Life Sciences Research for Lifelong Health

Publications lipidomics

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MS-based lipidomics of human blood plasma - a community-initiated position paper to develop accepted guidelines.
Burla B, Arita M, Arita M, Bendt AK, Cazenave-Gassiot A, Dennis EA, Ekroos K, Han X, Ikeda K, Liebisch G, Lin MK, Loh TP, Meikle PJ, Orešič M, Quehenberger O, Shevchenko A, Torta F, Wakelam MJO, Wheelock CE, Wenk MR

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.

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Journal of lipid research, , 1539-7262, , 2018

PMID: 30115755

Open Access

Host lipidome analysis during rhinovirus replication in human bronchial epithelial cells identifies potential therapeutic targets.
Nguyen A, Guedan A, Mousnier A, Swieboda D, Zhang Q, Horkai D, Le Novere N, Solari R, Wakelam MJO

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.

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Journal of lipid research, , 1539-7262, , 2018

PMID: 29946055

Open Access

Mitochondria maintain controlled activation state of epithelial-resident T lymphocytes.
Konjar Š, Frising UC, Ferreira C, Hinterleitner R, Mayassi T, Zhang Q, Blankenhaus B, Haberman N, Loo Y, Guedes J, Baptista M, Innocentin S, Stange J, Strathdee D, Jabri B, Veldhoen M

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.

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Science immunology, 3, 2470-9468, , 2018

PMID: 29934344

Flotillin proteins recruit sphingosine to membranes and maintain cellular sphingosine-1-phosphate levels.
Riento K, Zhang Q, Clark J, Begum F, Stephens E, Wakelam MJ, Nichols BJ

Sphingosine-1-phosphate (S1P) is an important lipid signalling molecule. S1P is produced via intracellular phosphorylation of sphingosine (Sph). As a lipid with a single fatty alkyl chain, Sph may diffuse rapidly between cellular membranes and through the aqueous phase. Here, we show that the absence of microdomains generated by multimeric assemblies of flotillin proteins results in reduced S1P levels. Cellular phenotypes of flotillin knockout mice, including changes in histone acetylation and expression of Isg15, are recapitulated when S1P synthesis is perturbed. Flotillins bind to Sph in vitro and increase recruitment of Sph to membranes in cells. Ectopic re-localisation of flotillins within the cell causes concomitant redistribution of Sph. The data suggest that flotillins may directly or indirectly regulate cellular sphingolipid distribution and signalling.

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PloS one, 13, 1932-6203, e0197401, 2018

PMID: 29787576

Open Access

Sequence-dependent attack on peptides by photoactivated platinum anticancer complexes.
Wootton CA, Sanchez-Cano C, Lopez-Clavijo AF, Shaili E, Barrow MP, Sadler PJ, O'Connor PB

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.

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Chemical science, 9, 2041-6520, 2733-2739, 2018

PMID: 29732057

Open Access

Deciphering lipid structures based on platform-independent decision rules.
Hartler J, Triebl A, Ziegl A, Trötzmüller M, Rechberger GN, Zeleznik OA, Zierler KA, Torta F, Cazenave-Gassiot A, Wenk MR, Fauland A, Wheelock CE, Armando AM, Quehenberger O, Zhang Q, Wakelam MJO, Haemmerle G, Spener F, Köfeler HC, Thallinger GG

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.

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Nature methods, , 1548-7105, , 2017

PMID: 29058722

Dietary restriction protects from age-associated DNA methylation and induces epigenetic reprogramming of lipid metabolism.
Hahn O, Grönke S, Stubbs TM, Ficz G, Hendrich O, Krueger F, Andrews S, Zhang Q, Wakelam MJ, Beyer A, Reik W, Partridge L

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.

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Genome biology, 18, 1474-760X, 56, 2017

PMID: 28351387

Open Access

Autotaxin-lysophosphatidic acid receptor signalling regulates hepatitis C virus replication.
Farquhar MJ, Humphreys IS, Rudge SA, Wilson GK, Bhattacharya B, Ciaccia M, Hu K, Zhang Q, Mailly L, Reynolds GM, Aschcroft M, Balfe P, Baumert TF, Roessler S, Wakelam MJ, McKeating JA

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.

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Journal of hepatology, , 1600-0641, , 2017

PMID: 28126468

Runx1 Orchestrates Sphingolipid Metabolism and Glucocorticoid Resistance in Lymphomagenesis.
Kilbey A, Terry A, Wotton S, Borland G, Zhang Q, Mackay N, McDonald A, Bell M, Wakelam MJ, Cameron ER, Neil JC

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.

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Journal of cellular biochemistry, 118, 1097-4644, 1432-1441, 2017

PMID: 27869314

Open Access

Using lipidomics analysis to determine signalling and metabolic changes in cells.
Nguyen A, Rudge SA, Zhang Q, Wakelam MJ

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.

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Current opinion in biotechnology, 43, 1879-0429, 96-103, 2017

PMID: 27816901

The Phospholipase D2 Knock Out Mouse Has Ectopic Purkinje Cells and Suffers from Early Adult-Onset Anosmia.
Vermeren MM, Zhang Q, Smethurst E, Segonds-Pichon A, Schrewe H, Wakelam MJ

Phospholipase D2 (PLD2) is an enzyme that produces phosphatidic acid (PA), a lipid messenger molecule involved in a number of cellular events including, through its membrane curvature properties, endocytosis. The PLD2 knock out (PLD2KO) mouse has been previously reported to be protected from insult in a model of Alzheimer's disease. We have further analysed a PLD2KO mouse using mass spectrophotometry of its lipids and found significant differences in PA species throughout its brain. We have examined the expression pattern of PLD2 which allowed us to define which region of the brain to analyse for defect, notably PLD2 was not detected in glial-rich regions. The expression pattern lead us to specifically examine the mitral cells of olfactory bulbs, the Cornus Amonis (CA) regions of the hippocampus and the Purkinje cells of the cerebellum. We find that the change to longer PA species correlates with subtle architectural defect in the cerebellum, exemplified by ectopic Purkinje cells and an adult-onset deficit of olfaction. These observations draw parallels to defects in the reelin heterozygote as well as the effect of high fat diet on olfaction.

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PloS one, 11, 1932-6203, e0162814, 0

PMID: 27658289

Open Access

Exosomes bind autotaxin and act as a physiological delivery mechanism to stimulate LPA receptor signalling in cells.
Jethwa SA, Leah EJ, Zhang Q, Bright NA, Oxley D, Bootman MD, Rudge SA, Wakelam MJ

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.

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Journal of cell science, , 1477-9137, , 2016

PMID: 27557622

Open Access

C13orf31 (FAMIN) is a central regulator of immunometabolic function.
Cader MZ, Boroviak K, Zhang Q, Assadi G, Kempster SL, Sewell GW, Saveljeva S, Ashcroft JW, Clare S, Mukhopadhyay S, Brown KP, Tschurtschenthaler M, Raine T, Doe B, Chilvers ER, Griffin JL, Kaneider NC, Floto RA, D'Amato M, Bradley A, Wakelam MJ, Dougan G, Kaser A

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.

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Nature immunology, , 1529-2916, , 2016

PMID: 27478939

Inhibition of fatty acid desaturation is detrimental to cancer cell survival in metabolically compromised environments.
Peck B, Schug ZT, Zhang Q, Dankworth B, Jones DT, Smethurst E, Patel R, Mason S, Jiang M, Saunders R, Howell M, Mitter R, Spencer-Dene B, Stamp G, McGarry L, James D, Shanks E, Aboagye EO, Critchlow SE, Leung HY, Harris AL, Wakelam MJ, Gottlieb E, Schulze A

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.

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Cancer & metabolism, 4, 2049-3002, 6, 2016

PMID: 27042297

Open Access

Disallowance of Acot7 in β-Cells Is Required for Normal Glucose Tolerance and Insulin Secretion.
Martinez-Sanchez A, Pullen TJ, Chabosseau P, Zhang Q, Haythorne E, Cane MC, Nguyen-Tu MS, Sayers SR, Rutter GA

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.

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Diabetes, 65, 1939-327X, 1268-82, 2016

PMID: 26861785

Purification, characterization and crystallization of the F-ATPase from Paracoccus denitrificans.
Morales-Rios E, Watt IN, Zhang Q, Ding S, Fearnley IM, Montgomery MG, Wakelam MJ, Walker JE

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.

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Open biology, 5, 2046-2441, , 2015

PMID: 26423580

Cell cycle progression is an essential regulatory component of phospholipid metabolism and membrane homeostasis.
Sanchez-Alvarez M, Zhang Q, Finger F, Wakelam MJ, Bakal C

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.

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Open biology, 5, 2046-2441, , 2015

PMID: 26333836

Open Access

Phosphoinositide 3-kinase-related overgrowth: cellular phenotype and future therapeutic options.
Parker VE, Knox RG, Zhang Q, Wakelam MJ, Semple RK

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.

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Lancet (London, England), 385 Suppl 1, 1474-547X, S77, 2015

PMID: 26312899

Speed and sensitivity of phototransduction in Drosophila depend on degree of saturation of membrane phospholipids.
Randall AS, Liu CH, Chu B, Zhang Q, Dongre SA, Juusola M, Franze K, Wakelam MJ, Hardie RC

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.

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The Journal of neuroscience : the official journal of the Society for Neuroscience, 35, 1529-2401, 2731-46, 2015

PMID: 25673862

Open Access

Acetyl-CoA Synthetase 2 Promotes Acetate Utilization and Maintains Cancer Cell Growth under Metabolic Stress.
Schug ZT, Peck B, Jones DT, Zhang Q, Grosskurth S, Alam IS, Goodwin LM, Smethurst E, Mason S, Blyth K, McGarry L, James D, Shanks E, Kalna G, Saunders RE, Jiang M, Howell M, Lassailly F, Thin MZ, Spencer-Dene B, Stamp G, van den Broek NJ, Mackay G, Bulusu V, Kamphorst JJ, Tardito S, Strachan D, Harris AL, Aboagye EO, Critchlow SE, Wakelam MJ, Schulze A, Gottlieb E

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.

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Cancer cell, 27, 1878-3686, 57-71, 2015

PMID: 25584894

Open Access

Unexpected crosslinking and diglycation as advanced glycation end-products from glyoxal.
Lopez-Clavijo AF, Duque-Daza CA, Soulby A, Canelon IR, Barrow M, O'Connor PB

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.

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Journal of the American Society for Mass Spectrometry, 25, 1879-1123, 2125-33, 2014

PMID: 25315462

Melanoma Cells Break Down LPA to Establish Local Gradients That Drive Chemotactic Dispersal.
Muinonen-Martin AJ, Susanto O, Q Zhang, Smethurst E, Faller WJ, Veltman DM, Kalna G, Lindsay C, Bennett DC, Sansom OJ, Herd R, Jones R, Machesky LM, Wakelam MJ, Knecht DA, Insall RH

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.

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PLoS biology, 12, 1545-7885, e1001966, 2014

PMID: 25313567

Open Access

Fatty Acid Uptake and Lipid Storage Induced by HIF-1α Contribute to Cell Growth and Survival after Hypoxia-Reoxygenation.
Bensaad K, Favaro E, Lewis CA, Peck B, Lord S, Collins JM, Pinnick KE, Wigfield S, Buffa FM, Li JL, Q Zhang, MJ Wakelam, Karpe F, Schulze A, Harris AL

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. In 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 β-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.

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Cell reports, 9, 2211-1247, 349-65, 2014

PMID: 25263561

Open Access

Modulation of triglyceride and cholesterol ester synthesis impairs assembly of infectious hepatitis C virus.
Liefhebber JM,Hague CV,Zhang Q,Wakelam MJ,McLauchlan J

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.

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The Journal of biological chemistry, 289, 1083-351X, 21276-88, 2014

PMID: 24917668

Open Access

Lipid droplet formation in response to oleic acid in Huh-7 cells is mediated by the fatty acid receptor FFAR4.
Rohwedder A,Zhang Q,Rudge SA,Wakelam MJ

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.

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Journal of cell science, 127, 1477-9137, 3104-15, 2014

PMID: 24876224

Open Access