Anabolism - Steroid Biosynthesis Lecture

OUTLINE: ANABOLISM II OUTLINE: Introduction and review Transport Glycogenolysis Glycolysis Other sugars Pasteur: Anaerobic vs Aerobic Biosynthesis of Fatty Acids and Lipids Fatty Acids Exam-1 material Fermentations Pyruvate Krebs’ Cycle Oxidative Phosphorylation Exam-2 material Electron transport Chemiosmotic theory: Phosphorylation Fat Catabolism Fatty acid Catabolism Exam-3 material Mobilization from tissues (mostly adipose) Activation of fatty acids Transport; carnitine Oxidation: b-oxidation, 4 steps: Protein Catabolism Amino-Acid Degradation Dealing with the nitrogen; Urea Cycle Dealing with the carbon; Seven Families Nucleic Acid & Nucleotide Degradation PHOTOSYNTHESIS: Overview and Key experiments: Light Reactions energy in a photon/pigments Reaction center &Photosystems (PSII & PSI) Proton Motive Force – ATP Carbon Assimilation – Calvin Cycle Rubisco/Oxygenase (Glycolate cycle) remaking Ru 1,5P 2 Overview and regulation C4 versus C3 plants Kornberg cycle - glyoxylate Carbohydrate Biosynthesis in Animals precursors/Cori cycle Gluconeogenesis reversible steps irreversible steps – four Glycogen Synthesis UDP-Glc/Glycogen synthase/branching Pentose-Phosphate Pathway oxidative-NADPH non-oxidative-Ribose 5-P contrasts location & transport Synthesis acetyl-CoA carboxylase fatty acid synthase ACP priming 4 steps Exam-4 material Control of fatty acid metabolism Diversification of fatty acids elongation desaturation Eicosanoids Prostaglandins and Thromboxane Triacylglycerides Membrane lipids Glycerophospholipids Sphingolipids Isoprene lipids: Cholesterol Ketone body synthesis Mevalonate Cholesterol bile acids steroids metabolism control of cholesterol biosynthesis ANABOLISM III OUTLINE: Biosynthesis of Amino Acids and Nucleotides Nitrogen fixation nitrogenase Regulation of Carbohydrate Metabolism Anaplerotic reactions Exam-5 material ANABOLISM II: Biosynthesis of Fatty Acids & Lipids 1. Biosynthesis of fatty acids 2. Regulation of fatty acid degradation and synthesis 3. Diversification of fatty acids a. Elongation/desaturation b. Eicosanoids (Prostaglandins and Thromboxane) 4. Assembly of fatty acids into triacylglycerol and phospholipids 5. Metabolism of isoprenes a. Ketone bodies and Isoprene biosynthesis b. Isoprene polymerization i. ii. iii. iv. Cholesterol Steroids & other molecules Regulation Role of cholesterol in human disease 1 Cholesterol and Steroid Biosynthesis Fates of Cholesterol After Synthesis •In vertebrates, most cholesterol is synthesized in the liver, then exported. -They are exported as bile acids, biliary cholesterol, or cholesteryl esters. -Bile is stored in the gall bladder and secreted into the small intestine after fatty meal. -Bile acids such as taurocholic acid emulsify fats. –They surround droplets of fat, increasing surface area for attack by lipases. Vitamin D pre-VitD 3 7-dehydro-Chol ( Oxysterols) •Other tissues convert cholesterol into steroid hormones and Vitamin D From degradation of Cys Lecithin-Cholesterol Acyl TransferaseCatalyzed Reaction Occurs in HDL (LCAT) •Adrenal gland-synthesized steroids: – mineralcorticoids • control electrolyte balance, reabsorption of Na+, Cl−, HCO3− from kidney – glucocorticoids • regulate gluconeogenesis, reduce inflammation •Gonad-synthesized steroids: Oxysterols are involved in transcriptional control, signaling – progesterone, in development, apoptosis, inflammation, and sterol transport testosterone, estrogens Steroid Biosynthesis Steroid Hormones Derived from Cholesterol • Takes place in mitochondria • The “side chain” on C-17 of the D ring is modified, then cleaved. C-20 C-22 • Two adjacent carbons are hydroxylated. • Desmolase cleaves • All three use mixedfunction oxidases, NADPH and cytochrome P450 2 Steroid Biosynthesis Steroid Hormones Derived from Cholesterol • Takes place in mitochondria • The “side chain” on C-17 of the D ring is modified, then cleaved. ox=C3O & iso=C4/5 C-11 C-17 C-21 C-21 C-11 C-17 –acetateC-20/21 –C-19 aromatase C-20 C-22 C-18 ox-C18O • Two adjacent carbons are hydroxylated. • Desmolase cleaves • All three use mixedfunction oxidases, NADPH and cytochrome P450 Cholesterol and Steroid Biosynthesis TABLE 21-1 Major Classes of Human Plasma Lipoproteins: Some Properties Composition (wt %) Lipoprotein Density (g/ml) Protein PL Free cholesterol Cholesteryl esters Triacylglycerols Chylomicrons <1.006 2 (ApoB-48,-E,-CII) 9 1 3 85 VLDL 0.95–1.006 10 (ApoC-I,CII) 18 7 12 50 LDL 1.006–1.063 23 (ApoB-100) 20 8 37 10 HDL 1.063–1.210 55 (ApoA-I,-II) 24 2 15 4 Source: Data from D. Kritchevsky, Nutr. Int. 2:290, 1986. • Lipids are carried through the plasma on spherical particles. –surface is made of protein (called apolipoprotein*) and a phospholipid monolayer –interior contains cholesterol, TAGs, and cholesteryl esters, which are more nonpolar than cholesterol • Named based on position of sedimentation (density) in centrifuge *“apolipoprotein” refers to the protein part of a lipoprotein particle. • Composition varies between class of lipoprotein • Includes four major classes shown in Table 3 Cholesterol and Steroid Biosynthesis Biological Roles and Characteristics of Lipoproteins TABLE 21-2 Apolipoproteins of the Human Plasma Lipoproteins Apolipoprotein Polypeptide molecular weight Lipoprotein association Function (if known) ApoA-I 28,100 HDL Activates LCAT; interacts with ABC transporter ApoA-II 17,400 HDL Inhibits LCAT ApoA-IV 44,500 Chylomicrons, HDL Activates LCAT; cholesterol transport/clearance ApoB-48 242,000 Chylomicrons Cholesterol transport/clearance ApoB-100 Binds to LDL receptor 512,000 VLDL, LDL ApoC-I 7,000 VLDL, HDL ApoC-II 9,000 Chylomicrons, VLDL, HDL Activates lipoprotein lipase ApoC-III 9,000 Chylomicrons, VLDL, HDL Inhibits lipoprotein lipase ApoD 32,500 HDL ApoE 34,200 Chylomicrons, VLDL, HDL Triggers clearance of VLDL and chylomicron remnants ApoH 50,000 Possibly VLDL, binds cardiolipin Roles in coagulation, lipid metabolism, apoptosis, inflammation Cholesterol and Steroid Biosynthesis Biological Roles of Lipoproteins in Trafficking Cholesterol and TAGs Receptor-Mediated Endocytosis IN: Exogenous (diet) ❶–❹ Pathway HDL Pathway 10 – 13 OUT: Endogenous Pathway Enterohepatic Pathway ❺–❾ 14 4 Cholesterol and Steroid Biosynthesis Cholesterol and Steroid Biosynthesis Regulation of Cholesterol Metabolism •Major regulation is by gene expression: Three levels – Sterol regulatory element-binding proteins (SREBPs) • When sterol levels are high, oxysterols are produced, which retain SREBPs in the ER membrane with other proteins. ⊕ • When sterol levels fall, the complex is cleaved and HMG-CoA HMG-CoA mRNA ↓ mRNA moves to the nucleus. • It activates transcription of HMG-CoA reductase and transcription ↑ LDL receptor, as well as other genes. ↑ N o transcription ↑ oxysterol 5 Cholesterol and Steroid Biosynthesis Regulation of Cholesterol Metabolism •Major regulation is by gene expression: Three levels – Sterol regulatory element-binding proteins (SREBPs) • When sterol levels are high, oxysterols are produced, which retain SREBPs in the ER membrane with other proteins. ⊕ ↑ ↑ • When sterol levels fall, the complex is cleaved and HMG-CoA HMG-CoA mRNA ↓ mRNA moves to the nucleus. • It activates transcription of HMG-CoA reductase and LDL receptor, as well as other genes. ↑ – Translational control on mRNA stability ↑ • Increased mevalonate and other isoprenes destabilize the HMG-CoA mRNA • Decrease in mevalonate stabilizes the mRNA; N o transcription – Post-translational control • • • • High cholesterol produces oxysterol, which allosterically binds HMG-CoA reductase Destabilizes by a conformational change that allows ubiquitination Proteolytic degradation of HMG-CoA reductase Also inhibits RME Cholesterol and Steroid Biosynthesis Regulation of Cholesterol Metabolism •Major regulation is by gene expression: Three levels – Sterol regulatory element-binding proteins (SREBPs) • When sterol levels are high, oxysterols are produced, which retain SREBPs in the ER membrane with other proteins. ⊕ • When sterol levels fall, the complex is cleaved and ↓ mRNAHMG-CoA moves to the nucleus. • It activates transcription of HMG-CoA reductase and LDL receptor, as well as other genes. – Translational control on mRNA stability • Increased mevalonate and other isoprenes destabilize the HMG-CoA mRNA • Decrease in mevalonate stabilizes the mRNA; – Post-translational control •Minor regulatory mechanisms: Phosphorylation/de-phosphorylation – Covalent modification provides short-term regulation – Glucagon, epinephrine Other effects of high cholesterol Activation of ACAT, which increases esterification for storage Transcriptional regulation of the LDL • Also, AMP-dependent protein kinase receptor gene activation when AMP rises, kinase phosphorylates the enzyme à activity ¯, cholesterol synthesis ¯ • cascades lead to phosphorylation, ¯ activity – Insulin: cascades lead to dephosphorylation,­ activity 6 Cholesterol and Steroid Biosynthesis Regulation of Cholesterol Synthesis by Sterol regulatory elementbinding protein (SREBP) Cholesterol ↓ Oxysterol ↓ HMG-CoA reductase↓ • Insig (insulin-induced gene protein) senses cholesterol levels through oxysterol. HMG-CoA reductase↑ LDL receptor↑ – Also helps sequester SREBP in ER – oxysterol triggers ubiquination of HMG-CoA reductase • targets the enzyme for degradation by the proteosome – Loss of oxysterol triggers ubiquination of Insig • targets the protein for degradation by the proteosome Clinical Correlations Cardiovascular Disease (CVD) • Multifactorial • Very high LDL-cholesterol levels tend to correlate with atherosclerosis. – although many heart attack victims have normal cholesterol, and many people with high cholesterol do not have heart attacks • • Low HDL-cholesterol levels are negatively associated with heart disease. Reverse Cholesterol Transport by HDL explains why HDL is cardioprotective 7 Clinical Correlations Cardiovascular Disease (CVD) How Plaques Form • HDL picks up cholesterol from nonliver tissues, including foam cells at growing plaques. • HDL carries cholesterol back to the liver Participation of ABCA1and ABCG1 transporters and SR-B1 receptors • Clinical Correlations Familial Hypercholesterolemia Cardiovascular Disease (CVD) • Due to genetic mutation in LDL receptor • Impairs receptor-mediated uptake of cholesterol from LDL • Cholesterol accumulates in the blood and in foam cells. • Regulation mechanisms based on cholesterol sensing Brown and Goldstein inside the cell don’t work. Nobel Prize in 1985 • Homozygous individuals can experience severe CVD as youths. Statin Drugs Inhibit HMG-CoA Reductase to Lower Cholesterol Synthesis • Statins resemble mevalonate à competitive inhibitors of HMGCoA reductase • First statin, lovastatin, found in fungi – lowers serum cholesterol by tens of percent • Also reported to improve circulation, stabilize plaques by removing cholesterol from them, and reduce vascular inflammation 8 Cholesterol and Steroid Biosynthesis We learned that: Summary • synthesis of fatty acids is a multistep process starting from acetyl-CoA and its carboxylated product, malonyl-CoA; it uses a poly-enzyme protein • After synthesis of palmityl-CoA, fatty acids are elongated and desaturated; PUFAs are used for prostaglandin, thromboxane, and leukotriene synthesis • Phosphatidic acid is a precursor to phospholipids and TAGs • phospholipids and TAGs are built on a glycerol backbone that can be derived from dihydroxyacetone phosphate or glycerol • head groups are attached using one of two methods, both use CDP carrier • pathways to the synthesis of specific head groups vary by organism and may use salvage pathways • Ketone-body synthesis goes through HMG-CoA, made from multiple acetyl-CoAs • cholesterol is derived from isoprene units, which comes from HMG-CoA via the mevalonate pathway • cholesterol can be metabolized and modified in a variety of ways • cholesterol and TAGs are trafficked in lipoproteins; classified by density • There are regulatory mechanisms of lipid biosynthesis….both FA and chol. ANABOLISM III: Biosynthesis Amino Acids & Nucleotides 9 ANABOLISM III: Biosynthesis Amino Acids & Nucleotides 1) Nitrogen fixation: N2 à +NH4 2) Nitrogen assimilation: incorporation of inorganic nitrogen (nitrates, nitrites, and ammonia) into biomolecules 3) Biosynthesis of amino acids a) non-essential b) essential 4) Biosynthesis of nucleotides 5) Control of nitrogen metabolism 6) Biosynthesis and degradation of heme; other 2° products of amino acids Biosynthesis Amino Acids & Nucleotides • Nitrogen (after H, O, and C) is a major element of living organisms • Most nitrogen is inert in the atmosphere as dinitrogen • Making dinitrogen useful is not easy Atmosphere is 80% N2 , but is chemically inert need N2 + 3 H2 à 2 NH3 Even though DG′°= –33.5 kJ/mol… breaking a triple bond has high activation energy (i.e., SLOW, kinetically stable), this can be accomplished using non-biological processes: N2 and O2 à NO via lightning N2 and H2 à NH3 via the industrial Haber-Bosch process requires T>400 °C, P>300 atm Industrial synthesis of NH3 via the Haber process is one of mankind’s most significant chemical processes. 10 Biosynthesis Amino Acids & Nucleotides • Haber process Biosynthesis Amino Acids & Nucleotides Catabolism Anabolism Purines, pyrimidines, Nucleotides porphorins neurotransmitters hormones glutathione CO2 cofactors SAM/THF (C1) a- [Q,N,R, + W(In),H(Im)] NH4 [24 other Ns] a-Ketoglutarate N2 Aspartate a-Keto acids Glutamate Sugars Oxaloacetate Fatty acids 11 Biosynthesis Amino Acids & Nucleotides Chemistry of Molecular Nitrogen Review: Oxidation States of Nitrogen Compounds Nitrate • • • • NO3 – N+5 O3–6 Nitrate Also Nitric acid (HNO3) and Dinitrogen pentoxide (N2O5) • “ate” is the higher oxidation state. Nitrogen(IV) dioxide • NO2 • N+4 O2–4 • Nitrogen dioxide • gas Nitrite NO2– N+3 O2–4 Na-Nitrite Also Nitrous acid (HONO) • “ite” is light on oxygen and oxidation state. • • • • Nitric oxide Nitrogen(I) Nitrogen (Nitrogen(II)) oxide • • • • NO N+2 O–2 Non-salt Gas • Physiologically important 2° messenger and paracrine signal • • • • N2O N+1 O–2 Non-salt gas • N2 • N0 • Covalent triple bond • gas Ammonia • NH3 • N–3 H3+3 • NH3: N has oxidation state of –3. • Ammonium cation salts • gas Biology of Molecular Nitrogen: The Nitrogen Cycle 12