Goslin 2.0 Implements the Recent Lipid Shorthand Nomenclature for MS-Derived Lipid Structures.

Goslin is the first grammar-based computational library for the recognition/parsing and normalization of lipid names following the hierarchical lipid shorthand nomenclature. The new version Goslin 2.0 implements the latest nomenclature and adds an additional grammar to recognize systematic IUPAC-IUB fatty acyl names as stored, e.g., in the LIPID MAPS database and is perfectly suited to update lipid names in LIPID MAPS or HMDB databases to the latest nomenclature. Goslin 2.0 is available as a standalone web application with a REST API as well as C++, C#, Java, Python 3, and R libraries. Importantly, it can be easily included in lipidomics tools and scripts providing direct access to translation functions. All implementations are open source.

Update on LIPID MAPS classification, nomenclature, and shorthand notation for MS-derived lipid structures.

A comprehensive and standardized system to report lipid structures analyzed by MS is essential for the communication and storage of lipidomics data. Herein, an update on both the LIPID MAPS classification system and shorthand notation of lipid structures is presented for lipid categories Fatty Acyls (FA), Glycerolipids (GL), Glycerophospholipids (GP), Sphingolipids (SP), and Sterols (ST). With its major changes, i.e., annotation of ring double bond equivalents and number of oxygens, the updated shorthand notation facilitates reporting of newly delineated oxygenated lipid species as well. For standardized reporting in lipidomics, the hierarchical architecture of shorthand notation reflects the diverse structural resolution powers provided by mass spectrometric assays. Moreover, shorthand notation is expanded beyond mammalian phyla to lipids from plant and yeast phyla. Finally, annotation of atoms is included for the use of stable isotope-labeled compounds in metabolic labeling experiments or as internal standards. This update on lipid classification, nomenclature, and shorthand annotation for lipid mass spectra is considered a standard for lipid data presentation.

Deciphering lipid structures based on platform-independent decision rules.

We achieve automated and reliable annotation of lipid species and their molecular structures in high-throughput data from chromatography-coupled tandem mass spectrometry using decision rule sets embedded in Lipid Data Analyzer (LDA; http://genome.tugraz.at/lda2). Using various low- and high-resolution mass spectrometry instruments with several collision energies, we proved the method's platform independence. We propose that the software's reliability, flexibility, and ability to identify novel lipid molecular species may now render current state-of-the-art lipid libraries obsolete.

Phthalates efficiently bind to human peroxisome proliferator activated receptor and retinoid X receptor α, ß, γ subtypes: an in silico approach.

This exhaustive in silico study looks into the molecular interactions of phthalates and their metabolites with human peroxisome proliferator-activated receptor (hPPAR) and retinoid X receptor (hRXR) α, ß and γ subtypes--the nuclear receptor proteins function as transcription factors by regulating the expression of downstream genes. Apart from the much discussed plasticizer bisphenol A, we examined the binding affinities of 15 common diphthalates and their monophthalates, natural (linoleic acid, conjugated linoleic acid) and synthetic (bezafibrate, pioglitazone, GW 50156) ligands with hPPARs. In addition to these phthalates, specific natural (retinoic and phytanic acids) and synthetic (bexarotene, rosiglitazone) ligands were examined with hRXRs. The Maestro, Schrödinger Suite 2012 was used for the molecular docking study. In general, natural ligands of hPPAR showed less binding efficiencies than phthalic acid esters and drugs. The diphthalate di-iso-decyl phthalate showed the highest G score (-9.99) with hPPAR (γ), while its monophthalate (mono-iso-decyl phthalate) showed a comparatively less G score (-9.56). Though the PPAR modulator GW 50156 showed strong affinity with all hPPAR subtypes, its highest G score (-12.43) was with hPPARß. Hazardous di(2-ethylhexyl)phthalate generally showed a greater preference to hRXRs than hPPARs, but its highest G score (-10.87) was with hRXRα; while its monophthalate (Mono(2-ethylhexyl)phthalate) showed a lesser G score (-8.59). The drug bexarotene showed the highest G score (-13.32) with hRXRß. Moreover, bisphenol A showed more affinity towards hRXR. Briefly, this study gives an overview on the preference of phthalic acid esters, natural and synthetic ligands on to hPPAR and hRXR subtypes, which would lead to further in vitro mechanistic as well as in vivo preclinical and clinical studies.

Shorthand notation for lipid structures derived from mass spectrometry.

There is a need for a standardized, practical annotation for structures of lipid species derived from mass spectrometric approaches; i.e., for high-throughput data obtained from instruments operating in either high- or low-resolution modes. This proposal is based on common, officially accepted terms and builds upon the LIPID MAPS terminology. It aims to add defined levels of information below the LIPID MAPS nomenclature, as detailed chemical structures, including stereochemistry, are usually not automatically provided by mass spectrometric analysis. To this end, rules for lipid species annotation were developed that reflect the structural information derived from the analysis. For example, commonly used head group-specific analysis of glycerophospholipids (GP) by low-resolution instruments is neither capable of differentiating the fatty acids linked to the glycerol backbone nor able to define their bond type (ester, alkyl-, or alk-1-enyl-ether). This and other missing structural information is covered by the proposed shorthand notation presented here. Beyond GPs, we provide shorthand notation for fatty acids/acyls (FA), glycerolipids (GL), sphingolipids (SP), and sterols (ST). In summary, this defined shorthand nomenclature provides a standard methodology for reporting lipid species from mass spectrometric analysis and for constructing databases.

The impact of genetic stress by ATGL deficiency on the lipidome of lipid droplets from murine hepatocytes.

We showed earlier that nutritional stress like starvation or high-fat diet resulted in phenotypic changes in the lipidomes of hepatocyte lipid droplets (LDs), representative for the pathophysiological status of the mouse model. Here we extend our former study by adding genetic stress due to knockout (KO) of adipocyte triglyceride lipase (ATGL), the rate limiting enzyme in LD lipolysis. An intervention trial for 6 weeks with male wild-type (WT) and ATGL-KO mice was carried out; both genotypes were fed lab chow or were exposed to short-time starvation. Isolated LDs were analyzed by LC-MS/MS. Triacylglycerol, diacylglycerol, and phosphatidylcholine lipidomes, in that order, provided the best phenotypic signatures characteristic for respective stresses applied to the animals. This was evidenced at lipid species level by principal component analysis, calculation of average values for chain-lengths and numbers of double bonds, and by visualization in heat maps. Structural backgrounds for analyses and metabolic relationships were elaborated at lipid molecular species level. Relating our lipidomic data to nonalcoholic fatty liver diseases of nutritional and genetic etiologies with or without accompanying insulin resistance, phenotypic distinction in hepatocyte LDs dependent on insulin status emerged. Taken together, lipidomes of hepatocyte LDs are sensitive responders to nutritional and genetic stress.

Lipidomic analysis of lipid droplets from murine hepatocytes reveals distinct signatures for nutritional stress.

Liver steatosis can be induced by fasting or high-fat diet. We investigated by lipidomic analysis whether such metabolic states are reflected in the lipidome of hepatocyte lipid droplets (LDs) from mice fed normal chow diet (FED), fasted (FAS), or fed a high-fat diet (HFD). LC-MS/MS at levels of lipid species profiles and of lipid molecular species uncovered a FAS phenotype of LD enriched in triacylglycerol (TG) molecular species with very long-chain (VLC)-PUFA residues and an HFD phenotype with less unsaturated TG species in addition to characteristic lipid marker species. Nutritional stress did not result in dramatic structural alterations in diacylglycerol (DG) and phospholipid (PL) classes. Moreover, molecular species of bulk TG and of DG indicated concomitant de novo TG synthesis and lipase-catalyzed degradation to be active in LDs. DG species with VLC-PUFA residues would be preferred precursors for phosphatidylcholine (PC) species, the others for TG molecular species. In addition, molecular species of PL classes fitted the hepatocyte Kennedy and phosphatidylethanolamine methyltransferase pathways. We demonstrate that lipidomic analysis of LDs enables phenotyping of nutritional stress. TG species are best suited for such phenotyping, whereas structural analysis of TG, DG, and PL molecular species provides metabolic insights.

Lipid Data Analyzer: unattended identification and quantitation of lipids in LC-MS data.

MOTIVATION: The accurate measurement of the lipidome permits insights into physiological and pathological processes. Of the present high-throughput technologies, LC-MS especially bears potential of monitoring quantitative changes in hundreds of lipids simultaneously. In order to extract valuable information from huge amount of mass spectrometry data, the aid of automated, reliable, highly sensitive and specific analysis algorithms is indispensable. RESULTS: We present here a novel approach for the quantitation of lipids in LC-MS data. The new algorithm obtains its analytical power by two major innovations: (i) a 3D algorithm that confines the peak borders in m/z and time direction and (ii) the use of the theoretical isotopic distribution of an analyte as selection/exclusion criterion. The algorithm is integrated in the Lipid Data Analyzer (LDA) application which additionally provides standardization, a statistics module for results analysis, a batch mode for unattended analysis of several runs and a 3D viewer for the manual verification. The statistics module offers sample grouping, tests between sample groups and export functionalities, where the results are visualized by heat maps and bar charts. The presented algorithm has been applied to data from a controlled experiment and to biological data, containing analytes distributed over an intensity range of 10(6). Our approach shows improved sensitivity and an extremely high positive predictive value compared with existing methods. Consequently, the novel algorithm, integrated in a user-friendly application, is a valuable improvement in the high-throughput analysis of the lipidome. IMPLEMENTATION AND AVAILABILITY: The Java application is freely available for non-commercial users at http://genome.tugraz.at/lda. Raw data associated with this manuscript may be downloaded from ProteomeCommons.org Tranche using the following hash: ZBh3nS5bXk6I/Vn32tB5Vh0qnMpVIW71HByFFQqM0RmdF4/4Hcn H3Wggh9kU2teYVOtM1JWwHIeMHqSS/bc2yYNFmyUAAAAAAACl DQ ==

α-Linolenic acid and product octadecanoids in Styrian pumpkin seeds and oils: How processing impacts lipidomes of fatty acid, triacylglycerol and oxylipin molecular structures.

Styrian pumpkin seed oil is a conditioned green-colored oil renowned for nutty smell and taste. Due to α-linolenic acid (ALA) contents below 1% of total fatty acids and the prospect of nutritional health claims based on its potential oxidation products, we investigated the fate of ALA and product oxylipins in the course of down-stream processing of seeds and in oils. Lipidomic analyses with Lipid Data Analyzer 2.8.1 revealed: Processing did not change (1) main fatty acid composition in the oils, (2) amounts of triacylglycerol species, (3) structures of triacylglycerol molecular species containing ALA. (4) Minor precursor ALA in fresh Styrian and normal pumpkins produced 6 product phytoprostanes in either cultivar, quantitatively more in the latter. (5) In oil samples 7 phytoprostanes and 2 phytofurans were detected. The latter two are specific for their presence in pumpkin seed oils, of note, quantitatively more in conditioned oils than in cold-pressed native oils.

A comprehensive method for lipid profiling by liquid chromatography-ion cyclotron resonance mass spectrometry.

This work aims to combine chromatographic retention, high mass resolution and accuracy, MS/MS spectra, and a package for automated identification and quantitation of lipid species in one platform for lipidomic analysis. The instrumental setup elaborated comprises reversed-phase HPLC coupled to a Fourier transform ion cyclotron resonance mass spectrometer (LTQ-FT), and Lipid Data Analyzer (LDA) software. Data analysis for lipid species quantification in this platform is based on retention time, mass resolution of 200,000, and mass accuracy below 2 ppm. In addition, automatically generated MS/MS spectra provide structural information at molecular level. This LC/MS technology allows analyzing complex biological samples in a quantitative manner as shown here paradigmatically for murine lipid droplets having a huge surplus of triacylglycerol species. Chromatographic preseparation of the bulk lipid class alleviates the problem of ion suppression of lipid species from other classes. Extension of 1D to 2D chromatography is possible, yet time consuming. The platform affords unambiguous detection of lipid species as low as 0.1‰ within major lipid classes. Taken together, a novel lipidomic LC/MS platform based on chromatographic retention, high mass resolution and accuracy, MS/MS analysis, and quantitation software enables analysis of complex samples as demonstrated for lipid droplets.

Activation of PPARgamma reverses a defect of surfactant synthesis in mice lacking two types of fatty acid binding protein.

Lung surfactant is a lipid-protein-film covering the inner alveolar surface. We have previously shown that double knock-out (d-ko) mice lacking both the epidermal-type (E-) and the heart-type (H-) fatty acid binding protein (FABP) exhibit a defect of surfactant synthesis in alveolar type II cells that can be corrected by feeding pioglitazone, a drug that activates peroxisome proliferator-activated receptor gamma (PPARgamma). Here, we demonstrate first that healthy surfactant at collapse pressure produces protrusions composed of bilayers but not folds, second that the d-ko effect profoundly perturbs lipid/hydrophobic protein composition, pressure-area isotherm, and structural organisation of the surfactant at nanoscale, parameters that are critical for the normal breathing cycle. In support of these data in vivo measurements of lung function reveal that maximum compliance in d-ko vs. wild-type mice is significantly reduced. Further, we show that the biophysical phenotype can be corrected substantially with pioglitazone. Finally, we show that d-ko alveolar cells up-regulate liver-type (L-) FABP, a member of the FABP family that we have previously shown to interact with PPARgamma. Taken together, these data suggest that PPARgamma agonists could be a tool to repair surfactant damage caused by dysfunctional alveolar lipid metabolism, and provide in vivo support for L-FABP aided signaling.

Localization of fatty acid binding protein of epidermal type common to dendritic cells and presumptive macrophages in Peyer's patches and epithelial M cells of mouse intestine.

Fatty acid binding protein of epidermal type (E-FABP) was expressed/localized in most, if not all, populations of the dendritic cells in the subepithelial domes, follicles and interfollicular regions of Peyer's patches and presumptive macrophages in their germinal centers, and all M cells in the follicle-associated epithelium of mouse intestine. The immunoreactivity in both of the cell populations makes it easy to recognize the accumulation of DCs in the subepithelial domes in close proximity to the base of M cells, which is essential for luminal antigens to be transported to Peyer's patches. E-FABP may play some important roles in the mucosal immune reaction through Peyer's patches and associated structures.

Adipocyte-type fatty acid-binding protein as inter-compartmental shuttle for peroxisome proliferator activated receptor gamma agonists in cultured cell.

We and others showed earlier that liver-type, epidermal-type and adipocyte-type (A-) fatty acid-binding proteins (FABPs) mediate peroxisome proliferator activated receptor (PPAR) dependent gene expression by channelling their ligand (fatty acid or drug) to the nuclear receptors via direct protein/protein interaction. To clarify mechanistic details of this signaling path, we address here A-FABP import into the nucleus and its interaction with PPARgamma. Making use of COS cells transfected with wild-type or mutant A-FABPs, we exclude posttranslational modification of A-FABP as import signal and provide evidence for both, ligand-dependent and ligand-independent nuclear translocation. With the aid of in vitro pull down assay we demonstrate that specific interaction of A-FABP with PPARgamma isoforms does not require ligand. Moreover, A-FABP binds not only to the ligand-binding domain including hinge domain (domains DEF), but also to the DNA-binding domain including AB domains (domains ABC) of PPARgamma.

Recombinant purple acid phosphatase isoform 3 from sweet potato is an enzyme with a diiron metal center.

Purple acid phosphatases (PAPs) from sweet potato (sp) have been classified on the basis of their primary structure and the dinuclear metal center into isoforms spPAP1 [Fe(III)-Zn(II)] and spPAP2 [Fe(III)-Mn(II)]; for spPAP3 only the cDNA is known. With the aim of unraveling the character of the dinuclear metal center we report here the characterization of this isoform at the protein level. We cloned spPAP3 cDNA in a baculovirus and overexpressed this enzyme in Sf9 insect cells. Preparation of recombinant spPAP3 in two steps afforded pure enzyme with yields of 4.5 mg.L(-1) culture medium. This enzyme is a dimeric, disulfide-linked PAP of 110 kDa, similar to known PAP isoforms from higher plants. Enzymatic studies and spectroscopic properties (max. absorption at 550-565 nm) indicated a diiron enzyme; quantitative and semiquantitative metal analysis using ICP-OES and TOF-SIMS, respectively, revealed the presence of only iron in purified spPAP3. Metal replacement in the second metal-binding site upon preparation of the semiapo-enzyme with Fe(II), Zn(II), or Mn(II) showed highest activities with Fe(II). The data show that recombinant spPAP3 has a diiron metal center. Site-directed mutagenesis was conducted to check catalytic efficiency at the atomic level. Tyr291 at the substrate-binding site in spPAP3 was mutated to His and Ala, the respective residues found in spPAP1 and spPAP2. Kinetic analysis showed that conversion of Tyr291 to His further optimized the performance of this protein as a diiron enzyme, whereas the Ala mutation weakened the catalytic efficiency regardless of the metal present in the second binding site.

Fatty acid-binding proteins--insights from genetic manipulations.

Fatty acid-binding proteins (FABPs) belong to the conserved multigene family of the intracellular lipid-binding proteins (iLBPs). These proteins are ubiquitously expressed in vertebrate tissues, with distinct expression patterns for the individual FABPs. Various functions have been proposed for these proteins, including the promotion of cellular uptake and transport of fatty acids, the targeting of fatty acids to specific metabolic pathways, and the participation in the regulation of gene expression and cell growth. Novel genetic tools that have become available in recent years, such as transgenic cell lines, animals, and knock-out mice, have provided the opportunity to test these concepts in physiological settings. Such studies have helped to define essential cellular functions of individual FABP-types or of combinations of several different FABPs. The deletion of particular FABP genes, however, has not led to gross phenotypical changes, most likely because of compensatory overexpression of other members of the iLBP gene family, or even of unrelated fatty acid transport proteins. This review summarizes the properties of the various FABPs expressed in mammalian tissues, and discusses the transgenic and ablation studies carried out to date in a functional context.

Henk van den Bosch: chemist and biochemist.

Henk van den Bosch is a native of The Netherlands and recently retired from his position as Professor at Utrecht University. This article summarizes the many scientific achievements of Dr. van den Bosch. He enjoys an international reputation for his research on phospholipases A, cardiolipin biosynthesis in eukaryotes, lysophospholipases, phosphatidylcholine biosynthesis for lung surfactant, plasmalogen biosynthesis in peroxisomes, diagnosis of peroxisomal disorders and most recently his work on alkyl-dihydroxyacetone phosphate synthase. During his research career Henk van den Bosch published approximately 280 articles and presented 110 invited lectures.

Phenotype of palmitic acid transport and of signalling in alveolar type II cells from E/H-FABP double-knockout mice: contribution of caveolin-1 and PPARgamma.

Based on the assumption that fatty-acid-binding proteins (FABPs) of the epidermal-type (E-FABP) and heart-type (H-FABP) in murine alveolar type II (TII) cells mediate the synthesis of dipalmitoyl phosphatidylcholine (DPPC), the main surfactant phospholipid, we analysed TII cells isolated from wild-type (wt) and E/H-FABP double-knockout (double-ko) mice. Application of labelled palmitic acid to these cells revealed a drop in uptake, beta-oxidation, and incorporation into neutral lipids and total phosphatidylcholine (PC) of TII cells from double-ko mice. Whereas incorporation of labelled palmitic acid into DPPC remained unchanged, degradation studies demonstrated a substantial shift in DPPC synthesis from de novo to reacylation. In addition, increased expression of mRNAs and proteins of caveolin-1 and PPARgamma, and an increase of the mRNA encoding fatty acid translocase (FAT) was observed in the double-ko phenotype. As caveolin-1 interacted with PPARgamma, we assumed that FAT, caveolin-1, and PPARgamma form a signalling chain for fatty acid or drug. Consequently, PPARgamma-selective pioglitazone was added to the diet of double-ko mice. We found that further activation of PPARgamma could 'heal' the E/H-FABP double-ko effect in these TII cells as transport and utilisation of labelled palmitic acid restored a wt phenocopy. This indicated that E-FABP and/or H-FABP are involved in the mediation of DPPC synthesis in wt TII cells.

Cloning, cellular localization, genomic organization, and tissue-specific expression of the TGFbeta1-inducible SMAP-5 gene.

SMAP-5 is a member of the five-pass transmembrane protein family localizing in the Golgi apparatus and the endoplasmic reticulum. These proteins have been implicated in intracellular trafficking, in secretion and in vesicular transport. Phylogenetic analyses revealed that SMAP-5 is a member of a small Rab GTPase interacting factor protein family. The human SMAP-5 gene spans about 12.5 kb and comprises 6 exons on chromosomal locus 5q32. The proximal 5'-flanking region of the gene lacks a TATA box and is highly GC rich. Consistent with this, the SMAP-5 gene is expressed in all tissues. The highest level of expression was found in coronary smooth muscle cells, in which expression of the SMAP-5 gene was induced by transforming growth factor beta1, thus indicating that this protein may play an important role in inflammation.

Inhibition of TNFalpha in vivo prevents hyperoxia-mediated activation of caspase 3 in type II cells.

BACKGROUND: The mechanisms during the initial phase of oxygen toxicity leading to pulmonary tissue damage are incompletely known. Increase of tumour necrosis factor alpha (TNFalpha) represents one of the first pulmonary responses to hyperoxia. We hypothesised that, in the initial phase of hyperoxia, TNFalpha activates the caspase cascade in type II pneumocytes (TIIcells). METHODS: Lung sections or freshly isolated TIIcells of control and hyperoxic treated rats (48 hrs) were used for the determination of TNFalpha (ELISA), TNF-receptor 1 (Western blot) and activity of caspases 8, 3, and 9 (colorimetrically). NF-kappaB activation was determined by EMSA, by increase of the p65 subunit in the nuclear fraction, and by immunocytochemistry using a monoclonal anti-NF-kappaB-antibody which selectively stained the activated, nuclear form of NF-kappa B. Apoptotic markers in lung tissue sections (TUNEL) and in TIIcells (cell death detection ELISA, Bax, Bcl-2, mitochondrial membrane potential, and late and early apoptotic cells) were measured using commercially available kits. RESULTS: In vivo, hyperoxia activated NF-kappaB and increased the expression of TNFalpha, TNF-receptor 1 and the activity of caspase 8 and 3 in freshly isolated TIIcells. Intratracheal application of anti-TNFalpha antibodies prevented the increase of TNFRI and of caspase 3 activity. Under hyperoxia, there was neither a significant change of cytosolic cytochrome C or of caspase 9 activity, nor an increase in apoptosis of TIIcells. Hyperoxia-induced activation of caspase 3 gradually decreased over two days of normoxia without increasing apoptosis. Therefore, activation of caspase 3 is a temporary effect in sublethal hyperoxia and did not mark the "point of no return" in TIIcells. CONCLUSION: In the initiation phase of pulmonary oxygen toxicity, an increase of TNFalpha and its receptor TNFR1 leads to the activation of caspase 8 and 3 in TIIcells. Together with the hyperoxic induced increase of Bax and the decrease of the mitochondrial membrane potential, activation of caspase 3 can be seen as sensitisation for apoptosis. Eliminating the TNFalpha effect in vivo by anti-TNFalpha antibodies prevents the pro-apoptotic sensitisation of TIIcells.

Occurrence of immunoreactivity for adipocyte-type fatty acid binding protein in degenerating granulosa cells in atretic antral follicles of mouse ovary.

The localization of adipocyte-type fatty acid binding protein (A-FABP) in the mature mouse ovary was examined by immuno-light and electron microscopy. Solitary round cells showing the distinct immunoreactivity for A-FABP were detected in 1-6 antral follicles. In sets of two consecutive sections in a mirror alignment on slide glasses which were treated for immunoreactivity for A-FABP and TUNEL reaction separately, cells immunoreactive for A-FABP appeared in the same antral follicles as containing cells exhibiting TUNEL-reaction. In immunoelectron microscopy, A-FABP-immunopositive cells were found to contain highly electron-dense nuclei of round, irregular or crescent shapes together with cytoplasmic remnants without any features of macrophages or cells of extrinsic origin. Therefore the cells were identified as apoptotic granulosa cells. The apoptotic cells immunoreactive for A-FABP were often seen to be enclosed/engulfed in adjacent cells exhibiting normal ultrastructures without containing numerous lysosomes. The present findings suggest that A-FABP is involved in the apoptosis of ovarian granulosa cells, probably through its interaction with peroxisome proliferator activated receptors.