No abstract

In this era of genomics, transcriptomics, and proteomics, metabolomics is emerging as an important component of the omics evolution ( 1 ). Of the four kinds of biological molecules that comprise the human body, i.e., nucleic acids, amino acids (proteins), carbohydrates (sugars), and lipids (fats), lipids stand out among the various cellular metabolites in the sheer number of distinct molecular species. Using state-of-the-art lipidomics approaches made possible by newly developed instrumentation, protocols, and bioinformatics tools ( 2 ), the LIPID MAPS Consortium Abstract The focus of the present study was to defi ne the human plasma lipidome and to establish novel analytical methodologies to quantify the large spectrum of plasma lipids. Partial lipid analysis is now a regular part of every patient's blood test and physicians readily and regularly prescribe drugs that alter the levels of major plasma lipids such as cholesterol and triglycerides. Plasma contains many thousands of distinct lipid molecular species that fall into six main categories including fatty acyls, glycerolipids, glycerophospholipids, sphingolipids, sterols, and prenols. The physiological contributions of these diverse lipids and how their levels change in response to therapy remain largely unknown. As a fi rst step toward answering these questions, we provide herein an in-depth lipidomics analysis of a pooled human plasma obtained from healthy individuals after overnight fasting and with a gender balance and an ethnic distribution that is representative of the US population. In total, we quantitatively assessed the levels of over 500 distinct molecular species distributed among the main lipid categories. As more information is obtained regarding the roles of individual lipids in health and disease, it seems likely that future blood tests will include an ever increasing number of these lipid molecules. -Quehenberger, O., A.

A number of clinical isolates of Pseudomonas aeruginosa are cytotoxic to mammalian cells due to the action of the 74-kDa protein ExoU, which is secreted into host cells by the type III secretion system and whose function is unknown. Here we report that the swift and profound cytotoxicity induced by purified ExoU or by an ExoUexpressing strain of P. aeruginosa is blocked by various inhibitors of cytosolic (cPLA 2 ) and Ca 2؉ -independent (iPLA 2 ) phospholipase A 2 enzymes. In contrast, no cytoprotection is offered by inhibitors of secreted phospholipase A 2 enzymes or by a number of inhibitors of signal transduction pathways. This suggests that phospholipase A 2 inhibitors may represent a novel mode of treatment for acute P. aeruginosa infections. We find that 300 -600 molecules of ExoU/cell are required to achieve half-maximal cell killing and that ExoU localizes to the host cell plasma membrane in punctate fashion. We also show that ExoU interacts in vitro with an inhibitor of cPLA 2 and iPLA 2 enzymes and contains a putative serine-aspartate catalytic dyad homologous to those found in cPLA 2 and iPLA 2 enzymes. Mutation of either the serine or the aspartate renders ExoU non-cytotoxic. Although no phospholipase or esterase activity is detected in vitro, significant phospholipase activity is detected in vivo, suggesting that ExoU requires one or more host cell factors for activation as a membrane-lytic and cytotoxic phospholipase.

The Group IVA cytosolic phospholipase A(2) (GIVA PLA(2)) is a particularly attractive target for drug development because it is the rate-limiting provider of proinflammatory mediators. We previously reported the discovery of novel 2-oxoamides that inhibit GIVA PLA(2) [Kokotos, G.; et al. J. Med. Chem. 2002, 45, 2891-2893]. In the present work, we have further explored this class of inhibitors and found that the 2-oxoamide functionality is more potent when it contains a long 2-oxoacyl residue and a free carboxy group. Long-chain 2-oxoamides based on gamma-aminobutyric acid and gamma-norleucine are potent inhibitors of GIVA PLA(2). Such inhibitors act through a fast and reversible mode of inhibition in vitro, are able to block the production of arachidonic acid and prostaglandin E(2) in cells, and demonstrate potent in vivo anti-inflammatory and analgesic activity.

The outer monolayer of the outer membrane of Gram-negative bacteria consists of the lipid A component of lipopolysaccharide (LPS), a glucosamine-based saccharolipid that is assembled on the inner surface of the inner membrane. The first six enzymes of the lipid A pathway are required for bacterial growth and are excellent targets for the development of new antibiotics. Following assembly, the ABC transporter MsbA flips nascent LPS to the periplasmic side of the inner membrane, whereupon additional transport proteins direct it to the outer surface of the outer membrane. Depending on the bacterium, various covalent modifications of the lipid A moiety may occur during the transit of LPS to the outer membrane. These extra-cytoplasmic modification enzymes are therefore useful as reporters for monitoring LPS trafficking. Because of its conserved structure in diverse Gram-negative pathogens, lipid A is recognized as foreign by the TLR4/MD2 receptor of the mammalian innate immune system, resulting in rapid macrophage activation and robust cytokine production

2 . This increased affinity is accompanied by an increase in substrate hydrolysis of a similar magnitude. The binding studies and kinetic analysis indicate that PtdIns(4,5)P 2 binds to cPLA 2 in a 1:1 stoichiometry. The magnitude of the effect of PtdIns(4,5)P 2 is unique among anionic phospholipids and larger than that for other polyphosphate phosphatidylinositols. The effect of PtdIns(4,5)P 2 on the activity of cPLA 2 is at least an order of magnitude larger than the concomitant changes in the fraction of the enzyme associated with lipid membranes. Striking parallels between the interaction of cPLA 2 with PtdIns(4,5)P 2 and the interaction of the pleckstrin homology domain of phospholipase C␦ 1 with PtdIns(4,5) 2 combined with sequence analysis of cPLA 2 lead us to propose the existence and location of a pleckstrin homology domain in cPLA 2 . We further show that the very nature of the interaction of proteins such as cPLA 2 with multiple ligands incorporated into membranes follows a specific model which necessitates the use of an experimental methodology suitable for a membrane interface to allow for a meaningful analysis of the data.Cytosolic phospholipase A 2 (cPLA 2 ) 1 has unique structural and regulatory properties within the PLA 2 superfamily (1, 2). Current widespread interest in the cytosolic phospholipase A 2 stems from its putative role as a key enzyme involved in the inflammatory response, a provider of arachidonic acid, the precursor for prostaglandins and leukotrienes (3, 4). This enzyme was also implicated in the regulation of several other processes such as platelet activation, cell proliferation, and the generation of several second messengers (3-5). A mobilization of intracellular Ca 2ϩ was implicated in the translocation of cPLA 2 to cellular membranes resulting in the subsequent specific liberation of arachidonic acid from the sn-2 position of phospholipids (6, 7). This scenario of activation of cPLA 2 was validated by the discovery that the N terminus of this enzyme contains an autonomous calcium and lipid binding domain, CaLB (7), homologous to the C2 domain (8) of protein kinase C and several other proteins shown to associate with lipid membranes in a Ca 2ϩ -dependent fashion (7, 9). Both the native enzyme and its CaLB domain were shown to associate with fragments of cellular membranes and synthetic lipid vesicles at physiologically relevant Ca 2ϩ concentrations (6, 7, 10, 11). Thus far, the CaLB domain is the only recognized regulatory domain of cPLA 2 (5).It was noted early on (12) that several anionic phospholipids including phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2 ) activated cPLA 2 . This stimulatory effect was hypothesized to result from an enhancement of the partitioning of cPLA 2 into lipid membranes caused in general by all anionic lipids. We have now employed an approach that was specifically designed for the characterization of proteins that bind to membranes through multiple attachment points to demonstrate that cPLA 2 binds in a 1:1 stoichiometry with high affinit...

A novel class of potent human cytosolic phospholipase A(2) (GIVA PLA(2)) inhibitors was developed. These inhibitors were designed to contain the 2-oxoamide functionality and a free carboxyl group. Among the compounds tested, a long-chain 2-oxoamide containing L-gamma-norleucine was the most potent inhibitor, causing a 50% decrease in GIVA PLA(2) activity at 0.009 mole fraction.

The Group IVA cytosolic phospholipase A2 (GIVA cPLA2) is a key provider of substrates for the production of eicosanoids and platelet-activating factor. We explored the structure-activity relationship of 2-oxoamide-based compounds and GIVA cPLA2 inhibition. The most potent inhibitors are derived from delta- and gamma-amino acid-based 2-oxoamides. The optimal side-chain moiety is a short nonpolar aliphatic chain. All of the newly developed 2-oxoamides as well as those previously described have now been tested with the human Group V secreted PLA2 (GV sPLA2) and the human Group VIA calcium-independent PLA2 (GVIA iPLA2). Only one 2-oxoamide compound had appreciable inhibition of GV sPLA2, and none of the potent GIVA cPLA2 inhibitors inhibited either GV sPLA2 or GVIA iPLA2. Two of these specific GIVA cPLA2 inhibitors were also found to have potent therapeutic effects in animal models of pain and inflammation at dosages well below the control nonsteroidal anti-inflammatory drugs.