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BIOM 209/CHEM 210/PHARM 209 Sphingolipid and Sterol 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 © Sphingolipid definitions Sphingosine: a family of compounds, with the most common found in mammals being this 18-carbon amino alcohol with a trans double bond; the starting point for ceramides. Ceramide: a sphingosine molecule connected to a fatty acid by an amide bond. Ceramides are the starting point for sphingomyelin, cerebrosides and gangliosides. Sphingomyelin: a ceramide that has a phosphorylcholine head group in place of its hydroxyl. Present in most mammalian cells, and rich in myelin sheaths around nerves. E.A. DENNIS 2016 © Palmitoyl CoA + Serine = Sphingosine Serine Palmitoyl-CoA 1. Serine donates 2 carbons and an amino group 2. Reduction of the carbonyl to a hydroxyl 3. Acyl group added to convert to a dihydroceramide 4. Then, oxidation to add a double bond Look familiar? Resembles both b-oxidation and D4 desaturation N-acyl-Sphingosine (Ceramide) E.A. DENNIS 2016 © Sphingosine-1-phosphate +H N 3 Sphingosine kinase Sphingosine phosphatase • Sphingosine can be phosphorylated by sphingosine kinases, ubiquitous enzymes in the cytosol, ER and nucleus to make sphingosine-1-phosphate (S1P). • Sphingosine-1-phosphate, a lysophospholipid, acts as a potent messenger molecule that operates both intra- and inter-cellularly. • Within the cell, it promotes mitosis and inhibits apoptosis. It also regulates calcium mobilization and cell growth in response to a variety of extracellular stimuli. +H N 3 • • Outside the cell, S1P exerts many of its effects through interaction with five specific G protein-coupled receptors on cell surfaces. Different cells have different receptor profiles. S1P is vital to the function of several immune cells. It is a major regulator of T cell development, B and T cell recirculation, tissue homing patterns, and chemotactic responses to chemokines. E.A. DENNIS 2016 © Sphingosine + Fatty Acid = Ceramide Sphingosine Fatty acyl-CoA R = (CH2)n-CH3 Simple acyl transfer, but to an amide bond instead of the typical ester Ceramide E.A. DENNIS 2016 © Ceramide + Choline = Sphingomyelin Phosphocholine head group gives sphingomyelin a hydrophilic end Choline carries a positive charge the whole molecule becomes more amphoteric The phosphocholine headgroup is transferred to ceramide from PC Choline E.A. DENNIS 2016 © Comparison of Sphingomyelin and PC At least one fatty acid of PC is usually unsaturated or polyunsaturated, whereas, SM is usually saturated or mono-unsaturated; therefore, SM rich membranes are less “fluid” than typical PC-rich membranes. E.A. DENNIS 2016 © Comparison of S-1-P and LPA Sphingosine-1-phosphate (neutral zwitterion; net charge 0) +H N 3 Lysophosphatidic acid (Example: 1-myristoyl-sn-glycerophosphate) (net negative charge) E.A. DENNIS 2016 © More Definitions Galactose (polar head) Ceramide (non-polar tail) Glycosidic bond Cerebrosides: a ceramide that has a sugar added to the head group. Most commonly, the sugar is glucose (Glu) or galactose (Gal). Sialic acid Gangliosides: a ceramide that has multiple sugars including at least 1 sialic acid residue added to the head group. Increased variety and complexity. E.A. DENNIS 2016 © Ceramide + Sugar = Cerebroside Ceramide UDP-Glucose Sugar is activated by UDP UDP Addition of sugar occurs at the C1 OH group of ceramide Cerebroside (Example:glucosyl-ceramide) E.A. DENNIS 2016 © Ceramide + (Many Sugars) = Gangliosides GM1 GM2 GM3 Sugars are activated by UDP (sialic acid by CMP) Each sugar is added individually Gangliosides can have varied, complex structures They often function as antigens and surface markers Stearic acid (C18) N-acyl chain Trivia: Do you know your blood type? Is it A+? B-? O? The letters refer to the specific multi-sugar structures are attached to gangliosides and proteins on the surface of your red blood cells. E.A. DENNIS 2016 © Degradation of Sphingolipids • The amide bond of sphingolipids does not break down easily – which is why they make good membrane components • Enzymatic degradation is used for turnover – LOTS of degradation enzymes exist • it’s a long, complicated bunch of pathways • Genetic defects in these enzymes cause a long list of diseases – – – – all involve unhealthy accumulation of some sphingolipid most are rare, but more common in specific ethnicities key diseases: Gaucher’s, Tay-Sachs’, Fabry’s and Niemann-Pick Resources: (Online Mendelian Inheritance in Man) • OMIM Web site: www.ncbi.nih.gov/OMIM/searchomim.html E.A. DENNIS 2016 © Degradation of Sphingolipids GM1 Sulfatide Globoside GM1 b-galactosidase Hexosaminidase A/B GM1 Gangliosidosis Gal GM2 GM3 Metachromatic leukodystrophy GalNAc Trihexosylceramide GalNAc Fabry’s disease Lactosylceramide Ganglioside neuraminidase Galactocerebrosidase Krabbe’s disease Gal Glucocerebroside b-galactosidase NANA Sphingomyelin SO42- Galactocerebroside a-galactosidase A Hexosaminidase A Tay-Sachs disease Sandhoff’s disease Arylsulfatase A Gal Gal Ceramide Glucocerebrosidase Gaucher’s disease Glc Sphingomyelinase Phosphocholine Fatty acid + Niemann-Pick disease Sphingosine Ceramidase Farber’s disease E.A. DENNIS 2016 © Tay-Sachs’ Disease • Incidence: Like Gaucher’s but rarer – ~1:30 Ashkenazi Jews are carriers – ~1:500 carriers in general population • Symptoms:Neurodegenerative – mental retardation and seizures – listlessness, fixed gaze, hypotonia – cherry-red spot on retina (see picture) • Mechanism: Genetic – Lack of GM2 hexosaminidase A Cherry-red spot on a patient’s retina, a common finding in patients with TaySachs’ disease. • Auto recessive, OMIM #272800 – Ganglioside GM2 • Builds up in CNS • Treatments: No good therapy yet Trivia: Injections of recombinant hexosaminidase A do not help Tay-Sachs’ patients because it cannot cross the blood-brain barrier. – Supportive and symptomatic – Patients die by age 5 – Gene therapy target (future) E.A. DENNIS 2016 © Gaucher’s Disease • Incidence: Uncommon in most groups – ~1:13 Ashkenazi Jews are carriers • Symptoms: – enlarged liver and spleen (see picture) – easy bruising and bone fractures – hyperpigmentation of skin – sometimes: anemia • Mechanism: Genetic – Lack of working b-glucosidase • Auto recessive, OMIM #230800 – Glucosyl acylsphingosine • Builds up in liver, spleen & bone Magic marker outlines of the enlarged liver and spleen in a school-aged boy with Gaucher’s disease. Note also the hyperpigmented skin. • Treatments: – Recombinant acid b-glucosidase – Symptomatic support – Gene therapy target (future) E.A. DENNIS 2016 © Niemann-Pick Disease Type A • Incidence: Type A is the most severe of the 5 subtypes of Niemann-Pick Disease – ~1:90 Ashkenazi Jews are carriers • Symptoms: Neurodegenerative – Large abdomen within 3-6 mos. and jaundice – Progressive loss of early motor skills, progressive spasticity, developmental delay – Cherry red spot in the eye – (Generally) a very rapid decline leading to death by two to three years of age. • Mechanism: Genetic – Lack of Sphingomyelinase • Auto recessive, OMIM #257200 – Sphingomyelin Patient with Niemann Pick Disease • builds up in CNS, liver and lungs • Treatments: – Supportive and symptomatic – Patients die by age 3 – No effective therapy to date E.A. DENNIS 2016 © Summary of Today’s Sphingolipids Molecule(s) Synthesis Scheme Significance Sphingosine Palmitoyl CoA + Serine Brings in the amine group Important signaling molecule Ceramides Sphingosine + Fatty Acid Amide bond, hydrophobicity Important signaling molecule Sphingomyelins Ceramide + PhosphoCholine Amphoteric & charged, diseases Membrane component Cerebrosides Ceramide + (Mono)saccharides Amphoteric & neutral, diseases Rich in brain Gangliosides Ceramide + Polysaccharides + Sialic acid Complexity, diseases Rich in brain E.A. DENNIS 2016 © Lipid Biochemistry - The Big Picture Today’s Topic 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 © Why Do We Care about Cholesterol? Heart Disease – #1 killer in US – largely preventable – strongly linked to cholesterol – overall deaths linked as well (see graph) Figure: Levine, NEJM, 332, 512-21 (1995). E.A. DENNIS 2016 © Cholesterol Structure and Numbering E.A. DENNIS 2016 © Cholesterol Stereochemistry Stereochemical diagram of sterol nucleus E.A. DENNIS 2016 © Cholesterol Synthesis in a Nutshell 3 acetyl CoA’s HMG-CoA HMG-CoA reductase Mevalonate Isopentyl Pyrophosphate Squalene Cholesterol E.A. DENNIS 2016 © The Business End of Cholesterol Synthesis thiolase HMG-CoA synthase Regulated Step! HMG-CoA reductase E.A. DENNIS 2016 © The Later Steps in Cholesterol Synthesis Isoprene group • Steps are irreversible after isopentenyl pyrophosphate • Starting with squalene, everything is hydrophobic – carrier proteins! Isopentyl Pyrophosphate Squalene Squalene E.A. DENNIS 2016 © Cholesteryl Esters - ACAT Style Cholesterol Acyl-CoA-cholesterol acyl transferase (ACAT) Fatty Acyl-CoA CoA-SH • ACAT = “acyl CoA cholesterol acyl transferase” • It acts mainly inside cells • Donor acyl comes from a free fatty acyl CoA Cholesteryl ester E.A. DENNIS 2016 © Cholesteryl Esters - LCAT Style Cholesterol Phosphatidylcholine (lecithin) Lecitincholesterol acyltransferase (LCAT) Cholesterol Ester Lyso-phosphatidylcholine (lysolecithin) • LCAT = “lecithin-cholesterol acyl transferase” • Two-thirds of plasma cholesterol is esterfied by LCAT • Acyl chain is donated from sn-2 position of phosphatidylcholine E.A. DENNIS 2016 © Bile Acids and Bile Salts Cholesterol 7a-hydroxylase Choline Cholic Acid • Made in the Liver (where the cholesterol is being made) • Made directly from cholesterol • Secreted into the bile ducts and GI tract • Reabsorbed and recycled Cholic Acid – a bile acid Glycine Glycocholic Acid – a bile salt E.A. DENNIS 2016 © Steroid Hormone Synthesis • A wide range of hormones are made from cholesterol – – – – Figure: Lehninger AL, Nelson DL, Cox MM (1993), Principles of Biochemistry, 2 nd ed. Worth Publishers, Inc. estrogen testosterone cortisol aldosterone E.A. DENNIS 2016 © Regulation of Cholesterol Synthesis • HMG-CoA reductase is the regulated enzyme • Inhibitors – Glucagon – XOL (negative feedback) • Promotor: Insulin • Intracellular synthesized cholesterol downregulates LDL receptors so cell takes up less extracellular LDL cholesterol Figure: Lehninger AL, Nelson DL, Cox MM (1993), Principles of Biochemistry, 2 nd ed. Worth Publishers, Inc. E.A. DENNIS 2016 © Removal of Bile Acids Upregulates 7α-Hydroxylase Cholesterol 7a-hydroxylase Choline • Net result: decrease in intracellular cholesterol in liver • Causes upregulation of LDL receptors so increases removal of circulating LDL • Results in overall lowering of plasma cholesterol Cholic Acid E.A. DENNIS 2016 © Statin-Class Drugs (Zocor) • Lovastatin was first • They inhibit HMG-CoA reductase • Decreases intracellular cholesterol and upregulates LDL receptors • Mimic HMG-CoA; competitive inhibitors • Very effective at lowering cholesterol – 25-40% drop is common • Widely prescribed – Currently, 30% of people over 65 years use a statin. This is up from 12% in 1997. E.A. DENNIS 2016 © Sterol Regulated Promoters SRE (Sterol regulatory element) SREBP (SRE binding protein) Figure: Brown, Nature, 343, 425-30 (1990 ). E.A. DENNIS 2016 © Proteolytic Release of SREBPs and Role of SCAP SCAP: SREBP cleavage activating protein Figure: Horton, JCI 109:1125-31 (2002) E.A. DENNIS 2016© Regulation of Cellular Sterol Content • HMG CoA reductase is controlled in several ways: – The sterol regulated element binding protein (SREBP) controls the rate of synthesis of HMG CoA reductase RNA. This transcription factor binds to the Sterol regulatory element -1 (SRE-1), a short DNA sequence on the 5’ side of the gene. When inactive, it is in the ER associated with SCAP (SREBP cleavage activating protein), a cholesterol sensor. When the cholesterol level falls, SCAP escorts SREBP into small membrane vesicles in the Golgi, and it is released via two cleavages. Then, it migrates to the nucleus and binds SRE to enhance transcription. As the cholesterol level rises, cleavage is blocked and SREBP in the nucleus is degraded, halting transcription. – Translation of HMG CoA reductase mRNA is inhibited by nonsterol metabolites derived from mevalonate and dietary cholesterol. – Degradation of HMG CoA reductase is strictly controlled. – Phosphorylation decreases the activity of the reductase. E.A. DENNIS 2016 © SREBP Regulated Promoters: Different Architecture, Distinct “Co-regulators” LDL Receptor 5’ - - 3’ HMG CoA Synthase 5’ - - 3’ HMG CoA Reductase 5’ - - 3’ Farnesyl Diphosphate Synthase 5’ - - 3’ Squalene Synthase 5’ - - 3’ Acetyl CoA Carboxylase 5’ - - 3’ Fatty Acid Synthase 5’ - - 3’ SREBP-2 5’ - - 3’ SREBPs = Sp1 = NF-Y/CBF = CREB/ATF = E.A. DENNIS 2016 © Role of SREBPs in Global Regulation of Lipid Metabolism SREBP1a works equally at all promoters Figure: Horton, JCI 109:1125-31 (2002) E.A. DENNIS 2016 © Summary: Regulation of Cellular Sterol Content • Sterol control of transcription affects more than 30 genes involved in the biosynthesis of cholesterol, triacylglycerols, phospholipids and fatty acids. • The regulation of these events is primarily due to sterol-regulated transcription of key rate limiting enzymes and by the regulated degradation of HMG CoA reductase. • Activation of transcriptional control occurs via the cleavage of the membrane-bound transcription factor sterol regulated element binding protein (SREBP). • Sterol regulatory element -1 (SRE-1) is in a gene that is required for transcriptional control. E.A. DENNIS 2016 ©