro-Michael addition of GSH conjugates, and the antioxidant function of low concentrations of lipid alkenals, we hypothesize that GSTA4-4 contributes a homeostatic role in lipid metabolism. Enzymatic kinetic parameters for retro-Michael addition with trans-2-Nonenal (NE) reveal the chemical competence of GSTA4-4 in this putative role. The forward GSTA4-4-catalyzed Michael addition occurs with the rapid exchange of the C2 proton of NE in D2O as observed by NMR. The isotope exchange was completely dependent on the presence of GSH. The overall commitment to catalysis, or the ratio of first order kcat,f for 'forward' Michael addition to the first order kcat,ex for H/D exchange is remarkably low, approximately 3:1. This behavior is consistent with the possibility that GSTA4-4 is a regulatory enzyme that contributes to steady-state levels of lipid alkenals, rather than a strict 'one way' detoxication enzyme.

Protective Effect of NO2-OA on Oxidative Stress, Gliosis, and Pro-Angiogenic Response in Müller Glial Cells.

Inflammation and oxidative and nitrosative stress are involved in the pathogenesis of proliferative retinopathies (PR). In PR, a loss of balance between pro-angiogenic and anti-angiogenic factors favors the secretion of vascular endothelial growth factor (VEGF). This vascular change results in alterations in the blood-retinal barrier, with extravasation of plasma proteins such as α2-macroglobulin (α2M) and gliosis in Müller glial cells (MGCs, such as MIO-M1). It is well known that MGCs play important roles in healthy and sick retinas, including in PR. Nitro-fatty acids are electrophilic lipid mediators with anti-inflammatory and cytoprotective properties. Our aim was to investigate whether nitro-oleic acid (NO2-OA) is beneficial against oxidative stress, gliosis, and the pro-angiogenic response in MGCs. Pure synthetic NO2-OA increased HO-1 expression in a time- and concentration-dependent manner, which was abrogated by the Nrf2 inhibitor trigonelline. In response to phorbol 12-myristate 13-acetate (PMA) and lipopolysaccharide (LPS), NO2-OA prevented the ROS increase and reduced the gliosis induced by α2M. Finally, when hypoxic MGCs were incubated with NO2-OA, the increase in VEGF mRNA expression was not affected, but under hypoxia and inflammation (IL-1β), NO2-OA significantly reduced VEGF mRNA levels. Furthermore, NO2-OA inhibited endothelial cell (BAEC) tubulogenesis. Our results highlight NO2-OA's protective effect on oxidative damage, gliosis; and the exacerbated pro-angiogenic response in MGCs.

Endothelial Cell Protein Targeting by Myeloperoxidase-Derived 2-Chlorofatty Aldehyde.

Neutrophils are important cellular mediators of injury and repair in diseases including ischemic heart disease, atherosclerosis, and sepsis. Myeloperoxidase-derived (MPO)-oxidants released from neutrophils are potential mediators of endothelial injury in disease. MPO-derived HOCl attacks plasmalogen phospholipid to liberate 2-chlorofatty aldehyde (2-ClFALD). Both 2-ClFALD and its oxidation product, 2-chlorofatty acid (2-ClFA), are electrophilic lipids, and both probably react with proteins through several mechanisms. In the present study, we investigate protein modification specifically by 2-ClFALD under non-reducing conditions (e.g., without stabilizing Schiff base bonds), which likely reflects nucleophilic targeting of the electrophilic chlorinated carbon. Protein modification by the ω-alkyne analog of 2-chlorohexadecanal (2-ClHDA), 2-ClHDyA, was compared to that with the ω-alkyne analog of 2-chlorohexadecanoic acid (2-ClHA), 2-ClHyA, in multiple cell lines, which demonstrated 2-ClFALD preferentially modifies proteins compared to 2-ClFA. The 2-ClHDyA modified proteins from EA.hy926 cells and human lung microvascular endothelial cells analyzed by shotgun proteomics and over-representation analysis included adherens junction, cell adhesion molecule binding, and cell substrate junction enrichment categories. It is possible that proteins in these groups may have roles in previously described 2-ClFALD-elicited endothelial barrier dysfunction.

Allosteric Regulation of the Soluble Epoxide Hydrolase by Nitro Fatty Acids: a Combined Experimental and Computational Approach

The human soluble epoxide hydrolase (hsEH) is a key regulator of epoxy fatty acid (EpFA) metabolism. Inhibition of sEH can maintain endogenous levels of beneficial EpFAs and reduce the levels of their corresponding diol products, thus ameliorating a variety of pathological conditions including cardiovascular, central nervous system and metabolic diseases. The quest for orthosteric drugs that bind directly to the catalytic crevice of hsEH has been prolonged and sustained over the past decades, but the disappointing outcome of clinical trials to date warrants alternative pharmacological approaches. Previously, we have shown that hsEH can be allosterically inhibited by the endogenous electrophilic lipid 15-deoxy-Δ12,14-Prostaglandin-J2, via covalent adduction to two cysteines, C423 and C522. In this study, we explore the properties and behaviour of three electrophilic lipids belonging to the class of the nitro fatty acids, namely 9- and 10-nitrooleate and 10-nitrolinoleate. Biochemical and biophysical investigations revealed that, in addition to C423 and C522, nitro fatty acids can covalently bind to additional nucleophilic residues in hsEH C-terminal domain (CTD), two of which predicted in this study to be latent allosteric sites. Systematic mapping of the protein mutational space and evaluation of possible propagation pathways delineated selected residues, both in the allosteric patches and in other regions of the enzyme, envisaged to play a role in allosteric signalling. The responses elicited by the ligands on the covalent adduction sites supports future fragment-based design studies of new allosteric effectors for hsEH with increased efficacy and selectivity.

Structural Modifications Yield Novel Insights Into the Intriguing Pharmacodynamic Potential of Anti-inflammatory Nitro-Fatty Acids.

Endogenous nitro-fatty acids (NFA) are potent electrophilic lipid mediators that exert biological effects in vitro and in vivo via selective covalent modification of thiol-containing target proteins. The cytoprotective, anti-inflammatory, and anti-tumorigenic effects of NFA in animal models of disease caused by targeted protein nitroalkylation are a valuable basis for the development of future anti-phlogistic and anti-neoplastic drugs. Considering the complexity of diseases and accompanying comorbidities there is an urgent need for clinically effective multifunctional drugs. NFA are composed of a fatty acid backbone containing a nitroalkene moiety triggering Michael addition reactions. However, less is known about the target-specific structure–activity relationships and selectivities comparing different NFA targets. Therefore, we analyzed 15 NFA derivatives and compared them with the lead structure 9-nitro-oleic acid (9NOA) in terms of their effect on NF-κB (nuclear factor kappa B) signaling inhibition, induction of Nrf-2 (nuclear factor erythroid 2-related factor 2) gene expression, sEH (soluble epoxide hydrolase), LO (lipoxygenase), and COX-2 (cyclooxygenase-2) inhibition, and their cytotoxic effects on colorectal cancer cells. Minor modifications of the Michael acceptor position and variation of the chain length led to drugs showing increased target preference or enhanced multi-targeting, partly with higher potency than 9NOA. This study is a significant step forward to better understanding the biology of NFA and their enormous potential as scaffolds for designing future anti-inflammatory drugs.

Chemoproteomic profiling reveals cellular targets of nitro-fatty acids

Nitro-fatty acids are a class of endogenous electrophilic lipid mediators with anti-inflammatory and cytoprotective effects in a wide range of inflammatory and fibrotic disease models. While these beneficial biological effects of nitro-fatty acids are mainly attributed to their ability to form covalent adducts with proteins, only a small number of proteins are known to be nitro-alkylated and the scope of protein nitro-alkylation remains undetermined. Here we describe the synthesis and application of a clickable nitro-fatty acid probe for the detection and first global identification of mammalian proteins that are susceptible to nitro-alkylation. 184 high confidence nitro-alkylated proteins were identified in THP1 macrophages, majority of which are novel targets of nitro-fatty acids, including extended synaptotagmin 2 (ESYT2), signal transducer and activator of transcription 3 (STAT3), toll-like receptor 2 (TLR2), retinoid X receptor alpha (RXRα) and glucocorticoid receptor (NR3C1). In particular, we showed that 9-nitro-oleate covalently modified and inhibited dexamethasone binding to NR3C1. Bioinformatic analyses revealed that nitro-alkylated proteins are highly enriched in endoplasmic reticulum and transmembrane proteins, and are overrepresented in lipid metabolism and transport pathways. This study significantly expands the scope of protein substrates targeted by nitro-fatty acids in living cells and provides a useful resource towards understanding the pleiotropic biological roles of nitro-fatty acids as signaling molecules or as multi-target therapeutic agents.

Synthesis of 9- and 12-nitro conjugated linoleic acid: Regiospecific isomers of naturally occurring conjugated nitrodienes

Conjugated diene-containing fatty acids (rumenic and rumelenic acids) are major substrates for nitration under physiological conditions. Their nitrated products are present in human urine. These nitrodiene-containing lipid electrophiles contain a strongly electron-withdrawing pair of conjugated double bonds amenable to nucleophilic attack in biological milieu, which affords them pluripotent signaling capabilities. We report synthetic methods to obtain useful quantities of three main biological nitrated fatty acids (9- and 12-nitro-conjugated linoleic acids and 9-nitro-conjugated linolenic acid) in six or seven steps from commercially available starting materials, for biological evaluation of these naturally occurring biomolecules.

A thiol redox sensor in soluble epoxide hydrolase enables oxidative activation by intra-protein disulfide bond formation

Soluble epoxide hydrolase (sEH), an enzyme that broadly regulates the cardiovascular system, hydrolyses epoxyeicosatrienoic acids (EETs) to their corresponding dihydroxyeicosatrienoic acids (DHETs). We previously showed that endogenous lipid electrophiles adduct within the catalytic domain, inhibiting sEH to lower blood pressure in angiotensin II-induced hypertensive mice. As angiotensin II increases vascular H2O2, we explored sEH redox regulation by this oxidant and how this integrates with inhibition by lipid electrophiles to regulate vasotone. Kinetics analyses revealed that H2O2 not only increased the specific activity of sEH but increased its affinity for substrate and increased its catalytic efficiency. This oxidative activation was mediated by formation of an intra-disulfide bond between C262 and C264, as determined by mass spectrometry and substantiated by biotin-phenylarsinate and thioredoxin-trapping mutant assays. C262S/264S sEH mutants were resistant to peroxide-induced activation, corroborating the disulfide-activation mechanism. The physiological impact of sEH redox state was determined in isolated arteries and the effect of the pro-oxidant vasopressor angiotensin II on arterial sEH redox state and vasodilatory EETs indexed in mice. Angiotensin II induced the activating intra-disulfide in sEH, causing a decrease in plasma EET/DHET ratios that is consistent with the pressor response to this hormone. Although sEH C262–C264 disulfide formation enhances hydrolysis of vasodilatory EETs, this modification also sensitized sEH to inhibition by lipid electrophiles. This explains why angiotensin II decreases EETs and increases blood pressure, but when lipid electrophiles are also present, that EETs are increased and blood pressure lowered.

Nitric Oxide and Electrophilic Cyclopentenone Prostaglandins in Redox signaling, Regulation of Cytoskeleton Dynamics and Intercellular Communication.

Nitric oxide (NO) and electrophilic cyclopentenone prostaglandins (CyPG) are local mediators that modulate cellular response to oxidative stress in different pathophysiological processes. In particular, there is increasing evidence about their functional role during inflammation and immune responses. Although the mechanistic details about their relationship and functional interactions are still far from resolved, NO and CyPG share the ability to promote redox-based post-translational modification (PTM) of proteins that play key roles in cellular homeostasis, signal transduction and transcription. NO-induced S-nitrosylation and S-glutathionylation as well as cyclopentenone-mediated adduct formation, are a few of the main PTMs by which intra- and inter-cellular signaling are regulated. There is a growing body of evidence indicating that actin and actin-binding proteins are susceptible to covalent PTM by these agents. It is well known that the actin cytoskeleton is key for the establishment of interactions among leukocytes, endothelial and muscle cells, enabling cellular activation and migration. In this review we analyze the current knowledge about the actions exerted by NO and CyPG electrophilic lipids on the regulation of actin dynamics and cytoskeleton organization, and discuss some open questions regarding their functional relevance in the regulation of intercellular communication.

Protein Lipoxidation: Basic Concepts and Emerging Roles.

Protein lipoxidation is a non-enzymatic post-translational modification that consists of the covalent addition of reactive lipid species to proteins. This occurs under basal conditions but increases in situations associated with oxidative stress. Protein targets for lipoxidation include metabolic and signalling enzymes, cytoskeletal proteins, and transcription factors, among others. There is strong evidence for the involvement of protein lipoxidation in disease, including atherosclerosis, neurodegeneration, and cancer. Nevertheless, the involvement of lipoxidation in cellular regulatory mechanisms is less understood. Here we review basic aspects of protein lipoxidation and discuss several features that could support its role in cell signalling, including its selectivity, reversibility, and possibilities for regulation at the levels of the generation and/or detoxification of reactive lipids. Moreover, given the great structural variety of electrophilic lipid species, protein lipoxidation can contribute to the generation of multiple structurally and functionally diverse protein species. Finally, the nature of the lipoxidised proteins and residues provides a frameshift for a complex interplay with other post-translational modifications, including redox and redox-regulated modifications, such as oxidative modifications and phosphorylation, thus strengthening the importance of detailed knowledge of this process.

Regulation of arachidonic acid oxidation and metabolism by lipid electrophiles

Arachidonic acid (AA) is a precursor of enzymatic and non-enzymatic oxidized products such as prostaglandins, thromboxanes, leukotrienes, lipoxins, and isoprostanes. These products may exert signaling or damaging roles during physiological and pathological conditions, some of them being markers of oxidative stress linked to inflammation. Recent data support the concept that cyclooxygenases (COX), lipoxygenases (LOX), and cytochrome P450 (CYP450) followed by cytosolic and microsomal dehydrogenases can convert AA to lipid-derived electrophiles (LDE). Lipid-derived electrophiles are fatty acid derivatives bearing an electron-withdrawing group that can react with nucleophiles at proteins, DNA, and small antioxidant molecules exerting potent signaling properties. This review aims to describe the formation, sources, and electrophilic anti-inflammatory actions of key mammalian LDE.

Nitro-oleic acid, a ligand of CD36, reduces cholesterol accumulation by modulating oxidized-LDL uptake and cholesterol efflux in RAW264.7 macrophages

Macrophages play a pivotal role in the early stages of atherosclerosis development; they excessively accumulate cholesterol in the cytosol in response to modified Low Density Lipoprotein (mLDL). The mLDL are incorporated through scavenger receptors. CD36 is a high-affinity cell surface scavenger receptor that facilitates the binding and uptake of long-chain fatty acids and mLDL into the cell. Numerous structurally diverse ligands can initiate signaling responses through CD36 to regulate cell metabolism, migration, and angiogenesis. Nitro-fatty acids are endogenous electrophilic lipid mediators that react with and modulate the function of multiple enzymes and transcriptional regulatory proteins. These actions induce the expression of several anti-inflammatory and cytoprotective genes and limit pathologic responses in experimental models of atherosclerosis, cardiac ischemia/reperfusion, and inflammatory diseases. Pharmacological and genetic approaches were used to explore the actions of nitro-oleic acid (NO2-OA) on macrophage lipid metabolism. Pure synthetic NO2-OA dose-dependently increased CD36 expression in RAW264.7 macrophages and this up-regulation was abrogated in BMDM from Nrf2-KO mice. Ligand binding analysis revealed that NO2-OA specifically interacts with CD36, thus limiting the binding and uptake of mLDL. Docking analysis shows that NO2-OA establishes a low binding energy interaction with the alpha helix containing Lys164 in CD36. NO2-OA also restored autophagy flux in mLDL-loaded macrophages, thus reversing cholesterol deposition within the cell. In aggregate, these results indicate that NO2-OA reduces cholesterol uptake by binding to CD36 and increases cholesterol efflux by restoring autophagy.