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The lipoamino acids and endovanilloids have multiple roles in nociception, pain, and inflammation, yet their biological reactivity has not been fully characterized. Cyclooxygenases (COXs) and lipoxygenases (LOs) oxygenate polyunsaturated fatty acids to generate signaling molecules. The ability of COXs and LOs to oxygenate arachidonyl-derived lipoamino acids and vanilloids was investigated. COX-1 and COX-2 were able to minimally metabolize many of these species. However, the lipoamino acids were efficiently oxygenated by 12S- and 15S-LOs. The kinetics and products of oxygenation by LOs were characterized. Whereas 15S-LOs retained positional specificity of oxygenation with these novel substrates, platelet-type 12S-LO acted as a 12/15-LO. Fatty acid oxygenases may play an important role in the metabolic inactivation of lipoamino acids or vanilloids or may convert them to bioactive derivatives.

Cyclooxygenase-2 (COX-2) can oxygenate the endocannabinoids, arachidonyl ethanolamide (AEA) and 2-arachidonylglycerol (2-AG), to prostaglandin-H2-ethanolamide (PGH2-EA) and -glycerol ester (PGH2-G), respectively. Further metabolism of PGH2-EA and PGH2-G by prostaglandin synthases produces a variety of prostaglandin-EA's and prostaglandin-G's nearly as diverse as those derived from arachidonic acid. Thus, COX-2 may regulate endocannabinoid levels in neurons during retrograde signaling or produce novel endocannabinoid metabolites for receptor activation. Endocannabinoid-metabolizing enzymes are important regulators of their action, so we tested whether PG-G levels may be regulated by monoacylglycerol lipase (MGL) and fatty acid amide hydrolase (FAAH). We found that PG-Gs are poor substrates for purified MGL and FAAH compared to 2-AG and/or AEA. Determination of substrate specificity demonstrates a 30-100- and 150-200-fold preference of MGL and FAAH for 2-AG over PG-Gs, respectively. The substrate specificity of AEA compared to those of PG-Gs was approximately 200-300 fold higher for FAAH. Thus, PG-Gs are poor substrates for the major endocannabinoid-degrading enzymes, MGL and FAAH.

Sterol carrier protein-2 (SCP-2) facilitates cholesterol (Ch) and phospholipid (PL) transfer/exchange between membranes and appears to play a key role in intracellular lipid trafficking. Whether SCP-2 can also facilitate lipid hydroperoxide (LOOH) transfer between membranes and thereby potentially enhance dissemination of peroxidative damage was examined in this study. Transfer kinetics of photochemically generated cholesterol hydroperoxide (ChOOH) species (5alpha-OOH, 6alpha/6beta-OOH, 7alpha/7beta-OOH) and phospholipid hydroperoxide (PLOOH) families (PCOOH, PEOOH, PSOOH) were determined, using HPLC with electrochemical detection for peroxide analysis. LOOH donor/acceptor pairs employed in transfer experiments included (i) all liposomes (e.g., agglutinable SUVs/ nonagglutinable LUVs); (ii) photoperoxidized erythrocyte ghosts/SUVs or vice versa; and (iii) SUVs/mitochondria. In a SUV/ghost system at 37 degrees C, the rate constant for total ChOOH spontaneous transfer was approximately 8 times greater than that for unoxidized Ch. Purified bovine liver and human recombinant SCP-2 exhibited an identical ability to stimulate overall ChOOH transfer, 0.5 unit/mL (based on [(14)C]Ch transfer) increasing the first-order rate constant (k) approximately 7-fold. SCP-2-enhanced translocation of individual ChOOHs increased with increasing hydrophilicity in the following order: 6beta-OOH6alpha-OOH5alpha-OOH7alpha/7beta-OOH. Likewise, SCP-2 stimulated PCOOH, PEOOH, or PSOOH transfer approximately 6-fold, but the net k was 1/5 that of 5alpha-OOH and 1/10 that of 7alpha/7beta-OOH. Donor membrane properties favoring SCP-2-enhanced LOOH transfer included (i) increasing PL unsaturation and (ii) increasing net negative charge imposed by phosphatidylserine. Cytotoxic relevance was demonstrated by showing that SCP-2 accelerates 7alpha-OOH transfer from SUVs to isolated mitochondria and that this enhances peroxide-induced loss of the mitochondrial membrane potential. On the basis of these findings, we postulate that SCP-2, by trafficking ChOOHs and PLOOHs in addition to parent lipids, might exacerbate cell injury under oxidative stress conditions.

Under oxidative pressure in the vascular circulation, erythrocytes and phagocytic cells may accumulate membrane lipid hydroperoxides (LOOHs), including cholesterol- and phospholipid-derived species (ChOOHs, PLOOHs). LOOH translocation from cells to low-density lipoprotein (LDL) might sensitize the latter to free radical-mediated oxidative modification, an early event associated with atherogenesis. To test this, we examined the spontaneous transfer kinetics of various ChOOH species (5 alpha-OOH, 6 alpha-OOH, 6 beta-OOH, 7 alpha/7 beta-OOH) and various PLOOH groups (PCOOH, PEOOH, PSOOH, SMOOH) using photoperoxidized erythrocyte ghosts as model donors and freshly prepared LDL as an acceptor. LOOH departure or uptake was monitored by reverse-phase HPLC with reductive electrochemical detection. Mildly peroxidized ghost membranes transferred overall ChOOH and PLOOH to LDL with apparent first-order rate constants approximately 60 and approximately 35 times greater than those of the respective parent lipids. Individual ChOOH rate constants decreased in the following order: 7 alpha/7 beta-OOH5 alpha-OOH6 alpha-OOH6 beta-OOH. Kinetics for reverse transfer from LDL to ghosts followed the same trend, but rates were significantly higher for all species and their combined activation energy was lower (41 vs 85 kJ/mol). PLOOH transfer rate constants ranged from 4- to 15-fold lower than the composite ChOOH constant, their order being as follows: PCOOH approximately PEOOH approximately PSOOHSMOOH. Similar PLOOH transfer kinetics were observed when LDL acceptor was replaced by unilamellar liposomes, consistent with desorption from the donor membrane being the rate-limiting step. The susceptibility of transfer LOOH-enriched LDL to Cu2+-induced chain peroxidative damage was assessed by monitoring the accumulation of conjugated dienes and products of free radical-mediated cholesterol oxidation. In both cases, transfer-acquired LOOHs significantly reduced the lag time for chain initiation relative to that observed using nonperoxidized ghosts. These findings are consistent with the idea that LDL can acquire significant amounts of "seeding" LOOHs via translocation from various donors in the circulation.

Lipid hydroperoxides (LOOHs) can be generated in cells when cholesterol (Ch) and other unsaturated lipids in cell membranes are degraded under conditions of oxidative stress. If LOOHs escape reductive detoxification by glutathione-dependent selenoperoxidases, they may undergo iron-catalyzed one-electron reduction to free radical species, thus triggering peroxidative chain reactions which exacerbate oxidative membrane damage. LOOHs are more polar than parent lipids and much longer-lived than free radical precursors or products. Accordingly, intermembrane transfer of LOOHs (analogous to that of unoxidized precursors) might be possible, and this could jeopardize acceptor membranes. We have investigated this possibility, using photoperoxidized [(14)C]Ch-labeled erythrocyte ghosts as cholesterol hydroperoxide (ChOOH) donors and unilamellar liposomes [e.g., dimyristoyl-phosphatidylcholine/Ch, 9:1 mol/mol] as acceptors. ChOOH material consisted mainly of 5alpha-hydroperoxide, a singlet oxygen adduct. Time-dependent transfer of ChOOH versus Ch at 37 degrees C was determined, using high-performance liquid and thin-layer chromatographic methods to analyze liposomal extracts for these species. A typical experiment in which the starting ChOOH/Ch mol ratio in ghosts was approximately 0.05 showed that the initial transfer rate of ChOOH was approximately 16 times greater than that of parent Ch. Using [(14)C]Ch as a reporter in liposome acceptors, we found that transfer-acquired ChOOHs, when exposed to a lipophilic iron chelate and ascorbate, could trigger strong peroxidative chain reactions, as detected by accumulation of [(14)C]Ch oxidation products. These findings support the hypothesis that intermembrane transfer of ChOOHs can contribute to their prooxidant membrane damaging and cytotoxic potential.

Polyunsaturated fatty acids are primary targets during oxidative stress. Diffusible electrophilic α,β-unsaturated aldehydes, such as 4-hydroxynonenal (HNE), have been shown to modify proteins that mediate cell signaling and modify gene expression pathways. We describe a global strategy for identifying the protein targets of HNE modification. A similar approach can be used for any electrophiles derived from an oxidized lipid. Curr. Protoc. Toxicol. 40:17.11.1-17.11.13. © 2009 by John Wiley & Sons, Inc. Keywords: electrophile; click chemistry; protein modification

Polyunsaturated fatty acids (PUFA) are primary targets of free radical damage during oxidative stress. Diffusible electrophilic alpha,beta-unsaturated aldehydes, such as 4-hydroxynonenal (HNE), have been shown to modify proteins that mediate cell signaling (e.g., IKK and Keap1) and alter gene expression pathways responsible for inducing antioxidant genes, heat shock proteins, and the DNA damage response. To fully understand cellular responses to HNE, it is important to determine its protein targets in an unbiased fashion. This requires a strategy for detecting and isolating HNE-modified proteins regardless of the nature of the chemical linkage between HNE and its targets. Azido or alkynyl derivatives of HNE were synthesized and demonstrated to be equivalent to HNE in their ability to induce heme oxygenase induction and induce apoptosis in colon cancer (RKO) cells. Cells exposed to the tagged HNE derivatives were lysed and exposed to reagents to effect Staudinger ligation or copper-catalyzed Huisgen 1,3 dipolar cycloaddition reaction (click chemistry) to conjugate HNE-adducted proteins with biotin for subsequent affinity purification. Both strategies yielded efficient biotinylation of tagged HNE-protein conjugates, but click chemistry was found to be superior for the recovery of biotinylated proteins from streptavidin-coated beads. Biotinylated proteins were detected in lysates from RKO cell incubations with azido-HNE at concentrations as low as 1 microM. These proteins were affinity purified with streptavidin beads, and proteomic analysis was performed by linear ion trap mass spectrometry. Proteomic analysis revealed a dose-dependent increase in labeled proteins with increased sequence coverage at higher concentrations. Several proteins involved in stress signaling (heat shock proteins 70 and 90 and the 78-kDa glucose-regulated protein) were selectively adducted by azido- and alkynyl-HNE. The use of azido and alkynyl derivatives in conjunction with click chemistry appears to be a valuable approach for the identification of the protein targets of HNE.

Whereas spontaneous and protein-mediated transfer/exchange of cholesterol (Ch) between membranes has been widely studied, relatively little is known about the translocation of Ch oxidation products, particularly hydroperoxide species (ChOOHs), which can act as cytotoxic prooxidants. A major aim of the present study was to examine and compare the intermembrane transfer characteristics of several biologically relevant ChOOH isomers, including singlet oxygen-derived 5alpha-OOH, 6alpha-OOH, and 6beta-OOH and free radical-derived 7alpha-OOH and 7beta-OOH. These species were generated in [(14)C]Ch-labeled donor membranes [erythrocyte ghosts or unilamellar DMPC/Ch (1.0:0.8 mol/mol) liposomes] by means of dye-sensitized photoperoxidation. Spontaneous transfer to nonoxidized acceptor membranes (DMPC liposomes or ghosts, respectively) at 37 degrees C was monitored by thin-layer chromatography with phosphorimaging radiodetection (HPTLC-PI) or liquid chromatography with mercury cathode electrochemical detection [HPLC-EC(Hg)]. The former allowed measurement of total (unresolved) ChOOH along with parent Ch, whereas the latter allowed measurement of individual ChOOHs. Ghost membranes in which approximately 4% of the Ch had been peroxidized, giving mainly 5alpha-OOH, transferred total ChOOH and Ch to liposomes in apparent first-order fashion, the rate constant for ChOOH being approximately 65 times greater. Like Ch desorption, ChOOH desorption from donor membranes was found to be rate limiting, and rate varied inversely with size when liposomal donors were used. For individual ChOOHs, rate constant magnitude (7alpha/7beta-OOH > 5alpha-OOH > 6alpha-OOH > 6beta-OOH) correlated inversely with reverse-phase HPLC retention time, suggesting that faster transfer reflects greater hydrophilicity. Liposome-borne ChOOHs exhibited the same order of toxicity toward COH-BR1 cells, which are deficient in ability to detoxify these peroxides. The prospect of disseminating oxidative cell injury via translocation of ChOOHs and other lipid hydroperoxides is readily apparent from these findings.

P14 systeMs aPProacHes to estaBlisHing tHe relationsHiP Between Protein ModiFication and cellUlar resPonses By liPid electroPHiles Lawrence J. Marnett1, Aaron Jacobs2, Andrew Vila1, Colleen McGrath1, Mariana Boiani3, Jody Ullery3, Keri Tallman3, Ned A. Porter1, Bing Zhang1, and Daniel C. Liebler1 1Vanderbilt University School of Medicine, 2University of Hawaii, 3Vanderbilt University, USA Electrophilic products of lipid peroxidation form covalent adducts with proteins and DNA that lead to alterations in cell signaling. We compared the ability of 4-hydroxynonenal (HNE), 4-oxononeal (ONE) and several oxidative metabolites to induce apoptosis and cell signaling in a human colon cancer cell line, RKO, and to inhibit cytokine synthesis in a human macrophage cell line, THP-1. To explore the molecular basis of electrophile signaling, we developed a strategy for post-hoc tagging of modified proteins that enables a comprehensive determination of their identities. HNE was substituted at its ω-termini with azido or alkynyl groups, which enabled derivatization via Click chemistry to attach biotin moieties. Exposed cells are lysed, protein extracts treated to attach biotin moieties, then tagged proteins adsorbed to streptavidin beads for affinity purification. Adsorbed proteins were digested to peptides that were identified by liquid chromatography/ mass spectrometry. In parallel, RNA was prepared from exposed cells and the pattern of gene expression determined by microarray analysis. These two datasets the identity of modified proteins and the transcriptional changes induced were generated for each electrophile exposure. Informatic analysis enabled linkages of these two datasets and generation of testable hypotheses regarding the transcriptional consequences of protein modification. This analysis led to the identification of several important cellular responses to electrophile damage resulting from the activation of fourteen and inhibition of four transcriptional pathways. An important activated pathway was the heat-shock response. siRNA-mediated knockdown of the transcription factor, Hsf1, eliminated HNE induction of heat-shock gene expression and increased the sensitivity of cells to its toxicity. Increased sensitivity was due to reduction in the levels of the anti-apoptotic proteins, Bcl-XL, Mcl-1, and to some extent, Bcl-2. The reduced levels of the Bcl-2 family members were due to the inability of the Hsf1-knocked-down cells to express the co-chaperone, Bag3. Additional studies revealed that Bag3 plays an important role in controlling the turnover of the anti-apoptotic Bcl-2 family proteins. Systems level integration of protein modification and transcriptional regulation enables elucidation of the biological consequences of individual protein modification by electrophilic products of lipid peroxidation. Supported by P01ES013125.