Lecture_36_37_Raetz - Chris Raetz Department of...

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Unformatted text preview: Chris Raetz Department of Biochemistry Sands Building Room 269 684-3384 [email protected] Biochemistry 227 December 1 and 3, 2010 Biosynthesis of Prenols and Sterols Lehninger (5th Edition) Ch. 21 pp. 831-845 For discussion sections: Endo et al. FEBS Letters 72, 323-26, 1976 1 Eukaryotic Lipid Biochemistry - The Big Picture Sterols Prenols 2 Importance of Cholesterol and Related Molecules A unique class of lipids found in all eucaryotes • Rigid small molecules with multiple asymmetric centers (m.w. ~ 400) Rigid • Very hydrophobic with limited water solubility Very • Enriched in plasma membranes (~ 109 molecules/cell) Enriched • Required for eucaryotic growth but absent in bacteria and arachaea Required • Many metabolites exist with diverse functions Many • Covalently attached to C-terminus of a few proteins (hedgehog) Covalently • Minor structural variations: Minor Cholesterol (animal cells) Ergosterol (yeast) Stigmasterol (plants) 3 Structures of cholesterol and ergosterol M. Chevreul, 1815 H. Wieland, 1926 4 Three views of cholesterol 5 5 Functions of Cholesterol As a membrane component: • Modulates physical properties (liquid crystalline transition temperature) Modulates • Enriched in plasma membrane “lipid rafts” Enriched As a precursor of other substances: • Bile acids (detergents that facilitate lipid absorption) Bile • Steroid hormones (bind to specific nuclear receptors/transcription factors) Steroid Androgens and estrogens (testes and ovary) Glucocorticoids and mineralocorticoids (adrenal) Vitamin D3 and its active hydroxylated metabolites 6 Cholesterol conversion to bile salts (liver) Many Steps 7 Cholesterol as a precursor of steroid hormones 8 Cholesterol side chain cleavage Cholesterol and biosynthesis of steroid hormones steroid 1 3 5 20 22 17 Cholesterol 20,22-Desmolase (cleaves C-C bond) Pregnenolone 3ß-OH-Steroid Dehydrogenase Progesterone 17α-Hydroxylase 17α -OH-Progesterone 17,20-Desmolase Androstenedione 17ß-OH-Steroid Dehydrogenase Testes Adrenal Cortisol Aldosterone Ovary Testosterone 5 Prostate/Skin Aromatase 5α-Reductase DihydroTestosterone Estradiol 9 Cholesterol homeostasis Total body cholesterol ~50 gm Plasma cholesterol ~200mg/100ml (5 mM) Dietary cholesterol ~ 200 - 400 mg/day Endogenous cholesterol biosynthesis Endogenous (mainly liver) ~ 700 - 1000 mg/day 700 Cholesterol lost from the body (bile acids and cholesterol) ~ 900 - 1400 mg/day 10 Cholesterol as a risk factor for atherosclerosis 11 Atherosclerotic lesions contain large amounts of cholesterol Atherosclerotic crystals crystals 12 12 Cholesterol Structure and Numbering 13 13 Cholesterol biosynthesis: overview (see “Hitler’s Gift”, E. P. Kennedy, J. Biol. Chem. 276, 42619, 2001 (see J. An unusual pathway that starts with acetate: • Unique enzymatic reaction mechanisms Unique - carbonium ion intermediates - complex carbons skeleton rearrangements • Early branch points leading to non-sterol products Early • Later steps (sterol formation) are O2 dependent Later • Regulation mainly at the level of transcription Regulation • Important medical genetics and pharmacology Important • “Big Bucks” 14 Konrad Bloch Konrad (1912-2000) (1912-2000) Cholesterol biosynthesis: origin of carbons O C H3 C O- 2 x NADPH 3 x Acetyl-CoA Acetyl-CoA (2C) (2C) HMG-CoA (6C) Mevalonate (6C) Mevalonate Mevalonate (6C) Isopentenyl-PP Isopentenyl-PP (5C) Farnesyl-PP (15C) (15C) Squalene (30C) Dimethylallyl-PP O2 (5C) “Cyclase” O2 Squalene (30C) Squalene epoxide (30C) Lanosterol (30C) Cholesterol (27C) 15 HO Sources of acetate (two carbon) fragments Ingestion of ethanol or acetate From glucose via pyruvate dehydrogenase Fatty acid oxidation 16 Formation of mevalonate and ketone bodies MEVALONATE + HS-CoA HMGCoA reductase 2 x NADPH During Ketoacidosis β -keto-butyrate acetone β -OH-butyrate 17 HMG-Coenzyme A Mechanism and Mechanism “Statin” Inhibitors HO COOH O (HMG-CoA) S CoA NADPH HO COOH OH H S CoA HO COOH O C oA SH H Mevaldehyde NADPH HO COOH OH Ki ~ 2-10 nM See Endo et al. FEBS Letters 1978 Mevalonate 18 Biochemists who Discovered and Developed Statins 19 Conversion of mevalonate to IPP and DPP O O H MV L N T E AO AE O 1 2 3 4 AP T AP T 5 O H O O P O OP O P O O- O O H O P O O O O O- O O Hs Dimethylallyl-PP (DPP) AP T O O P O O O O P O O P O O CO 2, P i O P O- O P O O- O O O O O- Hs HR Isopentenyl-PP (IPP) 20 Conversion of IPP and DPP to Farnesyl-PP (P) IP 4 O P OP --- (P) IP 4 OP O - - -P H S H R ( P) DP H S H R 1 ' H S O P OP --- O - P - O- P Geranyl-PP (GPP) O- P- O P - HS HS + H S H S H R O- P- O P - + HR , - O - P - O- P + O- P- O P H S H R HS O - P - O- P H S O- P- O P - Farnesyl -PP (FPP) HR + , - HS O - P - O- P HS Some prenyl transferases can do additional cycle(s) Some O - P - O- P HS 21 HS Geranyl-PP (GPP) Squalene synthase: a two step reaction H P O P O CH2 1 2 3 FPP CH3 H C H CH3 P O P O 1' FPP P O P O PP OO H3 2 C H 3 C H 3 C H 2 1 1 ' C H Presqu a le n e-PP N ADPH P O P O + N AD P Intermediate 1 Sq ualene 22 1' Squalene epoxidase (1) and lanosterol synthase (2) (1) Squalene Squalene epoxide (2) H+ Enz-AH+ Squalene epoxide Protosterol cation (transient, rearranges) Lanosterol 23 Alternative routes to cholesterol 1 2 3 Demethylation Lanosterol NADPH and O2 Double bond shifts Desmosterol Inhibited by Triparanol Inhibited (withdrawn -1958) (withdrawn (Causes Cyclopia) NADPH Reduction 7-Dehydrocholesterol UV light Bile acids Vitamin D3 Cholesterol Steroid hormones 24 Stages of cholesterol biosynthesis Early Acetyl-CoA Steroid Hormones, Bile etc. Middle Late HMG-CoA lovastatin Certain Proteins Certain (Ras) (Ras) Isopentenyl-PP Farnesyl-PP Dimethylallyl-PP Dolichol Squalene Mevalonate Cholesterol Some tRNAs Quinones Modified Proteins 25 25 Protein farnesyl transferase Ras Protein 26 26 Stages of cholesterol biosynthesis Early Acetyl-CoA Steroid Hormones, Bile etc. Middle Late HMG-CoA lovastatin Certain Proteins Certain (Ras) (Ras) Isopentenyl-PP Farnesyl-PP Dimethylallyl-PP Dolichol Squalene Mevalonate Cholesterol Some tRNAs Quinones Modified Proteins 27 27 Cholesterol attachment to sonic hedgehog protein BBA 1529, 188-202, 2000 Science 304, 1755-59, 2004 28 28 Lipid modified hedgehog relieves Ptc inhibition of Smo Hh, hedgehog; Ptc, patched; Smo, smoothened. Inhibits Smo directly 29 29 Newborn sheep with cyclopia following exposure Newborn of the pregnant mother to cyclopamine of Veratrum californicum 30 Selective potent inhibitors of the sterol pathway Target HMG-CoA Reductase Inhibitors Lovastatin, Pravastatin Simvastatin, Atorvastatin etc. Ezetimibe Uses or Effects Cholesterol Lowering Cholesterol Lowering Lowering Topical Antifungal Topical Oral Antifungal (Candida/AIDS) Cholesterol absorption Squalene epoxidase Terbinafene Lanosterol 14-demethylase Fluconazole and other “azoles” Combined market value of the statins (2008) ~ $ 34,000,000,000 ! Combined ______________________________________________________________________ ______________________________________________________________________ Protein farnesyl transferase SCH66336 Smoothened receptor Cyclopamine and others Anti-cancer? (Phase III) Anti-cancer? Causes cylopia Anti-cancer? 31 ...
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This note was uploaded on 12/19/2010 for the course BIOCHEM 227 taught by Professor Coggins during the Fall '10 term at Duke.

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