1. 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 ©

2. 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 ©

3. Palmitoyl CoA + Serine = Sphingosine Palmitoyl-CoA Serine E.A. DENNIS 2016 © N-acyl-Sphingosine (Ceramide)

4. Sphingosine-1-phosphate 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. 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 kinase Sphingosine phosphatase +H3N+H3N +H3N+H3N

5. Sphingosine + Fatty Acid = Ceramide Simple acyl transfer, but to an amide bond instead of the typical ester E.A. DENNIS 2016 © Sphingosine Fatty acyl-CoA Ceramide R = (CH 2 ) n -CH 3

6. 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 ©

7. Comparison of Sphingomyelin and PC E.A. DENNIS 2016 © 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.

8. Comparison of S-1-P and LPA E.A. DENNIS 2016 © Sphingosine-1-phosphate (neutral zwitterion; net charge 0) Lysophosphatidic acid (Example: 1-myristoyl-sn-glycerophosphate) +H3N+H3N (net negative charge)

9. More Definitions Ceramide (non-polar tail) Galactose (polar head) Glycosidic bond Cerebrosides: a ceramide that has a sugar added to the head group. Most commonly, the sugar is glucose (Glu) or galactose (Gal). Gangliosides: a ceramide that has multiple sugars including at least 1 sialic acid residue added to the head group. Increased variety and complexity. Sialic acid E.A. DENNIS 2016 ©

10. Ceramide + Sugar = Cerebroside Sugar is activated by UDP Addition of sugar occurs at the C1 OH group of ceramide Ceramide Cerebroside ( Example : glucosyl-ceramide ) E.A. DENNIS 2016 © UDP-Glucose UDP

11. Ceramide + (Many Sugars) = Gangliosides 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. Gangliosides can have varied, complex structures They often function as antigens and surface markers Sugars are activated by UDP (sialic acid by CMP) Each sugar is added individually GM 1 GM 2 GM 3 Stearic acid (C18) N-acyl chain E.A. DENNIS 2016 ©

12. 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 ©

13. GM 1 GM 2 GM 3 GM 1 Gangliosidosis GM 1  -galactosidase Tay-Sachs disease Hexosaminidase A Sandhoff’s disease Hexosaminidase A/B Fabry’s disease  -galactosidase A Globoside Trihexosylceramide LactosylceramideGlucocerebroside Galactocerebrosidase Ganglioside neuraminidase  -galactosidase Ceramide Glucocerebrosidase Gaucher’s disease Arylsulfatase A Krabbe’s disease Metachromatic leukodystrophy Galactocerebroside Sulfatide Sphingosine Ceramidase Farber’s disease Niemann-Pick disease Sphingomyelinase Sphingomyelin Fatty acid + Gal GalNAc NANAGal GalNAc Gal SO 4 2- Gal Glc Phosphocholine Degradation of Sphingolipids E.A. DENNIS 2016 ©

14. 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 GM 2 hexosaminidase A Auto recessive, OMIM #272800 –Ganglioside GM 2 Builds up in CNS Treatments: No good therapy yet –Supportive and symptomatic –Patients die by age 5 –Gene therapy target (future) Cherry-red spot on a patient’s retina, a common finding in patients with Tay- Sachs’ disease. Trivia: Injections of recombinant hexosaminidase A do not help Tay-Sachs’ patients because it cannot cross the blood-brain barrier. E.A. DENNIS 2016 ©

15. 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  -glucosidase Auto recessive, OMIM #230800 –Glucosyl acylsphingosine Builds up in liver, spleen & bone Treatments: –Recombinant acid  -glucosidase –Symptomatic support –Gene therapy target (future) Magic marker outlines of the enlarged liver and spleen in a school-aged boy with Gaucher’s disease. Note also the hyperpigmented skin. E.A. DENNIS 2016 ©

16. 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 builds up in CNS, liver and lungs Treatments: –Supportive and symptomatic –Patients die by age 3 –No effective therapy to date Patient with Niemann Pick Disease E.A. DENNIS 2016 ©

17. Summary of Today’s Sphingolipids Molecule(s)Synthesis SchemeSignificance SphingosinePalmitoyl CoA + SerineBrings in the amine group Important signaling molecule CeramidesSphingosine + Fatty AcidAmide bond, hydrophobicity Important signaling molecule SphingomyelinsCeramide + PhosphoCholineAmphoteric & charged, diseases Membrane component CerebrosidesCeramide + (Mono)saccharidesAmphoteric & neutral, diseases Rich in brain GangliosidesCeramide + PolysaccharidesComplexity, diseases + Sialic acidRich in brain E.A. DENNIS 2016 ©

18. Lipid Biochemistry - The Big Picture Today’sTopic E.A. DENNIS 2016 © Figure: Voet, D, Voet JG, Pratt CW (2006), Fundamentals of Biochemistry: Life at the Molecular Level, 2 nd ed. Reprinted with permission of John Wiley & Sons, Inc.

19. 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 ©

20. Cholesterol Structure and Numbering E.A. DENNIS 2016 ©

21. Cholesterol Stereochemistry E.A. DENNIS 2016 © Stereochemical diagram of sterol nucleus

22. Cholesterol Synthesis in a Nutshell 3 acetyl CoA’s HMG-CoA Mevalonate Isopentyl Pyrophosphate Squalene Cholesterol HMG-CoAreductase E.A. DENNIS 2016 ©

23. The Business End of Cholesterol Synthesis RegulatedStep! E.A. DENNIS 2016 © HMG-CoA reductase HMG-CoA synthase thiolase

24. The Later Steps in Cholesterol Synthesis Isopentyl Pyrophosphate Squalene Isoprene group E.A. DENNIS 2016 ©

25. Cholesteryl Esters - ACAT Style ACAT = “acyl CoA cholesterol acyl transferase” It acts mainly inside cells Donor acyl comes from a free fatty acyl CoA Cholesterol Cholesteryl ester Fatty Acyl-CoA CoA-SH Acyl-CoA-cholesterol acyl transferase (ACAT) E.A. DENNIS 2016 ©

26. Cholesteryl Esters - LCAT Style 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 © Lecitin- cholesterol acyltransferase (LCAT) Cholesterol Cholesterol Ester Phosphatidylcholine (lecithin) Lyso-phosphatidylcholine (lyso- lecithin)

27. Bile Acids and Bile Salts 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 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 Cholesterol 7  -hydroxylase Choline Cholic Acid Cholic Acid – a bile acid Glycocholic Acid – a bile salt Glycine E.A. DENNIS 2016 ©

28. Steroid Hormone Synthesis A wide range of hormones are made from cholesterol –estrogen –testosterone –cortisol –aldosterone E.A. DENNIS 2016 © Figure: Lehninger AL, Nelson DL, Cox MM (1993), Principles of Biochemistry, 2 nd ed. Worth Publishers, Inc.

29. 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 E.A. DENNIS 2016 © Figure: Lehninger AL, Nelson DL, Cox MM (1993), Principles of Biochemistry, 2 nd ed. Worth Publishers, Inc.

30. Removal of Bile Acids Upregulates 7α-Hydroxylase Cholesterol 7  -hydroxylase Choline Cholic Acid E.A. DENNIS 2016 ©

31. Statin-Class Drugs 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. ( Zocor) E.A. DENNIS 2016 ©

32. Sterol Regulated Promoters SRE (Sterol regulatory element) SREBP (SRE binding protein) E.A. DENNIS 2016 © Figure: Brown, Nature, 343, 425-30 (1990 ).

33. Proteolytic Release of SREBPs and Role of SCAP SCAP: SREBP cleavage activating protein E.A. DENNIS 2016 © Figure: Horton, JCI 109:1125-31 (2002)

34. 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 ©

35. SREBP Regulated Promoters: Different Architecture, Distinct “Co-regulators”

36. Role of SREBPs in Global Regulation of Lipid Metabolism SREBP1a works equally at all promoters E.A. DENNIS 2016 © Figure: Horton, JCI 109:1125-31 (2002)

37. 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 ©