Polyunsaturated fatty acids (PUFA) are primary targets of free radical damage during oxidative stress. Diffusible electrophilic R, -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 streptavidincoated beads. Biotinylated proteins were detected in lysates from RKO cell incubations with azido-HNE at concentrations as low as 1 µM. 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 glucoseregulated 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.

No abstract

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.

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-OOH > 5 alpha-OOH > 6 alpha-OOH > 6 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 PSOOH > SMOOH. 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.

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-OOH < 6alpha-OOH < 5alpha-OOH < 7alpha/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.

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 lipoaminoacids 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-H 2 -ethanolamide (PGH 2 -EA) and -glycerol ester (PGH 2 -G), respectively. Further metabolism of PGH 2 -EA and PGH 2 -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 ∼200-300 fold higher for FAAH. Thus, PG-Gs are poor substrates for the major endocannabinoid-degrading enzymes, MGL and FAAH.COX-2 can oxygenate arachidonylethanolamide (AEA) 1 and 2-arachidonylglycerol (2-AG) to prostaglandin-H 2 -ethanolamide (PGH 2 -EA) and -glycerol ester (PGH 2 -G), respectively (1, 2). Prostaglandin isomerases/synthases metabolize the PGH 2 -derived species to the corresponding PGE 2 , PGD 2 , PGF 2R , and PGI 2 , (but not TxA 2 ) ethanolamides or glyceryl esters in vitro (3). Peritoneal macrophages synthesize PGE 2 -G and PGI 2 -G in response to zymosan treatment, and RAW 264.7 cells synthesize PGD 2 -G when exposed to ionomycin following lipopolysaccharide and interferon-γ pretreatment (4, 5). An important consideration when assessing the impact of PG-G synthesis in cells or tissues is the rate of degradation of this bioactive lipid. For example, primary macrophages do not significantly degrade PG-Gs over several hours, but PGE 2 -G is rapidly metabolized to its free acid species in rat plasma [t 1/2 ∼ 14 s (6)]. In contrast to PG-Gs, PG-EAs are relatively stable to hydrolysis in rat whole blood (6). Instead, PGE 2 -EA undergoes slow dehydration and/or isomerization to produce PGB 2 -EA (approximately two-thirds of the starting material converted over 40 h at 37°C) (6).Although previous cellular studies demonstrate PG-G formation (4, 5), evidence for the production of these species in intact tissue is lacking. However, it has been shown that COX-2 participates in regulating 2-AG levels in neurons during retrograde signaling (7), and COX-2 metabolites of 2-AG metabolism (PGE 2 -G, D 2 -G, and F 2R -G) increase the frequency of miniature inhibitory postsynaptic currents in primary hippocampal neurons (8). This latter effect is independent of endocannabinoid and prostanoid signaling pathways. However, it appears that COX-2 inhibitors specifically block inhibitory retrograde signaling in the hippocampus. Others show that COX-2 does n...

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.