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4-Hydroxynonenal (HNE) is a major aldehydic product of lipid peroxidation known to exert several biological effects. Normal and malignant cells of the same origin express different sensitivity to HNE. We used human osteosarcoma cells (HOS) in different stages of differentiation in vitro, showing differences in mitosis, DNA synthesis, and alkaline phosphatase (ALP) staining. Differentiated HOS cells showed decreased proliferation (3H-thymidine incorporation), decreased viability (thiazolyl blue tetrazolium bromide-MTT), and increased apoptosis and necrosis (nuclear morphology by staining with 4′,6-diamidino-2-phenylindole-DAPI). Differentiated HOS also had less expressed c-MYC, but the same amount of c-FOS (immunocytochemistry). When exposed to HNE, differentiated HOS produced more reactive oxygen species (ROS) in comparison with undifferentiated HOS. To clarify this, we measured HNE metabolism by an HPLC method, total glutathione (GSH), oxidized GSH (ox GSH), glutathione transferase activity (GST), proteasomal activity by enzymatic methods, HNE-protein adducts by genuine ELISA and fatty acid composition by GC-MS in these cell cultures. Differentiated HOS cells had less GSH, lower HNE metabolism, increased formation of HNE-protein adducts, and lower proteasomal activity, in comparison to undifferentiated counterpart cells, while GST and oxGSH were the same. Fatty acids analyzed by GC-MS showed that there is an increase in C20:3 in differentiated HOS while the amount of C20:4 remained the same. The results showed that the cellular machinery responsible for protection against toxicity of HNE was less efficient in differentiated HOS cells. Moreover, differentiated HOS cells contained more C20:3 fatty acid, which might make them more sensitive to free radical-initiated oxidative chain reactions and more vulnerable to the effects of reactive aldehydes such as HNE. We propose that HNE might act as natural promotor of decay of malignant (osteosarcoma) cells in case of their differentiation associated with alteration of the lipid metabolism.

In conjugative elements such as integrating conjugative elements (ICEs) or conjugative plasmids (CPs) transcription of DNA transfer genes is a prerequisite for cells to become transfer competent, i.e., capable of delivering plasmid DNA via bacterial conjugation into new host bacteria. In the large family of F-like plasmids belonging to the MobF12A group, transcription of DNA transfer genes is tightly controlled and dependent on the activation of a single promoter, designated PY. Plasmid encoded TraJ and chromosomally encoded ArcA proteins are known activators, whereas the nucleoid associated protein heat-stable nucleoid structuring (H-NS) silences the PY promoter. To better understand the role of these proteins in PY promoter activation, we performed in vitro DNA binding studies using purified H-NS, ArcA, and TraJR 1 (TraJ encoded by the conjugative resistance plasmid R1). All proteins could bind to R1PY DNA with high affinities; however, only ArcA was found to be highly sequence specific. DNase I footprinting studies revealed three H-NS binding sites, confirmed the binding site for ArcA, and suggested that TraJ contacts a dyad symmetry DNA sequence located between -51 and -38 in the R1PY promoter region. Moreover, TraJR 1 and ArcA supplied together changed the H-NS specific protection pattern suggesting that these proteins are able to replace H-NS from R1PY regions proximal to the transcription start site. Our findings were corroborated by PY-lacZ reporter fusions with a series of site specific R1PY promoter mutations. Sequential changes of some critical DNA bases in the TraJ binding site (jbs) from plasmid R1 to plasmid F led to a remarkable specificity switch: The PY promoter became activatable by F encoded TraJ whereas TraJR 1 lost its activation function. The R1PY mutagenesis approach also confirmed the requirement for the host-encoded response-regulator ArcA and indicated that the sequence context, especially in the -35 region is critical for PY regulation and function.

During inflammation, activated leukocytes release cytotoxic mediators that compromise blood–brain barrier (BBB) function. Under inflammatory conditions, myeloperoxidase (MPO) is critically involved in inflicting BBB damage. We used genetic and pharmacological approaches to investigate whether MPO induces aberrant lipid homeostasis at the BBB in a murine endotoxemia model. To corroborate findings in a human system we studied the impact of sera from sepsis and non-sepsis patients on brain endothelial cells (hCMEC/D3). In response to endotoxin, the fatty acid, ceramide, and sphingomyelin content of isolated mouse brain capillaries dropped and barrier dysfunction occurred. In mice, genetic deficiency or pharmacological inhibition of MPO abolished these alterations. Studies in metabolic cages revealed increased physical activity and less pronounced sickness behavior of MPO−/− compared to wild-type mice in response to sepsis. In hCMEC/D3 cells, exogenous tumor necrosis factor α (TNFα) potently regulated gene expression of pro-inflammatory cytokines and a set of genes involved in sphingolipid (SL) homeostasis. Notably, treatment of hCMEC/D3 cells with sera from septic patients reduced cellular ceramide concentrations and induced barrier and mitochondrial dysfunction. In summary, our in vivo and in vitro data revealed that inflammatory mediators including MPO, TNFα induce dysfunctional SL homeostasis in brain endothelial cells. Genetic and pharmacological inhibition of MPO attenuated endotoxin-induced alterations in SL homeostasis in vivo, highlighting the potential role of MPO as drug target to treat inflammation-induced brain dysfunction.

Hereditary spastic paraplegias, a group of neurodegenerative disorders, can be caused by loss-of-function mutations in the protein spartin. However, the physiological role of spartin remains largely elusive. Here we show that heterologous expression of human or Drosophila spartin extends chronological lifespan of yeast, reducing age-associated ROS production, apoptosis, and necrosis. We demonstrate that spartin localizes to the proximity of mitochondria and physically interacts with proteins related to mitochondrial and respiratory metabolism. Interestingly, Nde1, the mitochondrial external NADH dehydrogenase, and Pda1, the core enzyme of the pyruvate dehydrogenase complex, are required for spartin-mediated cytoprotection. Furthermore, spartin interacts with the glycolysis enhancer phospo-fructo-kinase-2,6 (Pfk26) and is sufficient to complement for PFK26-deficiency at least in early aging. We conclude that mitochondria-related energy metabolism is crucial for spartin’s vital function during aging and uncover a network of specific interactors required for this function.

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.

Conjugative transfer plays a major role in the transmission of antibiotic resistance in bacteria. pIP501 is a Gram-positive conjugative model plasmid with the broadest transfer host-range known so far and is frequently found in Enterococcus faecalis and Enterococcus faecium clinical isolates. The pIP501 type IV secretion system is encoded by 15 transfer genes. In this work, we focus on the VirB1-like protein TraG, a modular peptidoglycan metabolizing enzyme, and the VirB8-homolog TraM, a potential member of the translocation channel. By providing full-length traG in trans, but not with a truncated variant, we achieved full recovery of wild type transfer efficiency in the traG-knockout mutant E. faecalis pIP501ΔtraG. With peptidoglycan digestion experiments and tandem mass spectrometry we could assign lytic transglycosylase and endopeptidase activity to TraG, with the CHAP domain alone displaying endopeptidase activity. We identified a novel interaction between TraG and TraM in a bacterial-2-hybrid assay. In addition we found that both proteins localize in focal spots at the E. faecalis cell membrane using immunostaining and fluorescence microscopy. Extracellular protease digestion to evaluate protein cell surface exposure revealed that correct membrane localization of TraM requires the transmembrane helix of TraG. Thus, we suggest an essential role for TraG in the assembly of the pIP501 type IV secretion system.

The majority of Merkel cell carcinoma, a highly aggressive neuroendocrine cancer of the skin, is associated with Merkel cell polyomavirus infection. Polyomavirus binding, internalization, and infection are mediated by glycosphingolipids. Besides receptor function, bioactive sphingolipids are increasingly recognized as potent regulators of several hallmarks of cancer. Merkel cell polyomavirus+ and Merkel cell polyomavirus– cells express serine palmitoyl transferase subunits and sphingosine kinase (SK) 1/2 mRNA. Induced expression of Merkel cell polyomavirus–large tumor antigen in human lung fibroblasts resulted in upregulation of SPTLC1-3 and SK 1/2 expression. Therefore, we exploited pharmacological inhibition of sphingolipid metabolism as an option to interfere with proliferation of Merkel cell polyomavirus+ Merkel cell carcinoma cell lines. We used myriocin (a serine palmitoyl transferase antagonist) and two SK inhibitors (SKI-II and ABC294640). In MKL-1 and WaGa cells myriocin decreased cellular ceramide, sphingomyelin, and sphingosine-1-phosphate content. SKI-II increased ceramide species but decreased sphingomyelin and sphingosine-1-phosphate concentrations. Aberrant sphingolipid homeostasis was associated with reduced cell viability, increased necrosis, procaspase-3 and PARP processing, caspase-3 activity, and decreased AKTS473 phosphorylation. Myriocin and SKI-II decreased tumor size and Ki-67 staining of xenografted MKL-1 and WaGa tumors on the chorioallantoic membrane. Our data suggest that pharmacological inhibition of sphingolipid synthesis could represent a potential therapeutic approach in Merkel cell carcinoma.

Summary Membrane phospholipids typically contain fatty acids (FAs) of 16 and 18 carbon atoms. This particular chain length is evolutionarily highly conserved and presumably provides maximum stability and dynamic properties to biological membranes in response to nutritional or environmental cues. Here, we show that the relative proportion of C16 versus C18 FAs is regulated by the activity of acetyl-CoA carboxylase (Acc1), the first and rate-limiting enzyme of FA de novo synthesis. Acc1 activity is attenuated by AMPK/Snf1-dependent phosphorylation, which is required to maintain an appropriate acyl-chain length distribution. Moreover, we find that the transcriptional repressor Opi1 preferentially binds to C16 over C18 phosphatidic acid (PA) species: thus, C16-chain containing PA sequesters Opi1 more effectively to the ER, enabling AMPK/Snf1 control of PA acyl-chain length to determine the degree of derepression of Opi1 target genes. These findings reveal an unexpected regulatory link between the major energy-sensing kinase, membrane lipid composition, and transcription., Highlights • AMPK/Snf1 inhibition of acetyl-CoA carboxylase controls fatty acyl-chain length • Opi1 repressor preferentially binds to C16 rather than C18 acyl-chains in PA • Acyl-chain length tunes Opi1 sequestration to the ER and target gene derepression • AMPK/Snf1 thus uses its effect on acyl-chain length to control Opi1 target genes, Hofbauer et al. find that yeast Snf1/AMPK regulation of acetyl-CoA carboxylase affects acyl-chain length (C16 versus C18) in membrane phospholipids. Moreover, preferential binding of the transcriptional repressor Opi1 to C16-containing phosphatidic acid controls Opi1 sequestration and target gene derepression. Snf1/AMPK regulation of membrane-lipid composition thus determines a key transcriptional output.

A bioassay-guided phytochemical analysis of the ethanolic extract of Grindelia argentina Deble & Oliveira-Deble (Asteraceae) allowed the isolation of a known flavone, hispidulin, and three new oleanane-type saponins, 3-O-β-D-xylopyranosyl-(1→3)-β-D-glucopyranosyl-2β,3β,16α,23-tetrahydroxyolean-12-en-28-oic acid 28-O-β-D-xylopyranosyl-(1→2)-β-D-apiofuranosyl-(1→3)-β-D-xylopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyl ester (2), 3-O-β-D-glucopyranosyl-2β,3β,23-trihydroxyolean-12-en-28-oic acid 28-O-β-D-xylopyranosyl-(1→2)-β-D-apiofuranosyl-(1→3)-β-D-xylopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyl ester, (3) and 3-O-β-D-xylopyranosyl-(1→3)-β-D-glucopyranosyl-2β,3β,23-trihydroxyolean-12-en-28-oic acid 28-O-β-D-xylopyranosyl-(1→2)-β-D-apiofuranosyl-(1→3)-β-D-xylopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyl ester (4), named grindeliosides A-C, respectively. Their structures were determined by extensive 1D- and 2D-NMR experiments along with mass spectrometry and chemical evidence. The isolated compounds were evaluated for their inhibitory activities against LPS/IFN-γ-induced NO production in RAW 264.7 macrophages and for their cytotoxic activities against the human leukemic cell line CCRF-CEM and MRC-5 lung fibroblasts. Hispidulin markedly reduced LPS/IFN-γ-induced NO production (IC50 51.4 μM), while grindeliosides A-C were found to be cytotoxic, with grindelioside C being the most active against both CCRF-CEM (IC50 4.2±0.1 μM) and MRC-5 (IC50 4.5±0.1 μM) cell lines.

Adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL) are key enzymes involved in intracellular degradation of triacylglycerols. It was the aim of this study to elucidate how the deficiency in one of these proteins affects the residual lipolytic proteome in adipose tissue. For this purpose, we compared the lipase patterns of brown and white adipose tissue from ATGL (-/-) and HSL (-/-) mice using differential activity-based gel electrophoresis. This method is based on activity-recognition probes possessing the same substrate analogous structure but carrying different fluorophores for specific detection of the enzyme patterns of two different tissues in one electrophoresis gel. We found that ATGL-deficiency in brown adipose tissue had a profound effect on the expression levels of other lipolytic and esterolytic enzymes in this tissue, whereas HSL-deficiency hardly showed any effect in brown adipose tissue. Neither ATGL- nor HSL-deficiency greatly influenced the lipase patterns in white adipose tissue. Enzyme activities of mouse tissues on acylglycerol substrates were analyzed as well, showing that ATGL-and HSL-deficiencies can be compensated for at least in part by other enzymes. The proteins that responded to ATGL-deficiency in brown adipose tissue were overexpressed and their activities on acylglycerols were analyzed. Among these enzymes, Es1, Es10, and Es31-like represent lipase candidates as they catalyze the hydrolysis of long-chain acylglycerols.

2000 ribosomes have to be synthesized in yeast every minute. Therefore the fast production of ribosomal proteins, their efficient delivery to the nucleus and correct incorporation into ribosomal subunits are prerequisites for optimal growth rates. Here, we report that the ankyrin repeat protein Yar1 directly interacts with the small ribosomal subunit protein Rps3 and accompanies newly synthesized Rps3 from the cytoplasm into the nucleus where Rps3 is assembled into pre-ribosomal subunits. A yar1 deletion strain displays a similar phenotype as an rps3 mutant strain, showing an accumulation of 20S pre-rRNA and a 40S export defect. The combination of an rps3 mutation with a yar1 deletion leads to an enhancement of these phenotypes, while increased expression of RPS3 suppresses the defects of a yar1 deletion strain. We further show that Yar1 protects Rps3 from aggregation in vitro and increases its solubility in vivo. Our data suggest that Yar1 is a specific chaperone for Rps3, which serves to keep Rps3 soluble until its incorporation into the pre-ribosome.

SummaryNumerous studies in humans link a nonsynonymous genetic polymorphism (I148M) in adiponutrin (ADPN) to various forms of fatty liver disease and liver cirrhosis. Despite its high clinical relevance, the molecular function of ADPN and the mechanism by which I148M variant affects hepatic metabolism are unclear. Here we show that ADPN promotes cellular lipid synthesis by converting lysophosphatidic acid (LPA) into phosphatidic acid. The ADPN-catalyzed LPA acyltransferase (LPAAT) reaction is specific for LPA and long-chain acyl-CoAs. Wild-type mice receiving a high-sucrose diet exhibit substantial upregulation of Adpn in the liver and a concomitant increase in LPAAT activity. In Adpn-deficient mice, this diet-induced increase in hepatic LPAAT activity is reduced. Notably, the I148M variant of human ADPN exhibits increased LPAAT activity leading to increased cellular lipid accumulation. This gain of function provides a plausible biochemical mechanism for the development of liver steatosis in subjects carrying the I148M variant.

Carboxylesterases constitute a large enzyme family in insects, which is involved in diverse functions such as xenobiotic detoxi fi cation, lipid metabolism and reproduction. Phylogenetically, many insect carbox- ylesterases are represented by multienzyme clades, which are encoded by evolutionarily ancient gene clusters such as the a -Esterase cluster. Much in contrast to the vital importance attributed to carbox- ylesterases in general, the in vivo function of individual a -Esterase genes is largely unknown. This study employs a functional proteomics approach to identify esterolytic enzymes of the vinegar fl y Drosophila melanogaster fat body. One of the fat body carboxylesterases, a -Esterase-7 , was selected for mutational analysis by gene targeting to generate a deletion mutant fl y. Phenotypic characterization of a -Esterase-7 null mutants and transgenic fl ies, which overexpress a chimeric a -Esterase-7:EGFP gene, reveals important functions of a -Esterase-7 in insecticide tolerance, lipid metabolism and lifespan control. The presented fi rst deletion mutant of any a -Esterase in the model insect D. melanogaster generated by gene targeting not only provides experimental evidence for the endogenous functions of this gene family. It also offers an entry point for in vivo structure-function analyses of a -Esterase- 7, which is of central importance for naturally occurring insecticide resistance in wild populations of various dipteran insect species.

One decade after the genomic and the proteomic life science revolution, new ‘omics’ fields are emerging. The metabolome encompasses the entity of small molecules—Most often end products of a catalytic process regulated by genes and proteins—with the lipidome being its fat soluble subdivision. Within recent years, lipids are more and more regarded not only as energy storage compounds but also as interactive players in various cellular regulation cycles and thus attain rising interest in the bio-medical community. The field of lipidomics is, on one hand, fuelled by analytical technology advances, particularly mass spectrometry and chromatography, but on the other hand new biological questions also drive analytical technology developments. Compared to fairly standardized genomic or proteomic high-throughput protocols, the high degree of molecular heterogeneity adds a special analytical challenge to lipidomic analysis. In this review, we will take a closer look at various mass spectrometric platforms for lipidomic analysis. We will focus on the advantages and limitations of various experimental setups like ‘shotgun lipidomics’, liquid chromatography—Mass spectrometry (LC-MS) and matrix assisted laser desorption ionization-time of flight (MALDI-TOF) based approaches. We will also examine available software packages for data analysis, which nowadays is in fact the rate limiting step for most ‘omics’ workflows.

International audience; Mycosporine-like compounds, comprising mycosporines and mycosporine-like amino acids (MAAs) are UV protecting secondary metabolites described in organisms such as fungi, algae, cyanobacteria or animals. Lichens however, were only poorly investigated for such constituents so far. Here, a method for the characterization of mycosporines and MAAs in purified aqueous extracts, involving HPTLC coupled to spectrophotodensitometry, HPLC-DAD-MS(n) and UPLC-HRMS analysis, is described. This optimized protocol was validated on three algae and one cyanolichen containing known MAAs and mycosporines, and then applied to 18 cyanolichen species. Analyses revealed the presence of five already described mycosporine-like compounds in the investigated species, including mycosporine serinol in Lichina and Peltigera species and mycosporine glutamicol in Degelia plumbea. Apart from that, eight unknown mycosporine-like compounds were detected and tentatively characterized on the basis of their DAD spectra and their MS(n) and HRMS data: two in the alga Porphyra dioica and six in cyanolichen species belonging to the genera Degelia, Nephroma and Stereocaulon. From Nephroma laevigatum, the mycosporine hydroxyglutamicol was preparatively isolated and identified through HRMS, 1D and 2D NMR spectroscopic data. The optimized analytical protocol allowed the characterization of mycosporine-like compounds in small amounts of material and confirmed the potential of cyanolichens as a source of mycosporine compounds. It should also be applicable to investigate lichen species with green algae photobionts for mycosporine-like compounds.

The dichloromethane extract of the flowers of Helianthus angustifolius L. (Asteraceae, Heliantheae) was investigated in vitro for its cytotoxic activity using human cancer cell lines: CCRF-CEM leukemia, MDA-MB-231 breast cancer, U251 glioblastoma, HCT 116 colon cancer cells, and the human lung fibroblast cell line MRC-5. Cytotoxicity-guided fractionation led to the isolation of four related heliangolide-type sesquiterpene lactones. The structures were elucidated by means of NMR spectroscopy and high-resolution mass spectrometry. Of the investigated compounds, 8-methacrylyl-4,15-iso-atriplicolide (1) showed the highest activity against all tested cancer cell lines with IC₅₀ values ranging from 0.26 ± 0.01 µM for CCRF-CEM cells to 4.22 ± 0.26 µM for MRC-5 cells.

Mitochondrial outer membrane permeabilization is a watershed event in the process of apoptosis, which is tightly regulated by a series of pro- and anti-apoptotic proteins belonging to the BCL-2 family, each characteristically possessing a BCL-2 homology domain 3 (BH3). Here, we identify a yeast protein (Ybh3p) that interacts with BCL-XL and harbours a functional BH3 domain. Upon lethal insult, Ybh3p translocates to mitochondria and triggers BH3 domain-dependent apoptosis. Ybh3p induces cell death and disruption of the mitochondrial transmembrane potential via the mitochondrial phosphate carrier Mir1p. Deletion of Mir1p and depletion of its human orthologue (SLC25A3/PHC) abolish stress-induced mitochondrial targeting of Ybh3p in yeast and that of BAX in human cells, respectively. Yeast cells lacking YBH3 display prolonged chronological and replicative lifespans and resistance to apoptosis induction. Thus, the yeast genome encodes a functional BH3 domain that induces cell death through phylogenetically conserved mechanisms.

Oxidative modification of Trigonopsis variabilis d -amino acid oxidase in vivo is traceable as the conversion of Cys108 into a stable cysteine sulfinic acid, causing substantial loss of activity and thermostability of the enzyme. To simulate native and modified oxidase each as a microheterogeneity-resistant entity, we replaced Cys108 individually by a serine (C108S) and an aspartate (C108D), and characterized the purified variants with regard to their biochemical and kinetic properties, thermostability, and reactivity towards oxidation by hypochlorite. Tandem MS analysis of tryptic peptides derived from a hypochlorite-treated inactive preparation of recombinant wild-type oxidase showed that Cys108 was converted into cysteine sulfonic acid, mimicking the oxidative modification of native enzyme as isolated. Colorimetric titration of protein thiol groups revealed that in the presence of ammonium benzoate (0.12 mM), the two muteins were not oxidized at cysteines whereas in the wild-type enzyme, one thiol group was derivatized. Each site-directed replacement caused a conformational change in d -amino acid oxidase, detected with an assortment of probes, and resulted in a turnover number for the O2-dependent reaction with D -Met which in comparison with the corresponding wild-type value was decreased two- and threefold for C108S and C108D, respectively. Kinetic analysis of thermal denaturation at 50 °C was used to measure the relative contributions of partial unfolding and cofactor dissociation to the overall inactivation rate in each of the three enzymes. Unlike wild-type, C108S and C108D released the cofactor in a quasi-irreversible manner and were therefore not stabilized by external FAD against loss of activity. The results support a role of the anionic side chain of Cys108 in the fine-tuning of activity and stability of d -amino acid oxidase, explaining why C108S was a surprisingly poor mimic of the native enzyme.

Paired, sac-like and typically large opisthonotal glands (syn. oil glands), mainly considered for chemical protection and communication, characterize the so-called ‘glandulate Oribatida’ which include the Parhyposomata, Mixonomata, Desmonomata and Brachypylina but also the Astigmata. Among these groups distinct evolutionary trends affect the morphology of glands and their secretion profiles, thereby rendering them highly informative characters with phylogenetic significance. One striking tendency, convergently occurring in a few glandulate groups, leads to the degeneration or even complete regression of opisthonotal glands. In this study, a first example of coherent evolutionary steps towards opisthonotal gland degeneration is described by using desmonomatan Camisiidae as a model: Opisthonotal glands in representatives of genus Platynothrus still show morphologically and chemically ancient conditions with fairly-well developed glandular reservoirs. Secretion patterns mainly consist of a characteristic set of terpenes and aromatics (‘astigmatid compounds’) as found in outgroups such as desmonomatan Trhypochthoniidae. Progressive states of regression of opisthonotal glands, along with a reduction of component-richness and amounts of secretions, occur in representatives of Heminothrus and, more conspicuously, in species of Camisia, most likely indicating a consistent evolutionary trend. This trend towards opisthonotal gland atrophy may be due to novel alternative and cheap strategies of passive defense in more-derivative camisiids – such as mechanical protection by encrustation of the cuticle – that possibly compensate for the lack of chemical defenses.

In eukaryotes, S-adenosyl-L-homocysteine hydrolase (Sah1) offers a single way for degradation of S-adenosyl-L-homocysteine, a product and potent competitive inhibitor of S-adenosyl-L-methionine (AdoMet)-dependent methyltransferases. De novo phosphatidylcholine (PC) synthesis requires three AdoMet-dependent methylation steps. Here we show that down-regulation of SAH1 expression in yeast leads to accumulation of S-adenosyl-L-homocysteine and decreased de novo PC synthesis in vivo. This decrease is accompanied by an increase in triacylglycerol (TG) levels, demonstrating that Sah1-regulated methylation has a major impact on cellular lipid homeostasis. TG accumulation is also observed in cho2 and opi3 mutants defective in methylation of phosphatidylethanolamine to PC, confirming that PC de novo synthesis and TG synthesis are metabolically coupled through the efficiency of the phospholipid methylation reaction. Indeed, because both types of lipids share phosphatidic acid as a precursor, we find in cells with down-regulated Sah1 activity major alterations in the expression of the INO1 gene as well as in the localization of Opi1, a negative regulatory factor of phospholipid synthesis, which binds and is retained in the endoplasmic reticulum membrane by phosphatidic acid in conjunction with VAMP/synaptobrevin-associated protein, Scs2. The addition of homocysteine, by the reversal of the Sah1-catalyzed reaction, also leads to TG accumulation in yeast, providing an attractive model for the role of homocysteine as a risk factor of atherosclerosis in humans.

Saccharomyces cerevisiae, as well as other eukaryotes, preserves fatty acids and sterols in a biologically inert form, as triacylglycerols and steryl esters. The major triacylglycerol lipases of the yeast S. cerevisiae identified so far are Tgl3p, Tgl4p, and Tgl5p (Athenstaedt, K., and Daum, G. (2003) YMR313c/TGL3 encodes a novel triacylglycerol lipase located in lipid particles of Saccharomyces cerevisiae. J. Biol. Chem. 278, 23317–23323; Athenstaedt, K., and Daum, G. (2005) Tgl4p and Tgl5p, two triacylglycerol lipases of the yeast Saccharomyces cerevisiae, are localized to lipid particles. J. Biol. Chem. 280, 37301–37309). We observed that upon cultivation on oleic acid, triacylglycerol mobilization did not come to a halt in a yeast strain deficient in all currently known triacylglycerol lipases, indicating the presence of additional not yet characterized lipases/esterases. Functional proteome analysis using lipase and esterase inhibitors revealed a subset of candidate genes for yet unknown hydrolytic enzymes on peroxisomes and lipid droplets. Based on the conserved GXSXG lipase motif, putative functions, and subcellular localizations, a selected number of candidates were characterized by enzyme assays in vitro, gene expression analysis, non-polar lipid analysis, and in vivo triacylglycerol mobilization assays. These investigations led to the identification of Ayr1p as a novel triacylglycerol lipase of yeast lipid droplets and confirmed the hydrolytic potential of the peroxisomal Lpx1p in vivo. Based on these results, we discuss a possible link between lipid storage, lipid mobilization, and peroxisomal utilization of fatty acids as a carbon source.

Efficient catabolism of cellular triacylglycerol (TG) stores requires the TG hydrolytic activity of adipose triglyceride lipase (ATGL). The presence of comparative gene identification-58 (CGI-58) strongly increased ATGL-mediated TG catabolism in cell culture experiments. Mutations in the genes coding for ATGL or CGI-58 in humans cause neutral lipid storage disease characterized by TG accumulation in multiple tissues. ATGL gene mutations cause a severe phenotype especially in cardiac muscle leading to cardiomyopathy that can be lethal. In contrast, CGI-58 gene mutations provoke severe ichthyosis and hepatosteatosis in humans and mice, whereas the role of CGI-58 in muscle energy metabolism is less understood. Here we show that mice lacking CGI-58 exclusively in muscle (CGI-58KOM) developed severe cardiac steatosis and cardiomyopathy linked to impaired TG catabolism and mitochondrial fatty acid oxidation. The marked increase in ATGL protein levels in cardiac muscle of CGI-58KOM mice was unable to compensate the lack of CGI-58. The addition of recombinant CGI-58 to cardiac lysates of CGI-58KOM mice completely reconstituted TG hydrolytic activities. In skeletal muscle, the lack of CGI-58 similarly provoked TG accumulation. The addition of recombinant CGI-58 increased TG hydrolytic activities in control and CGI-58KOM tissue lysates, elucidating the limiting role of CGI-58 in skeletal muscle TG catabolism. Finally, muscle CGI-58 deficiency affected whole body energy homeostasis, which is caused by impaired muscle TG catabolism and increased cardiac glucose uptake. In summary, this study demonstrates that functional muscle lipolysis depends on both CGI-58 and ATGL.

The nuclear export of the preribosomal 60S (pre-60S) subunit is coordinated with late steps in ribosome assembly. Here, we show that Bud20, a conserved C(2)H(2)-type zinc finger protein, is an unrecognized shuttling factor required for the efficient export of pre-60S subunits. Bud20 associates with late pre-60S particles in the nucleoplasm and accompanies them into the cytoplasm, where it is released through the action of the Drg1 AAA-ATPase. Cytoplasmic Bud20 is then reimported via a Kap123-dependent pathway. The deletion of Bud20 induces a strong pre-60S export defect and causes synthetic lethality when combined with mutant alleles of known pre-60S subunit export factors. The function of Bud20 in ribosome export depends on a short conserved N-terminal sequence, as we observed that mutations or the deletion of this motif impaired 60S subunit export and generated the genetic link to other pre-60S export factors. We suggest that the shuttling Bud20 is recruited to the nascent 60S subunit via its central zinc finger rRNA binding domain to facilitate the subsequent nuclear export of the preribosome employing its N-terminal extension.

Microglia, the immunocompetent cells of the CNS, are rapidly activated in response to injury and microglia migration towards and homing at damaged tissue plays a key role in CNS regeneration. Lysophosphatidic acid (LPA) is involved in signaling events evoking microglia responses through cognate G protein-coupled receptors. Here we show that human immortalized C13NJ microglia express LPA receptor subtypes LPA(1), LPA(2), and LPA(3) on mRNA and protein level. LPA activation of C13NJ cells induced Rho and extracellular signal-regulated kinase activation and enhanced cellular ATP production. In addition, LPA induced process retraction, cell spreading, led to pronounced changes of the actin cytoskeleton and reduced cell motility, which could be reversed by inhibition of Rho activity. To get an indication about LPA-induced global alterations in protein expression patterns a 2-D DIGE/LC-ESI-MS proteomic approach was applied. On the proteome level the most prominent changes in response to LPA were observed for glycolytic enzymes and proteins regulating cell motility and/or cytoskeletal dynamics. The present findings suggest that naturally occurring LPA is a potent regulator of microglia biology. This might be of particular relevance in the pathophysiological context of neurodegenerative disorders where LPA concentrations can be significantly elevated in the CNS.

Comparative gene identification-58 (CGI-58), also designated as alpha/beta-hydrolase domain containing-5 (ABHD-5), is a lipid droplet-associated protein that activates adipose triglyceride lipase (ATGL) and acylates lysophosphatidic acid. Activation of ATGL initiates the hydrolytic catabolism of cellular triacylglycerol (TG) stores to glycerol and nonesterified fatty acids. Mutations in both ATGL and CGI-58 cause "neutral lipid storage disease" characterized by massive accumulation of TG in various tissues. The analysis of CGI-58-deficient (Cgi-58(-/-)) mice, presented in this study, reveals a dual function of CGI-58 in lipid metabolism. First, systemic TG accumulation and severe hepatic steatosis in newborn Cgi-58(-/-) mice establish a limiting role for CGI-58 in ATGL-mediated TG hydrolysis and supply of nonesterified fatty acids as energy substrate. Second, a severe skin permeability barrier defect uncovers an essential ATGL-independent role of CGI-58 in skin lipid metabolism. The neonatal lethal skin barrier defect is linked to an impaired hydrolysis of epidermal TG. As a consequence, sequestration of fatty acids in TG prevents the synthesis of acylceramides, which are essential lipid precursors for the formation of a functional skin permeability barrier. This mechanism may also underlie the pathogenesis of ichthyosis in neutral lipid storage disease patients lacking functional CGI-58.

Triacylglycerol accumulation in insulin target tissues is associated with insulin resistance. Paradoxically, mice with global targeted deletion of adipose triglyceride lipase (ATGL), the rate-limiting enzyme in triacylglycerol hydrolysis, display improved glucose tolerance and insulin sensitivity despite triacylglycerol accumulation in multiple tissues. To determine the molecular mechanisms for this phenotype, ATGL-deficient (ATGL(-/-)) and wild-type mice were injected with saline or insulin (10 units/kg, intraperitoneally), and then phosphorylation and activities of key insulin-signaling proteins were determined in insulin target tissues (liver, adipose tissue, and muscle). Insulin signaling and/or glucose transport was also evaluated in isolated adipocytes and skeletal muscle ex vivo. In ATGL(-/-) mice, insulin-stimulated phosphatidylinositol 3-kinase and Akt activities as well as phosphorylation of critical residues of IRS1 (Tyr(P)-612) and Akt (Ser(P)-473) were increased in skeletal muscle in vivo. Insulin-stimulated phosphatidylinositol 3-kinase activity and total insulin receptor and insulin receptor substrate 1, but not other parameters, were also increased in white adipose tissue in vivo. In contrast, in vivo measures of insulin signaling were decreased in brown adipose tissue and liver. Interestingly, the enhanced components of insulin signaling identified in skeletal muscle and white adipose tissue in vivo and their expected downstream effects on glucose transport were not present ex vivo. ATGL deficiency altered intramyocellular lipids as well as serum factors known to influence insulin sensitivity. Thus, skeletal muscle, rather than other tissues, primarily contributes to enhanced insulin sensitivity in ATGL(-/-) mice in vivo despite triacylglycerol accumulation, and both local and systemic factors contribute to tissue-specific effects of global ATGL deficiency on insulin action.

The mitochondrial dimeric phospholipid cardiolipin is characterized by a high degree of unsaturation of its acyl chains, which is important for its functional interaction with mitochondrial enzymes. The unusual fatty acid composition of cardiolipin molecular species emerges from a de novo synthesized "premature" species by extensive acyl chain remodeling that involves as yet only partially identified acyltransferases and phospholipases. Recently, the yeast protein Taz1p was shown to function as a transacylase, which catalyzes the reacylation of monolysocardiolipin to mature cardiolipin. A defect in the orthologous human TAZ gene is associated with Barth syndrome, a severe genetic disorder, which may lead to cardiac failure and death in childhood. We now identified the protein encoded by reading frame YGR110W as a mitochondrial phospholipase, which deacylates de novo synthesized cardiolipin. Ygr110wp has a strong substrate preference for palmitic acid residues and functions upstream of Taz1p, to generate monolysocardiolipin for Taz1p-dependent reacylation with unsaturated fatty acids. We therefore rename the Ygr110wp as Cld1p (cardiolipin-specific deacylase 1).

In lipid metabolism, the liver acts as a buffer for transient energy fluctuations. It temporarily stores fatty acids as triacylglycerol and secretes them as very low density lipoprotein into the circulation when the period of maximum lipid load has passed. The lipolytic enzymes responsible for mobilization of internal lipid stores in the liver have not been identified yet. We introduced active site-directed chemical probes for lipolytic activity profiling in complex mixtures, known as activity-based proteomics, and employed it for global analysis and functional annotation of lipolytic proteins in mouse adipose tissue. Here we report the combined application of two approaches using fluorescent and biotinylated probes for discovery and discrimination of lipolytic and esterolytic enzymes in mouse liver subproteomes. Proteomes labeled with the fluorescent probes were analyzed by 2-DE while proteomes labeled with the biotinylated probe were subjected to avidin-affinity isolation. Of 37 totally identified proteins, 15 were detected using both approaches while 14 and 8 were solely identified by 2-DE and avidin-affinity isolation, respectively. Moreover, 12 enzymes were classified as potential lipases and/or cholesteryl esterases by their reaction with probes specific for the respective activities directly in their proteomes.

Hydrolysis of triacylglycerols and cholesteryl esters is a key event in energy homeostasis of animals. However, many lipolytic activities still await their molecular identification. Here we report on a novel tool for concomitant analysis of lipases in complex proteomes. Fluorescent activity tags mimicking lipid substrates were used to label the proteome of mouse adipose tissue. Analysis by two-dimensional gel electrophoresis and LC-MS/MS led to the identification of all known intracellular lipases as well as a number of novel candidates. One of them was recently shown to be involved in triacylglycerol mobilization in adipocytes and therefore named adipose triglyceride lipase. Functional characterization of expressed enzymes demonstrated that lipolytic and esterolytic activities could be well discriminated. Thus our results show the first map of the lipolytic proteome of mouse adipose tissue and demonstrate the general applicability of our method for rapid profiling and identification of lipolytic activities in complex biological samples.

Globotriaosylceramide is a neutral glycolipid containing the trihexoside Gal(alpha1-4)Gal(ss1-4)Glc(ss1-1') covalently bound to N-acylsphingosine. It was identified as the main storage substance in the kidney of patients with Fabry disease, an X-linked deficiency of lysosomal alpha-galactosidase A which can significantly be ameliorated by enzyme replacement therapy. Unlike hemizygote males, affected heterozygote females cannot be identified by enzyme assays and therefore may remain untreated. A quantitation of urinary globotriaosylceramides was proposed as an alternative method for their diagnosis. However, the required studies on physiological and pathological variations in the excretion of trihexosides so far have been prevented by a lack of suitable methods. A validated, robust and quick high-throughput method for the quantitative analysis of globotriaosylceramide isoforms using stable-isotope-dilution/internal standardization and electrospray ionization mass spectrometry (ESI-MS) was developed. An internal standard, stearoyl-d35-globotriaosylceramide, was synthesized by enzymatic coupling of d35-stearic acid to the corresponding lyso-ceramidetrihexoside. Glycolipid isoforms of high purity were obtained from a 5-mL urine portion by extraction on C18 solid-phase columns and a novel washing protocol. ESI-MS analysis was performed in full and neutral loss scan modes. Urinary trihexosyl- and some of the di- and monohexosylceramide isoforms can be quantified within a single experiment. All glycolipid isoforms were above detection limit in healthy male and female subjects (n = 63). Prominent elevations of tetracosanoyl-(C24:0 plus C24:1)-globotriaosylceramides were found in urines from female (>2.5-fold above normals) or male Fabry patients (>5.8-fold above normals), but not among controls. Globotriaosylceramide isoforms shall now be analyzed in Fabry patients, non-genetic kidney disease and healthy subjects to define the conditions for a safe diagnosis of heterozygotes.

The truncated phospholipids 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC) and 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC) are oxidation products of 1-palmitoyl-2-arachidonoyl phosphatidylcholine. Depending on concentration and the extent of modification, these compounds induce growth and death, differentiation and inflammation of vascular cells thus playing a role in the development of atherosclerosis. Here we describe the import of fluorescent POVPC and PGPC analogs into cultured RAW 264.7 macrophages and the identification of their primary protein targets. We found that the fluorescent oxidized phospholipids were rapidly taken up by the cells. The cellular target sites depended on the chemical reactivity of these compounds but not on the donor (aqueous lipid suspension, albumin or LDL). The great differences in cellular uptake of PGPC and POVPC are a direct consequence of the subtle structural differences between both molecules. The former compound (carboxyl lipid) can only physically interact with the molecules in its immediate vicinity. In contrast, the aldehydo-lipid covalently reacts with free amino groups of proteins by forming covalent Schiff bases, and thus becomes trapped in the cell surface. Despite covalent binding, POVPC is exchangeable between (lipo)proteins and cells, since imines are subject to proton-catalyzed base exchange. Protein targeting by POVPC is a selective process since only a limited subfraction of the total proteome was labeled by the fluorescent aldehydo-phospholipid. Chemically stabilized lipid–protein conjugates were identified by MS/MS. The respective proteins are involved in apoptosis, stress response, lipid metabolism and transport. The identified target proteins may be considered primary signaling platforms of the oxidized phospholipid., Highlights ► Identification of primary protein targets of truncated phospholipids. ► Covalent modification of proteins by aldehydophospholipids. ► Exchange of fluorescent aldehydophospholipids between cells, lipoproteins and albumin. ► Imaging of fluorescent oxidized phospholipids in live cells.