cholesterol structure most plasma cholesterol is in the esterified form (not found in cells or membranes) cholesterol functions in all membranes (drives formation of lipid microdomains) cholesterol is the precursor for steroid hormones note 4 fused rings, single dbl bond, single hydroxyl, acyl chain at C17 1 Cholesterol FAQs Cholesterol is the second most abundant fraction in blood besides glucose. All the carbon atoms in cholesterol come from Acetyl-CoA. Energy for synthesis comes from hydrolysis of thioester bonds of acetyl CoA and ATP hydrolysis. Synthesis occurs in the cytoplasm, with key enzymes found in the membrane of the ER. 2 Cholesterol promotes the liquid-ordered phase of membranes cholesterol has limited flexibility and is amphipathic. it stiffens the membrane and regulates permeability. it can interact with and affect the structure of integral membrane proteins (e.g. lipid rafts) 3
S.R. Wassall, W. Stillwell / Chemistry and Physics of Lipids 153 (2008) 57 63 Cholesterol is less soluble in (artificial) membranes high in unsaturated acyl chains 4 Journal of Lipid Research, Vol. 44, 655-667, April 2003 5 Lynen F. DerWegvonder. AktiviertenEssigsa üse zuden terpenen und den fettsa uren. Les Prix Nobel. Stockholm: Norstedt & Sons, 1965: 205 45. 6
Cholesterol synthesis initially follows that of ketone bodies 3 cytoplasmic acetyl CoA molecules are sequentially condensed to form HMG CoA (6 carbons) 7 cytoplasmic HMG-CoA Synthase mitochondrial HMG-CoA Synthase Cholesterol biosynthesis includes over 30 enzymatic steps that occur in the cytoplasm and outer membrane of the ER 8 Mito_HMG- CoA Synthase Cyto_HMG- CoA Synthase 9
10 11 12
13 Biochemistry, Vol. 39, No. 8, 2000 Beginning with squalene, sterolcarrier-protein keeps the remaining intermediates soluble 14 Squalene epoxide is converted to lanosterol in concerted fashion 15
16 The committed step of de novo cholesterol biosynthesis is catalyzed by HMG CoA reductase The reduction of HMG CoA by HMG CoA reductase results in the oxidation of two NADPH and results in mevalonate. HMG CoA reductase is a membrane protein of the ER: catalytic domain projects into the cytoplasm. Target of statin drugs 17 The committed step of de novo cholesterol biosynthesis is catalyzed by HMG CoA reductase 18
HMG-CoA-reductase forms a homotetramer and utilizes NADPH and CoA cofactors 19 Cell Research (2008) 18:609 621 HMG-CoA-r has a cytoplasmic catalytic domain and a membrane sterol sensing domain 20 HMG-CoA-reductase utilizes two NADPH for each HMG-CoA 1. NADPH reduces HMG carbon. 2. His donates a proton and cleaves CoA from HMG. 3. Second NADPH protonates HMG carbonyl to form Mevalonate. 21
Fine control of cholesterol homeostasis Inhibitory phosphorylation of HMG-CoA-r at SER872 (e.g. by AMP-dependent protein kinase) and regulated by hormones. 22 Coarse control of cholesterol homeostasis ER Lumen Insig SCAP SREBP Nucleus Reg bhlh SRE Sterols bhlh Golgi bhlh bhlh Lumen S1P S2P 1. Regulation of the transcription of HMG-CoA-r (and others) by SCAP/SREBP (sterol-responseelement-binding-protein) 23 Coarse control of cholesterol homeostasis VCP Ubc7 gp78 Cytosol ER Insig Lumen HMG CoA reductase Insig binding Sterols Ubc7 gp78 VCP Ubiquitination Ufd1 Degradation VCP VCP Ubc7 Ubc7 gp78 Extraction gp78 Proteasome Geranylgeraniol OH 2. INSIG protein senses sterol concentration and regulates ubiquitin-mediated protein degradation of HMG-CoA-Reductase 24
Summary of cholesterol homeostasis Transcription HMG-CoA-r Degradation P HMG-CoA-r [sterol] compensatory regulation phosphorylation INSIG-mediated degradation dephosphorylation SREB-mediated transcription 25 SCIENCE VOL 292 11 MAY 2001 Statins are competitive inhibitors of HMG-CoA-reductase 26 HMG CoA NADP+ monomer-1 monomer-2 Statin binding blocks HMG-CoA binding site 27
Statin binding blocks the Coenzyme-A binding pocket 28 Plant sterols Ezetimibe Reduces amount of circulating cholesterol not compensated by increased de novo synthesis of cholesterol Reduces amount of circulating cholesterol (inhibits absorption) compensated for by increased de novo synthesis 29 Plant sterol margarines (Benecol, sitosterol) act through a different mechanism than Ezetimibe Recent evidence points to enhanced degradation of HMG-CoA-r. 30
Cholesterol is modified by cytochrome p450 enzymes to form steroid hormones 31 Degradation of Cholesterol Unlike fatty acids, sterols cannot be used as an energy source The sterol ring nucleus is eliminated from the body by conversion to bile acids and bile salts. 32 Degradation of Cholesterol The theme is for cholesterol to be converted to a relatively soluble amphipathic molecule. As a bonus, these molecules are used as emulsifying agents during digestion. 33
Lipoprotein particles transport lipids. Each lipoprotein recruits a different set of lipid transfer proteins 34 LDL delivers cholesterol directly to the interior of cells 35 HDL are secreted empty and scavenge cholesterol via Apo-A associated LCAT (lecithincholesteral acyltransferase) activity 36
Lecithin-cholesteryl-acyltransferase (LCAT) is recruited to HDL via Apolipoprotein A1(Apo A) LCAT helps to sequester cholesterol (amphipathic) by esterifying it to a (hydrophobic) fatty acid 37 Biophysical Journal, 88:548-556, 2005 apolipoprotein A1 homodimer forms an α-helical belt to capture lipids 38