Corinne M. Spickett

Oxidized Phospholipid Inhibition of Toll-like Receptor (TLR) Signaling Is Restricted to TLR2 and TLR4 ROLES FOR CD14, LPS-BINDING PROTEIN, AND MD2 AS TARGETS FOR SPECIFICITY OF INHIBITION

The generation of reactive oxygen species is a central feature of inflammation that results in the oxidation of host phospholipids. Oxidized phospholipids, such as 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine (OxPAPC), have been shown to inhibit signaling induced by bacterial lipopeptide or lipopolysac-charide (LPS), yet the mechanisms responsible for the inhibition of Toll-like receptor (TLR) signaling by OxPAPC remain incompletely understood. Here, we examined the mechanisms by which OxPAPC inhibits TLR signaling induced by diverse ligands in macrophages, smooth muscle cells, and epithelial cells. OxPAPC inhibited tumor necrosis factor-α production, IκBα degradation, p38 MAPK phosphorylation, and NF-κB-dependent reporter activation induced by stimulants of TLR2 and TLR4 (Pam3CSK4 and LPS) but not by stimulants of other TLRs (poly(I·C), flagellin, loxoribine, single-stranded RNA, or CpG DNA) in macrophages and HEK-293 cells transfected with respective TLRs and significantly reduced inflammatory responses in mice injected subcutaneously or intraperitoneally with Pam3CSK4. Serum proteins, including CD14 and LPS-binding protein, were identified as key targets for the specificity of TLR inhibition as supplementation with excess serum or recombinant CD14 or LBP reversed TLR2 inhibition by OxPAPC, whereas serum accessory proteins or expression of membrane CD14 potentiated signaling via TLR2 and TLR4 but not other TLRs. Binding experiments and functional assays identified MD2 as a novel additional target of OxPAPC inhibition of LPS signaling. Synthetic phospholipid oxidation products 1-palmitoyl-2-(5-oxovaleryl)-sn-glycero-3-phosphocholine and 1-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine inhibited TLR2 signaling from ∼30 μm. Taken together, these results suggest that oxidized phospholipid-mediated inhibition of TLR signaling occurs mainly by competitive interaction with accessory proteins that interact directly with bacterial lipids to promote signaling via TLR2 or TLR4.

Chemistry of phospholipid oxidation

The oxidation of lipids has long been a topic of interest in biological and food sciences, and the fundamental principles of non-enzymatic free radical attack on phospholipids are well established, although questions about detail of the mechanisms remain. The number of end products that are formed following the initiation of phospholipid peroxidation is large, and is continually growing as new structures of oxidized phospholipids are elucidated. Common products are phospholipids with esterified isoprostane-like structures and chain-shortened products containing hydroxy, carbonyl or carboxylic acid groups; the carbonyl-containing compounds are reactive and readily form adducts with proteins and other biomolecules. Phospholipids can also be attacked by reactive nitrogen and chlorine species, further expanding the range of products to nitrated and chlorinated phospholipids. Key to understanding the mechanisms of oxidation is the development of advanced and sensitive technologies that enable structural elucidation. Tandem mass spectrometry has proved invaluable in this respect and is generally the method of choice for structural work. A number of studies have investigated whether individual oxidized phospholipid products occur in vivo, and mass spectrometry techniques have been instrumental in detecting a variety of oxidation products in biological samples such as atherosclerotic plaque material, brain tissue, intestinal tissue and plasma, although relatively few have achieved an absolute quantitative analysis. The levels of oxidized phospholipids in vivo is a critical question, as there is now substantial evidence that many of these compounds are bioactive and could contribute to pathology. The challenges for the future will be to adopt lipidomic approaches to map the profile of oxidized phospholipid formation in different biological conditions, and relate this to their effects in vivo. This article is part of a Special Issue entitled: Oxidized phospholipids-their properties and interactions with proteins.

The lipid peroxidation product 4-hydroxy-2-nonenal: Advances in chemistry and analysis

4-Hydroxy-2-nonenal (HNE) is one of the most studied products of phospholipid peroxidation, owing to its reactivity and cytotoxicity. It can be formed by several radical-dependent oxidative routes involving the formation of hydroperoxides, alkoxyl radicals, epoxides, and fatty acyl cross-linking reactions. Cleavage of the oxidized fatty acyl chain results in formation of HNE from the methyl end, and 9-oxo-nonanoic acid from the carboxylate or esterified end of the chain, although many other products are also possible. HNE can be metabolized in tissues by a variety of pathways, leading to detoxification and excretion. HNE-adducts to proteins have been detected in inflammatory situations such as atherosclerotic lesions using polyclonal and monoclonal antibodies, which have also been applied in ELISAs and western blotting. However, in order to identify the proteins modified and the exact sites and nature of the modifications, mass spectrometry approaches are required. Combinations of enrichment strategies with targetted mass spectrometry routines such as neutral loss scanning are now facilitating detection of HNE-modified proteins in complex biological samples. This is important for characterizing the interactions of HNE with redox sensitive cell signalling proteins and understanding how it may modulate their activities either physiologically or in disease.

Advances in methods for the determination of biologically relevant lipid peroxidation products

Abstract Lipid peroxidation is recognized to be an important contributor to many chronic diseases, especially those of an inflammatory pathology. In addition to their value as markers of oxidative damage, lipid peroxidation products have also been shown to have a wide variety of biological and cell signalling effects. In view of this, accurate and sensitive methods for the measurement of lipid peroxidation products are essential. Although some assays have been described for many years, improvements in protocols are continually being reported and, with recent advances in instrumentation and technology, highly specialized and informative techniques are increasingly used. This article gives an overview of the most currently used methods and then addresses the recent advances in some specific approaches. The focus is on analysis of oxysterols, F2-isoprostanes and oxidized phospholipids by gas chromatography or liquid chromatography mass spectrometry techniques and immunoassays for the detection of 4-hydroxynonenal.

Pathways of phospholipid oxidation by HOCl in human LDL detected by LC-MS.

A wealth of evidence now indicates that low-density lipoprotein (LDL) must be modified to promote atherosclerosis, and that this may involve oxidants released by phagocytes. Many studies of oxidative damage in atherosclerosis previously have concentrated on damage by nonhalogenated oxidants, but HOCl is a highly toxic oxidant produced by myeloperoxidase in phagocytes, which is also likely to be important in the disease pathogenesis. Currently some controversy exists over the products resulting from reaction of HOCl with LDL lipids, in particular regarding whether predominantly chlorohydrins or lipid peroxides are formed. In this study LC-MS of phosphatidylcholines in human LDL treated either with HOCl or the myeloperoxidase system was used as a specific method to detect chlorohydrin and peroxide formation simultaneously, and with comparable sensitivity. Chlorohydrin products from lipids containing oleic, linoleic and arachidonic acids were detected, but no hydroperoxides of linoleoyl or arachidonoyl lipids could be observed. This study provides the first direct evidence that lipid chlorohydrins rather than peroxides are the major products of HOCl- or myeloperoxidase-treated LDL phospholipids. This in turn provides important information required for the study of oxidative damage in vivo which will allow the type and source of oxidants involved in the pathology of atherosclerosis to be investigated.

A comparison of five lipid extraction solvent systems for lipidomic studies of human LDL

Lipidome profile of fluids and tissues is a growing field as the role of lipids as signaling molecules is increasingly understood, relying on an effective and representative extraction of the lipids present. A number of solvent systems suitable for lipid extraction are commonly in use, though no comprehensive investigation of their effectiveness across multiple lipid classes has been carried out. To address this, human LDL from normolipidemic volunteers was used to evaluate five different solvent extraction protocols [Folch, Bligh and Dyer, acidified Bligh and Dyer, methanol (MeOH)-tert-butyl methyl ether (TBME), and hexane-isopropanol] and the extracted lipids were analyzed by LC-MS in a high-resolution instrument equipped with polarity switching. Overall, more than 350 different lipid species from 19 lipid subclasses were identified. Solvent composition had a small effect on the extraction of predominant lipid classes (triacylglycerides, cholesterol esters, and phosphatidylcholines). In contrast, extraction of less abundant lipids (phosphatidylinositols, lyso-lipids, ceramides, and cholesterol sulfates) was greatly influenced by the solvent system used. Overall, the Folch method was most effective for the extraction of a broad range of lipid classes in LDL, although the hexane-isopropanol method was best for apolar lipids and the MeOH-TBME method was suitable for lactosyl ceramides.

Physiological effects of oxidized phospholipids and their cellular signaling mechanisms in inflammation.

Oxidized phospholipids, such as the products of the oxidation of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine by nonenzymatic radical attack, are known to be formed in a number of inflammatory diseases. Interest in the bioactivity and signaling functions of these compounds has increased enormously, with many studies using cultured immortalized and primary cells, tissues, and animals to understand their roles in disease pathology. Initially, oxidized phospholipids were viewed largely as culprits, in line with observations that they have proinflammatory effects, enhancing inflammatory cytokine production, cell adhesion and migration, proliferation, apoptosis, and necrosis, especially in vascular endothelial cells, macrophages, and smooth muscle cells. However, evidence has emerged that these compounds also have protective effects in some situations and cell types; a notable example is their ability to interfere with signaling by certain Toll-like receptors (TLRs) induced by microbial products that normally leads to inflammation. They also have protective effects via the stimulation of small GTPases and induce up-regulation of antioxidant enzymes and cytoskeletal rearrangements that improve endothelial barrier function. Oxidized phospholipids interact with several cellular receptors, including scavenger receptors, platelet-activating factor receptors, peroxisome proliferator-activated receptors, and TLRs. The various and sometimes contradictory effects that have been observed for oxidized phospholipids depend on their concentration, their specific structure, and the cell type investigated. Nevertheless, the underlying molecular mechanisms by which oxidized phospholipids exert their effects in various pathologies are similar. Although our understanding of the actions and mechanisms of these mediators has advanced substantially, many questions do remain about their precise interactions with components of cell signaling pathways.

Detection of phospholipid oxidation in oxidatively stressed cells by reversed-phase HPLC coupled with positive-ionization electrospray MS

Measurement of lipid peroxidation is a commonly used method of detecting oxidative damage to biological tissues, but the most frequently used methods, including MS, measure breakdown products and are therefore indirect. We have coupled reversed-phase HPLC with positive-ionization electrospray MS (LC-MS) to provide a method for separating and detecting intact oxidized phospholipids in oxidatively stressed mammalian cells without extensive sample preparation. The elution profile of phospholipid hydroperoxides and chlorohydrins was first characterized using individual phospholipids or a defined phospholipid mixture as a model system. The facility of detection of the oxidized species in complex mixtures was greatly improved compared with direct-injection MS analysis, as they eluted earlier than the native lipids, owing to the decrease in hydrophobicity. In U937 and HL60 cells treated in vitro with t-butylhydroperoxide plus Fe2+, lipid oxidation could not be observed by direct injection, but LC-MS allowed the detection of monohydroperoxides of palmitoyl-linoleoyl and stearoyl-linoleoyl phosphatidylcholines. The levels of hydroperoxides observed in U937 cells were found to depend on the duration and severity of the oxidative stress. In cells treated with HOCl, chlorohydrins of palmitoyloleoyl phosphatidylcholine were observed by LC-MS. The method was able to detect very small amounts of oxidized lipids compared with the levels of native lipids present. The membrane-lipid profiles of these cells were found to be quite resistant to damage until high concentrations of oxidants were used. This is the first report of direct detection by LC-MS of intact oxidized phospholipids induced in cultured cells subjected to oxidative stress.

Direct observation of lipid hydroperoxides in phospholipid vesicles by electrospray mass spectrometry

Positive ion electrospray ionization mass spectrometry was used to obtain a lipid profile of vesicles prepared from egg yolk lethicin and enriched with arachidonylstearoyl phosphatidylcholine and dipalmitoyl phosphatidylcholine. The vesicles were oxidized by treatment with tert-butylhydroperoxide and iron (II) sulfate, and the formation of hydroperoxides of the polyunsaturated lipid arachidonylstearoyl phosphatidylcholine was observed. The native lipid signal at 832 a.m.u. decreased and new signals appeared at 864, 896, and 928 a.m.u., corresponding to the addition of one (+32), two (+64), and three (+96) molecules of dioxygen. The dihydroperoxide was found to be the most favourable peroxide product, but it appeared that a degradation of the hydroperoxides was occurring concomitant with their formation, and only their net formation was observed. The rate of depletion of the polyunsaturated lipid and the rate of accumulation of the hydroperoxides was found to increase with the Fe2+ concentration between 10 microM and 2 mM, and was also dependent on the tert-butylhydroperoxide concentration. This is the first report of analysis of lipid hydroperoxides by electrospray mass spectrometry, showing that technique offers a sensitive, direct, and informative approach to the study of oxidative damage to biological membranes.

Post-translational modifications and mass spectrometry detection

In this review, we provide a comprehensive bibliographic overview of the role of mass spectrometry and the recent technical developments in the detection of post-translational modifications (PTMs). We briefly describe the principles of mass spectrometry for detecting PTMs and the protein and peptide enrichment strategies for PTM analysis, including phosphorylation, acetylation and oxidation. This review presents a bibliographic overview of the scientific achievements and the recent technical development in the detection of PTMs is provided. In order to ascertain the state of the art in mass spectrometry and proteomics methodologies for the study of PTMs, we analyzed all the PTM data introduced in the Universal Protein Resource (UniProt) and the literature published in the last three years. The evolution of curated data in UniProt for proteins annotated as being post-translationally modified is also analyzed. Additionally, we have undertaken a careful analysis of the research articles published in the years 2010 to 2012 reporting the detection of PTMs in biological samples by mass spectrometry.