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BIOM 209/CHEM 210/PHARM 209 Glycerolipid and Phospholipid Metabolism, Signaling and Lipidomics 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 © Types of Biological Lipids • Lipids derived from ketoacyl units – Fatty acids (inc. Prostaglandins and wax esters) – Glycerolipids – Glycerophospholipids – Sphingolipids – Saccharolipids – Polyketides • Lipids derived from isoprene units – Sterols – Prenols (inc. terpenes and Fat Soluble Vitamins) • Mixtures – Lipoproteins E.A. DENNIS 2016 © Definitions Glycerophospholipid = an amphipathic lipid in which two fatty acyl groups are attached to a glycerol-3-phosphate whose phosphate group is linked to a polar group X=head group Usually a saturated FA Phosphatidic acid = the simplest glycerophospholipid -the precursor to other phospholipids and to triacylglycerols. Also called diacylglycerol-3-phosphate. Usually an unsaturated FA Triacylglycerol = a lipid in which three fatty acids are esterified by a glycerol backbone. It is the major form of energy storage in humans. Also called a triglyceride. E.A. DENNIS 2016 © Glycerol-3-Phosphate Synthesis • sn-glycerol-3-phosphate is the backbone of triglycerides and phospholipids • sn-glycerol-3-phosphate has the R configuration at C2 Backbone of phospholipids and triglycerides E.A. DENNIS 2016 © Biosynthesis of Phosphatidic Acid • Precursors – Fatty acids – sn-glycerol-3-phosphate • sn-glycerol-3-phosphate is produced from the – Reduction of DHAP by glycerol phosphate dehydrogenase OR – Phosphorylation of glycerol by glycerol kinase and ATP • Acyl transferases perform two successive esterifications with fatty acyl Co A to generate phosphatidic acid E.A. DENNIS 2016 © Biosynthesis of Triacylglycerol • Phosphatidic acid phosphatase removes the phosphate producing 1,2Diacylglycerol • An acyl transferase transfers an acyl CoA to position 3. E.A. DENNIS 2016 © Biosynthesis of Glycerophospholipids glycerol phosphodiester • Glycerophospholipids (or phospholipids) can be made from – Phosphatidic acid OR – Diacylglycerol • There are many different head groups which can be linked to the C3 of glycerol by a phosphodiester bond • Cytidine triphosphate (CTP) provides the synthetic energy in the synthesis of all PLs E.A. DENNIS 2016 © Strategies for Phospholipid Synthesis Strategy 1: Headgroup activated with CDP Strategy 1: The polar head group is activated before being attached to the lipid – Used during the synthesis of PE and PC Strategy 2: The hydrophobic tail of diacylglycerol is activated rather than the polar head group – Used during the synthesis of PI and PG Strategy 2: Diacylglycerol activated with CDP E.A. DENNIS 2016 © De Novo Synthesis of Phosphatidylcholine (PC) • PC is the most abundant phospholipid in eukaryotic cells • PC is also known as lecithin De Novo Synthesis • Choline is phosphorylated • Cytidyltransferase makes CDPcholine • C3 OH groups of DAG attacks the phosphoryl groups of the activated CDP-choline displacing CMP and yielding the glycerophospholipid E.A. DENNIS 2016 © De Novo Synthesis of PE • PE is the second most abundant phospholipid in eukaryotic cells De Novo Synthesis • Ethanolamine is phosphorylated • Cytidyltransferase makes CDP-ethanolamine • C3 OH groups of DAG attacks the phosphoryl groups of the activated CDP-ethanolamine or displacing CMP and yielding the glycerophospholipid E.A. DENNIS 2016 © Synthesis of PS Phosphatidylserine (PS) is synthesized from PE by a head group exchange Phosphatidylserine synthase 2 Bacteria can make PS de novo because they have a PS synthase which adds serine to diacylglycerol(Strategy 1 mechanism) Mammals do not make PS de novo because they lack this type of PS synthase E.A. DENNIS 2016 © Interconversion of PS, PE and PC • PS decarboxylase in the mitochondria can convert PS to PE – Bacteria can do this too! • A calcium-activated transferase can exchange ethanolamine for the serine of PS – This reaction occurs in the ER and Golgi • In mammals, PE can undergo 3 successive methylations to yield PC – This reaction occurs in the ER of liver – S-adenosylmethionine is the methyl donor E.A. DENNIS 2016 © De Novo Synthesis of PI Synthesis of PI Phosphatidic acid attacks the phosphoryl group of CTP to form activated CDP-diacylglycerol • Inositol attacks CDPdiacylglycerol E.A. DENNIS 2016 © Phosphatidylinositol Phosphorylation These OH groups can also be esterified with PO32Phosphatidylinositol (PI) • PI can be phosphorylated to different degrees • PIP2 = phosphatidylinositol 4,5-bisphosphate is very important in signal transduction – When a receptor G protein is activated it can mediate the cleavage of PIP2 to DG and IP3 – DG activates protein kinase C which adds phosphates to certain proteins – IP3 mobilizes intracellular Ca and activates certain cell processes E.A. DENNIS 2016 © De Novo Synthesis of PG Synthesis of PG • The C1 OH group of glycerol3-phosphate attacks CDPdiacylglycerol • The phosphoryl group is hydrolyzed to form PG E.A. DENNIS 2016 © Cardiolipin • First isolated from heart tissue • Many autoimmune diseases, such as lupus, are associated with anti-cardiolipin antibodies for unknown reasons • Formed by the condensation of two molecules of PG with the elimination of one molecule of glycerol E.A. DENNIS 2016 © Plasmalogens • About 20% of eukaryotic glycerophospholipids are plasmalogens. They are found in varying amounts in different tissues. • Plasmalogens contain a hydrocarbon chain linked to glycerol C1 by a vinyl ether linkage. E.A. DENNIS 2016 © Major Components of Membranes • Proteins – – – – Channels & Pumps Structural proteins Receptors Reaction enzymes • Lipids – Sterols (Cholesterol) – Glycerophospholipids – Glycerolipids (neutral lipids) – “Surfactant” – Sphingolipids E.A. DENNIS 2016 © Lipid Bilayers • In aqueous solution, amphiphilic molecules form micelles to eliminate the contact of the hydrophobic tails with water, but allow the polar heads to be in contact with it. • The diameter of the micelle depends upon the length of the tail. • A suspension of PLs can form liposomes, closed, self-sealing solvent-filled vesicles, that have only a single bilayer. • Liposomes serve as models of biological membranes. Figures: Voet, D, Voet JG, Pratt CW (2006), Fundamentals of Biochemistry: Life at the Molecular Level, 2nd ed. Reprinted with permission of John Wiley & Sons, Inc. E.A. DENNIS 2016 © Movement in Bilayers Key Principle: Things on one side tend to stay on that side... …unless specifically moved by a carrier protein called a “flippase.” Some Implications: • Lipid populations on each side can be different • Receptors aim out • Embedded/anchored enzymes localize reactions to only one side • Ion pumps move the same ions the same direction • Etc. Figure: Voet, D, Voet JG, Pratt CW (2006), Fundamentals of Biochemistry: Life at the Molecular Level, 2nd ed. Reprinted with permission of John Wiley & Sons, Inc. E.A. DENNIS 2016 © DPPC = Surfactant • Dipalmitoyl phosphatidylcholine (DPPC) • Main function: reduces surface tension at the alveoli-air interface (increases compliance) E.A. DENNIS 2016 © Diagnostics: the L/S Ratio • Phosphatidylcholine (PC) is produced by type II alveolar epithelial cells in the fetal lung. During gestation it is found in the amniotic fluid. PC 22 18 Amniotic Fluid14 Concentration (mg/dl) 10 • Sphingomyelin remains at a low level throughout gestation. It can be used as a baseline for comparison • The L/S ratio is close to 1:1 until the 30-35th week of gestation. At this time, lecithin content increases dramatically attaining a ratio of greater than 2:1. This indicates pulmonary maturity. Sphingomyelin 6 2 18 22 26 30 34 38 Term Gestation (weeks) • This test is very accurate in determining the absence of RDS; but patients with a ratio <2:1 may not have RDS. E.A. DENNIS 2016 © Digestion of Fats Figure: Lehninger AL, Nelson DL, Cox MM (1993), Principles of Biochemistry, 2nd ed. Worth Publishers, Inc. E.A. DENNIS 2016 © Bile Acids/Salts R2 = OH R2 = NH – CH2 – COOH R2 = NH – CH2 – CH2 – SO3H R1 = OH Cholic acid Glycocholic acid Taurocholic acid R1 = H Chenodeoxycholic acid Glycochenodeoxycholic acid Taurochenodeoxycholic acid • Bile salts act as detergents in the digestive tract to emulsify triglycerides and phospholipids into micelles. Bile salts are a highly oxidized form of cholesterol. E.A. DENNIS 2016 © Micelles • The hydrophobic surface of the bile salt associates with TAGs, and a number of these aggregate to form a micelle. • This allows the association of pancreatic lipase which liberates free fatty acids in a smaller micelle which can be absorbed through the mucosa. E.A. DENNIS 2016 © Definitions • Lipases or Acyl Hydrolases – Triacylglycerol Lipase – the general term. – Lingual Lipase- found in saliva for pre-digestion. – Pancreatic Lipase- produced by the pancreas for digestion. – Lipoprotein lipase- found on capillary endothelial cells. It hydrolyzes TAG in chylomicrons and VLDLs. – Hormone Sensitive Lipase- generates FAs when energy is needed. Found in adipose tissue. E.A. DENNIS 2016 © Definitions • Pancreatic Lipase – Only removes FAs from the 1 and 3 positions – Single polypeptide chain of 48,000 MW – Requires colipase, a 10,000 MW cofactor, which helps the association of pancreatic lipase with the lipid/water interface E.A. DENNIS 2016 © Lineweaver-Burk Plot of Lipase Activity • When TAG is mixed with water, it forms two layers. • When the water and TAG are shaken, they mix together forming microemulsions. • The finer the emulsion, the greater the activity of the enzyme due to increased surface area • 1/v versus surface area is the same for both Lipase Action Coarse emulsion Fine emulsion 1/v 1/[S] Fine Coarse 1/v 1/[Surface Area] E.A. DENNIS 2016 © Phospholipase Sites of Action 1-palmitoyl, 2-oleoyl-phosphatidylinositol- 4’,5’ bisphosphate (PIP2) E.A. DENNIS 2016 © More Definitions • Pancreatic Phospholipase A2 – Removes FAs from the 2 position – 124 aa single polypeptide chain of 14,000 MW – Produced as a zymogen. Trypsin hydrolyzes a peptide bond at position 7 of the zymogen to make the active enzyme. E.A. DENNIS 2016 © Lipases and Phospholipases Lipases are unique because their substrates are lipids, not small molecules. • At low concentrations, DiC7PC forms monomers • At higher concentrations, it forms micelles. • The concentration at which micelles form is called the critical micelle concentration (CMC). DiC7PC PLA2 on DiC7PC Mixed Micelles with detergent Micelles V Figure: Voet, D, Voet JG, Pratt CW (2006), Fundamentals of Biochemistry: Life at the Molecular Level, 2nd ed. Reprinted with permission of John Wiley & Sons, Inc. Monomers [S] CMC E.A. DENNIS 2016 © New Thematic Review Series on Phospholipases in Lipid Signaling and Disease