Reactive oxygen species and NO-derived oxidizing, nitrosating and nitrating products mediate diverse cell signaling and pathologic processes in cardiovascular, pulmonary, and neurodegenerative diseases (1, 2). These reactive inflammatory mediators chemically modify carbohydrates, DNA bases, amino acids, and unsaturated fatty acids to form oxidized, nitrosated and nitrated derivatives. For example, accumulation of inflammatory-induced protein tyrosine nitration products represents a shift from the physiological signal-transducing actions of NO to an oxidative, nitrative, and potentially pathogenic pathway (1).Recently, it has been reported that nitration products of unsaturated fatty acids (nitroalkenes) are formed via NO-dependent oxidative reactions (3-5). These derivatives were initially viewed to be, like nitrotyrosine, a "footprint" of NO-dependent redox reactions (3, 6). More recently, we have observed that nitrolinoleate (LNO 2 ) 7 mediates pluripotent cell signaling actions, since it induces relaxation of phenylephrine-preconstricted rat aortic rings, inhibits thrombin-induced Ca 2ϩ elevations and aggregation of human platelets, and attenuates human neutrophil superoxide generation, degranulation, and integrin expression. These cell responses are mediated by both cGMP-and cAMP-dependent and -independent mechanisms (7-9).LNO 2 positional isomers, including 9-, 10-, 12-, and 13-nitro-9,12-cis-octadecadienoic acids, have been identified as free acids in human plasma and red blood cells and as esterified components of plasma lipoproteins and red blood cell membranes (10). In addition, plasma and red cell free and esterified nitrooleate (OA-NO 2 , isomers 9-and 10-nitro-9-cis-octadecenoic acid) was also identified in healthy human blood (11).Current knowledge indicates that enzymatically oxidized unsaturated fatty acid-derived products, such as prostaglandins, thromboxanes, leukotrienes, epoxyeicosatrienoic acids, hydroxyeicosatetraenoic acids, lipoxins, and resolvins serve as lipid mediators or autacoids. These signaling mediators act within a local microenvironment to orchestrate both physiological and pathological events, including platelet aggregation, constriction of vascular smooth muscle, neonatal development, wound healing, and resolution of inflammation (12, 13). In this context, endogenous nitrated fatty acid derivatives, such as * This work was supported by National Institutes of Health Grants HL068878, HL075397, and S06GM08248 (to Y. E. C.), HL70146 (to R. P. P.), and HL58115 and HL64937 (to B. A. F.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. □ S The on-line version of this article (available at http://www.jbc.org) contains supplemental Table S1, Fig. 1, and Movie 1. 1 Supported by American Diabetes Association Grant JFA 7-05-JF-12. 2 These authors contributed equally to this work. 3 Supported by the postdoctoral f...

Nitric oxide ( ⅐ NO)-derived reactive species nitrate unsaturated fatty acids, yielding nitroalkene derivatives, including the clinically abundant nitrated oleic and linoleic acids. The olefinic nitro group renders these derivatives electrophilic at the carbon ␤ to the nitro group, thus competent for Michael addition reactions with cysteine and histidine. By using chromatographic and mass spectrometric approaches, we characterized this reactivity by using in vitro reaction systems, and we demonstrated that nitroalkene-protein and GSH adducts are present in vivo under basal conditions in healthy human red cells. Nitro-linoleic acid (9-, 10-, 12-, and 13-nitro-9,12-octadecadienoic acids) (m/z 324.2) and nitro-oleic acid (9-and 10-nitro-9-octadecaenoic acids) (m/z 326.2) reacted with GSH (m/z 306.1), yielding adducts with m/z of 631.3 and 633.3, respectively. At physiological concentrations, nitroalkenes inhibited glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which contains a critical catalytic Cys (Cys-149). GAPDH inhibition displayed an IC 50 of ϳ3 M M for both nitroalkenes, an IC 50 equivalent to the potent thiol oxidant peroxynitrite (ONOO ؊ ) and an IC 50 30-fold less than H 2 O 2 , indicating that nitroalkenes are potent thiol-reactive species. Liquid chromatography-mass spectrometry analysis revealed covalent adducts between fatty acid nitroalkene derivatives and GAPDH, including at the catalytic Cys-149. Liquid chromatography-mass spectrometry-based proteomic analysis of human red cells confirmed that nitroalkenes readily undergo covalent, thiol-reversible post-translational modification of nucleophilic amino acids in GSH and GAPDH in vivo. The adduction of GAPDH and GSH by nitroalkenes significantly increased the hydrophobicity of these molecules, both inducing translocation to membranes and suggesting why these abundant derivatives had not been detected previously via traditional high pressure liquid chromatography analysis. The occurrence of these electrophilic nitroalkylation reactions in vivo indicates that this reversible post-translational protein modification represents a new pathway for redox regulation of enzyme function, cell signaling, and protein trafficking. Nitric oxide ( ⅐ NO)5 exerts a broad influence on cell and inflammatory signaling via both cGMP-dependent and -independent oxidative, nitrosative, and nitrative reactions (1, 2). The nitration of polyunsaturated fatty acids present in both membranes and lipoproteins is now emerging as a novel mechanism for transducing ⅐ NO-dependent redox signaling (3, 4). Recent evidence indicates that all major unsaturated fatty acids present in human blood contain some proportion of alkenyl nitro derivatives (R 1 HCϭC(NO 2 )R 2 ), also termed nitroalkenes. Because of the prevalence of fatty acid nitroalkenes in healthy humans, these species are now appreciated as an abundant pool of bioactive oxides of nitrogen in the vasculature (5). The two most clinically abundant nitroalkene fatty acid derivatives, nitro-oleic acid (9-and 10-nitro-9-cis-octadeca...

Mass spectrometric analysis of human plasma and urine revealed abundant nitrated derivatives of all principal unsaturated fatty acids. Nitrated palmitoleic, oleic, linoleic, linolenic, arachidonic and eicosapentaenoic acids were detected in concert with their nitrohydroxy derivatives. Two nitroalkene derivatives of the most prevalent fatty acid, oleic acid, were synthesized (9-and 10-nitro-9-cis-octadecenoic acid; OA-NO 2 ), structurally characterized and determined to be identical to OA-NO 2 found in plasma, red cells, and urine of healthy humans. These regioisomers of OA-NO 2 were quantified in clinical samples using 13 C isotope dilution. Plasma free and esterified OA-NO 2 concentrations were 619 ؎ 52 and 302 ؎ 369 nM, respectively, and packed red blood cell free and esterified OA-NO 2 was 59 ؎ 11 and 155 ؎ 65 nM. The OA-NO 2 concentration of blood is ϳ50% greater than that of nitrated linoleic acid, with the combined free and esterified blood levels of these two fatty acid derivatives exceeding 1 M. OA-NO 2 is a potent ligand for peroxisome proliferator activated receptors at physiological concentrations. CV-1 cells co-transfected with the luciferase gene under peroxisome proliferator-activated receptor (PPAR) response element regulation, in concert with PPAR␥, PPAR␣, or PPAR␦ expression plasmids, showed dose-dependent activation of all PPARs by OA-NO 2 . PPAR␥ showed the greatest response, with significant activation at 100 nM, while PPAR␣ and PPAR␦ were activated at ϳ300 nM OA-NO 2 . OA-NO 2 also induced PPAR␥-dependent adipogenesis and deoxyglucose uptake in 3T3-L1 preadipocytes at a potency exceeding nitrolinoleic acid and rivaling synthetic thiazolidinediones. These data reveal that nitrated fatty acids comprise a class of nitric oxide-derived, receptor-dependent, cell signaling mediators that act within physiological concentration ranges.The oxidation of unsaturated fatty acids converts lipids, otherwise serving as cellular metabolic precursors and structural components, into potent signaling molecules including prostaglandins, leukotrienes, isoprostanes, and hydroxy-and hydroperoxyeicosatetraenoates. These enzymatic and auto-catalytic oxidation reactions yield products that orchestrate immune responses, neurotransmission, and the regulation of cell growth. For example, prostaglandins are cyclooxygenase-derived lipid mediators that induce receptor-dependent regulation of inflammatory responses, vascular function, initiation of parturition, cell survival, and angiogenesis (1). In contrast, the various isoprostane products of arachidonic acid auto-oxidation exert vasoconstrictive and pro-inflammatory signaling actions via receptor-dependent and -independent mechanisms (2). A common element of these diverse lipid signaling reactions is that nitric oxide ( ⅐ NO) 6 and other oxides of nitrogen significantly impact lipid mediator formation and bioactivities.The ability of ⅐ NO and ⅐ NO-derived species to oxidize, nitrosate, and nitrate biomolecules serves as the molecular basis for how ⅐ NO influences the sy...

Background: Nitroalkene fatty acids are electrophilic cell metabolites that mediate anti-inflammatory signaling actions. Results: Conjugated linoleic acid is the preferential unsaturated fatty acid substrate for nitration reactions during oxidative inflammatory conditions and digestion. Conclusion: Nitro-fatty acid formation in vivo occurs during metabolic and inflammatory reactions and modulates cell signaling. Significance: Nitro-conjugated linoleic acid transduces signaling actions of nitric oxide, nitrite, and conjugated linoleic acid.

The peroxisome proliferator-activated receptor-␥ (PPAR␥) binds diverse ligands to transcriptionally regulate metabolism and inflammation. Activators of PPAR␥ include lipids and antihyperglycemic drugs such as thiazolidinediones (TZDsThe rapidly expanding global burden of type II diabetes mellitus (DM) 3 and the concomitant increased risk for cardiovascular disease (1, 2) have motivated better understanding of relevant cell signaling pathways and potential therapeutic strategies. One major characteristic of metabolic syndrome and DM is insulin resistance, leading to hyperglycemia and dyslipidemia. Following initial clinical use of TZDs as anti-hyperglycemic agents to treat DM in the late 1990s, the nuclear receptor PPAR␥ was discovered as their molecular target. This receptor is expressed primarily in adipose tissue, muscle, and macrophages, where it regulates glucose uptake, lipid metabolism/ storage, and cell proliferation/differentiation (3-5). Thus, PPAR␥ ligands and allied downstream signaling events play a pivotal role in both the development and treatment of DM (6, 7). This is underscored by the observation that mutations in the C-terminal helix 12 of the ligand-binding domain (LBD) of PPAR␥ (e.g. P467L or V290M) are linked with severe insulin resistance and the onset of juvenile DM (8).The oxidizing inflammatory milieu contributing to the pathogenesis of obesity, diabetes, and cardiovascular disease also promotes diverse biomolecule oxidation, nitrosation, and nitration reactions by O 2 and ⅐ NO-derived species. Although oxidized fatty acids typically propagate proinflammatory conditions, the recently detected class of NO 2 -FA act as anti-inflammatory mediators. Nitroalkene derivatives of oleic acid (OA-NO 2 ) and linoleic acid (LNO 2 ) have been detected in healthy human blood and murine cardiac tissue. The levels of free/unesterified OA-NO 2 are ϳ1-3 nM in human plasma (9, 10), with OA-NO 2 produced at increased rates and present at higher concentrations during inflammatory and metabolic stress (11-13). The signaling actions of NO 2 -FA are primarily ascribed to the electrophilic olefinic carbon situated ␤ to the electron-withdrawing NO 2 substituent, facilitating kinetically rapid and reversible Michael addition with nucleophilic amino acids (i.e. Cys and His) (14). NO 2 -FA adduction of proteins and GSH occurs in model systems and clinically, with this reaction

Cytochrome c-dependent electron transfer and apoptosome activation require protein-protein binding, which are mainly directed by conformational and specific electrostatic interactions. Cytochrome c contains four highly conserved tyrosine residues, one internal (Tyr67), one intermediate (Tyr48), and two more accessible to the solvent (Tyr74 and Tyr97). Tyrosine nitration by biologically-relevant intermediates could influence cytochrome c structure and function. Herein, we analyzed the time course and site(s) of tyrosine nitration in horse cytochrome c by fluxes of peroxynitrite. Also, a method of purifying each (nitrated) cytochrome c product by cation-exchange HPLC was developed. A flux of peroxynitrite caused the time-dependent formation of different nitrated species, all less positively charged than the native form. At low accumulated doses of peroxynitrite, the main products were two mononitrated cytochrome c species at Tyr97 and Tyr74, as shown by peptide mapping and mass spectrometry analysis. At higher doses, all tyrosine residues in cytochrome c were nitrated, including dinitrated (i.e., Tyr97 and Tyr67 or Tyr74 and Tyr67) and trinitrated (i.e., Tyr97, Tyr74, and Tyr67) forms of the protein, with Tyr67 well represented in dinitrated species and Tyr48 being the least prone to nitration. All mono-, di-, and trinitrated cytochrome c species displayed an increased peroxidase activity. Nitrated cytochrome c in Tyr74 and Tyr67, and to a lesser extent in Tyr97, was unable to restore the respiratory function of cytochrome c-depleted mitochondria. The nitration pattern of cytochrome c in the presence of tetranitromethane (TNM) was comparable to that obtained with peroxynitrite, but with an increased relative nitration yield at Tyr67. The use of purified and well-characterized mono- and dinitrated cytochrome c species allows us to study the influence of nitration of specific tyrosines in cytochrome c functions. Moreover, identification of cytochrome c nitration sites in vivo may assist in unraveling the chemical nature of proximal reactive nitrogen species.

The aqueous decay and concomitant release of nitric oxide ( ⅐ NO) by nitrolinoleic acid (10-nitro-9,12-octadecadienoic acid and 12-nitro-9,12-octadecadienoic acid; LNO 2 ) are reported. Mass spectrometric analysis of reaction products supports a modified Nef reaction as the mechanism accounting for the generation of ⅐ NO by the aqueous reactions of fatty acid nitroalkene derivatives. Nitrolinoleic acid is stabilized by an aprotic milieu, with LNO 2 decay and ⅐ NO release strongly inhibited by phosphatidylcholine/cholesterol liposome membranes and detergents when present at levels above their critical micellar concentrations. The release of ⅐ NO from LNO 2 was induced by UV photolysis and triiodide-based ozone chemiluminescence reactions currently used to quantify putative protein nitrosothiol and N-nitrosamine derivatives. This reactivity of LNO 2 complicates the qualitative and quantitative analysis of biological oxides of nitrogen when applying UV photolysis and triiodidebased analytical systems to biological preparations typically abundant in nitrated fatty acids. The results reveal that nitroalkene derivatives of linoleic acid are pluripotent signaling mediators that act not only via receptor-dependent mechanisms, but also by transducing the signaling actions of ⅐ NO via pathways subject to regulation by the relative distribution of LNO 2 to hydrophobic versus aqueous microenvironments.Nitrolinoleic acid (10-nitro-9,12-octadecadienoic acid and 12-nitro-9,12-octadecadienoic acid; LNO 2 ) 1 is present in plasma lipoproteins and red blood cell membranes at concentrations of ϳ500 nM, rendering this species the most quantitatively abundant, biologically active oxide of nitrogen in the human vascular compartment (1). Nitrolinoleic acid is a product of nitric oxide ( ⅐ NO)-dependent linoleic acid nitration reactions that predominantly occur at the C-10 and C-12 alkene carbons. The positional isomer distribution of the LNO 2 alkenyl nitro group indicates that in vivo fatty acid nitration is a consequence of nucleophilic (nitronium group (NO 2 ϩ )) and/or radical (nitrogen dioxide ( ⅐ NO 2 )) addition reactions with olefinic carbons.Recent observations reveal that LNO 2 is a pluripotent signaling mediator that acts via both receptor-dependent and receptor-independent pathways. Nitrated fatty acids are specific and high affinity endogenous ligands for peroxisome proliferator-activated receptors (2) and serve to activate receptordependent gene expression at physiological concentrations. LNO 2 also activates cAMP-dependent protein kinase signaling pathways in neutrophils and platelets, serving to down-regulate the activation of these inflammatory cells (3, 4). Finally, LNO 2 induces vessel relaxation in an endothelium-independent manner (5). This LNO 2 -mediated relaxation of phenylephrine-preconstricted aortic rings is 1) a consequence of LNO 2 -induced stimulation of smooth muscle cell and aortic segment cGMP content, 2) inhibitable by the ⅐ NO scavenger oxyhemoglobin, and 3) 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-on...

Antigen B (EgAgB) is the most abundant and immunogenic antigen produced by the larval stage (metacestode) of Echinococcus granulosus. It is a lipoprotein, the structure and function of which have not been completely elucidated. EgAgB apolipoprotein components have been well characterised; they share homology with a group of hydrophobic ligand binding proteins (HLBPs) present exclusively in cestode organisms, and consist of different isoforms of 8-kDa proteins encoded by a polymorphic multigene family comprising five subfamilies (EgAgB1 to EgAgB5). In vitro studies have shown that EgAgB apolipoproteins are capable of binding fatty acids. However, the identity of the native lipid components of EgAgB remains unknown. The present work was aimed at characterising the lipid ligands bound to EgAgB in vivo. EgAgB was purified to homogeneity from hydatid cyst fluid and its lipid fraction was extracted using chloroform∶methanol mixtures. This fraction constituted approximately 40–50% of EgAgB total mass. High-performance thin layer chromatography revealed that the native lipid moiety of EgAgB consists of a variety of neutral (mainly triacylglycerides, sterols and sterol esters) and polar (mainly phosphatidylcholine) lipids. Gas-liquid chromatography analysis showed that 16∶0, 18∶0 and 18∶1(n-9) are the most abundant fatty acids in EgAgB. Furthermore, size exclusion chromatography coupled to light scattering demonstrated that EgAgB comprises a population of particles heterogeneous in size, with an average molecular mass of 229 kDa. Our results provide the first direct evidence of the nature of the hydrophobic ligands bound to EgAgB in vivo and indicate that the structure and composition of EgAgB lipoprotein particles are more complex than previously thought, resembling high density plasma lipoproteins. Results are discussed considering what is known on lipid metabolism in cestodes, and taken into account the Echinococcus spp. genomic information regarding both lipid metabolism and the EgAgB gene family.