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BIOM 209/CHEM 210/PHARM 209 Lipid Cell Signaling Genomics, Proteomics and Metabolomics January 5, 2016 Professor Edward A. Dennis Department of Chemistry and Biochemistry Department of Pharmacology, School of Medicine University of California, San Diego Copyright/attribution notice: You are free to copy, distribute, adapt and transmit this tutorial or individual slides (without alteration) for academic, non-profit and non-commercial purposes. Attribution: Edward A. Dennis (2010) “LIPID MAPS Lipid Metabolomics Tutorial” www.lipidmaps.org E.A. DENNIS 2016 © OMICS OVERVIEW: GENOMICS/PROTEOMICS/METABOLOMICS OF LIPID METABOLISM AND CELL SIGNALING AND IMPLICATIONS FOR HUMAN DISEASE PROFILING AND BIOMARKER DISCOVERY A. B. C. D. LIPID MAPS Initiative in Lipidomics Human Plasma Lipidome Eicosadomics of Macrophages Genomics and Proteomics Integration LIPID MAPS Lipid Metabolites And Pathways Strategy GENOMICS (TRANSCRIPTOMICS) 4 base side chains ~25,000 coding genes PROTEOMICS 20 amino acid side chains ~30,000 in databases METABOLOMICS nucleic acids, amino acids, sugars and fats: >105 ~42,000 in databases “LIPIDOMICS” Dennis (2009) Lipidomics Joins the Omics Evolution, PNAS, 106, 2089. 5 all the fats: >10 ~40,000 in LM database Number of Citations Increases in Omics Citations 1985-2009 Wenk MR (2010) Lipidomics: New Tools and Applications. Cell, 143, 888-895. Lipidomics Publications 2002-2015 Human Plasma Lipidomics NIST collected (pooled) fasting plasma from 100 individuals 50% female and 50% male; age 40-50 15% of the total taken from individuals of Hispanic origin Human Plasma Metabolites (mg/dL) Lipids Nucleic Acids Amino Acids Sugars Human Plasma Lipid Categories (M) Sterol Lipids Sphingolipids Fatty Acyls Glycerolipids Glycerophospholipids Prenols Lipid categories and Species in the Human Plasma *SRM Number of Species Sum (nmol/ml) Sum (mg/dl) Fatty Acyls 107 214 5.8 Glycerolipids 74 1110 93.7 Glycerophospholipids 160 2590 200 Sphingolipids 204 318 23.7 Sterol Lipids 35 3174 123 Prenol Lipids 8 5 3.7 588 7411 449.9 Lipid Category Total *SRM = standard reference material J Lipid Res 51, 3299-3305 (2010) SRM: Prostaglandins, Isoprostanes 3.0 2.5 pmol/ml 2.0 1.5 1.0 0.5 0.0 SRM: Sterols 8 7 nmol/ml 6 5 4 3 2 1 0 Cholesterol 3.8 umol/ml SRM: Cholesteryl Esters 2500 nmol/ml 2000 1500 1000 500 0 0 32:1 34:0 34:1 34:1p 34:2 34:2p 36:0 36:1 36:2 36:2e/36:1p 36:3 36:3e/36:2p 36:4 36:4e/36:3p 36:5 36:5e/36:4p 38:1 38:2 38:3 38:4 38:5 38:5e/38:4p 38:6 38:6e/38:5p 40:1 40:4 40:5 40:5e 40:6 40:6e 40:7 40:7e 42:1 42:5 42:5p 42:6 42:6p 42:7 nmol/ml SRM: Phosphatidylethanolamines 60 50 40 30 20 10 Human Plasma Lipid Diversity J Lipid Res, 51, 3299-3305 (2010) Plasma Lipids in the Metabolic Syndrome Quehenberger & Dennis, New Eng. J. Medicine, 365, 1812-23 (2011) Implications of Lipidomics for the Future of Clinical Medicine • Identification of metabolites in human plasma and other tissues for diagnostic purposes • Discovery of novel metabolites as biomarkers for disease states • Quantitation of metabolites which permit dynamic monitoring of disease pathophysiology over time • Evaluation of the efficacy of pharmacotherapeutic agents targeted to specific diseases which affect lipid metabolic pathways (statins) LIPID MAPS NIGMS Large Scale Collaborative Grant “Glue Grant” [NIH U54 GM 69338] Mouse Macrophage: Environmental Agonist: HPLC/Mass Spectrometer: Synthesis/Characterize: Bioinformatics: Website: RAW cell line & primary cell initially LPS, then oxidized LDL identify known & new, quantify new lipids, MS quantitative standards informatics and lipid networks LIPID MAPS -- Nature Lipidomics Gateway, http://www.lipidmaps.org LIPID MAPS TM “CLASS”: Comprehensive Lipidomics Analysis using Separation Simplification cells or tissues probe cells tissues a “divide-and-conquer” strategy medium sonicate homogenate category specific internal standards (deuterated, odd-chain carbon) extraction category optimized (liq-liq, SPE) extract category specific GC LC (GC, NP-HPLC, RP-HPLC, chiral, specialty) mass spectrometer Harkewicz & Dennis, “Applications of mass spectrometry to lipids and membranes” Ann Rev Biochem, 80: 301-325 (2011) (variables) 1. Mass spectrometer types 2. Ionization mode 3. Additives (for ionization) 4. Mass spectrometer monitoring modes Kdo2-Lipid A (KLA, LPS subspecies) A specific agonist of TLR-4 on RAW 264.7 macrophages Raetz et al., 2006, J. Lipid Res. 47: 1097 Nuclei – DAPI Mitochondria – MitoTracker Red O-Specific chain Polysaccharide Core Lipid A Glycophospholipid Kdo Dennis et al (2010) J. Biol. Chem, 51, 39976-85 Phospholipase A2 (PLA2) Function in Arachidonic Acid Release PLA2 AA FA PX HO FA PX Arachidonic Acid Aspirin NSAID Cyclooxygenase Prostaglandins Lipoxygenase Leukotrienes Dennis et al (2011) Chem Rev, 111, 6130-85 Eicosanoid Signaling Pathways in RAW264.7 Macrophage ATP ATP LPS (KLA) Numerous eicosanoid metabolites Buczynski et. al. (2007) JBC, 282, 22834 Basic Macrophage Experimental Scheme Eicosanoid enzyme mRNA & protein Macrophages ± Kdo2 Lipid A (TLR4) ± 2 mM ATP (P2X7) Solid Phase Extraction & LC-MS/MS LC-MS/MS Isolate Media & Solid Phase Extraction (Eicosanoids) Cellular Eicosanoid Metabolism Buczynski, Dumlao, Dennis (2009) JLR, 50, 1015-38 Cellular Eicosanoid Metabolism COX CYP(EET) CYP(ω) 12-LOX 5-LOX 15-LOX Buczynski, Dumlao, Dennis (2009) JLR, 50, 1015-38 Cellular eicosanoid metabolism CYP(w) COX CYP(EET) 15-LOX 12-LOX Buczynski, Dumlao, Dennis, 2009, JLR, 50: 1015-38 5-LOX Cellular eicosanoid metabolism COX KLA / RAW Macrophage: Time-course Eicosanoid (ng / 1x106 cells) 80 AA 60 3 40 2 20 1 0 0 0.5 Eicosanoid (ng / 1x106 cells) 4 11-HETE 0.4 0.3 0.2 PGF2a 60 PGJ2 50 15 PGE2 250 12 200 9 150 6 100 3 50 0 0 60 15D PGD2 50 40 40 30 30 20 20 0.1 10 10 0.0 0 0 0.5 0.5 0.5 PGD2 80 15D PGJ2 60 40 20 0 Eicosanoid (ng / 1x106 cells) 0 5-HETE LTC4 11t LTC4 4 8 12 16 20 24 Time (hr) 0.4 0.4 0.4 Extracellular 0.3 0.3 0.3 Intracellular 0.2 0.2 0.2 0.1 0.1 0.1 0.0 0.0 0.0 0 4 8 12 16 20 24 Time (hr) 0 4 8 12 16 20 24 Time (hr) PGD2 + metabolites (Extracellular) 0 4 8 12 16 20 24 Time (hr) Buczynski et al (2007) JBC, 282, 22834 Fluxomics: KLA / RAW Macrophage Time-course Gupta, Maurya, Stephens, Dennis, Subramaniam (2009) Biophys J, 96, 4542 Prediction of Eicosanoid Fluxes in TLR4-Primed/Purinergic-Stimulated BMDM COX pathway LO pathway www.lipidmaps.org Kihara et al, (2014) Biophys J, 106, 966-975 Good fit! Macrophage Phenotypes Resident Peritoneal (RPM) Thioglycolate-Elicited Peritoneal (TGEM) Sterile Inflammation Bone Marrow-Derived (BMDM) RAW264.7 Cell Line (RAW) Tumor M-CSF Ab-MuLV Eicosanoid Changes by Phenotype 8 Hr Fold Increase PGI2 PGE2 PGD2 TxB2 Fold Decrease Not detected COX-2 Metabolites and Transcripts PGE2/PGD2 vs PGES/PGDS Transcripts Proportionality of Eicosanoids and Transcripts RPM TGEM J. Leukocyte Biology, 90, 563-574 (2011) BMDM RAW Directed Proteomics on Enzymes of Eicosanoid Biosynthesis FPR1 FPR2 THIKA DHB4 NLTP ECHP PROSTAGLANDIN CATABOLIC PATHWAYS PE2R1 GPR44 PE2R2 PPARD PE2R3 PD2R ACOX1 PPARA PTGR2 PE2R4 ACOX2 PPARG PF2R PI2R PTGIS CYCLOOXYGENASE PATHWAY THAS PTGDS CBR1 PTGD2 CAO93 PTGR1 ACOX3 HYES PTGES TA2R PGES2 PGH2 CP4AA CP4FE Q8QZW 4 CP254 CP4CA CP255 Q924D 1 Q6IEF7 CP4F3 B0G0Y 1 CP2BJ CP238 CP239 CP240 CP2CT CP2J5 CP4AE CY250 PA24A CP237 PGH1 TEBP CP1B1 Arachidonic Acid PA2G5 PA2GA AL5AP PA2GX LOX5 GGT1 LTC4S GGT5 MGST3 LKHA4 DPEP1 DPEP2 5-LIPOXYGENASE PATHWAY A2RST1 LX12B CYTOCHROM P450 AHR LX12L LOX12 CBR1 LX15B LOXE3 PGDH RECEPTORS CLTR2 LT4R1 CLTR1 LX12E LT4R2 12-LIPOXYGENASE PATHWAY 15-LIPOXYGENASE PATHWAY ENZYMES Sabido, Quehenberger Dennis, Aebersold (2012) Mol Cell Proteomics 11: M111.014746 Protein Abundance after KLA Stimulation Eicosanoid Genes to Proteins to Metabolites Genes Proteins Eico Eicosanoid Metabolites Quehenberger & Dennis, New Eng. J. Medicine, 365, 1812-23 (2011) THE NEED FOR “METABOLOMICS” “Premiums in the shape of sensational discoveries may be hoped for, but cannot be assured even to the greatest genius. But what has to penetrate, relative to this question, more completely into the consciousness of pathologists, is this, that to understand zymoses, to be able to counteract them by rational, as distinguished from empirical or accidentally discovered means, is only possible by the aid of a complete knowledge of the chemical constitution of all the tissues, organs and juices of the body, and of all their possible products.” Johann Ludwig Wilhelm Thudichum (1829-1901) A Treatise on the Chemical Constitution of the Brain (1884) Quoted from [Joseph Needham (1971) The Chemistry of Life, Cambridge Univ Press p. 199] LIPID MAPS TM