Acyl-CoA synthetase 3 promotes lipid droplet biogenesis in ER microdomains.

Control of lipid droplet (LD) nucleation and copy number are critical, yet poorly understood, processes. We use model peptides that shift from the endoplasmic reticulum (ER) to LDs in response to fatty acids to characterize the initial steps of LD formation occurring in lipid-starved cells. Initially, arriving lipids are rapidly packed in LDs that are resistant to starvation (pre-LDs). Pre-LDs are restricted ER microdomains with a stable core of neutral lipids. Subsequently, a first round of"emerging" LDs is nucleated, providing additional lipid storage capacity. Finally, in proportion to lipid concentration, new rounds of LDs progressively assemble. Confocal microscopy and electron tomography suggest that emerging LDs are nucleated in a limited number of ER microdomains after a synchronized stepwise process of protein gathering, lipid packaging, and recognition by Plin3 and Plin2. A comparative analysis demonstrates that the acyl-CoA synthetase 3 is recruited early to the assembly sites, where it is required for efficient LD nucleation and lipid storage.

A user-friendly web portal for T-Coffee on supercomputers

Background: Parallel T-Coffee (PTC) was the first parallel implementation of the T-Coffee multiple sequence alignment tool. It is based on MPI and RMA mechanisms. Its purpose is to reduce the execution time of the large-scale sequence alignments. It can be run on distributed memory clusters allowing users to align data sets consisting of hundreds of proteins within a reasonable time. However, most of the potential users of this tool are not familiar with the use of grids or supercomputers. Results: In this paper we show how PTC can be easily deployed and controlled on a super computer architecture using a web portal developed using Rapid. Rapid is a tool for efficiently generating standardized portlets for a wide range of applications and the approach described here is generic enough to be applied to other applications, or to deploy PTC on different HPC environments. Conclusions: The PTC portal allows users to upload a large number of sequences to be aligned by the parallel version of TC that cannot be aligned by a single machine due to memory and execution time constraints. The web portal provides a user-friendly solution.

T-Coffee: a web server for the multiple sequence alignment of protein and RNA sequences using structural information and homology extension

This article introduces a new interface for T-Coffee, a consistency-based multiple sequence alignment program. This interface provides an easy and intuitive access to the most popular functionality of the package. These include the default T-Coffee mode for protein and nucleic acid sequences, the M-Coffee mode that allows combining the output of any other aligners, and template-based modes of T-Coffee that deliver high accuracy alignments while using structural or homology derived templates. These three available template modes are Expresso for the alignment of protein with a known 3D-Structure, R-Coffee to align RNA sequences with conserved secondary structures and PSI-Coffee to accurately align distantly related sequences using homology extension. The new server benefits from recent improvements of the T-Coffee algorithm and can align up to 150 sequences as long as 10 000 residues and is available from both http://www.tcoffee.org and its main mirror http://tcoffee.crg.cat.

Plant-derived phenolics inhibit the accrual of structurally characterised protein and lipid oxidative modifications

Epidemiological data suggest that plant-derived phenolics beneficial effects include an inhibition of LDL oxidation. After applying a screening method based on 2,4-dinitrophenyl hydrazine- protein carbonyl reaction to 21 different plant-derived phenolic acids, we selected the most antioxidant ones. Their effect was assessed in 5 different oxidation systems, as well as in other model proteins. Mass-spectrometry was then used, evidencing a heterogeneous effect on the accumulation of the structurally characterized protein carbonyl glutamic and aminoadipic semialdehydes as well as for malondialdehyde-lysine in LDL apoprotein. After TOF based lipidomics, we identified the most abundant differential lipids in Cu++-incubated LDL as 1-palmitoyllysophosphatidylcholine and 1-stearoyl-sn-glycero-3-phosphocholine. Most of selected phenolic compounds prevented the accumulation of those phospholipids and the cellular impairment induced by oxidized LDL. Finally, to validate these effects in vivo, we evaluated the effect of the intake of a phenolic-enriched extract in plasma protein and lipid modifications in a well-established model of atherosclerosis (diet-induced hypercholesterolemia in hamsters). This showed that a dietary supplement with a phenolic-enriched extract diminished plasma protein oxidative and lipid damage. Globally, these data show structural basis of antioxidant properties of plant-derived phenolic acids in protein oxidation that may be relevant for the health-promoting effects of its dietary intake. that a dietary supplement with a phenolic-enriched extract diminished plasma protein oxidative and lipid damage. Globally, these data show structural basis of antioxidant properties of plant-derived phenolic acids in protein oxidation that may be relevant for the health-promoting effects of its dietary intake.

Lipid rafts act as specialised domains for tetanus toxin binding and internalisation in neurons

Tetanus (TeNT) is a zinc protease that blocks neurotransmission by cleaving the synaptic protein vesicle-associated membrane protein/synaptobrevin. Although its intracellular catalytic activity is well established, the mechanism by which this neurotoxin interacts with the neuronal surface is not known. In this study, we characterize p15s, the first plasma membrane TeNT binding proteins and we show that they are glycosylphosphatidylinositol-anchored glycoproteins in nerve growth factor (NGF)-differentiated PC12 cells, spinal cord cells, and purified motor neurons. We identify p15 as neuronal Thy-1 in NGF-differentiated PC12 cells. Fluorescence lifetime imaging microscopy measurements confirm the close association of the binding domain of TeNT and Thy-1 at the plasma membrane. We find that TeNT is recruited to detergent-insoluble lipid microdomains on the surface of neuronal cells. Finally, we show that cholesterol depletion affects a raft subpool and blocks the internalization and intracellular activity of the toxin. Our results indicate that TeNT interacts with target cells by binding to lipid rafts and that cholesterol is required for TeNT internalization and/or trafficking in neurons.

Lipid phosphate phosphatase 3 participates in transport carrier formation and protein trafficking in the early secretory pathway

The inhibition of phosphatidic acid phosphatase (PAP) activity by propanolol indicates that diacylglycerol (DAG) is required for the formation of transport carriers at the Golgi and for retrograde trafficking to the ER. Here we report that the PAP2 family member lipid phosphate phosphatase 3 (LPP3, also known as PAP2b) localizes in compartments of the secretory pathway from ER export sites to the Golgi complex. The depletion of human LPP3: (i) reduces the number of tubules generated from the ER-Golgi intermediate compartment and the Golgi, with those formed from the Golgi being longer in LPP3-silenced cells than in control cells; (ii) impairs the Rab6-dependent retrograde transport of Shiga toxin subunit B from the Golgi to the ER, but not the anterograde transport of VSV-G or ssDsRed; and (iii) induces a high accumulation of Golgi-associated membrane buds. LPP3 depletion also reduces levels of de novo synthesized DAG and the Golgi-associated DAG contents. Remarkably, overexpression of a catalytically inactive form of LPP3 mimics the effects of LPP3 knockdown on Rab6-dependent retrograde transport. We conclude that LPP3 participates in the formation of retrograde transport carriers at the ER-Golgi interface, where it transitorily cycles, and during its route to the plasma membrane.

Treatment of rats with a self-selected hyperlipidic diet, increases the lipid content of the main adipose tissue sites in a proportion similar to that of the lipids in the rest of organs and tissues

Adipose tissue (AT) is distributed as large differentiated masses, and smaller depots covering vessels, and organs, as well as interspersed within them. The differences between types and size of cells makes AT one of the most disperse and complex organs. Lipid storage is partly shared by other tissues such as muscle and liver. We intended to obtain an approximate estimation of the size of lipid reserves stored outside the main fat depots. Both male and female rats were made overweight by 4-weeks feeding of a cafeteria diet. Total lipid content was analyzed in brain, liver, gastrocnemius muscle, four white AT sites: subcutaneous, perigonadal, retroperitoneal and mesenteric, two brown AT sites (interscapular and perirenal) and in a pool of the rest of organs and tissues (after discarding gut contents). Organ lipid content was estimated and tabulated for each individual rat. Food intake was measured daily. There was a surprisingly high proportion of lipid not accounted for by the main macroscopic AT sites, even when brain, liver and BAT main sites were discounted. Muscle contained about 8% of body lipids, liver 1-1.4%, four white AT sites lipid 28-63% of body lipid, and the rest of the body (including muscle) 38-44%. There was a good correlation between AT lipid and body lipid, but lipid in"other organs" was highly correlated too with body lipid. Brain lipid was not. Irrespective of dietary intake, accumulation of body fat was uniform both for the main lipid storage and handling organs: large masses of AT (but also liver, muscle), as well as in the"rest" of tissues. These storage sites, in specialized (adipose) or not-specialized (liver, muscle) tissues reacted in parallel against a hyperlipidic diet challenge. We postulate that body lipid stores are handled and regulated coordinately, with a more centralized and overall mechanisms than usually assumed.

Coffee polyphenols change the expression of STAT5b and ATF-2 modifying cyclin D1 levels in cancer cells

Background: Epidemiological studies suggest that coffee consumption reduces the risk of cancer, but the molecular mechanisms of its chemopreventive effects remain unknown. Objective: To identify differentially expressed genes upon incubation of HT29 colon cancer cells with instant caffeinated coffee (ICC) or caffeic acid (CA) using whole genome microarrays. Results: ICC incubation of HT29 cells caused the overexpression of 57 genes and the underexpression of 161, while CA incubation induced the overexpression of 12 genes and the underexpression of 32. Using Venn-Diagrams, we built a list of five overexpressed genes and twelve underexpressed genes in common between the two experimental conditions. This list was used to generate a biological association network in which STAT5B and ATF-2 appeared as highly interconnected nodes. STAT5B overexpression was confirmed at the mRNA and protein levels. For ATF-2, the changes in mRNA levels were confirmed for both ICC and CA, whereas the decrease in protein levels was only observed in CA-treated cells. The levels of cyclin D1, a target gene for both STAT5B and ATF-2, were dowregulated by CA in colon cancer cells and by ICC and CA in breast cancer cells. Conclusions: Coffee polyphenols are able to affect cyclin D1 expression in cancer cells through the modulation of STAT5B and ATF-2.

Assessment of the levels of degradation in fat co- and by-products for feed uses and their relationships with some lipid composition parameters

This paper discusses the levels of degradation of some co- and byproducts of the food chain intended for feed uses. As the first part of a research project, 'Feeding Fats Safety', financed by the sixth Framework Programme-EC, a total of 123 samples were collected from 10 European countries, corresponding to fat co- and byproducts such as animal fats, fish oils, acid oils from refining, recycled cooking oils, and other. Several composition and degradation parameters (moisture, acid value, diacylglycerols and monoacylglycerols, peroxides, secondary oxidation products, polymers of triacylglycerols, fatty acid composition, tocopherols, and tocotrienols) were evaluated. These findings led to the conclusion that some fat by- and coproducts, such as fish oils, lecithins, and acid oils, show poor, nonstandardized quality and that production processes need to be greatly improved. Conclusions are also put forward about the applicability and utility of each analytical parameter for characterization and quality control.

Randomized, crossover, double-blind, placebo-controlled trial to assess the lipid lowering effect of co-formulated TDF/FTC

Abstract INTRODUCTION: Previous studies have described improvements on lipid parameters when switching from other antiretroviral drugs to tenofovir (TDF) and impairments in lipid profile when discontinuing TDF. [1-3] It is unknown, however, if TDF has an intrinsic lipid-lowering effect or such findings are due to the addition or removal of other offending agents or other reasons. MATERIALS AND METHODS: RESULTS: 46 subjects with a median age of 43 (40-48) years were enrolled in the study: 70% were male, 56% received DRV/r and 44% LPV/r. One subject withdrew the study voluntarily at week 4 and another one interrupted due to diarrhoea at week 24. Treatment with TDF/FTC decreased total, LDL and HDL-cholesterol from 235.9 to 204.9 (p<0.001), 154.7 to 127.6 (p<0.001) and 50.3 to 44.5 mg/dL (p<0.001), respectively. In comparison, total, LDL and HDL-cholesterol levels remained stable during placebo exposure. Week 12 total cholesterol (p<0.001), LDL-cholesterol (p<0.001) and HDL-cholesterol (p=0.011) levels were significantly lower in TDF/FTC versus placebo. Treatment with TDF/FTC reduced the fraction of subjects with abnormal fasting total-cholesterol (≥200 mg/dL) from 86.7% to 56.8% (p=0.001) and LDL-cholesterol (≥130 mg/dL) from 87.8% to 43.9% (p<0.001), which was not observed with placebo. There were no virological failures, and CD4 and triglyceride levels remained stable regardless of exposure. CONCLUSION: Coformulated TDF/FTC has an intrinsic lipid-lowering effect, likely attributable to TDF.

Aeromonas hydrophila flagella glycosylation: involvement of a lipid carrier.

Polar flagellin proteins from Aeromonas hydrophila strain AH-3 (serotype O34) were found to be O-glycosylated with a heterogeneous glycan. Mutants unable to produce WecP or Gne enzymes showed altered motility, and the study of their polar flagellin glycosylation showed that the patterns of glycosylation differed from that observed with wild type polar flagellin. This suggested the involvement of a lipid carrier in glycosylation. A gene coding for an enzyme linking sugar to a lipid carrier was identified in strain AH-3 (WecX) and subsequent mutation abolished completely motility, flagella production by EM, and flagellin glycosylation. This is the first report of a lipid carrier involved in flagella O-glycosylation. A molecular model has been proposed. The results obtained suggested that the N-acetylhexosamines are N-acetylgalactosamines and that the heptasaccharide is completely independent of the O34-antigen lipopolysaccharide. Furthermore, by comparing the mutants with differing degrees of polar flagellin glycosylation, we established their importance in A. hydrophila flagella formation and motility.

Multidrug resistance protein 1 localization in lipid raft domains and prostasomes in prostate cancer cell lines

Background: One of the problems in prostate cancer (CaP) treatment is the appearance of the multidrug resistance phenotype, in which ATP-binding cassette transporters such as multidrug resistance protein 1 (MRP1) play a role. Different localizations of the transporter have been reported, some of them related to the chemoresistant phenotype. Aim: This study aimed to compare the localization of MRP1 in three prostate cell lines (normal, androgen-sensitive, and androgen-independent) in order to understand its possible role in CaP chemoresistance. Methods: MRP1 and caveolae protein markers were detected using confocal microscopy, performing colocalization techniques. Lipid raft isolation made it possible to detect these proteins by Western blot analysis. Caveolae and prostasomes were identified by electron microscopy. Results: We show that MRP1 is found in lipid raft fractions of tumor cells and that the number of caveolae increases with malignancy acquisition. MRP1 is found not only in the plasma membrane associated with lipid rafts but also in cytoplasmic accumulations colocalizing with the prostasome markers Caveolin-1 and CD59, suggesting that in CaP cells, MRP1 is localized in prostasomes. Conclusion: We hypothesize that the presence of MRP1 in prostasomes could serve as a reservoir of MRP1; thus, taking advantage of the release of their content, MRP1 could be translocated to the plasma membrane contributing to the chemoresistant phenotype. The presence of MRP1 in prostasomes could serve as a predictor of malignancy in CaP

Multidrug resistance protein 1 localization in lipid raft domains and prostasomes in prostate cancer cell lines

Background: One of the problems in prostate cancer (CaP) treatment is the appearance of the multidrug resistance phenotype, in which ATP-binding cassette transporters such as multidrug resistance protein 1 (MRP1) play a role. Different localizations of the transporter have been reported, some of them related to the chemoresistant phenotype. Aim: This study aimed to compare the localization of MRP1 in three prostate cell lines (normal, androgen-sensitive, and androgen-independent) in order to understand its possible role in CaP chemoresistance. Methods: MRP1 and caveolae protein markers were detected using confocal microscopy, performing colocalization techniques. Lipid raft isolation made it possible to detect these proteins by Western blot analysis. Caveolae and prostasomes were identified by electron microscopy. Results: We show that MRP1 is found in lipid raft fractions of tumor cells and that the number of caveolae increases with malignancy acquisition. MRP1 is found not only in the plasma membrane associated with lipid rafts but also in cytoplasmic accumulations colocalizing with the prostasome markers Caveolin-1 and CD59, suggesting that in CaP cells, MRP1 is localized in prostasomes. Conclusion: We hypothesize that the presence of MRP1 in prostasomes could serve as a reservoir of MRP1; thus, taking advantage of the release of their content, MRP1 could be translocated to the plasma membrane contributing to the chemoresistant phenotype. The presence of MRP1 in prostasomes could serve as a predictor of malignancy in CaP

Multidrug resistance protein 1 localization in lipid raft domains and prostasomes in prostate cancer cell lines

Background: One of the problems in prostate cancer (CaP) treatment is the appearance of the multidrug resistance phenotype, in which ATP-binding cassette transporters such as multidrug resistance protein 1 (MRP1) play a role. Different localizations of the transporter have been reported, some of them related to the chemoresistant phenotype. Aim: This study aimed to compare the localization of MRP1 in three prostate cell lines (normal, androgen-sensitive, and androgen-independent) in order to understand its possible role in CaP chemoresistance. Methods: MRP1 and caveolae protein markers were detected using confocal microscopy, performing colocalization techniques. Lipid raft isolation made it possible to detect these proteins by Western blot analysis. Caveolae and prostasomes were identified by electron microscopy. Results: We show that MRP1 is found in lipid raft fractions of tumor cells and that the number of caveolae increases with malignancy acquisition. MRP1 is found not only in the plasma membrane associated with lipid rafts but also in cytoplasmic accumulations colocalizing with the prostasome markers Caveolin-1 and CD59, suggesting that in CaP cells, MRP1 is localized in prostasomes. Conclusion: We hypothesize that the presence of MRP1 in prostasomes could serve as a reservoir of MRP1; thus, taking advantage of the release of their content, MRP1 could be translocated to the plasma membrane contributing to the chemoresistant phenotype. The presence of MRP1 in prostasomes could serve as a predictor of malignancy in CaP

Enhanced fatty acid oxidation in adipocytes and macrophages reduces lipid-induced triglyceride accumulation and inflammation

Lipid overload in obesity and type 2 diabetes is associated with adipocyte dysfunction, inflammation, macrophage infiltration, and decreased fatty acid oxidation (FAO). Here, we report that the expression of carnitine palmitoyltransferase 1A (CPT1A), the rate-limiting enzyme in mitochondrial FAO, is higher in human adipose tissue macrophages than in adipocytes and that it is differentially expressed in visceral vs. subcutaneous adipose tissue in both an obese and a type 2 diabetes cohort. These observations led us to further investigate the potential role of CPT1A in adipocytes and macrophages. We expressed CPT1AM, a permanently active mutant form of CPT1A, in 3T3-L1 CARΔ1 adipocytes and RAW 264.7 macrophages through adenoviral infection. Enhanced FAO in palmitate-incubated adipocytes and macrophages reduced triglyceride content and inflammation, improved insulin sensitivity in adipocytes, and reduced endoplasmic reticulum stress and ROS damage in macrophages. We conclude that increasing FAO in adipocytes and macrophages improves palmitate-induced derangements. This indicates that enhancing FAO in metabolically relevant cells such as adipocytes and macrophages may be a promising strategy for the treatment of chronic inflammatory pathologies such as obesity and type 2 diabetes.