Autophagy is a fundamental catabolic process that uses a unique post-translational modification, the conjugation of ATG8 protein to phosphatidylethanolamine (PE). ATG8 lipidation also occurs during non-canonical autophagy, a parallel pathway involving conjugation of ATG8 to single membranes (CASM) at endolysosomal compartments, with key functions in immunity, vision, and neurobiology. It is widely assumed that CASM involves the same conjugation of ATG8 to PE, but this has not been formally tested. Here, we discover that all ATG8s can also undergo alternative lipidation to phosphatidylserine (PS) during CASM, induced pharmacologically, by LC3-associated phagocytosis or influenza A virus infection, in mammalian cells. Importantly, ATG8-PS and ATG8-PE adducts are differentially delipidated by the ATG4 family and bear different cellular dynamics, indicating significant molecular distinctions. These results provide important insights into autophagy signaling, revealing an alternative form of the hallmark ATG8 lipidation event. Furthermore, ATG8-PS provides a specific "molecular signature" for the non-canonical autophagy pathway.
Lipoprotein lipase (LPL) is upregulated in atherosclerotic lesions and it may promote the progression of atherosclerosis, but the mechanisms behind this process are not completely understood. We previously showed that the phosphorylation of Akt within THP-1 macrophages is increased in response to the lipid hydrolysis products generated by LPL from total lipoproteins. Notably, the free fatty acid (FFA) component was responsible for this effect. In the present study, we aimed to reveal more detail as to how the FFA component may affect Akt signalling. We show that the phosphorylation of Akt within THP-1 macrophages increases with total FFA concentration and that phosphorylation is elevated up to 18 hours. We further show that specifically the palmitoleate component of the total FFA affects Akt phosphorylation. This is tied with changes to the levels of select molecular species of phosphoinositides. We further show that the total FFA component, and specifically palmitoleate, reduces apolipoprotein A-I-mediated cholesterol efflux, and that the reduction can be reversed in the presence of the Akt inhibitor MK-2206. Overall, our data support a negative role for the FFA component of lipoprotein hydrolysis products generated by LPL, by impairing macrophage cholesterol efflux via Akt activation.
Dietary restriction (DR) during adulthood can greatly extend lifespan and improve metabolic health in diverse species. However, whether DR in mammals is still effective when applied for the first time at old age remains elusive. Here, we report results of a late-life DR switch experiment employing 800 mice, in which 24 months old female mice were switched from ad libitum (AL) to DR or vice versa. Strikingly, the switch from DR-to-AL acutely increases mortality, whereas the switch from AL-to-DR causes only a weak and gradual increase in survival, suggesting a memory of earlier nutrition. RNA-seq profiling in liver, brown (BAT) and white adipose tissue (WAT) demonstrate a largely refractory transcriptional and metabolic response to DR after AL feeding in fat tissue, particularly in WAT, and a proinflammatory signature in aged preadipocytes, which is prevented by chronic DR feeding. Our results provide evidence for a nutritional memory as a limiting factor for DR-induced longevity and metabolic remodeling of WAT in mammals.
The circadian clock regulates immune responses to microbes and affects pathogen replication, but the underlying molecular mechanisms are not well understood. Here we demonstrate that the circadian components BMAL1 and REV-ERBα influence several steps in the hepatitis C virus (HCV) life cycle, including particle entry into hepatocytes and RNA genome replication. Genetic knock out of Bmal1 and over-expression or activation of REV-ERB with synthetic agonists inhibits the replication of HCV and the related flaviruses dengue and Zika via perturbation of lipid signaling pathways. This study highlights a role for the circadian clock component REV-ERBα in regulating flavivirus replication.
Lipids are increasingly recognized as dynamic, critical metabolites affecting human physiology and pathophysiology. LIPID MAPS is a free resource dedicated to serving the lipid research community.
Tumour cells exhibit altered lipid metabolism compared to normal cells. Cell signalling kinases are important for regulating lipid synthesis and energy storage. How upstream kinases regulate lipid content, versus direct targeting of lipid metabolising enzymes, is currently unexplored. We evaluated intracellular lipid concentrations in prostate and breast tumour spheroids, treated with drugs directly inhibiting metabolic enzymes FASN, ACC, DGAT and PDHK, or cell signalling kinase enzymes PI3K, AKT and mTOR with lipidomic analysis. We assessed whether baseline lipid profiles corresponded to inhibitors' effectiveness in modulating lipid profiles in 3D-growth, and their relationship to therapeutic activity. Inhibitors against PI3K, AKT and mTOR significantly inhibited MDA-MB-468 and PC3 cell growth in 2D and 3D spheroid growth, while moderately altering lipid content. Conversely, metabolism inhibitors against FASN and DGAT altered lipid content most effectively, while only moderately inhibiting growth compared to kinase inhibitors. The FASN and ACC inhibitors' effectiveness in MDA-MB-468, versus PC3, suggested the former depended more on synthesis whereas the latter may salvage lipids. Although baseline lipid profiles didn't predict growth effects, lipid changes on therapy matched the growth effects of FASN and DGAT inhibitors. Several phospholipids, including phosphatidylcholine, were also upregulated following treatment, possibly via the Kennedy pathway. As this promotes tumour growth, combination studies should include drugs targeting it. Two-dimensional drug screening may miss important metabolism inhibitors or underestimate their potency. Clinical studies should consider serial measurements of tumour lipids to prove target modulation. Pre-therapy tumour classification by de novo lipid synthesis versus uptake may help demonstrate efficacy.
Human blood is a self-regenerating, lipid-rich biologic fluid that is routinely collected in hospital settings. The inventory of lipid molecules found in blood plasma (plasma lipidome) offers insights into individual metabolism and physiology in health and disease. Disturbances in lipid metabolism also occur in conditions that are not directly linked to lipid metabolism; therefore, plasma lipidomics based on mass spectrometry (MS) is an emerging tool in an array of clinical diagnostics and disease management. However, challenges exist in the translation of such lipidomic data to clinical applications. These relate to the reproducibility, accuracy, and precision of lipid quantitation, study design, sample handling, and data sharing. This position paper emerged from a workshop that initiated a community-led process to elaborate and define a set of generally accepted guidelines for quantitative MS-based lipidomics of blood plasma or serum, with harmonization of data acquired on different instrumentation platforms in independent laboratories across laboratories as an ultimate goal. We hope that other fields may benefit from and follow such a precedent.
In patients with asthma or chronic obstructive pulmonary disease rhinovirus infections can provoke acute worsening of disease and limited treatment options exist. Viral replication in the host cell induces significant remodeling of intracellular membranes, but few studies have explored this mechanistically or as a therapeutic opportunity. We performed unbiased lipidomic analysis on human bronchial epithelial cells infected over a 6 hour period with the RV-A1b strain of rhinovirus to determine changes in 493 distinct lipid species. Through pathway and network analysis we identified temporal changes in the apparent activities of a number of lipid metabolizing and signaling enzymes. In particular, analysis highlighted fatty acid synthesis and ceramide metabolism as potential anti-rhinoviral targets. To validate the importance of these enzymes in viral replication, we explored the effects of commercially-available enzyme inhibitors upon RV-A1b infection and replication. Ceranib-1, D609 and C75 were the most potent inhibitors, which confirmed that fatty acid synthase and ceramidase are potential inhibitory targets in rhinoviral infections. More broadly, this study demonstrates the potential of lipidomics and pathway analysis to identify novel targets to treat human disorders.
Epithelial-resident T lymphocytes, such as intraepithelial lymphocytes (IELs) located at the intestinal barrier, can offer swift protection against invading pathogens. Lymphocyte activation is strictly regulated because of its potential harmful nature and metabolic cost, and most lymphocytes are maintained in a quiescent state. However, IELs are kept in a heightened state of activation resembling effector T cells but without cytokine production or clonal proliferation. We show that this controlled activation state correlates with alterations in the IEL mitochondrial membrane, especially the cardiolipin composition. Upon inflammation, the cardiolipin composition is altered to support IEL proliferation and effector function. Furthermore, we show that cardiolipin makeup can particularly restrict swift IEL proliferation and effector functions, reducing microbial containment capability. These findings uncover an alternative mechanism to control cellular activity, special to epithelial-resident T cells, and a novel role for mitochondria, maintaining cells in a metabolically poised state while enabling rapid progression to full functionality.
Octahedral platinum(iv) complexes such as ,,-[Pt(N)(OH)(pyridine)] () are stable in the dark, but potently cytotoxic to a range of cancer cells when activated by UVA or visible light, and active . Photoactivation causes the reduction of the complex and leads to the formation of unusual Pt(ii) lesions on DNA. However, radicals are also generated in the excited state resulting from photoactivation (J. S. Butler, J. A. Woods, N. J. Farrer, M. E. Newton and P. J. Sadler, , 2012, , 16508-16511). Here we show that once photoactivated, also can interact with peptides, and therefore proteins are potential targets of this candidate drug. High resolution FT-ICR MS studies show that reactions of activated by visible light with two neuropeptides Substance P, RPKPQQFFGLM-NH () and [Lys]-Bombesin, pEQKLGNQWAVGHLM-NH () give rise to unexpected products, in the form of both oxidised and platinated peptides. Further MS/MS analysis using electron-capture dissociation (ECD) dissociation pathways (enabling retention of the Pt complex during fragmentation), and EPR experiments using the spin-trap DEPMPO, show that the products generated during the photoactivation of depend on the amino acid composition of the peptide. This work reveals the multi-targeting nature of excited state platinum anticancer complexes. Not only can they target DNA, but also peptides (and proteins) by sequence dependent platination and radical mechanisms.
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.
Dietary restriction (DR), a reduction in food intake without malnutrition, increases most aspects of health during aging and extends lifespan in diverse species, including rodents. However, the mechanisms by which DR interacts with the aging process to improve health in old age are poorly understood. DNA methylation could play an important role in mediating the effects of DR because it is sensitive to the effects of nutrition and can affect gene expression memory over time.
Chronic hepatitis C is a global health problem with an estimated 170 million HCV infected individuals at risk of progressive liver disease and hepatocellular carcinoma (HCC). Autotaxin (ATX) is a phospholipase with diverse roles in physiological and pathological processes including inflammation and oncogenesis. Clinical studies have reported increased ATX expression in chronic hepatitis C, however, the pathways regulating ATX and its role in the viral life cycle are not well understood.
The three-membered RUNX gene family includes RUNX1, a major mutational target in human leukemias, and displays hallmarks of both tumor suppressors and oncogenes. In mouse models, the Runx genes appear to act as conditional oncogenes, as ectopic expression is growth suppressive in normal cells but drives lymphoma development potently when combined with over-expressed Myc or loss of p53. Clues to underlying mechanisms emerged previously from murine fibroblasts where ectopic expression of any of the Runx genes promotes survival through direct and indirect regulation of key enzymes in sphingolipid metabolism associated with a shift in the "sphingolipid rheostat" from ceramide to sphingosine-1-phosphate (S1P). Testing of this relationship in lymphoma cells was therefore a high priority. We find that ectopic expression of Runx1 in lymphoma cells consistently perturbs the sphingolipid rheostat, whereas an essential physiological role for Runx1 is revealed by reduced S1P levels in normal spleen after partial Cre-mediated excision. Furthermore, we show that ectopic Runx1 expression confers increased resistance of lymphoma cells to glucocorticoid-mediated apoptosis, and elucidate the mechanism of cross-talk between glucocorticoid and sphingolipid metabolism through Sgpp1. Dexamethasone potently induces expression of Sgpp1 in T-lymphoma cells and drives cell death which is reduced by partial knockdown of Sgpp1 with shRNA or direct transcriptional repression of Sgpp1 by ectopic Runx1. Together these data show that Runx1 plays a role in regulating the sphingolipid rheostat in normal development and that perturbation of this cell fate regulator contributes to Runx-driven lymphomagenesis. J. Cell. Biochem. 118: 1432-1441, 2017. © 2016 Wiley Periodicals, Inc.
Recent advances in lipidomics tools and software assist in the identification and quantification of lipid species detected by mass spectrometry. By integrating mass spectrometric lipid data into mapped pathways and databases, an entire network of lipid species which both demonstrates the complexity of lipid structures and biochemical interactions can be constructed. Here we demonstrate lipidomics analysis at both systematic and molecular levels. This review focuses on four points: how lipid data can be collected and processed with the support of tools, software and databases; how lipidomic analysis is performed at the molecular level; how to integrate data analysis into a biological context; how the results of such analysis predict enzyme activities and potential sites for therapeutic interventions or manipulation of enzyme activities.
Autotaxin (ATX) the lysophospholipase responsible for generating the lipid receptor agonist lysophosphatidic acid (LPA) is a secreted enzyme. Here we show that once secreted it can bind to the surface of cell secreted exosomes. Exosome-bound ATX is catalytically active and carries generated LPA. Once bound to a cell, through specific integrin interaction, ATX releases the LPA to activate cell surface G-protein coupled LPA receptors; inhibition of signaling by the receptor antagonist Ki1642 suggests these are either LPAR1 or LPAR3. The binding stimulates downstream signaling including AKT and MAPK phosphorylation, the release of intracellular stored calcium and cell migration. We propose that exosomal binding of LPA-loaded ATX provides a means of efficiently delivering the lipid agonist to cell surface receptors to promote signalling. We further propose that this is a means whereby autotaxin-LPA signaling operates physiologically.
Single-nucleotide variations in C13orf31 (LACC1) that encode p.C284R and p.I254V in a protein of unknown function (called 'FAMIN' here) are associated with increased risk for systemic juvenile idiopathic arthritis, leprosy and Crohn's disease. Here we set out to identify the biological mechanism affected by these coding variations. FAMIN formed a complex with fatty acid synthase (FASN) on peroxisomes and promoted flux through de novo lipogenesis to concomitantly drive high levels of fatty-acid oxidation (FAO) and glycolysis and, consequently, ATP regeneration. FAMIN-dependent FAO controlled inflammasome activation, mitochondrial and NADPH-oxidase-dependent production of reactive oxygen species (ROS), and the bactericidal activity of macrophages. As p.I254V and p.C284R resulted in diminished function and loss of function, respectively, FAMIN determined resilience to endotoxin shock. Thus, we have identified a central regulator of the metabolic function and bioenergetic state of macrophages that is under evolutionary selection and determines the risk of inflammatory and infectious disease.
Enhanced macromolecule biosynthesis is integral to growth and proliferation of cancer cells. Lipid biosynthesis has been predicted to be an essential process in cancer cells. However, it is unclear which enzymes within this pathway offer the best selectivity for cancer cells and could be suitable therapeutic targets.
Encoding acyl-CoA thioesterase-7 (Acot7) is one of ∼60 genes expressed ubiquitously across tissues but relatively silenced, or disallowed, in pancreatic β-cells. The capacity of ACOT7 to hydrolyze long-chain acyl-CoA esters suggests potential roles in β-oxidation, lipid biosynthesis, signal transduction, or insulin exocytosis. We explored the physiological relevance of β-cell-specific Acot7 silencing by re-expressing ACOT7 in these cells. ACOT7 overexpression in clonal MIN6 and INS1(832/13) β-cells impaired insulin secretion in response to glucose plus fatty acids. Furthermore, in a panel of transgenic mouse lines, we demonstrate that overexpression of mitochondrial ACOT7 selectively in the adult β-cell reduces glucose tolerance dose dependently and impairs glucose-stimulated insulin secretion. By contrast, depolarization-induced secretion was unaffected, arguing against a direct action on the exocytotic machinery. Acyl-CoA levels, ATP/ADP increases, membrane depolarization, and Ca(2+) fluxes were all markedly reduced in transgenic mouse islets, whereas glucose-induced oxygen consumption was unchanged. Although glucose-induced increases in ATP/ADP ratio were similarly lowered after ACOT7 overexpression in INS1(832/13) cells, changes in mitochondrial membrane potential were unaffected, consistent with an action of Acot7 to increase cellular ATP consumption. Because Acot7 mRNA levels are increased in human islets in type 2 diabetes, inhibition of the enzyme might provide a novel therapeutic strategy.
The structures of F-ATPases have been determined predominantly with mitochondrial enzymes, but hitherto no F-ATPase has been crystallized intact. A high-resolution model of the bovine enzyme built up from separate sub-structures determined by X-ray crystallography contains about 85% of the entire complex, but it lacks a crucial region that provides a transmembrane proton pathway involved in the generation of the rotary mechanism that drives the synthesis of ATP. Here the isolation, characterization and crystallization of an integral F-ATPase complex from the α-proteobacterium Paracoccus denitrificans are described. Unlike many eubacterial F-ATPases, which can both synthesize and hydrolyse ATP, the P. denitrificans enzyme can only carry out the synthetic reaction. The mechanism of inhibition of its ATP hydrolytic activity involves a ζ inhibitor protein, which binds to the catalytic F1-domain of the enzyme. The complex that has been crystallized, and the crystals themselves, contain the nine core proteins of the complete F-ATPase complex plus the ζ inhibitor protein. The formation of crystals depends upon the presence of bound bacterial cardiolipin and phospholipid molecules; when they were removed, the complex failed to crystallize. The experiments open the way to an atomic structure of an F-ATPase complex.
We show that phospholipid anabolism does not occur uniformly during the metazoan cell cycle. Transition to S-phase is required for optimal mobilization of lipid precursors, synthesis of specific phospholipid species and endoplasmic reticulum (ER) homeostasis. Average changes observed in whole-cell phospholipid composition, and total ER lipid content, upon stimulation of cell growth can be explained by the cell cycle distribution of the population. TORC1 promotes phospholipid anabolism by slowing S/G2 progression. The cell cycle stage-specific nature of lipid biogenesis is dependent on p53. We propose that coupling lipid metabolism to cell cycle progression is a means by which cells have evolved to coordinate proliferation with cell and organelle growth.
Somatic activating mutations in PIK3CA, which encodes the p110α catalytic subunit of phosphoinositide-3-kinase (PI3K) are frequently found in cancers and have been identified in a spectrum of mosaic overgrowth disorders ranging from isolated digit enlargement to more extensive overgrowth of the body, brain, or vasculature. We aimed to study affected dermal fibroblasts with a view to inform therapeutic studies, and to observe cancer-associated mutations in isolation.
Drosophila phototransduction is mediated via a G-protein-coupled PLC cascade. Recent evidence, including the demonstration that light evokes rapid contractions of the photoreceptors, suggested that the light-sensitive channels (TRP and TRPL) may be mechanically gated, together with protons released by PLC-mediated PIP2 hydrolysis. If mechanical gating is involved we predicted that the response to light should be influenced by altering the physical properties of the membrane. To achieve this, we used diet to manipulate the degree of saturation of membrane phospholipids. In flies reared on a yeast diet, lacking polyunsaturated fatty acids (PUFAs), mass spectrometry showed that the proportion of polyunsaturated phospholipids was sevenfold reduced (from 38 to ∼5%) but rescued by adding a single species of PUFA (linolenic or linoleic acid) to the diet. Photoreceptors from yeast-reared flies showed a 2- to 3-fold increase in latency and time to peak of the light response, without affecting quantum bump waveform. In the absence of Ca(2+) influx or in trp mutants expressing only TRPL channels, sensitivity to light was reduced up to ∼10-fold by the yeast diet, and essentially abolished in hypomorphic G-protein mutants (Gαq). PLC activity appeared little affected by the yeast diet; however, light-induced contractions measured by atomic force microscopy or the activation of ectopic mechanosensitive gramicidin channels were also slowed ∼2-fold. The results are consistent with mechanosensitive gating and provide a striking example of how dietary fatty acids can profoundly influence sensory performance in a classical G-protein-coupled signaling cascade.
A functional genomics study revealed that the activity of acetyl-CoA synthetase 2 (ACSS2) contributes to cancer cell growth under low-oxygen and lipid-depleted conditions. Comparative metabolomics and lipidomics demonstrated that acetate is used as a nutritional source by cancer cells in an ACSS2-dependent manner, and supplied a significant fraction of the carbon within the fatty acid and phospholipid pools. ACSS2 expression is upregulated under metabolically stressed conditions and ACSS2 silencing reduced the growth of tumor xenografts. ACSS2 exhibits copy-number gain in human breast tumors, and ACSS2 expression correlates with disease progression. These results signify a critical role for acetate consumption in the production of lipid biomass within the harsh tumor microenvironment.
Glyoxal-derived advanced glycation end-products (AGEs) are formed in physiological systems affecting protein/peptide function and structure. These AGEs are generated during aging and chronic diseases such as diabetes and are considered arginine glycating agents. Thus, the study of glyoxal-derived AGEs in lysine residues and amino acid competition is addressed here using acetylated and non-acetylated undecapeptides, with one arginine and one lysine residue available for glycation. Tandem mass spectrometry results from a Fourier transform ion cyclotron resonance mass spectrometer showed glycated species at both the arginine and lysine residues. One species with the mass addition of 116.01096 Da is formed at the arginine residue. A possible structure is proposed to explain this finding (Nδ-[2-(dihydroxymethyl)-2H,3aH,4H,6aH-[1,3]dioxolo[5,6-d]imidazolin-5-yl]-L-ornithine-derived AGE). The second species corresponded to intramolecular crosslink involving the lysine residue and its presence is checked with ion-mobility mass spectrometry.
The high mortality of melanoma is caused by rapid spread of cancer cells, which occurs unusually early in tumour evolution. Unlike most solid tumours, thickness rather than cytological markers or differentiation is the best guide to metastatic potential. Multiple stimuli that drive melanoma cell migration have been described, but it is not clear which are responsible for invasion, nor if chemotactic gradients exist in real tumours. In a chamber-based assay for melanoma dispersal, we find that cells migrate efficiently away from one another, even in initially homogeneous medium. This dispersal is driven by positive chemotaxis rather than chemorepulsion or contact inhibition. The principal chemoattractant, unexpectedly active across all tumour stages, is the lipid agonist lysophosphatidic acid (LPA) acting through the LPA receptor LPAR1. LPA induces chemotaxis of remarkable accuracy, and is both necessary and sufficient for chemotaxis and invasion in 2-D and 3-D assays. Growth factors, often described as tumour attractants, cause negligible chemotaxis themselves, but potentiate chemotaxis to LPA. Cells rapidly break down LPA present at substantial levels in culture medium and normal skin to generate outward-facing gradients. We measure LPA gradients across the margins of melanomas in vivo, confirming the physiological importance of our results. We conclude that LPA chemotaxis provides a strong drive for melanoma cells to invade outwards. Cells create their own gradients by acting as a sink, breaking down locally present LPA, and thus forming a gradient that is low in the tumour and high in the surrounding areas. The key step is not acquisition of sensitivity to the chemoattractant, but rather the tumour growing to break down enough LPA to form a gradient. Thus the stimulus that drives cell dispersal is not the presence of LPA itself, but the self-generated, outward-directed gradient.
An in vivo model of antiangiogenic therapy allowed us to identify genes upregulated by bevacizumab treatment, including Fatty Acid Binding Protein 3 (FABP3) and FABP7, both of which are involved in fatty acid uptake. InÂ vitro, both were induced by hypoxia in a hypoxia-inducible factor-1α (HIF-1α)-dependent manner. There was a significant lipid droplet (LD) accumulation in hypoxia that was time and O2 concentration dependent. Knockdown of endogenous expression of FABP3, FABP7, or Adipophilin (an essential LD structural component) significantly impaired LD formation under hypoxia. We showed that LD accumulation is due to FABP3/7-dependent fatty acid uptake while de novo fatty acid synthesis is repressed in hypoxia. We also showed that ATP production occurs via Î²-oxidation or glycogen degradation in a cell-type-dependent manner in hypoxia-reoxygenation. Finally, inhibition of lipid storage reduced protection against reactive oxygen species toxicity, decreased the survival of cells subjected to hypoxia-reoxygenation in-vitro, and strongly impaired tumorigenesis in-vivo.
In hepatitis C virus infection, replication of the viral genome and virion assembly are linked to cellular metabolic processes. In particular, lipid droplets, which store principally triacylglycerides (TAGs) and cholesterol esters (CEs), have been implicated in production of infectious virus. Here, we examine the effect on productive infection of triacsin C and YIC-C8-434, which inhibit synthesis of TAGs and CEs by targeting long-chain acyl-CoA synthetase and acyl-CoA:cholesterol acyltransferase, respectively. Our results present high resolution data on the acylglycerol and cholesterol ester species that were affected by the compounds. Moreover, triacsin C, which blocks both triglyceride and cholesterol ester synthesis, cleared most of the lipid droplets in cells. By contrast, YIC-C8-434, which only abrogates production of cholesterol esters, induced an increase in size of droplets. Although both compounds slightly reduced viral RNA synthesis, they significantly impaired assembly of infectious virions in infected cells. In the case of triacsin C, reduced stability of the viral core protein, which forms the virion nucleocapsid and is targeted to the surface of lipid droplets, correlated with lower virion assembly. In addition, the virus particles that were released from cells had reduced specific infectivity. YIC-C8-434 did not alter the association of core with lipid droplets but appeared to decrease production of infectious virus particles, suggesting a block in virion assembly. Thus, the compounds have antiviral properties, indicating that targeting synthesis of lipids stored in lipid droplets might be an option for therapeutic intervention in treating chronic hepatitis C virus infection.
It is unclear how changes in lipid droplet size and number are regulated - for example, it is not known whether this involves a signalling pathway or is directed by cellular lipid uptake. Here, we show that oleic acid stimulates lipid droplet formation by activating the long-chain fatty acid receptor FFAR4, which signals through a pertussis-toxin-sensitive G-protein signalling pathway involving phosphoinositide 3-kinase (PI3-kinase), AKT (also known as protein kinase B) and phospholipase D (PLD) activities. This initial lipid droplet formation is not dependent upon exogenous lipid, whereas the subsequent more sustained increase in the number of lipid droplets is dependent upon lipid uptake. These two mechanisms of lipid droplet formation point to distinct potential intervention points.
The advent of mass spectrometric methods has facilitated the determination of multiple molecular species of cellular lipid classes including the polyphosphoinositides, though to date methods to analyse and quantify each of the individual three PtdInsP and three PtdInsP2 species are lacking. The use of imaging methods has allowed intracellular localization of the phosphoinositide classes but this methodology does not determine the acyl structures. The range of molecular species suggests a greater complexity in polyphosphoinositide signaling than yet defined but elucidating this will require further method development to be achieved. This article is part of a Special Issue entitled Tools to study lipid functions.
The binding sites of two ruthenium(II) organometallic complexes of the form [(η(6)-arene)Ru(N,N)Cl](+), where arene/N,N = biphenyl (bip)/bipyridine (bipy) for complex AH076, and biphenyl (bip)/o-phenylenediamine (o-pda) for complex AH078, on the peptides angiotensin and bombesin have been investigated using Fourier transform ion cyclotron resonance (FTICR) mass spectrometry. Fragmentation was performed using collisionally activated dissociation (CAD), with, in some cases, additional data being provided by electron capture dissociation (ECD). The primary binding sites were identified as methionine and histidine, with further coordination to phenylalanine, potentially through a π-stacking interaction, which has been observed here for the first time. This initial peptide study was expanded to investigate protein binding through reaction with insulin, on which the binding sites proposed are histidine, glutamic acid, and tyrosine. Further reaction of the ruthenium complexes with the oxidized B chain of insulin, in which two cysteine residues are oxidized to cysteine sulfonic acid (Cys-SO3H), and glutathione, which had been oxidized with hydrogen peroxide to convert the cysteine to cysteine sulfonic acid, provided further support for histidine and glutamic acid binding, respectively.
Glycation is a post-translational modification (PTM) that affects the physiological properties of peptides and proteins. In particular, during hyperglycaemia, glycation by α-dicarbonyl compounds generate α-dicarbonyl-derived glycation products also called α-dicarbonyl-derived advanced glycation end products. Glycation by the α-dicarbonyl compound known as glyoxal was studied in model peptides by MS/MS using a Fourier transform ion cyclotron resonance mass spectrometer. An unusual type of glyoxal-derived AGE with a mass addition of 21.98436 Da is reported in peptides containing combinations of two arginine-two lysine, and one arginine-three lysine amino acid residues. Electron capture dissociation and collisionally activated dissociation results supported that the unusual glyoxal-derived AGE is formed at the guanidino group of arginine, and a possible structure is proposed to illustrate the 21.9843 Da mass addition.
The post-translational modification known as glycation affects the physiological properties of peptides and proteins. Glycation is particularly important during hyperglycaemia where α-dicarbonyl compounds are generated. These compounds react with proteins to generate α-dicarbonyl-derived glycation products, which are correlated with diabetic complications such as nephropathy, retinopathy, and neuropathy, among others. One of these α-dicarbonyl compounds is ethanedial, also known as glyoxal. Thereby, glyoxal binding to protein/peptides is studied by electron capture dissociation (ECD) and collisionally activated dissociation (CAD).
Regulation of lipid metabolism via activation of sterol regulatory element binding proteins (SREBPs) has emerged as an important function of the Akt/mTORC1 signaling axis. Although the contribution of dysregulated Akt/mTORC1 signaling to cancer has been investigated extensively and altered lipid metabolism is observed in many tumors, the exact role of SREBPs in the control of biosynthetic processes required for Akt-dependent cell growth and their contribution to tumorigenesis remains unclear.
OBJECTIVE: Decreased expression of diacylglycerol kinase delta (DGKÎ´) has been linked to insulin resistance in humans and mice and it is abundantly expressed in adipose tissue. Therefore, its role in adipogenesis was examined. DESIGN AND METHODS: 3T3-L1 pre-adipocytes were generated in which DGKÎ´ expression had been knocked down and the effect of this on adipogenesis was determined. Lipidomic analyses were performed to determine levels of the DGKÎ´ product phosphatidic acid (PA), its substrate diacylglycerol (DAG) and triglyceride (TG). RESULTS: Inhibiting DGKÎ´ expression prevents adipogenesis. DGKÎ´ knockdown in differentiating adipocytes blunted the increase in total levels of PA and DAG but did not affect the early rise in TG levels. DAG or PA species acting as TG precursors were only modestly reduced by DGKÎ´ knockdown which significantly impaired the accumulation of DAG or PA species implicated in intracellular signaling. The DAG activated kinase PKCÎ´ was also stimulated in DGKÎ´ knockdown cells, despite no increase in detectable species of DAG. CONCLUSIONS: DGKÎ´ is a novel regulator of adipogenesis and phosphorylates a quantitatively small pool of signaling DAG important for differentiation and indirectly affects overall levels of signaling DAG and PA species distinct from those acting as precursors for TG synthesis.
Rotaviruses (RVs) cause acute gastroenteritis in infants and young children, and are globally distributed. Within the infected host cell, RVs establish replication complexes in viroplasms ('viral factories') to which lipid droplet organelles are recruited. To further understand this recently discovered phenomenon, the lipidomes of RV-infected and uninfected MA104 cells were investigated. Cell lysates were subjected to equilibrium ultracentrifugation through iodixanol gradients. Fourteen different classes of lipids were differentiated by mass spectrometry. The concentrations of virtually all lipids were elevated in RV-infected cells. Fractions of low density (1.11-1.15 g mlâ»Â¹), in which peaks of the RV dsRNA genome and lipid droplet- and viroplasm-associated proteins were observed, contained increased amounts of lipids typically found concentrated in the cellular organelle lipid droplets, confirming the close interaction of lipid droplets with viroplasms. A decrease in the ratio of the amounts of surface to internal components of lipid droplets upon RV infection suggested that the lipid droplet-viroplasm complexes became enlarged.
The functions and morphology of cellular membranes are intimately related and depend not only on their protein content but also on the repertoire of lipids that comprise them. In the absence of in vivo data on lipid asymmetry in endomembranes, it has been argued that motors, scaffolding proteins or integral membrane proteins rather than non-lamellar bilayer lipids such as diacylglycerol (DAG), are responsible for shaping of organelles, local membrane curvature and fusion. The effects of direct alteration of levels of such lipids remain predominantly uninvestigated. Diacylglycerol (DAG) is a well documented second messenger. Here we demonstrate two additional conserved functions of DAG: a structural role in organelle morphology, and a role in localised extreme membrane curvature required for fusion for which proteins alone are insufficient. Acute and inducible DAG depletion results in failure of the nuclear envelope (NE) to reform at mitosis and reorganisation of the ER into multi-lamellar sheets as revealed by correlative light and electron microscopy and 3D reconstructions. Remarkably, depleted cells divide without a complete NE, and unless rescued by 1,2 or 1,3 DAG soon die. Attenuation of DAG levels by enzyme microinjection into echinoderm eggs and embryos also results in alterations of ER morphology and nuclear membrane fusion. Our findings demonstrate that DAG is an in vivo modulator of organelle morphology in mammalian and echinoderm cells, indicating a fundamental role conserved across the deuterostome superphylum.
Glycation by endogenous dicarbonyl metabolites such as glyoxal is an important spontaneous post-translational (PTM) modification of peptides and proteins associated with structural and functional impairment. The aim of this study was to investigate types and site of PTM of glyoxal-derived advanced glycation end-products-in the neuropeptide substance P by ultrahigh-resolution Fourier transform ion cyclotron resonance (FTICR), mass spectrometry, and tandem mass spectrometry (MS/MS) experiments. The main site of PTM by glyoxal was the side chain guanidine moiety of the arginine residue. Binding site identification has been achieved by electron capture dissociation, double-resonance electron capture dissociation, and collision-activated dissociation, with assignment of the modified amino acid residue with mass error <1 ppm.
Epidemiologic and genetic evidence links type 2 diabetes, obesity, and cancer. The tumor-suppressor phosphatase and tensin homologue (PTEN) has roles in both cellular growth and metabolic signaling. Germline PTEN mutations cause a cancer-predisposition syndrome, providing an opportunity to study the effect of PTEN haploinsufficiency in humans.
The phosphatidylinositol 3-kinase (PI3K)-AKT signaling pathway is critical for cellular growth and metabolism. Correspondingly, loss of function of PTEN, a negative regulator of PI3K, or activating mutations in AKT1, AKT2 or AKT3 have been found in distinct disorders featuring overgrowth or hypoglycemia. We performed exome sequencing of DNA from unaffected and affected cells from an individual with an unclassified syndrome of congenital progressive segmental overgrowth of fibrous and adipose tissue and bone and identified the cancer-associated mutation encoding p.His1047Leu in PIK3CA, the gene that encodes the p110Î± catalytic subunit of PI3K, only in affected cells. Sequencing of PIK3CA in ten additional individuals with overlapping syndromes identified either the p.His1047Leu alteration or a second cancer-associated alteration, p.His1047Arg, in nine cases. Affected dermal fibroblasts showed enhanced basal and epidermal growth factor (EGF)-stimulated phosphatidylinositol 3,4,5-trisphosphate (PIP(3)) generation and concomitant activation of downstream signaling relative to their unaffected counterparts. Our findings characterize a distinct overgrowth syndrome, biochemically demonstrate activation of PI3K signaling and thereby identify a rational therapeutic target.
Inhibition of Î±vÎ²3 integrin or expression of oncogenic mutants of p53 promote invasive cell migration by enhancing endosomal recycling of Î±5Î²1 integrin under control of the Rab11 effector Rab-coupling protein (RCP). In this paper, we show that diacylglycerol kinase Î± (DGK-Î±), which phosphorylates diacylglycerol to phosphatidic acid (PA), was required for RCP to be mobilized to and tethered at the tips of invasive pseudopods and to allow RCP-dependent Î±5Î²1 recycling and the resulting invasiveness of tumor cells. Expression of a constitutive-active mutant of DGK-Î± drove RCP-dependent invasion in the absence of mutant p53 expression or Î±vÎ²3 inhibition, and conversely, an RCP mutant lacking the PA-binding C2 domain was not capable of being tethered at pseudopod tips. These data demonstrate that generation of PA downstream of DGK-Î± is essential to connect expression of mutant p53s or inhibition of Î±vÎ²3 to RCP and for this Rab11 effector to drive the trafficking of Î±5Î²1 that is required for tumor cell invasion through three-dimensional matrices.
The polyphosphoinositides are key signaling lipids whose levels are tightly regulated within cells. As with other cellular lipids multiple species exist with distinct acyl chain makeups. There are methods which analyze the phosphoinositides as their deacylated derivatives which cannot address these distinct forms. Lipidomic analysis of the polyphosphoinositides has been hampered by difficulties with extraction and problems associated with binding of the lipids to surfaces. This review outlines the available MS methodologies, highlighting the difficulties associated with each. However, at present, no single methodology is available that can successfully and reproducibly quantitate each inositol phospholipid.
The signalling lipid phosphatidic acid (PA) is generated by the hydrolysis of phosphatidylcholine (PC), which is catalysed by phospholipase D (PLD) enzymes. Neutrophils, important cells of the innate immune system, maintain the body's defence against infection. Previous studies have implicated PLD-generated PA in neutrophil function; these have relied heavily on the use of primary alcohols to act as inhibitors of PA production. The recent development of isoform-selective small molecule inhibitors and the generation of a knockout mouse model provide us with accurate tools to study the role of PLDs in neutrophil responses. We show that PLD1 is a regulator of phorbol-ester-, chemoattractant, adhesion-dependent and FcÎ³-receptor-stimulated production of reactive oxygen species (ROS) in neutrophils. Significantly we found that this role of PLD is isoform specific: the absence of PLD2 does not negatively affect these processes. Contrary to expectation, other functions required for an efficient immune response operate effectively in Pld2-deficient neutrophils or when both isoforms are inhibited pharmacologically. We conclude that although PLD1 does have important regulatory roles in neutrophils, the field has been confused by the use of primary alcohols; now that gold standard Pld-knockout mouse models are available, previous work might need to be reassessed.
The Runx genes (Runx1, 2, and 3) regulate cell fate in development and can operate as either oncogenes or tumor suppressors in cancer. The oncogenic potential of ectopic Runx expression has been shown in transgenic mice that develop lymphoma in potent synergy with overexpressed Myc, and in established fibroblasts that display altered morphology and increased tumorigenicity. Candidate oncogenic functions of overexpressed Runx genes include resistance to apoptosis in response to intrinsic and extrinsic stresses. In a search for gene targets responsible for this aspect of Runx phenotype, we have identified three key enzymes in sphingolipid metabolism (Sgpp1, Ugcg, and St3gal5/Siat9) as direct targets for Runx transcriptional regulation in a manner consistent with survival and apoptosis resistance. Consistent with these changes in gene expression, mass spectrometric analysis showed that ectopic Runx reduces intracellular long-chain ceramides in NIH3T3 fibroblasts and elevated extracellular sphingosine 1 phosphate. Runx expression also opposed the activation of c-Jun-NH(2)-kinase and p38(MAPK), key mediators of ceramide-induced death, and suppressed the onset of apoptosis in response to exogenous tumor necrosis factor alpha. The survival advantage conferred by ectopic Runx could be partially recapitulated by exogenous sphingosine 1 phosphate and was accompanied by reduced phosphorylation of p38(MAPK). These results reveal a novel link between transcription factor oncogenes and lipid signaling pathways involved in cancer cell survival and chemoresistance.
Phosphatidic acid (PA) is postulated to have both structural and signaling functions during membrane dynamics in animal cells. In this study, we show that before a critical time period during rhabdomere biogenesis in Drosophila melanogaster photoreceptors, elevated levels of PA disrupt membrane transport to the apical domain. Lipidomic analysis shows that this effect is associated with an increase in the abundance of a single, relatively minor molecular species of PA. These transport defects are dependent on the activation state of Arf1. Transport defects via PA generated by phospholipase D require the activity of type I phosphatidylinositol (PI) 4 phosphate 5 kinase, are phenocopied by knockdown of PI 4 kinase, and are associated with normal endoplasmic reticulum to Golgi transport. We propose that PA levels are critical for apical membrane transport events required for rhabdomere biogenesis.
In 2005, the International Lipid Classification and Nomenclature Committee under the sponsorship of the LIPID MAPS Consortium developed and established a "Comprehensive Classification System for Lipids" based on well-defined chemical and biochemical principles and using an ontology that is extensible, flexible, and scalable. This classification system, which is compatible with contemporary databasing and informatics needs, has now been accepted internationally and widely adopted. In response to considerable attention and requests from lipid researchers from around the globe and in a variety of fields, the comprehensive classification system has undergone significant revisions over the last few years to more fully represent lipid structures from a wider variety of sources and to provide additional levels of detail as necessary. The details of this classification system are reviewed and updated and are presented here, along with revisions to its suggested nomenclature and structure-drawing recommendations for lipids.