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Cellular and Molecular Pathology: Mechanisms of Cellular injury Part 1 Mark R. Ackermann Department of Veterinary Pathology College of Veterinary Medicine Iowa State University Ames Iowa 50011-1250 [email protected] 515 294 3647 Summer 2008 Copyright © Kinases. Kinases are proteins that are enzymes. Kinases also add phosphates to other things, like fructose. Pyruvate kinase actually transfer a P group for phosphoenolpyruvate to ADP; so it is adding a P onto ADP to make ATIP (see below). Kinases also activate other enzymes by adding phosphates groups; in a few cases, addition of the phosphate turns off an enzyme. kinase Sugar (no P group) ------Æ Sugar-P kinase Enzyme (not active) -------Æ Enzyme-P (now active) (in a few cases, the P actually inactivates the enzyme). Phosphatases take off the P group and generally inactivate enzymes enzymes, etc etc. Phosphatase Enzyme-P (active) -----Æ Enzyme (no P and not active, generally) Specific examples: Hexokinase (hexose is a six-carbon sugar) Glucose ---------------------Æ Glucose 6 P (with P on, the glucose cannot be transported out of the cell; it also commits the Glucose to GLYCOLYSIS OR GLYCOGEN SYNTHESIS) Pyruvate kinase Phosphoenolpyruvate and ADP ----------------Æ pyruvate plus ATP Tyrosine kinase (a receptor activated by a growth factor binding) Cell signaling molecule (no P) -----------------------------------------------------------Æ Cell signaling molecule-P Injury resulting in ATP loss • • • • • • • • • Hypoxia/ischemia Burn, cold Radiation Chemicals/drugs/toxins Infectious agents Immunologic/autoimmune Inflammation Nutrition Genetics 1 Normal cell volume control • Na+ is extracellular • K+ is intracellular • Membrane permeability – Selective passage • Water, carbon dioxide, oxygen, benzene, urea, glycerol – No passge • H+, sodium, bicarbonate (HCO3), potassium (K+), calcium, chloride, magnesium, glucose (some cells), sucrose • Many ions and molecules are regulated by: – Transportors, Channels, and ATPase pumps Transportors • Transportors are often – Slow, 102 to 104 molecules/second – Do not use ATP Na+-glucose • Unitransportors – Transport T t one ion/molecule i / l l Symport • Coupled transportors – Transport two ions/molecules Na+ • Symport (both transported in the same direction) • Antiport (transported in opposite directions) Antiport glucose Transportors • Unitransportors – Glucose • Can also be coupled with Na+ in the small intestine and renal tubules – Fructose • Intestine and liver • Coupled transportors – GABA, Norepinephrine, glutamate • Symport with Na+ – Peptides and amino acids • Symport with Na+/H • Transportors also for: – Pyruvate, fatty acyl Co A, and ATP 2 Channels • Channels are often – – – – Fast 107 to 108 ions/second Do not use ATP Connect cytosol to cell exterior through narrow passages (ie. pores) Selective • • • • Mainly transport Na+, K+, Ca2+ and Cl- Transport efficiency is greater than carrier proteins Transport is always passive S Secondary d tto membrane b d depolarization l i ti (voltage-gated) ( lt t d) – Examples • Na+/K+ channels for nerve fiber conduction • Ca++ channels in sarcoplasmic reticulum of muscle • Chloride (Cl-) channels – – – – – – Nearly 20 disorders known involving defects in skeletal muscle to nervous system Long QT syndrome » Loss of K+ channel function or gain of Na+ channel function Defects in Ryanodine receptor » malignant hyperthermia Ligand-gated channels in synapses, CFTR channels, and ClC channels (numerous functions) Defects in CFTR Cl- channel result in cystic fibrosis » Although CFTR is a ABC pump that utilizes ATP, it is considered a channel due to the rapid influx of chloride it can allow Defects in voltage-gated CLC chloride channels » Myoclonia congenita, Dent’s disease, Bartter’s disease, osteopetrosis Figure 1 Inherited mutations alter ion channel function and structure and cause human disease. Mutations may alter the permeation pathway (A) to inhibit the movement of ions through an open channel pore and may also alter ion channel gating by changing either the process by which channels open (activate) (B) or the process by which they inactivate (C) (C). Transitions from the open to the inactivated state reduce the number of channels that are available to conduct ions. Mutations that destabilize the inactivated, nonconducting state of the channel are gain-of-function mutations and are common to diverse diseases, including LQTS, certain forms of epilepsy, and muscle disorders such as hyperkalemic paralysis. Kass RS: J Clin Invest 8:1986-1989, 2005 Na+ K+ Ca+ Cl+ Kass RS: J Clin Invest 8:1986-1989, 2005 3 Inherited loss of channel function: Malignant hyperthermia • Dysfunctional ryanodine receptor (RYR 1) activity – A calcium channel • Sensitive to halothane/stress • Muscle contraction – Myosin ATPase activity, glycogenolysis, glucolysis, further release of calcium, excessive heat, lactic acidosis, ATP loss Inherited loss of channel function: Cystic fibrosis • Inherited defect of Chloride channel – CFTR (cystic fibrosis transmembrane conductance regulator) • DeltaF508 mutation in allele – 90% of CF patients have this mutation – Results in CFTR remaining in ER and being degraded by ubiquitin Cystic fibrosis • Sweat test is done clinically, but not definitive • Loss of ions in respiratory secretions – Mucus layer becomes thick – Also affects gastrointestinal secretions and pancreatic secretions • Increased susceptibility to respiratory infections, especially Pseudomonas and Burkholderi which are almost impossible to eliminate – Loss of ionic concentrations – Loss of antimicrobial peptide (defensin activity) – CFTR a receptor for Pseudomonas 4 CLC Chloride Channels • Nine mammalian genes encode for channels denoted CLCN1 to CLCN7 and CLCNKa and CLCNKb • Present in plasma membrane and membrane of intracellular organelles • Function to stabilize membrane potential, synaptic inhibition, cell fluid volume regulation, transepithelial transport, extracellular and vesicular acidification and endocytotic trafficking CLC Chloride Channel Structure • Double-barreled channels • Each pore is gated and can be opened and closed individually; gating is independent • Common gate exists to close both pores simultaneously • Single gate mutations • Common gate mutations 5 Myotonia Congenita • Impairment of skeletal muscle relaxation after contraction • Mutations in CLC1 which can cause either dominant ((Thomsen’s)) or recessive (Becker-type) disease • Recessive form more common and more severe • Dominant mutations act on common gate affecting both pores Myotonia Congenita • Treatment usually not necessary • Mexiletine (sodium channel blocker) Myotonia 6 Bartter Syndrome III • Renal tubular disease characterized by loss of NaCl reabsorption; PU/PD, bouts of severe dehydration, failure to thrive • Mutation in CLC-Kb with autosomal recessive i h it inheritance • Deafness not present because both CLC-Ka and CLC-Kb are present in inner ear • Mutation in barttin (beta subunit present in both CLC-Ka and CLC-Kb) leads to deafness Bartter Syndrome III • Treatment based on clinical presentation, not genetic mutations • Na+ and K+ supplements • ACE inhibitors • Indomethacin • Growth hormone • Response to therapy variable Dent’s Disease • X-linked recessive gene mutation in CLC5 with proteinuria, hyperphosphaturia, hypercalciuria and nephrocalcinosis h l i i • Endocytosis is impaired • Luminal parathyroid hormone increases causing decrease of Na-PO4 cotransporter • Serum concentrations of active vitD3 variable 7 Dent’s Disease • Treatment for nephrocalcinosis may slow progression to end-stage renal failure • Thiazide diuretics used which enhance Ca2+ reabsorption Dent’s Disease Osteopetrosis • Reduced or absent acidification of resorption lacuna leading to failed bone resorption by osteoclasts which results in brittle bones • Autosomal A t l recessive i or d dominant i t lilinked k d mutation in CLCN7 • CNS neurodegeneration resembling lysosomal storage diseases produced in knockout mice • Retinal degeneration 8 Osteopetrosis • Treatment success variable • Bone marrow transplant • Vitamin D administration (calcitriol) to enhance bone resorption • Prednisone given to improve hematological alterations (neutropenia and anemia) Osteopetrosis Conclusions Numerous diseases associated with CLC defects attributes to their wide range of physiologic functions and emphasizes their medical importance 9 ATPase pumps • ATPase pumps are often – Slow, 10 to 103 ions/molecules/second – All require ATP (by definition) • ATP ATPase ADP+Pi – ½ of all ATP is used to maintain transport gradients • Types of ATPase pumps – P, F, V, ABC ATPase pumps • P type ATPase pumps – Na/K, H, Ca transported – Locations • Plasma membrane for Na/K – 3 Na+ out, 2 K+ in – For cell homeostasis • Apical plasma membrane of stomach (H+/K+) • Plasma membrane of all cells (Ca++) • Sarcoplasmic reticulum (Ca++) ATPase pumps • F type ATPase – H+ ion only • Inner mitochondrial membrane – And chloroplast thylakoid membrane (plants) 10 ATPase pumps • V type ATPase – H ion only • Especially for pH regulation – Locations • Endosome, lysosome, secretory vesicles • Plasma membrane of osteoclasts, renal epithelial cells, neutrophils, macrophages – Activities • Endosomal killing/degradation, bone resorption, breakdown of ligand/receptor complexes (thus controlling regulation of receptor density), release of enzymes from Mannose-6-phosphate receptors (thus regulating enzyme activity) ATPase pumps • ABC type ATPase pumps – Pump ions (many types) and lipophilic substances – Location and substance • Endoplasmic reticulum – Peptide transport for MHC/Ag processing • Plasma membrane – MDR (P-glycoprotein) » Transports lipid-like toxins/drugs • Macrophages – ABCA1 C 1 » Transports cholesterol and phospholipids – defects contribute to atherosclerosis • Rod photoreceptors – ABCA4 » Transports retinoyl derivates » Defects result in macular degeneration • Liver – ABCB11 (BSEP) » Transports bile salts Cellular energy: required for ATP Glucose/glycogen Proteins Pyruvate Fatty acid Fatty Acyl Co A ACETYL CO A KREBS (produces NADH, FADH2 for Oxidative phosphorylation) OXIDATIVE PHOSPHORYLATION + NADH +02 =ATP Mitochondria ATP for: ATPase pumps, signaling, etc. A transporter is required for: Fatty Acyl Co A, pyruvate and ATP Cell plasma membrane 11 Mitochondrial ATP formation • The outer mitochondrial membrane is permeable to H+ ions • The inner mitochondrial membrane is impermeable • NADH generated in Kreb’s cycle – Releases electrons (H+) (H ) that enters oxidative phophorylation • H+ ion is moved from the inner mitochondrial matrix to the area between the inner and outer mitochondrial membranes • Oxygen enters, is converted to water • H+ enters the F1 ATPase – Converts ADP to ATP as H+ passes back to the inner mitochondrial area Mitochondrial formation of ATP Cytoplasm Outer mitochondrial membrane H+ H+ H+ FO/F1 ATPase Impermeable to H+ Inner mitochondrial membrane H+ NADH+ ½ O2 NAD H 20 H+ ADP Pi ATP Mitochondrial matrix Sites of inhibition to ATP synthesis H+ H+ NADH/CoQ: ROTENONE H+ FO/F1 ATPase H+ NADH+ NAD Cytochrome oxidase: CN, AZIDE, HYDROGEN SULFIDE HERBICIDES, NO ½ O2 ADP Pi Loss of membrane potential: DNP, SALICYLATE, VALINOMYCIN, GRANICIDIN H 20 H+ ATP FO/FA ATPase: DDT, other drugs Lack of oxygen: RESPIRATORY PARALYSIS HYPOXIA, ISCHEMIA (ergot alkaloids, Infarct, cocaine toxicity), CO, MET Hb 12 Impairment of mitochondrial synthesis of ATP • Inhibition of NADH production • Lack of glucose • Fatty acid oxidation • Loss of protein Effect of ischemia on cells • • • • • • • • • Decreased oxidative phosphorylation Decreased ATP synthesis Decreased ATPase pump activity Loss of inner mitochondrial membrane potential Loss of K+ from the cytoplasm Influx of Na+ Increased intracellular Ca++ Glucose/glycogen breakdown Lactate formation – decreased pH • Decreased transport vesicles • Decreased synthesis of proteins, lipids, loss of phospholipid turnover in membranes Increased intracellular calcium • Cytosol levels normally are 10-100nM (extracellular levels are 1-2 mM) • • Inositol triphosphate (IP3) and other factors can induce release Increased Ca++ levels can pass the external mitochondrial membrane – – – Intracellular storage in smooth and rough endoplasmic reticulum Storage kept in check with Ca++ATPase pumps If persistent, the calcium can then induce the internal mitochondrial membrane to form pores • • • • Pores are formed with adenine nucleotide translocase (ANT), voltage-dependent anion channel (VDAC), and cyclophilin Mitochondrial permeability transition, a state of calcium permeability by the inner membrane Enhances oxidative phosphorylation temporarily and free radical formation Eventually damages the inner mitochondrial area and calcium aggregates form • Simultaneously, calcium activates phospholipases and calpain • • Reduces ATPase activity Enhances calcium-dependent endonuclease activity and DNA degradation – – Phospholipase A2 degrades plasma membranes Calpain is a cysteine protease 13 Robbins and Contran Pathologic basis of disease, 7th edition Cell injury: reversible/irreversible changes Robbins and Contran Pathologic basis of disease, 7th edition Brown fat utilization and thermogenesis • Cold increases – PGC1 (a powerful transcriptional coactivator) for PPAR gamma (peroxisome proliferator activator receptor) – TR (thyroid hormone receptor) – RAR (retinoic acid receptor) – ER (estrogen receptor) • PGC1 PGC1, TR TR, RAR and d ER activate ti t UCP ((uncoupling li proteins) t i ) and d genes of mitochondrial respiratory chain for ATP synthesis (ATPase and cytochrome oxidase C) • The UCP (uncoupling proteins) are in the inner mitochondrial membrane along with F0/F1 ATPase. • The UCP allow H+ passage with loss of gradient resulting in heat with reduced ATP. – PGC1 also activates NRF 1and 2 (nuclear respiratory factors) for mitochondrial biosynthesis and increases conversion of type I to type II muscle fibers • Increased mitochondria for more heat 14 Radical formation • Radicals, reactive oxygen species (ROS) – Form secondary to: • Ultraviolet light, cell metabolism, inflammation – Cell metabolism • Of all oxygen used by cells – 2% is converted to ROS by mitochondrial electron transport system (ETS) – 1/100 oxygen molecules cause protein damage – 1/200 oxygen molecules cause DNA damage – Oxidation: loss of an electron – Reductant: Donates electrons via addition of a hydrogen ion or removal of an oxygen molecule. Free radicals • Free radicals . : – An unpaired electron in an outer orbital – Example: hydroxy radical : : • :O:H hydroxy radical (.0H) a radical • :O:H hydroxy ion (-OH) not a radical • The dot denotes an unpaired electron, but no inference about charge; the negative sign denotes charge • Singlet oxygen (O2) . • Superoxide anion (O2 -) • Hydroxy radical (.OH) • Peroxyl (ROO.) and alkoxyl radicals (RO.) An unpaired electron Nonradicals that are reactive and contribute to radicals • • • • • • • Hydrogen peroxide (H2O2) Hypochlorous acid (HCLO) Organic peroxides Aldehydes Ozone Singlet oxygen (1O2) Peroxynitrite (ONOOH) 15 Terms • Reactive oxygen species (ROS), oxygenderived species (ODS), oxidants, and reactive nitrogen species (RNS) – These are all often used interchangeably. Free Radical Formation • Reactions for Formation » Fe++ Fe +++ • Fenton Reaction H2O2 Æ OH. • Haber Weiss reaction H2O2 + O2. Æ OH. • Electron transport system Æ O2. - = O = O. - (unpaired electron) • Cytochrome P450, xanthine oxidase Æ O2. • Ionizing Radiation H20 Æ .OH • NADPH oxidase NADPH -2 e’s + 2O2Æ NADP+ + H+ + 2 O2 - • Lysyl oxidase-crosslinks collagen • NOS nitric oxide synthtase 1, 2, 3 (neuronal, endothelial, macrophage) Æ (peroxynitrite) OONO. • Radical + Nonradical = radical • Reperfusion injury – xanthine oxidase Properties of free radicals • Hydroxy radical (.OH) – The most reactive radical known in chemistry – Diffuses a short distance, 3 nm, short-lived (10-10 sec) • So active that it doesn’t travel far • Must be made close to DNA; H2O2 is the latent form that gets close to DNA and then is converted to hydroxy radical through the fenton reaction • Damages deoxyribose, all four DNA bases, phosphate backbone, lipids • . Superoxide anion (O2 -) – Diffuses a short distance, short-lived – Affects guanine selectively • • Alkoxyl (RO.)(10-6 sec) and peroxyl (ROO.)(17 seconds) Nitric oxide (NO.)/peroxynitrite (ONOO- or ONO2) – ONOO- diffuses a longer distance, 9 um, long-lived (minutes) • Can deaminate or nitrate (add nitrogen) to nucleotides • Especially damaging to guanine – Results in formation of 8-oxo-deoxyguanine (easy to detect) » ONOO- is more damaging to 8-oxodeoxyguanine than guanine – Also induces formation of 8-nitro-deoxyguanine (difficult to detect) • Non radicals, also a short livespan (seconds to hours) 16 Exogenous and endogenous sources • Exogenous sources: – Gamma radiation, UV, ultrasound, food, drugs, g p pollutants, xenobiotics, toxins • Endogenous sources: – below Exogenous sources • Reactive oxygen species (ROS) sources: – – – – – – – – – – Ionizing and non-ionizing radiation Phorbol esters Bleomycin, paraquat (these induce fibrosis) Organic peroxides H Heavy metals l ((next slide) lid ) Asbestos (fibers) Smoke Silica Nanoparticles Gases • Ozone • di-oxygen (O2) (oxygen itself) – and many others Metals and free radical formation • Metals generate free radicals via the Fenton reaction • Reduction/oxidation-active metals – Iron, copper, chromium, and cobalt • Increase ROS through a Fenton-like reaction • Reduction/oxidation-inactive metals – Lead, cadmium, mercury, nickel • Deplete cells of antioxidants – Especially thiol-containing antioxidants (glutathione reductase) through binding of these metals to SH groups • Activation of transcription factors: NF-kappa B, AP-1 and p53 – Metal-induced formation of ROS activate these transcription factors, all of which are redox-sensitive • Metals induce mutagenic lipid products. – Lipid peroxides, formed from ROS action on phospholipids (more later), further react with metals to produce mutagenic lipids (malondialdehyde, 4-hydroxynonenal, exocyclic DNA adducts (etheno adducts)). • Antioxidants may be important for heavy metal poisoning 17 Endogenous location of free radical formation • Endoplasmic reticulum (rER, sER) – sER biometabolism • Cytochrome P450, conversion of carbon tetrachloride to radical CCl4 to CCl3 • Mitochondria • Ubiquinone • Cytosol – Fenton reaction, xanthine oxidase, U.V. • Peroxisome – oxidases • Leukocyte granules – NADPH oxidase • Extracellular matrix – Lysyl oxidase (cross links collagen) Principal locations of radical formation in cells Cytosol: Nitric oxide synthetase UV: hydroxy radical Fenton reaction Reperfusion injury Granules: NADPH oxidase sER: Cytochrome p450 -biotransformation Mitochondria: Mit h di Oxidative phosphorylation Peroxisomes: Catalase, oxidases Extracellular matrix: Lysyl oxidase Robbins and Contran Pathologic basis of disease, 7th edition 18 Reperfusion injury • Reperfusion injury occurs in sites of ischemia that once again receive oxygen yg – Examples: • myocardial infarction • stroke • dissolution of a thrombus (clot) Reperfusion injury • Exuberant free radical formation occurs with reperfusion due to: – Xanthine oxidase pathway – Mitochondrial electron transport chain – Conversion of NOS to produce superoxide (rather than NO) – NADPH oxidase from infiltrating leukocytes 19 Free radical formation during reperfusion: xanthine oxidase Cell ischemia/loss of oxygen leads to ATP degradation. If perfusion is re-established, oxidative radicals are formed. ATP ISCHEMIA ADP Xanthine dehydrogenase AMP Adenosine Inosine Xanthine oxidase O2 O2 - Xanthine oxidase Xanthine Hypoxanthine SOD Uric acid H2O2 Fe++ REPERFUSION Tissue injury . OH Proximity of muscle and other cells to endothelial cells in reperfusion injury • Direct contact between myofibers and other cells to endothelial cells allows passage p g of nucleotides via nucleotide transport proteins (NTP) that can be used in the xanthine oxidase pathway Muscle or other cell ATP-ADP-AMP-Adenosine-Inosine NTP NTP Adenosine-Inosine X.O. pathway Endothelial cell Mitochondrial activity contribution to ROS in reperfusion injury • Distal to NADPH dehydrogenase in the mitochondrial electron transport chain – Ubiquinone (CoQ) increases ROS f formation ti • ROS induce mitochondrial permeability transition (MPT) • These contribute to ROS formation and reperfusion injury 20 Increased superoxide generation from NOS during reperfusion injury • All three NOS (nNOS, eNOS and iNOS) can switch from NO to superoxide generation – This occurs with depletion of NOS substrate ( i i and (arginine d BH4) • Both arginine and BH4 are depleted by ROS (vicious cycle) • The increase superoxide production contributes to ROS damage and reperfusion injury Leukocyte infiltration with reperfusion injury • Hypoxic tissues, including myocytes and endothelial cells increase expression of leukocyte adhesion molecules l l – Enhanced leukocyte (neutrophil) infiltration with reperfusion – Increased activity of NADPH oxidase by infiltrating leukocytes • NADPH oxidase produces additional ROS Reperfusion injury therapy • Therapies – Needed to reduce ROS damage but yet allowing gp perfusion and return to normoxia 21 ROS lipoperoxidation • ROS commonly cause lipoperoxidation – Linoleic acid, the most common polyunsaturated fatty acid in cells – One radical reaction can oxidize 60 molecules of linoleic acid – One radical reaction can oxidize 200 arachidonic acid molecules • Lipoperoxidated fatty acids are short lived – Reduced by glutathione peroxidase to a nonradical, or – Interact with a metal to form: • Aldehydes: malondialdehyde and 4hydroxynonenal (HNE) • 4-HNE can interact with a metal to form an epoxide Free radical damage: targets • Predilection sites of radical formation/damage • Mitochondria – ETS-major site of free radical formation • Phagosomes, peroxisomes • Enzymes with divalent cations – CU++, Zn++, Fe++ – Via Fenton-like reaction, damage occurs at the enzymatic active site • Endoplasmic reticulum membranes • Damage to DNA – O OH- formed f in nucleus when H2O2, 2O2 a latent fform, diffuses ff to nucleus where it can be converted to hydroxy radical by the Fenton reaction – Peroxynitrite, diffuses longer distances and can cause DNA damage • Lipid • Lipoperoxidation, fatty acid loss • Protein and glycoconjugates • Oxidizes SH groups to S-S and crosslinks often at cysteine or methionine residues • NO – Nitration: NO binding to an amino acid – Nitrosation NO binding to SH, amine, or hydroxyl group • Oxidized proteins: – Reduced binding of transcription factors, p53, Rb, and enzymes such as kinases, phosphatases, and DNA repair enzymes. Oxidative effects on cell signaling 22 Free Radical Scavengers • Enzymatic – Superoxide dismutase, SOD • Converts (O2.-)superoxide anion to H2O2 • SOD1, cytoplasm (zinc/copper) • SOD2, mitochondria (mangenese) • SOD3, extracellular – Glutathione peroxidase peroxidase, GPX, GPX glutathione reductase (repairs S S-S) S) • Converts H202 to water (H2O); also reduces glutathione – Thioredoxin (repair S-S) – Catalase • Converts H2O2 to H20 ROS scavengers • Nonenzymatic (scavenge radicals) – – – – – Alpha tocopherol (vitamin E) Ascorbic acid (vitamin C) Retinoic acids (vitamin A) Lycopenes (Vitamin A-like) Flavinoids, genistein, resperpins, resveratrol, quercetins— electron donors – Ferritin, lactoferrin, ceruloplasm, hemoglobin, metallothionen, uric acid, histidine residues – Bind divalent cations or serve as electron donors – Glutathione (glutamic acid-cysteine-glycine), melatonin (an – NH group), histidine dipeptides (caronsine, anserine) Nonenzymatic scavengers • Nonenzymatic scavengers – Also termed low molecular weight antioxidants (LMWA) – Donate an electron to the radicals but not reactive themselves • A wide spectrum of activity--nonspecific – Better permeability throughout the cells than enzymes • Can penetrate cell membranes • Allows radical scavenging in more areas of the cell 23 Repair of lipoperoxidation of phospholipids: Vitamin E and C PL Fatty acyl CoA TOC-OH Lipoperoxidation O-ASC-OH PL-OO O=ASC=O HO-ASC-OH lysophospholipid PL-OOH 2GR SS 2GR-SS 2GSH GR-[SH2] GSSG TOC-O GRO-SS GRO-[SH2] FA-OOH GPX FA-OH 2GSH GSSG Phospholipid membrane 2GR-SS GR-[SH2] NADPH+ + H+ PL phospholipid PL-OO phospholipid that is oxidized (lipoperoxidation) TOC alpha tocopherol, Vitamin E ASC ascorbic acid, Vitamin C NADP+ GRO glutaredoxin GSH glutathione (GSSG [oxidized]) GR glutathione reductase GPX glutathione peroxidase Free radicals and the “penumbra zone” • Penumbra zone – A region around an area of necrosis, infarction that is hypoxic yp – Occurs stroke, myocardial infarction or other inflammatory disease • The tissue in the penumbra zone may or may or may not die, depending upon the amount of hypoxia and level of free radicals Penumbra zone 24 Free radical theory of aging • Harman Denhan – 1956 – Developed the “free free radical theory of aging” • A concept that tissue damage caused by free radicals results in a “wear and tear” type of cell injury that is prolonged and over the years, leads to aging changes in cells Oxidative stress versus reductive stress • Redox (reduction/oxidation) potential – The balance between reductants and oxidants – The “redox state” is tightly regulated in the cell, like pH • Free radical theory of aging and suggests that all oxidative stress is damaging – But this is not always true, some oxidative stress is needed for cellular physiology • Oxidation is needed for thyroglobulin binding to iodine and thus thyroxine activity • Oxidation needed for neutrophil/leukocyte killing and lysosomes – Many studies to prevent cancer and disease by use of antioxidant nutrients do not show great improvement • Reductive stress, the opposite of oxidative stress can also be overdone • A balance in oxidative and reductive stress is likely optimal – Eat a balanced diet and you’ll be ok • “Everything in moderation, including moderation itself” – Michael Tassler, 1984 Cell signaling and oxidative changes • GTP-binding protein families – RAS and RHO • RHO – Induces actin and cytoskeletal changes • RAS – Active when bound to GTP – Hras, Nras and Kras, three subtypes » Kras most ubiquitous expression – Farnesyltransferase modifies RAS post-translationally » Allows RAS to be come functional » Farnesyltransferase is targeted for therapies to reduce RAS activity – Epidermal growth factor (EGF) induces RAS activity » Leads to: RAF/MEK/ERK/C-jun/AP-1 (more following) » Also leads to: PI3K/AKT and inhibition of BAD (cell survival) » Also leads to: FALGDS and PLC/protein kinase C (cell prolifeartion) » All of the above increase cell survival, cell cycle progression and calcium signaling 25 RAS • RAS/RAF/MEK/ERK and RAS/MEK/JNK activate cJun pathway – C-Jun a part of AP-1 (activated protein 1) • AP-1 is a dimeric basic region-leucine zipper transcription factor – AP-1 is composed of c-jun and c-fos » Other Oth such h transcription t i ti factors f t are Maf M f and d ATF • AP-1 and these transcription factors bind – TPA response elements (TRE) and c-AMP response elements (CRE) – TRE and CRE are promoter regions of genes that encode: » Other transcription factors, matrix degrading proteins, cyclins, cell adhesion molecules, and cytokines. Mechanistic basis of free radical activity of signalling • Oxidative radicals can increase signaling pathways • Any step in these pathways can become mutated and increase activity – Acti Activation ation of gro growth th factor receptor (listed abo above) e) – Activate NFKappa B » OH- can induce release of NFKappaB inhibitory subunit – increased phosphorylation of an enzyme (kinase) – decreased dephosphorylation (decreased phosphatase activity) » ONOO- can nitrate (add N) to tyrosine (on tyrosine kinases) and block phosphorylation – Damage to cysteine residue on active enzymatic sites of regulatory phosphotases such as PTEN (inhibits PI3K) – Activate oncogenes » C-jun is activated by H2O2 as is MAPK Cell proliferation 26 Cell cycle • Induction from Go to G1 – Growth factors – Signaling • Cyclins • Checkpoints and inhibitors – G1 to S – G2 to M • Checkpoint control • Transcription and passage through G1/S – E2F activation Response of cells to injury DNA injury Dividing cells Non-dividing cells Inhibition of proliferation Apoptosis/death (Failure of repair) Cancer Repair Mutations Signaling pathways and receptors: some initiate cell proliferation • • • • • • PI3 kinase MAP-kinase IP3 pathway cAMP AMP pathway th Steroid pathway JAK/STAT pathway • Growth factor receptors – PI3K – MAPK – IP3 • Seven transmembrane receptor – cAMP – IP3 • Steroid receptor • Cytokine receptor 27 7-transmembrane receptor: Chemokines, (nor)epinephrine glucagon, serotonin, vasopressin, histamine, calcitonin, rhodopsin, parathyroid hormone Growth factor receptors: EGF, KGF, ILGF, PDGF, FGF, TGF alpha, VEGF, c-Kit PLC IP3-DAG Ras (GTP/GDP, Raf) PI3 K MKKK, MEK, ERK (MAPK) PLC Cytokine receptor: Interleukins, Interferons, EPO, G-CSF Hormone receptor: Thyroid hormone, Vitamin D, retinoids JAK JAK G proteins-ras Ca++ cAMP PKC PK A STAT Steroid receptor STAT-P C t k l t l proteins Cytoskeletal t i Ca++/K+ pumps Calpain Ca++ BP-calmodulin PKB/AKT Ion channels, vision, olfactory Steroid TF cMyc, cJun, cFos, Foxo3 PPAR NF kappa B Cyclins Cell proliferation Cell metabolism/differentiation Inflammatory/immune responses Abbreviations of signaling molecules • • • • • • • • • • • • • • • • • • • AKT—protein kinase B (below) RAF—(MKKK) MEK—(MKK) ERK—(MK) MAPK—mitogen activated protein kinase IP3 inositol 1,4,5 triphosphate PI3—phosphoinositol three kinase DAG--diacylglycerol PLC phospholipase C PLC—phospholipase PKA—protein kinase A (AKT) PKB—protein kinase B PKC—protein kinase C cAMP—adenocyl 3,5, cylic monophosphate JAK—janus activating kinases STAT—signal transducers and activation of transcription Ca++ BP—calcium binding protein Steroid TF—transcription factor PPAR—peroxisome proliferation activating receptor cMyc, c-Jun, c-Fos—transcription factors for cell proliferation • • • • • • • • • • EGF—epidermal growth factor KGF—keratinocyte growth factor ILGF—insulin-like growth factor PDGF—platelet derived growth factor FGF-fibroblast growth factor TGF—transforming growth factor VEGF V VEGF—Vascular l endothelial d th li l growth th factor C-Kit—stem cell factor EPO—erythropoietin G-CSF—Granulocyte-colony stimulating factor Ras gene induction of cell proliferation 28 Ras/Rho • Small GTPases • RAS subfamily – H-ras, N-ras, E-ras, r-ras, Rap, Ral, rit, rheb • Rho subfamily – Rho A, Rho B, Rho C, Rac 1, Rac 2 Cdc42/G25k • Ras mediates – PI-3 pathway – RAF/MEKK pathway • These induce transcription factors that increase transcription of Cyclin D Transcription factor regulation of proliferation by Myc • Myc – c-Myc, n-Myc, and L-myc – Leucine zipper transcription factors – Exacerbate or reduce gene expression • Rather than turning “on on or off” off – If growth conditions are good, Myc promote proliferation through: • Enhanced expression of: – D and E cyclins – Cyclin dependent kinase 4 (CDK 4) • Reduced expression of: – P21 (CIP1) and p15 (INK 4B) – If growth conditions are poor, Myc also may enhance apoptosis (see apoptosis) Cyclins • Proteins that complex with cyclin dependent kinases • When complexed and phosphorylated the CDK’s become active serine/threonine kinases – CDK’s are expressed constitutively • Cyclins rise and are degraded by the ubiquitin pathway rapidly • Cyclins allow cells to pass through the Cyclinmajor A points of the cell cycle: CDK 1 – Go-G1-S-G2-M 29 Cell cycle checkpoints • G1 checkpoint – Major checkpoint after cell signaling activation • prevent cells from entering cell synthesis • Best understood checkpoint • Synthesis checkpoint – Reduction R d ti iin DNA synthesis th i • Regulated by ATM • Cyclin A and E remain inactive • Least understood checkpoint • G2 checkpoint – Inhibitory phosphorylations of cdc2 • Prevents cell proliferation right before chromosomal separation • Cdc25C is complexed with 14-3-3 and unable to activate cdc2 – Cdc25C is a phosphataste inactivated by 14-3-3 • Partially understood checkpoint – better than S, less than G1 Cell injury The Cell Cycle p53 G0- “inactive” Cyclin B CIP/KIP CDK 1 EGF M Cdc25c-14-3-3 Cdc2 FGF 1 hr G2/M checkpoint + G1 IGF 8 hr Nutrients TGF B G2 - 2 hr G1/S checkpoint S checkpoint Cell injury p53 Cyclin D Cyclin A ATM CDK 1 CDK 4 S Cyclin A CDK 2 Cyclin D 8 hr INK4 Cyclin E CIP/KIP CIP/KIP CDK 6 p53 Cell injury p53 CDK 2 Ras/nutrient induction of cell proliferation: Getting cells out of Go Nutrients Growth factors TOR RAS S6K PI3K S6 phosphorylation 40S 60S TOR eIF4E S6 phosphorylation 40S 60S Cyclin D ERK S6K eIF4E Cyclin D Cyclin D Cell proliferation 30 Cyclin activation of transcription and passage through G1/S checkpoint p130 released allowing E2F activity p130 Transcription and passage through G1/S checkpoint DP p130 ATP E2F Cyclin D DP CDK 4 Cyclin D CDK 6 CD kinase activity Cyclin D phosphorylates RB E2F RB P P P RB-P CDK 2 P P P Phosphorylated RB releases E2F and also opens chromatin structure for transcription. P phosphorylation RB denotes retinoblastoma protein Histone acetylase phosphorylate RB, decrease E2F and close chromatin structure. E2F a transcription factor that enhances cell proliferation and passage across the G1/S checkpoint P130 inactivates E2F and prevents Go cells from undergoing proliferation DP a DNA binding protein Cell activities needed for proliferation and activated by cyclin/CDK’s and E2F transcription • • • • Nuclear envelope breakdown Centrosome function Spindle assembly Chromosome condensation 31 With cell injury: Inhibition of proliferation to allow repair • If cells have DNA mutations and continue replication the mutation is fixed and p passed to the daughter g cell • If proliferation is ceased, cells can then attempt repair P53: The King. Guardian of the Genome p53 activation CK1 CK2 DNA damage induces activation of: ATM (ataxia telangiectasia) ATR (ATM and Rad3-related protein kinases) DNA-PK (DNA protein kinase) p53 p53 phosphorylated* P53 phosphorylation at a.a. site 20 allows binding to MDM2 but prevents export to the cytoplasm where degradation normally occurs. Activated p53 ARF MDM2 Ubiquitylation of p53 by MDM2 (a Ubiquitin ligase) and increased cell proliferation GADD45alpha (DNA repair) p21 Inhibition of cell proliferation Apoptosis Repair Senescence *P53 levels are normally low in cells and bound to MDM2 (a ubiquitin ligase). Phosphorylation of p53 at site 15, 37 greatly enhances transcriptional activity. ARF – induced by c Myc, reduces MDM-2 mediated degradation of p53, thereby inhibiting proliferation and promoting repair or apoptosis 32 P53 modifications • Mutations in p53 itself – 18,000 – Especially occur in the DNA binding portion • Residues 98-292 • Post-translational modifications – Phosphorylation p y of serine and theronine residues – Acetylation • Phosphorylation and acetylation “stabilizes” p53 in the nucleus – Can also occur on mutated p53 resulting in nuclear accumulation • Upon dephosphorylation and deacetylation p53 binds DNA – Ubiquitylation, sumoylation (small ubiquitin-like proteins) p53 inhibition of cell proliferation: INK4 and CIP/KIP inhibition of cyclins • p53 enhances – INK4 inhibitors • • • • INK4a (p16) INK4b b (p (p15) 5) INK 4c (p18) INK 4d (p19) – INK inhibitors: • Reduce activity of: – Cyclin D by: – Prevention of cyclin D binding to CDK4 • p53 enhances – CIP/KIP inhibitors • WAF (p21) • KIP (p27) ( 27) • KIP2 (p57) – CIP/KIP inhibitors: • Reduce activity of: – Cyclin D, E, A, B by: – Forming heterodimers with the cyclin/CDK complex Therapeutic strategies to inhibit CDK • • • • • • Direct inhibitors CDK Prevention of CDK/cyclin binding Enhancement of CDK-I Inhibition of CDK-I degradation Inhibition of cyclin synthesis Promotion of cyclin ubiquitization (degradation) • Inhibition of CDK activating kinases and cdc25 phosphatases • Stimulation of CDK-I activation 33 Markers of cell proliferation Marker BrdU Method of detection Flow, IHC Sensitivity S phase 3H thymidine radioactivity S phase Ploidy Flow Other thymidine analoge Can’t distinguish G2 from M phase Ki-67 Flow, IHC PCNA Flow, IHC all proliferative cells broad Cyclins Flow, IHC All phases PHK2 Flow Sensitive AgNOR Light microscopy Not precise labile antigen stabile antigen, DNA lesions increase Used in vet med? easy BrdU = bromodeoxyuridine; ploidy = number of chromosomes [aneuploidy, diploidy, Tetraploidy]; PCNR = proliferating cell nuclear antigen; AgNOR = silver stain of Nucleolar organizing region Cellular senescence • Senescent cells, especially in stem cells, reduces proliferation and therefore, neoplastic transformation • With DNA damage – Lymphoid y p cells often undergo g apoptosis p p – Epithelial and mesenchymal cells often undergo senescence • Molecular mechanisms of senescence: – Telomere shortening • p53 activated and reduces cell proliferation – p16 (INK 4a)/pRB expressed (in response to oncogenes such as Ras, BRAF) • inhibition of cell proliferation • Without senescence, injured cells proliferate and more easily contribute to cancer development 34 Neosis • Some senescence may be “leaky” – A few cells that are senescent and destined to die by apoptosis may escape the senescent “mitotic crisis” • Senescent mitotic block by short telomeres, no telomerase, and of tumor suppressor genes such p53, pRB and p16 (INK 4a) • Neosis cells have no telomeres, but p53, pRB and p16 (INK4a) are inhibited (allowing proliferation) – These cells with no telomerase may get abnormal chromosomal joining – Telomerase may then also become active—resulting in additional lifespan/growth – Mutations readily increase – Escaped cells undergo nuclear budding and cytokinesis – Escaped cells undergo endomitosis of polyploid DNA, DNA misrepair, chromatin modulation • Nucleus forms small buds (karyokinesis) that end up in small regions of cytoplasm (cytokinesis) » “Raju cells” Cell Death and Apoptosis Response of cells to injury DNA injury Dividing cells Non-dividing cells Inhibition of proliferation Apoptosis/death (Failure of repair) Cancer Repair Mutations 35 Apoptosis • Occurs physiologically in: – Embryogenesis, hormone-induced involution (p (prostate secondary y to castration), breast tissue after lactation, cell deletion (gut crypts, thymic selectin of lymphocytes), tumor cells, cytotoxic T cells, secondary to toxins and viruses Apoptosis: cellular features Apoptosis Cells shrinkage, chromatin condensation, cytoplasmic blebs, phagocytosis, caspase activity, transglutaminase activity, DNA degradation dependent on calcium and magnesium, DNA ladders, annexin V expression, i phosphatidylserine h h tid l i expression i on outer membrane leaflet, lack of neutrophil infiltration Necrosis Plasma membrane damage, dilation of cytocavitary network, peripheralization of chromatin, infiltration of neutrophils Apoptosis Robbins and Contran Pathologic basis of disease, 7th edition 36 Apoptosis Robbins and Contran Pathologic basis of disease, 7th edition Apoptosis: 180-200 bp ladder A. Control B. Apoptosis C. Necrosis Robbins and Contran Pathologic basis of disease, 7th edition Apoptotic Thymocytes Susan Elomore: Toxic Pathol 35:495-516, 2007 37 Cytoplasmic Budding Susan Elomore: Toxic Pathol 35:495-516, 2007 Tingible Body Macrophage Susan Elomore: Toxic Pathol 35:495-516, 2007 Apoptosis: initiation/prevention • Initiators of apoptosis: – TNF, nitric oxide, fas ligand, granzyme, viral infection, radiation, corticosteroids, DNA damage • Extrinsic E t i i pathway th • Intrinsic pathway • Inhibition of apoptosis: – Growth factors, differentiation factors, adequate intracellular nutrition, insulin, others – Activates “survival signaling pathway” 38 p53 activation DNA damage induces activation of: ATM (ataxia telangiectasia) ATR (ATM and Rad3-related protein kinases) DNA-PK (DNA protein kinase) CK1 CK2 p53 p53 phosphorylated* P53 phosphorylation at a.a. site 20 allows binding to MDM2 but prevents export to the cytoplasm where degradation normally occurs. Activated p53 ARF MDM2 Ubiquitylation of p53 by MDM2 (a Ubiquitin ligase) and increased cell proliferation GADD45alpha (DNA repair) p21 Inhibition of cell proliferation Apoptosis *P53 levels are normally low in cells and bound to MDM2 (a ubiquitin ligase). Repair Senescence Phosphorylation of p53 at site 15, 37 greatly enhances transcriptional activity. ARF – induced by c Myc, reduces MDM-2 mediated degradation of p53, thereby inhibiting proliferation and promoting repair or apoptosis Susan Elomore: Toxic Pathol 35:495-516, 2007 Transcription factor regulation of apoptosis • Myc – c-Myc, n-Myc, and L-myc – Leucine zipper transcription factors – Exacerbate or reduce gene expression • Rather than turning “on or off” – If growth conditions are good, Myc promotes proliferation (see cell proliferation) – If growth conditions are poor, cells do not achieve the “survival threshold” and Myc enhances apoptosis 39 Myc enhancement of apoptosis • Myc can enhance apoptosis by: – Activation of the intrinsic death pathway (Cytochrome C release) • Through: – R Reduced d d BCL2 expression i (BCL2 iis antiapoptotic) ti t ti ) – Enhanced PUMA expression (PUMA is proapoptotic) – Synergy with death receptor signalling – Generation of ROS – Increased ARF gene expression • ARF inhibits MDM2 allowing increased p53 activity, increased p21 and other proteins that inhibit of cell proliferation – Allows p53 to induce apoptosis (above) Extrinsic/intrinsic apoptosis signaling • Perforin/Granzyme pathway – Granzyme A and B (serine proteases) • B activates Caspase 10, ICAD and cleaves Bid • A DNA nicking • Extrinsic signaling – Ligand/receptors • TNF alpha/TNFR1, Fas/CD95L(Fas Ligand)), Apo3L/DR3, Apo2L/DR2, Apo2L/DR5 • Death domains of 80 amino acids – Activates Procaspase 8 to caspase 8 (active) – Inhibited by cFLIP which binds adaptor protein FADD and caspase 8 and Toso which blocks the Fas pathway • Intrinsic signaling – DNA damage, UV damage, viral infection, toxins, hyperthermia, free radicals, cell injury • ATM/ATR-p53 activation – Direct activation of BAX – Puma, Noka inhibition of BCL-2 Apoptosis TNF alpha APO-2,3L Trail receptor 1-4 DR 2,3,5 Cytotoxic T cell expressing FasL (CD95 Ligand) TNF receptor 1 Fas (CD95) DNA damage/cell damage Procaspase 8 ATM/ATR Granzyme B p53 PETN PI3K Survival Signaling: EGF, PDGF, IL-2, 3, Insulin PDK1,2 AKT perforin Cytotoxic T cell: Perforin, Granzyme A, B Caspase 10, BID Caspase 8 Caspase 12 perforin BID Nucleus Mitochondria Caspase 12 Cytochrome C AIF, CAD, Endo G Granzyme A Initiator caspases: 2, 8, 9, 10, 12 SET BAD (binds BCL2) (AKT phosphorylates BAD, BAD-P binds 14-3-3 releases BCL-2) BAX Caspases 3, 6, 7 “Effector caspases” Transglutaminase X-link proteins Cytokeratins, PARP, Gelosin (actin cleavage) Fodrin, NuMA 14-3-3 Puma, Noxa Endoplasmic reticulum ICAD CAD mammalian endonuclease Endonuclease G AIF DNA degradation Effector caspases: 3, 6, 7 BCL 2 released BAX (forms pore) SMAC, Diablo Apaf-1 ATP Procaspase 9 Apaf-1 Aven IAP Survivin Caspase 9 Components of the “apoptosome” ATP Cytochrome C Denotes inhibition Denotes activity or transition Denotes movement Apaf-1 Procaspase 9 40 Caspases • Caspases – Precursor-pro-caspases • Removal of N-terminal domain during activation – Caspase 1, 4, 5 • Proteolytic activation of IL-1b, not as active with apoptosis • Involved with inflammation – Caspases 2, 3, 6-11, 12, 14 • Cysteine proteases involved with apoptosis – Caspase 12 • Endoplasmic reticulum-released apoptotic factor initiated by amyloid beta • Released from endoplasmic reticulum and induces activation of effector caspases – Caspase 13 • Bovine gene – Caspase 14 • Embryonic tissues – Initiator caspases • 2, 8, 9, 10, 12 – Effector caspases • 3, 6, 7 • There is no one key enzyme, protein, or signal responsible for the ultimate death and lethal blow to a cell. – In other words, apoptosis factors occur simultaneously BCL-2 families • Anti-apoptotic – Contain all BCL-2 homology (BH) domains, BH1-4 • Includes membrane anchoring, channel formation, and regulation domains • BCL2 prevents Bax formation of a pore – Reduces release of Cytochrome C and reduces apoptosome formation – Members: • Bcl-2, Bcl-xl, Bcl-w, Mcl-1, Boo/Diva • Pro-apoptotic – Fewer BH domains – Members: • Bax, Bak, Bok/Mtd, Bcl-xs (BH3, BH1, BH2 domains) • Blk, Bad, Bmf, Bid, Puma, Noxa (BH3 only domains) – BH3 proteins sense cell damage and act only through BAX and Bak Uptake of apoptotic cells • Appears to contribute to killing – Tingle-body macrophages form • These are macrophages with abudant cytoplasm and internalized portions of apoptotic cells • Apoptotic A t ti cells ll express – Phosphatidylcholine (PC) and phosphatidylserine (PS) on the outer surface • PC allows detection of apoptotic cells (“find me”) by macrophages • PS allows internalizatoin (“eat me”) by macrophages 41 a. “Find me” b. “Eat me” LPC = lipophosphatidylcholine. PtdSer = Phosphatidylserine, ox LDL = oxidized low density lipoprotein; CD36 = scavenger Receptor; BAI1 = brain angiogenesis inhibitor; ICAM-3 = intercellular adhesion molecule -3; CD14 = Lipopolysaccharide binding protein receptor; alpha v beta 3 intergrin; MFGE8 = milk fat globule EGF factor 8; MER = receptor tyrosine kinase; GAS6 = growth arrest-specific 6; Nature Rev: Immunol 7:964-973, 2007 Non-apoptotic cell death • Resistance to apoptosis is a hallmark of cancer cells • Defects in non-apoptotic p p cell death are associated with cancer • Non-apoptotic cell death: – Senescence – Necrosis – Autophagy – Mitotic catastrophe Senescence • From Previous slide during cell cycle lecture: • Senescent cells, especially stem cells, reduces proliferation and therefore, neoplastic transformation • Occurs by: – Telomere shortening – p53 activated and reduces cell proliferation – p16 (INK 4a)/pRB expressed (in response to oncogenes such as Ras, BRAF) – inhibition of cell proliferation • Also, new today: – Senescent cells have: • Flattened cytoplasm, increased granularity, changes in metabolism • Induction of senescence-associated beta galactosidase (SA-beta Gal). 42 Necrosis • Unregulated cell death with release of intracellular components – membrane enlargement (stretching) due to swelling – cell swelling • dilation of cytocavitary network – vacuoles, nuclear membrane, sER, rER, mitochondria – fragmentation of the nuclear chromatin ultrastructurally – Failure of ion transport, ATP production, and pH balance – Inflammation (neutrophils, inflammatory mediators) and damage of surrounding tissue Autophagy • Type I programmed cell death • Type II programmed cell death – Apoptosis – Autophagosomes and autophagolysosomes accumulate • Occurs with – Growth factor withdrawl – Differentiation and developmental triggers » I do not know a specific example – Massive cell elimination – When phagocytes do not take up dying cells – – – – Caspase-independent Increased lysosomal activity Protein translocation to autophagosomes Signalling involves phosphotidylinositol 3-kinase (PI3K) and target of rapamycin (TOR) • Defects in this signaling pathway lead to cancer • Defects in beclin 1 gene, which works with PI3K to induce autophagy lead to cancer Autophagy • A normal cellular process • Contributes to cellular homeostasis for turnover of organelles and superfluous proteins • Maintains an amino acid p pool for g gluconeogenesis g and protein synthesis during starvation • Cell death (for when phagocytes cannot keep up) • Anti-aging mechanism for removing substances oxidized by free radicals • Triggered by nutrient depletion (starvation) 43 Autophagy • Type I programmed cell death – Apoptosis • Type II programmed cell death – Autophagosomes and autophagolysosomes accumulate • Occurs when – massive cell elimination occurs – When phagocytes do not take up dying cells – Autophagsomal activity reduces the cellular “mass” making type I apoptosis more efficient in developmental cell loss • Type I and type II programmed cell death are not mutually exclusive Cellular degradation of macromolecules and organelles • Microautophagy – Degradative material is delivered to the lysosomes by membrane invaginations of cytoplasm into the lysosome • Macroautophagy – Large organelles (especially mitochondria, endoplasmic reticulum and peroxisomes) within a double membrane vesicle that are degraded in autophagosomes that fuse with lysosomes • Chaperone-mediated autophagy – Degradation of specific proteins that are bound to chaperonens and internalized into the lysosome • Degraded by calcium-dependent cysteine proteases, calpains, proteasomes AVi-initial autophagosomal vacuole, contains a mitochondria and rER rER around the autophagosome Degraded material Partially degraded rER Kelekar: Ann NY Acad Sci 1066:259-271, 2005 44 Mechanisms of autophagosomal assembly • LC3-microtubule associated light chain arranges, structurally, the initial endosome • LAMP1/2 (lysosome-associated membrane protein) adhere to the phagosomal membrane as it matures and fuses with lysosome • Acid phosphatases and cathespins contribute to degradation degradation. • Inhibition of autophagosomes – Growth factors that activate TOR – TOR inhibits autophagosome formation • Induction of autophagosomes – Nutrient and amino acid starvation • Activate Beclin-1, p150 and Class III PI-3Kinase • Also activated ERK ½ and GAIP (which initiates GTP degradation to GDP); GDP induces autophagosome formation Kelekar: Ann NY Acad Sci 1066:259-271, 2005 Kelekar: Ann NY Acad Sci 1066:259-271, 2005 45 Autophagy and aging • Cells with limited mitotic activity • Accumulate more cell degradation products (garbage) – Can reduce cell adaptability, viability, and function • Cells can undergo apoptosis – Lipofuscin-is a main “waste” substance • Derived principally from incompletely degraded mitochondria – Mitochondria become damaged with time due to ROS production that injures mitochondrial DNA, mitochondrial proteins, and mitochondrial processes like fission/turnover • Lipofuscin and ceroid become difficult to degrade in lysosomes/autophagosomes and therefore accumulates Autophagy in disease • Especially important in diseases of non-dividing cells of the nervous system, muscle or when protein turnover is critical – Vacuolar myopathies • X-linked myopathy, inclusion body myositis, Marinesco-Sjorgren syndrome – Danon disease (cardiomyopathy and retardation) • Deficiency in LAMP-2 – Parkinson’s disease, Huntington’s disease, Alzheimer’s disease, and spongioform enephalopathies • Protein aggregates occur in these diseases; autophagosomes degrade the protein aggregates to a point Mitotic catastrophe • Death that can, and hopefully does occur when DNA damage does not induce cell arrest, DNA repair or apoptosis and G2 check point is defective – Allows cell to enter mitosis with mutations/defects or prematurely – Cell death • Can also occur with microtubule damage and disruption of the mitotic spindle • Mitotic giant cell formation/multiple micronuclei • But if cells in mitotic catastrophe escape death • Neosis • See above mitotic giant cells and micronuclei – These sound like karyo and cytokenesis and Raju cell formation • Survivin – Promotes cell proliferation and avoid mitotic catastrophe • Both of these can contribute to cancer – Loss of survivin promotes apoptosis • Inhibition of survivan may promote death and be a good therapy 46 Stem cells and cancer • Long-term residents – DNA lesion occur over decades – Especially lesions in pathways regulating stem cell renewal • Wnt and beta catenin, Hedgehog, Notch, Myc and PTEN – Stem cells (epithelial) grow in niches that have the right microenvironmental nourishment for replication • Symmetrical division – Two progeny cells in the niche • Asymmetrical division – One stem cell in the niche – One stem cell out of the niche » This one out of the niche losses it “stemness” and become differentiated or dies Genetic variation: SNPs. Epigenetic events: DNA methylation, HDACs,and polyamines (telomeres covered by Kuipel) SNP’s and cancer • Single nucleotide polymorphisms (SNP’s) – Single-base variations in DNA between individuals tied to traits and disease • Code red hair, freckles, pudginess, love of chocolate g cancer • Genetic risk for disease, including • There are 15 million locations in the genome where one base can differ between individuals – 3 million SNPs identified by HapMap – United Kingdom has heavily investigated: » Rheumatoid arthritis, bipolar disorder, coronary artery disease, type 1 diabetes, and Crohn’s disease • Other genetic variations – Gene copy number account for 20% of differences in gene activity; SNP’s account for many of the rest – Chromosomal inversions, insertions, deletions 47 SNPs and cancer • SNPedia – Website of SNPs, including cancer – www.snpedia.com – Visit during class • Science “Breakthrough of the Year” – Human Genetic Variation – Science 318:1842-1843, 2007 – Discuss during lecture • Sequenome • Projects – A company in San Diego that does high-throughput SNP identification through mass spectroscopy – SeatlleSNPs Variation Discovery Resource – Cancer Genome Anatomy Project • SNP500 Cancer project – NIH’s Pharmacogenetics Research Network – International HapMap project SNP’s • SNP – A SNP differs by a single base at a given position in the genome with a frequency of 1% in at least one population – Account for 90% of the total variation in the human genome • Other types of genetic variation include: – Nucleotide repeats, microsatellites, gene amplification, chromosomal amplification – Nonsynonymous SNP shifts from one amino acid to another – Synonymous SNPs have no amino acid change SNPs and disease (cancer) • SNPs associated with cancer susceptibility – N-acetyltransferase 2 (NAT2) – Glutathione S Transferase (GSTM1) • • • – Myeloperoxidase – Also SNPs in • • • • SNP in the promoter at G-463A is associated with decreased lung cancer risk in Caucasians cytochrome P4501A associated with lung cancer risk TNF and IL-1 associated with diffuse B cell lymphoma SNPs and cancer outcome – • Slow acetylations increase risk for bladder cancer, especially in smokers Rapid acetylations increase risk for colon cancer Homozygous individuals at increased risk for bladder and colorectal adenoma Cytochrome P450 enzyme CYP3A4 associated with increased long term survival Pharmacogenetics and cancer therapy SNPs – Thiopurine S methytransferase – Cytochrome P450 CYP2C • • – Metabolism of alkylating agents 5, 10 methylenetetrahydrofolate reductase (MTHFR) • – Increased thiopurine toxicity Increased methotrexate toxicity UDP-glucurosyltransferase (UGT1A1) • Increased irinotecan toxicity 48 Epigenetic inheritance • Definition: Cellular information, other than the DNA sequence itself, that is heritable during cell division. • Types of epigenetics information: • • DNA methylation. Histone Modifications. DNA methylation • Occurs in cytosines that precede guanines • Dinucleotide CpG. • 60-90% of all CpGs are methylated in mammals. • Unmethylated CpGs are grouped in clusters called “CpG islands” that are present in the 5' regulatory regions of many genes. DNA methylation and gene expression • DNA methylation – Covalent binding of a methyl group to the 5-carbon position of cytosine/guanine dinucleotides (CpG) • Termed m5CpG – 60-90% of cytokine/guanine sites are methylated in repetitive regions of DNA – Hypomethylated sites are usually in CpG-rich CpG rich regions (CpG islands) near promoter regions • Often near the core promoter and transcription start site • Transcriptions occurs if: – – – – Transcription factors are present The CpG island is unmethylated The chromatin state is “open” Histones are hyperacetylated (opens chromatin structure; more later) • Many promoters do lack CpG sites – By evolution, these were lost due to deamination of m5C and loss – Methylation of CpG islands reduces transcription • Inhibits binding of transcription factors • Promotes binding of methylated DNA binding proteins 49 DNA Methylation Reaction Catalyzed by DNA Methyltransferase (DNMT) DNA Hypomethylation Some toxic carcinogens act through methylation alterations: • Cadmium inhibits DNMT activity . • Arsenic induces Ras hypomethylation in mice. Dietary relation: • High dietary methionine increases methylation leading to low cancer incidence. DNA Hypomethylation and mechanisms of tumor formation Generation of chromosomal instability. • Hypomethylation causes recombination and chromosomal rearrangements leading to deletions and translocations. • Depletion of DNA methyl transferases leads to aneuploidy. 50 DNA Hypomethylation and mechanisms of tumor formation Genomic Imprinting. • It is the relative silencing of one parental allele compared to the other parental allele • Maintained partly by differentially methylated regions. • In cancer, hypomethylation disrupts imprinting called ‘Loss of imprinting (LOI)’. eg LOI of IGF-2 causes Wilms tumor. DNA Hypermethylation in tumors • Hypermethylation of CpG island in the promoter regions of tumor suppressor genes. • Results in silencing of gene. • First reported in Retinoblastoma tumor suppressor gene. • Followed by BRCA1, VHL, p16 INK4a. • DNA repair gene also silenced further increasing the chances of cancer. In normal cells (top) DNA methylation is concentrated in repetitive regions of the genome and most CpG island promoters are unmethylated. In tumor cells, the compartmentalization breaks down and repetitive DNA loses methylation while CpG island promoters acquire it, resulting in silencing of the associated gene. 51 DNA methylation and cancer cells • Tumor cells often have: – Global hypomethylation of repetitive DNA – Region-specific hypermethylation (esp CpG regions) • G Global oba hypomethylation ypo e y a o – Leads to chromosomal instability/mutation • Seen with oncogenes, c-myc, ras – Results in activation of these genes – Gene-specific hypomethylation also occurs • MAGE, S100A4 • Regional hypermethylation of CpG islands – Seen with tumor suppressor genes • Results in decreased tumor suppressor gene expression Inactivation of both alleles of a tumor suppressor gene. One allele can be inactivated by methylation and The second inactivated by point mutation, methylation or deletion. Gronbaek K, et al: APMIS 115: 1039-1059, 2007 52 DNA methylation pathway: SAM SAM-universal methyl donor to DNA, RNA, hormones, neurotransmitters, membrane lipids, proteins and other molecules DNA Methylated DNA (DNA methyltransferase) S adenosylhomocysteine (SAH) S adenosylmethionine (SAM) Adenosine Homocysteine THF Cobalamin (B12) Methionine pyridoxine (B6)) riboflavin (B2) Tetrahydrofolate (THF) Methyl Tetrahydrofolate (THF) Vitamins B2, B6 and B12 and folate (THF) involved above Diet Methyltransferases • 75% of methyl groups from SAM – Go to the formation of phosphatidylcholine • 25% of methyl groups from SAM – Go to methylation of DNA • SAM: – Is broken down to gluthathione – Has antioxidative properties in this regard DNA methylation • Can result in point mutations • Hypomethylation • Promoter hypermethylation – Loss of m5C and converstion to T – Can lead to up-regulation of non-desired genes • Cellular proliferation, decreased apoptosis – Reduces tumor suppressor gene activity • Examples: – – – – – – Autonomous growth (Ras, SOCS) Enhanced proliferation (p15, p16) Reduced apoptosis (DAPK) Tumor invasion (CDH1, TIMP) Angiogenesis (THBS1) Genome instability (MGMT, LMNA, MLH1, CHFR) • miRNA methylation • Methylation can also be a biomarker (hypermethylation of the above genes) 53 Histone Modifications • Histones undergo posttranslational modifications which alter their interaction with DNA and nuclear proteins. t i • The H3 and H4 histones have long tails protruding from the nucleosome which can be covalently modified at several places. • Modifications of the tail include methylation, acetylation, phosphorylation ubiquitination etc Histone Modifications • Acetylation is associated with transcriptional activation . • Effect of histone methylation depends on the amino acid residue and its position in the histone tail. Transcription active HAT adds Ac H3L4 trimethylation Transcription inactive H3L9 trimethylation MBD is a protein that binds m5CpG Attracts HDAC which removes Ac Gronbaek K, et al: APMIS 115: 1039-1059, 2007 54 Histone acetylases and histone deacetylases • Histone acetyltransferases (HAT) – Contributes to enhanced transcription of genes • Adds Ac • Relaxes the chromatin structure • Increased transcription • Histone deacetylases (HDAC) – Contributes to reduced transcription p of g genes byy • Ac removal • Tightens the chromatin structure • Decreased transcription HDAC, HAT, and HDAC inhibitors TF = denotes transcription factor Kim T-Y Bang Y-J, Robertson KD: Epigenetics 1:1 14-23, 2006 Histone acetylases and histone deacetylases • Histones – 146 nucleotide wrap around histones = nucleosome – H1 not involved with acetylation – H2A, H2B, H3, H4 • Lysine-rich tails are acetylated by HATs which removes the negative charge of lysine and allows relaxation of interaction between negative histones and the positive DNA – Histone acetyltransferases (HATs) • Acetylate lysine residues of histones – This enhances transcription – Transcription also enhanced by methylation of lysine 9 of histone 3 • Acetylation also of E2F (key to cell proliferation), proliferation) p53 p53, GATA – Regulates transcription of genes for cell proliferation – Increase cell arrest, apoptosis, and cell differentitation = decreased cancer – Histone deacetylases (HDACs) • Deacetylate lysine residues on histones • Transcription reduced by methylation of lysine 4 of histone 3 – May allow inhibition of tumor suppressor gene • Repress (decrease) gene transcription by removing the charge-neutral acetyl groups – Enhances gene transcription of other genes – Histone deacetylase inhibitors (HDAC-I) • Allows acetylation and thus gene transcription • Resultingly acts like HATs to decrease cell cycling (cell arrest), increasing apoptosis, and differentiation thus decreasing cancer • Later this semester: Cancer therapies; HDAC-I 55 HDAC inhibitors (HDACi) and transcription • Inhibition of HDAC (with HDACi) would conceiveably increase transcription of all genes – Due to increased acetylation and relaxed chromatin structure – For cancer therapy, could up regulate expression of a tumor suppressor gene • 20% of all genes are affected by HDACs • In fact, acetylation increases or decreases transcription – The ratio of up-regulated to down-regulated genes is 1:1 • Therefore, up and down regulation is equal • The HDACis may – Increase tumor suppressor gene expression – inhibit cell repair of neoplastic cells and also affect proliferation rates • http://www.methylgene.com/HDAC_animat.swf Acetylation effects on the cell • Acetylation occurs in: – Histones, as discussed • FYI, proteins can be acetylated, methylated, ubiquitinated, phosphorylated, ADPribosylated and deiminated. – Transcription factors • The acetylation occurs on lysine which is also the site of ubiquitin – Therefore, can affect degradation – Importin • Import export of proteins across the nuclear envelop – p53 • Regulating cell proliferation, repair, apoptosis – HSP90 • Protein stability – STAT3 • Cytokine signaling – Microtubules • Cell structure – Ku70 • Allows Bax to be free – Apoptosis – HMGB1 • A cytokine-like protein involved with inflammation, fever, and nausea Protein regulation by chaperone proteins • Chaperones proteins: – Constitutively expressed in normal cells • (1-2% of total cellular protein content) – Function as complexes with adaptor molecules and co-chaperone proteins – Do not covalently modify the substrate or “client molecules” on which they act • Interact with client proteins via cyclical binding pockets 56 HSP 90 HSP 70 HSP 70 HSP 60 HSP 10 Chaperones are required for essential housekeeping functions • Chaperones mediate – de novo protein folding during polypeptide-chain synthesis – translocation of protein across membranes – Quality control in the endoplasmic reticulum – Normal protein turnover – Post-translational regulation of signaling molecules – Assembly/disassembly of transcriptional complexes – Processing of immunogenic peptides by the immune system 57 a. Prevents aggregation b. Intracellular trafficking c. Maintains proteins in stable state for alterations such as ligand binding, phosphorylation or multi-subunit assembly d. Target proteins for degradation Chaperones as Heat Shock Proteins (HSP) • Upregulated in conditions of cellular stress – – – – – Heat Hypoxia Cellular Starvation Radiation Exposure Exposure to chemical mutagens • Upregulated in tumor cells – General hypoxic environment – Rapid cell proliferation – Function as biochemical buffers for extensive genetic heterogeneity that is characteristic in tumors Nature Reviews Cancer 5, 761-772 (2005); doi:10.1038/nrc1716 HSP90 AND THE CHAPERONING OF CANCER 58 Chaperone Proteins act on client proteins as complexes Drug Discovery Today Vol.9, No. 20 October 2004 ATP driven conformational changes is needed for chaperone/client protein interactions Stryer, Biochemistry, Fourth Edition HSP upregulation in tumor cells • HSP90 – Maintaining cellular signaling protein stability (hormone receptors and protein kinases) • HSP70 – Stability of multi-protein complexes • ER chaperones – Folding and maturation of immunoglobulins and MHC class I molecules – Calreticulin, calnexin, tapaisin, protein disulfide isomerase 59 Inhibiting HSP to treat cancer • Several experimental protocols are being used to target the chaperone proteins that are highly abundant in tumor cells and use them as therapeutic targets • HSP70 and HSP90 are the most abundant chaperone proteins identified in tumor cells, cells and the focus of most research • Many oncogene protein products are “clients” of HSP90 – ErbB2, EGFR, Bcr-Abl tyrosine kinase, Met tyrosine kinase, cRAf, b-Raf, androgen and estrogen receptors, HIF alpha and telomerase • Inhibition of HSP90 leads to degradation of the above oncoproteins Drug Inhibition of HSP90 RNA 60 • Much of the genome codes for mRNA – Some of this is spliced out as introns • 0.5% of the genome codes for rRNA • 0.2% of the genome codes for tRNA • 0.0?% codes for miRNA – Thus, most of the supercoiled genome codes for mRNA • A lot of the mRNA genes are not heavily transcribed • Whereas, Whereas rRNA and tRNA genes are often repeatedly transcribed • Of the transcription activity in a cell • mRNA > rRNA > tRNA – Of the transcripts that result in actual proteins: • rRNA > tRNA > mRNA • Thus much of mRNA is modified, spliced, or unstable and does not result in protein in comparison to r and t RNA – r and t RNA are more “routine” RNA’s and proteins RNA polymerases miRNA (more later) DNA RNA Polymerase I 39% DNA polymerases Pre-RNA: Pre-ribosomal RNA RNA Polymerase III 3% snRNA Pre-messenger RNA (hn RNA) 1. Modification (Sno RNA, nucleolus) 2 Processing 2. (Sno RNA) 3. Self splicing (group I intron) RNA: Gm7 5.8S RNAase P Pre-transfer RNA 1. Modification (poly A tail and 5’ cap) 2 Splicing 2. (sn RNA) 3. Editing (var.; gRNA) 4. Transport 5. Stability (ds RNA) 1. Modification (base, ribose) 2 Processing 2. (ribozyme) 3. Splicing (enzymatic) 4. Editing (enzymatic) mRNA rRNA 18S DNA synthesis and cell replication RNA Polymerase II 58% tRNA A (200) 28S 1% 17% 79% Protein RNA nomenclature Pre-rRNA: precursor of ribosomal RNAs, 5.8s, 18s, 28s Pre-mRNA: precursor of mRNAs, contains exons and introns Pre-tRNA: precursor of tRNA, contains several tRNA CAP: Modified guanosine at the 5’ end of all RNA polymerase transcripts A (200) Polyadenylated tail of mRNA RNAase P: sn RNA: catalytic (ribozyme) that processes the 5’ end of tRNA small nuclear RNA, U1-U6, generally U-rich, pre-mRNA splicing and pre-rRNA processing sno RNA: small nucleolar RNA, 200 different types involved with rRNA modification and processing Group I intron: self splicing RNA sequence in 28s ribosomal RNA RNA modifications: RNA stability (cap, poly A tail), formation of tertiary structure, interaction with RNA proteins (more later) Transcription of rRNA and tRNA as precursors: ensures equal amounts of RNA Pre-mRNA splicing: enlarges protein repertoire (more than alternative splicing), it has evolutionary advantages 61 RNA polymerase II holoenzyme for mRNA generation DNA arranged in a loop-like structure for RNA polymerase II transcription RTF RTF Adaptor 80 60 40 RTF-regulatory transcription factors 250 30a 110 30b RNA-Pol II 150 TBP +TFIIB +TFIIE +TFIIF +TFIIH Holoenzyme General transcription factors (GTF): TFIID, TFIIB, E, F, H mRNA stability and transport after synthesis • • • mRNP (mRNA proteins) are adaptors of mRNA to allow it to interface intracellular machinery for subcellular location, translation, decay with miRNA, and signal transduction – Some mRNP components are activators/others repressors Most mRNP components (proteins and miRNA for degradation) bind specific recognition elements in the untranslated 5 prime or 3 primer regions. A few mRNP components (proteins) target: – 7 methyguanosine cap, five prime end (CBC20/80) – Poly A tail at three prime end • PolyA binding protein nuclear • PolyA binging protein cytoplasmic (eIF4E, PABP) – 5’ end for transcription (eIF4G) – Y box proteins • Packing proteins along the length of mRNA – EJC (exon junction complexes) mRNA stability and transport after synthesis • mRNA export adaptor proteins – Target mRNA to nuclear pore for exit – Exportin 5 62 mRNA stability and transport after synthesis • mRNA export and fates in the cytoplasm – Exported by five prime end and immediately engaged with ribosome – Exported by non-five prime end, not engaged with ribosome • Transported to specific sites of cytoskeleton • Co-localized with other mRNA – Allows close interaction of protein subunits shortly after translation – Stored translationally silent • eIF4E, CPEB, and EJC attached mRNA moves to a cytoplasmic site for translation; co-localizes with other mRNA; promotes assembly of protein units NUCLEUS --mRNA exported non- 5’ first CYTOPLASM mRNA circular and translationally silent mRNA strand Nuclear pore eIF4G EJC (exons) mRNA export adaptor for nuclear pore Poly A binding proteins eIF4E, PABP; also CPEB, EJC --mRNA exported 5’ first and translated Poly A binding protein CBC20/80 ribosome mRNA degradation • mRNA degradation – – – – Polyadenylases degrade Poly A tail 5 prime cap removed by decapping enzymes RNA body y degraded g by y exonucleases mRNA endonuclease cleavage • Sequence specific • occurs by RISC in association with miRNA – Abberent mRNA’s • Premature translation stop signal (PTC’s discussed last week) • Lack a translation signal (nonstop RNA) – These occur by mutation, missplicing, premature polyadenylation • More comments on post-transcriptional regulation later • Stress cells and mRNA degradation (next slide) 63 mRNA stability and transport after synthesis • P bodies and Stress Granules • P bodies (PB): – Cytoplasmic processing bodies that form around aggregates of RNP not involved in translation – These RNP RNP’s s are targeted for PB’s PB s by miRNA/RISC • Stress granules – Retirement homes • Under stress, there is global translation arrest of “housekeeping” gene transcripts • Stress granules are composed of: – Inactive mRNP’s, 40S ribosomal units, mRNA binding proteins TIA-1 and TIAR » TIA-1 and TIAR have prion like domains that self oligomerize and promote assembly – When stress relieved, stress granules disassemble More on mRNA post transcriptional processing and it’s effect on RNA stability • mRNA structures that affect post transcriptional processing –5 5’ cap – 5’ untranslated region (5’ UTR) – Open reading frame (ORF) – 3’ untranslated region (3’ UTR) – 3’ poly adenine tail RNA post-transcriptional modifications for stability or degradation • 5’ Cap – Methylation of cap regulates expression – Cap prevents degradation • Increases stability of mRNA • 5’ UTR controls mRNA stability by – It’s ribosomal entry site – Actions on the 5’ UTR by upstream ORF – 5’ polypyrimidine sequences – RNA secondary structure 64 RNA post-transcriptional modifications for stability or degradation • ORF in mRNA regulate post transcriptional mRNA stability and activity by – Unusual codons • These can slow translation • 3 3’ UTRs affected by – RNA binding proteins (RNA BP) • Alter translation (can increase or decrease translation) • Tag mRNA for degradation – Decreased mRNA stabilithy • Protect UTR from nucleases – Increased mRNA stability – RNA BP (CBC20/80, eIF4G, eIF4E, PABP) altered by • Mutations of the RNA BP gene • Increased UTR target sequences for the RNA BP to bind • Decreased UTR target sequences for RNA BP binding RNA post-transcriptional modifications for stability or degradation • Poly AAAAA tails – Prevent degradation – Long tails have efficient translation – Short tails inefficient translation RNA post-transcriptional modifications for stability or degradation • Signaling affects on stability – MAPKK – Increase RNA BP • PABP-1 and TTP • These reduce deadenylation of the poly AAAA tail • This increases stability and increases translation 65 RNA silencing • Small RNA’s from inside or outside the cells are processed by iRNA machinery to inhibit g genes and p proteins by: y – Cleaving mRNA’s • siRNA fully complementary to mRNA – Blocking protein synthesis • miRNA partially matched with mRNA – Inhibiting transcription (RITS) • Complex enters nucleus and inhibits chromatin mi and siRNAs • iRNA’s – Term for microRNA (miRNA), small inhibitor RNA ((siRNA)) and intermediates – 20-26 nucleotides in length – Also: repeat-associated small interfering RNA’s (rasiRNA) miRNA – MicroRNAs (miRNAs) – RNA polymerase II makes miRNA • • • • • • • miRNA are double stranded 21-25 nucleotides long Derived from short hair-pin precursors 1000 miRNA genes (non-protein encoding) currently known 400 different types 30% of human genes regulated by miRNA Coded in introns from mRNA or snoRNA (small nucleolar RNA [sno RNA, a.k.a.: ribosomal RNA]) – miRNAs are pieces of mRNA from the non-protein coding regions • Pri-miRNA’s (folded) – Come off RNA polymerase II with mRNA and bind Drosha and Pasha – Double stranded (due to folding) • Pre-miRNA – – – – Formed after Drosha and Pasha cleavage Bound to exportin, transported to cytoplasm Cleaved by dicer Double stranded 66 CYTOPLASM NUCLEUS RNA poly II miRNA gene Pri-miRNA Pre-miRNA (Tails released by Drosha) Dicer miRNA • Dicer protein/enzyme – Converts pre-miRNA to mi RNA by cleaving g off the p pre-miRNA loop p end – Then eliminates one RNA strand • 5 prime end preserved and enters RISC complex – Less stable, unwinds more easily siRNA • Short-intefering or silencing RNA – Long ds RNA and mi RNA • si RNA sources of formation: – Endogenous • Not fully defined – By synthesis – By some viruses • Delivered for therapy by: – Gene (viral) vectors – Synthetic delivery 67 Wikipedia.com si and mi RNA activity on gene silencing Long ds RNA micro ds RNA Dicer R2D2 ATP Occurs spontaneously Occurs with viral infection Can occur experimentally Encoded in the genome Single stranded si RNA RISC is composed of: Argonaut protein and ss RNA Argonaut proteins Enters nucleus and inhibits transcription mRNA degradation if si RNA is fully complementary Inhibition of translation/protein synthesis if mRNA is not fully (only partially) complementary RISC • RISC complex – RNA induced silencing complex (RISC) • Composed of: – Argonaut A t protein t i and d smallll single i l strand t d off RNA • Mediates: – mRNA cleavage » si RNA fully complementary – Protein synthesis inhibition » mi RNA partially mismatched – Transcriptional gene silencing » RITS » A complex that enters the nucleus and affects chromatin 68 • mRNA cleavage by RISC – RISC (siRNA and argonaut protein) plus mRNA • siRNA is sequence specific to that mRNA • mRNA is destroyed • Blockage of protein synthesis – RISC ((mi RNA and argonaut g protein plus mRNA)) • mi RNA is only partially matched with mRNA • mRNA unable to enter ribosome for translation • Transcriptional gene silencing – RITS complex (RNA-induced transcriptional silencing (RITS) complex) • Composed of: – RISC (si RNA and argonaut protein) – Chp1 and Tas 3 proteins • RITS transported to the nucleus • siRNA within the RITS – Matches up with chromatin nucleotides – Also binds methylated lysine 9 histones • RITS/chromatin complex enlarges (other proteins bind) – TRANSCRIPTION (RNA polymerase binding) prevented How do iRNAs affect cancer? • miRNAs can enhance or repress mRNA translation – miR369-3 stimulates translation during arrested cells (non-proliferating) – miR369-3 reduces translation in dividing cells • Science 318:1877, 2007 • Improper activation of mRNA regulation in cancerous cells – Oncogenic miRNAs (oncomiRNAs) • miR-155 enhances cell proliferation • miR17-92 iR17 92 reduces d c-myc apoptosis t i actions ti • miR-21 inhibits TPM and apoptosis • miRNAs may alter transcription factors, enhance oncogenes, reduces tumor suppressor genes, and increase angiogenesis in tumors • American J Pathol 171:728, 2007 • Some miRNAs inhibit cancer – miR-15 and miR-16 inhibit BCL-2 allowing apoptosis – Let-7a inhibits RAS iRNA as a therapy • Therapy – siRNA delivery • High pressure tail vein – Recent work used low volume and normal pressure – Problem: excretion » There is rapid excretion » Degradation g is less of an issue • Vector delivery – Retrovirus, adenovirus, adeno-associated virus (AAV) with various promotors » Problems with non-specific delivery to unwanted sites, interferon reactions to the hairpin RNA • Benefit – Knocking down genes with precision » Other genes unaffected – siRNAs are more potent and longer-lasting than oligonucleotides (antisense DNA) and ribozymes » siRNAs have a greater potency in turning off genes • Hurdles: – Delivery to the correct neoplastic cell (“either” deleted) » This is true for systemic delivery or vector delivery – Continued activity over time (repeated administration needed possibly) 69 Chronic cell injury caused by intracellular accumulations of metals, bilirubin Ackermann Lipid accumulation Protein accumulation Lysosomal storage diseases Iron, copper, bilirubin, porphyria accumulation Robbins and Contran Pathologic basis of disease, 7th edition Other: hyperglycosylation Intracellular accumulation of metals • Iron (Fe++) and Copper (Cu++) • Accumulation of one or both results in cellular degeneration – Commonly hepatic degeneration/cirrhosis – Mechanism of toxicity: • Induction of free radical formation – Fenton reaction – Occurs in the region of the ion This region is often an important site for enzymatic activity, protein structure/conformation, signaling activity • Absorption and regulation – Iron body stores are regulated at the level of absorption by enterocytes (intestine) – Copper body stores are regulated at the level of excretion liver (excretion into bile) 70 Iron absorption Intestinal lumen Exfoliation of enterocyte Site of enterocyte exfoliation Intestinal wall Intestinal enterocytes Intestinal lumen Intestinal villi Intestinal crypt Intestinal villi-higher magnification Iron absorption Intestinal lumen containing dietary Iron (10-20 mg in diet/day); Only 1-2 mg required for absorption Fe++ Fe++ DMT-1 (divalent metal transporter) Enterocyte Labile iron (Ferritin) Nucleus Hepicidin-ferroportin complex degraded In lysosome-leads to reduced iron absorption Ferroportin-produced in enterocyte transferrin protein-carries absorbed iron (1-2 mg/day) Hepicidin-produced in liver Key iron proteins • Transferrin • Ferritin • DMT 1 • Ferroportin • Hepcidin – Carries iron in blood – Stores iron in cell – Transfers iron across the cell – Efflux of iron out of cell into the blood • The only efflux mechanism for iron – Binds ferroportin and induces degradation • Hemojuvelin • Iron regulatory protein 1 and 2 – Increases hepcidin synthesis – Bind iron regulatory elements (IRE 3’ and 5’) – IRE 3’ increases transcription of transferrin, ferritin, DMT1 and ferroportin – IRE5’ decreases transcription 71 Iron sources: From duodenum Scenescent rbcs Liver cells Syncyiotrophoblasts y y p for passage to fetus Fig. 4. Major iron flows are controlled by hepcidin–ferroportin interactions. Hepcidin Blocks outflow of iron from duodenum, liver, macrophages (and placenta) Hepcidin • Increased hepcidin – Decreased ferroportin – Decreased iron absorption – Hepcidin p increased with inflammation • Anemia of chronic inflammation • Keeps iron from microbes • Decreased hepcidin – Increased ferroportin – Increased iron absorption accumulation – Hemochromatosis • Hepatocellular damage and cirrhosis Iron accumulation • Normally – 1-2 mg/iron/day absorbed – Transferrin 30% saturated • If the body senses enough iron – Then transferrin does not pick up iron from the enterocyte – The enterocyte exfoliates • Iron stays in feces • With Hereditary hemochromatosis – 3-4 mg/iron/day absorbed – Transferrin 100% saturated 72 Robbins and Contran Pathologic basis of disease, 7th Edition, 2005 Causes of excessive iron accumulation • Primary – Hereditary hemochromatosis (adult and juvenile forms) • Juvenile form most severe – Man, Minah Birds, Birds of Paradise, Lampreys, Rock hydraxes • Secondary to another disease – Chronic liver disease • Alcoholic cirrhosis cirrhosis, chronic viral hepatitis hepatitis, shunts shunts, porphyria – Congenital atransferrinemia – Ineffective erythropoiesis (dyserythropoiesis; iron absorbed but not used) • Beta thallasemia, aplastic anemia, pyruvate kinase deficiency (Basenji dogs) – Idiopathic excessive parental iron injection • RBC transfusions, iron-dextran injection (sideroblastic anemia) – long term dialysis Treatment for iron storage • Hemochromatosis – Blood letting – Hepcidin • Costly • Thalassemias – Hepcidin? 73 Copper absorption Intestinal lumen containing dietary copper (1.5-4 mg/Cu++/day) Cu++ Enterocyte Liver Hepatocyte Hepatoc te Cu++ albumin Cu++ ceruloplasminSystemic delivery metallothionen Hepatocyte- copper bound to metallothionen and If excess in lysosomes lysosome Protein for copper excretion into bile Biliary excretion- copper is excreted into bile and eliminated (2-4 mg/day) Cu passes across the membrane with Ctlr protein, stored with MT or shuttled to the Golgi apparatus by HAH1 and uses a Cu atpase (ATP7b (WND) which is absent In Wilson’s disease) to pass across the membrane; Ccs proteins shuttles to SOD; Cox 17 shuttles to the mitochondria for its role with cytochrome C oxidase. Ceruloplasmin (Cp transfers copper to the plasma) Copper accumulation • Primary – Wilson’s disease • Defective WND – Amino acid defect at aa # 1069, commonly » Impairs biliary excretion of bile by inhibiting copper transport into the golgi Ceruloplasmin Serum copper Urinary copper Liver copper Wilson’s disease 0-200 19-64 100-1000 >250 Normal 200-350 70-152 <40 20-50 74 Copper accumulation • Primary – Wilson’s disease (previous slide) – Bedlingham terriers (BT) • Primary accumulation of copper – Metallothionen of BT remains neonatal-like • Secondary • In other breeds, copper may accumulate in liver secondary to bile stasis following liver damage BT 10000 Cu In liver Dalmation 7500 Doberman 5000 Skye Terrier Cirrhosis 3000 WHWT 2000 1000 500 250 0 Copper accumulation in sheep • Sheep on inbalanced diets can store excessive copper in liver – Sudden release is often seen • Copper damages hemoglobin – Free radical formation? • RBC’s lyse – Lost of deformability – Heinz body formation Metallothionien: Intracellular regulation of divalent cations • Metallothionien – Has 20 cysteine residues • SH groups can bind metals – Capable of binding (or releasing) • 12 copper ions • 7 zinc ions • 20 nitric oxide ions – MT binds physiological (Zn, Cu, Fe, Se) and xenobiotic (Cd, Hg, Ag) metals • Zinc – Vital for activity of some enzymes – Vital for inactivation of some enzymes – Vital for activation of binding of some DNA transcription factors 75 Metallothionein regulation by zinc and anti-oxidative activity Cytokines Glucocorticoid STAT GRE Methylation H2O2 Electrophiles MRE ARE :Up regulation :Down regulation Structural gene MTF-1 Apo MT Apo-MT Zn pool Zinc Finger Protein Inactive Protein Zn7-MT Apo-MT Oxidative Stress Degeneration Overview of metallothionein (MT) gene regulation The MT promoter has many elements that up-regulate transcription. These include the following: 1)metal response elements (MRE), which are activated by the metal-responsive transcription factor (MTF-1) after zinc occupancy; 2) glucocorticoid response elements (GRE); 3) elements activated by STAT (signal transducers and activators of transcription) proteins through cytokine signaling, and 4) the antioxidant (or electrophile) response elements (ARE) activated in response to redox status. Davis and Cousins, 2000. Bilirubin damage to cells • Unconjugated bilirubin (UCB) – – Formed by catabolism of heme by splenic macrophages not water soluble – UCB is conjugated with glucuronide by hepatocytes • • • – Therefore, transported by albumin Increases water solubility Most other cells lack the ability to conjugate bilirubin Hyperbilirubinemia in infants can occur with: • • Inherited deficiencies of glucuronidation enzymes Delayed development of glucuronidation enzymes • “Bili lights” UV light exposure in infants with elevated bilirubin levels – – Premature birth Bilirubin levels in serum increase with red blood cell lysis, bile stasis • • UCB readily enters cells UCB is antioxidant at modest elevations • UCB can induce injury at high levels – Absorbs electrons – Neurons – Astrocytes- • • • Apoptosis (next slide) UCB diffuses into the cell and alters mitochondrial membranes allowing Cytochrome C release reduced glutamate uptake – Prolonged glutamate at the synaptic cleft » Increased excitotoxicity (overstimulation of the NMDA receptors) » Na+, Ca++, Cl-, water influx » Apoptosis/necrosis of neurons Bilirubin neurotoxicity Ostrow JD, et al Trends in Mol Med 10 (2):65-69, 2004 76 Regulation of UCB levels in CNS tissue • UCB transportor across the blood brain barrier and choroid plexus • Protective – Organic anion transport proteins (OATP) • Transports UCB two directions – Into cells and into blood – Multidrug resistance receptors (p (p-glycoprotein) glycoprotein) • Transports UCB one direction – Into blood in BBB – Into blood and into CSF in choriod plexus » Could increased CSF UCB transport be detrimental? • These pumps likely explain why UCB accumulates in specific regions of brain – E.g. UCB remains in those regions lacking active pumps – CSF accumulation? • May allow increased levels around CSF UCB transporters Ostrow JD, et al Trends in Mol Med 10 (2):65-69, 2004 Effects of lipids and sugars on cellular degeneration Ackermann 77 Factors that mobilize fat • Intense lactation • Pregnancy toxemia • cattle, sheep, guinea pigs • • • • • • • • • Hyperlipidemia, ponies; hepatic lipidosis, cats Starvation Diabetes, hypothyroidism, Hyperadrenalcortisim Alcoholism Nephrotic syndrome Choline deficiency Anemia Fatal fasting syndrome, old world primates Hyperlipidemia diseases (more later); Schnauzers Robbins and Contran Pathologic basis of disease, 7th Edition, 2005 Cellular energy: required for ATP Glucose/glycogen Proteins Pyruvate Fatty acid Fatty Acyl Co A ACETYL CO A KREBS (produces NADH, FADH2 for Oxidative phosphorylation) OXIDATIVE PHOSPHORYLATION + NADH +02 =ATP Mitochondria ATP for: ATPase pumps, signaling, etc. A transporter is required for: Fatty Acyl Co A, pyruvate and ATP Cell plasma membrane 78 Fats and cholesterol • Are in the blood (as insoluble fatty forms) at low amounts: • Triglycerides, free fatty acids, glycerol (draw glycerol, mono di mono, di, and triglyceride) • Cholesterol, cholesterol esters • Phospholipids (draw; phosphatidylcholine) • Vitamins A, D, E, and K • Also transported as lipoproteins (LDL, VLDL, etc.) that contain apolipoproteins (Apo E, B, etc.) Lipid transport in blood • Lipoproteins • • • • Chylomicron, LDL, VLDL, HDL, IDL Center is lipid part Surface is protein part Classified by molecular size, electrophoretic mobility, and densities • Density: – Chylomicron (least dense), then VLDL, LDL, HDL (most dense) – Decreases with triglcyerides – Increases with protein and phospholipid content » Chylomicron has 83% triglyceride/2% protein » HDL has 8% triglyceride/33% protein Apolipoproteins • Bind lipids and become lipoproteins. Function in LDL’s, VLDL’s, chylmicrons, HDL’s, IDL’s. – If they are free of lipid, then called apoplipproteins • Serve as ligands for receptors • Apolipoprotein A – Major amount on: HDL’s • Apolipoproteins B – Major amounts on: chylomicrons, VLDL and LDL’s • Apolipoprotein C – Major amounts on: chylomicrons, VLDL, HDL • Apolipoprotein E – Major amounts on: VLDL and HDL, some on chylomicron – Three forms differ by one amino acid » E3 is the common form » E2 is associated with high triglycerides/cholesterol » E4 is associated with high cholesterol 79 B-48 E, C transfer Apo E, A, C HDL3 LCAT HDL2 Lipoprotein lipase (Triglyceride release) Cholesterol 75% of LDL 25% Other tissues Macrophage R: LDL R SR-A CD36 • • • • • • • 1% Robbins and Contran Pathologic basis of disease, 7th edition Lipoproteins Chylomicron – B 48, picks up E and C VLDL synthesized in hepatocyte • B 100, E and C • Dog also has B48 Nascent VLDL receives Apo E and C2 from HDL • This mature VLDL transports lipids LDL • B 100 • B 100 B 48 in dogs HDL1 • E, A, C • Dogs only; no HDL1 in man HDL2 • E, A, C • Dogs and man HDL3 • E, A, C • Dogs and man Lipoprotein and hepatic lipase • Lipoprotein lipase • Tissue capillaries • Activated by Apo C2 • Hydrolyzes triglycerides from VLDL • Hepatic lipase • Removes triglycerides in liver 80 Contents of LDL’s • • • • • Apolipoproteins and cholesterol Phosphatidylcholine (Phospholipid) Lysophophatidylcholine Sphingomyelin Fatty acids • Linoleic acid, palmitic acid, stearic acid, free fatty acids, triglycerides and trans fatty acids Oxidation/acetylation of LDL’s – Increases scavenger uptake by foam cells • 3-10 fold increase • Non-oxidized/acetylated (native) LDL’s are taken up by LDLR on hepatocyte and macrophage » Macrophage uptake of native LDL is slow and downregulated – LDL’s “trapped” pp byy proteoglycans p gy – Lipoperoxidation of phospholipids, fatty acids, and Apo B • Occurs by lipoxygenases, MPO, iNOS, ROS – Self propagation of the reaction – Decomposition to of fats to aldehydes, ketones – Apo B • Lysine residue oxidized • This inhibits binding of ApoB to LDLR Foam cell formation • Acetylated LDL’s • Rapidly taken up by macrophage LDLR (SRA-scavenger receptor A) – This receptor not down regulated by the cell • Oxidized LDL’s • Rapidly taken up by AGE receptors LDLR (SRA), CD36 and d other th receptors t – These receptors not down regulated by the cell • AGE receptors take up oxLDL’s and AGE • AGE (Advanced glycosylation endproducts) » RAGE (receptor for AGE), 80 K-H, OST-48, galectin-3, macrophage scavenger receptors type I and II (SR-A) » CD36 is a class B scavenger receptor, but like SR-A, takes up oxidized LDL’s and AGE 81 Foam cell Endothelial cells, smooth muscle cells, or macrophages, (or Cu++) Acetyl LDL Native LDL Fast ox LDL slow Fast Native LDL R (down regulated) Acetylated LDL R (SR-A) CD36, SR-A and (not down regulated ) Other ox LDL receptors (not down regulated) Steinberg: Nature medicine 8:1214, 2002 Foam cells fight back: Mechanisms to eliminate fat • OxLDL induce oxysterols in the cytoplasm • This activates transcription of » » » » » » » A E ffor HDLs ApoE HDL ABCA1- for cholesterol channel Fatty acid synthetase, for forming fats Fatty acids are made into free cholesterol Cholesterol bound to ApoA1 Cholesterol/ApoA1 goes to HDL Increase cholesterol outflow increases removal of esterified cholesterols also Cell protection against free cholesterol Ox LDL Apo E -carries FC out FC Oxysterols CE lysosome ABCA1 -transports FC HDL FC LXR RXR Apo E ABCA1 SREBP1c FAS LXR RXR RE’s FFA Apo A -picks up FC CE Lipid droplet LXR = liver X receptor RXR = Retinoic acid receptor LIVER CE = cholesterol ester FC = free cholesterol, ABCA1 = transporter, FAS = fatty acid synthesis, FFA = free fatty acid 82 Proatherogenic activity of oxidized LDL’s • • • • • • • • • • • • • • • Increase foam cells (increased scavenger uptake) Products are chemotactic to monocytes, T cells Cytotoxic, apoptosis Mitogenic for smooth muscle cells Increase scavenger receptor expression Increase PPAR actvity (gene fx, apoptosis) I Immunogenic; i autoantibody t tib d and d T cellll activation ti ti Increases LDL aggregation Substrate of spingomyelinase; aggregates LDL’s Procoagulant, increases tissue factor, platelet activating factor Increased monocyte CSF, MCF, IL-1, IL-8, chemokine receptors,NO Increased calcium and Nfkappa B Increased expression of matrix metalloproteinases Increased adhesion molecule expression Reduce vasomotor activity and vascular contractility Trans fatty acids • A double bond between carbons in a fatty acid that changes the conformation from cis to trans – Oleic and elaidic acid are 18 carbon • Oleic is cis; elaidic is trans • Trans fatty acids – IIncrease LDL LDLs, d decrease HDL HDLs, iincrease cholesterol:HDL h l t l HDL ratio ti and d increase blood triglyceride levels – Increase B100 size, secretion, lipid content – Decrease LDLapoB100 catabolism (increasing LDLs) – Increase cholestrol ester transfer from HDLs to LDLs • Thus decreasing HDL removal of cholesterol – Increase ICAM-1, VCAM-1 and E selectin on endothelial cells • Pro-inflammatory promoting atherosclerosis Atheroma formation • Diagram • Stages: – – – – – – – – – Fatty streaks in children Isolated foam f cells in adults Multiple foam cells Extracellular lipid accumulation Confluent lipid core Fibrous cap with smc proliferation Surface ulcer, thrombus Calcification Fibrosis Can regress Ab Above thi this liline 83 Robbins and Contran Pathologic basis of disease, 7th edition Robbins and Contran Pathologic basis of disease, 7th edition Robbins and Contran Pathologic basis of disease, 7th edition 84 Factors leading to atherosclerosis • Cigarette smoking • Two fold increase in atherosclerosis • Hypertension • Higher incidence blacks; 3% of Americans, Americans 75% of those over 75 years of age • Diabetes mellitus • 14 million americans • Serum cholesterol levels • Familial hypercholesterolemia – Increased cholesterol, types on next page • 50% of Americans (24-70 years) have >200 cholesterol levels LDL Receptor • LDL R family • Apo E R2, VLDL R, megalin, LDLR, LDL receptor-related protein • Genetic mutation can reduce expression – Multiple types of mutations identified in man • Results in increased circulating LDL’s, hypercholesterolemia and foam cell formation Genetic Causes of Hyperlipoproteinemias • I increased chylomicron • IIa increased LDL* • IIIa increases LDL/VLDL* • III increased chylomicrons • IV increased VLDL* • V increased VLDL/chylo *10, 40, 45% respectively • Others mutations in lp lipase mutation of LDL or Apo B mutation of LDL or Apo B mutation of Apo E mutation of lp lipase mut. Lpl or C2 • Decreased HDL; mutation in Apo AI • Tangier’s dz, mutation in cholesterol transport • Loss of HMG-CoA reductase 85 Schnauzers, Cats, Cows, Ewes • Schnauzers develop hyperlipidemia • Cats, cows and ewes, develop fatty livers – Cats, L-Carnitine defect? – Impaired ability to carry fats into the mitochondria and out of the hepatocyte • Cows and ewes – Often related to pregnancy/parturition/lactation Lipid disorders in dogs and cats Dogs Primary disorder Idiopathic hyperlipidemia hypercholesterolemia Secondary disorder high fat diet pancreatitis hypothyroidism yp y cholestasis hyperadrenocorticism nephrotic syndrome Cats Primary disorders inherited hyerchylomicronemia primary hypercholesterolemia Increased TG, cholesterol pancreatitis Increased cholesterol retinal degen increased cholesterol increased TG increased cholesterol increased cholesterol increased TG, cholesterol increased TG, cholesterol pancreatitis? pancreatitis atheroma insulin resistance increased TG, cholesterol Decreased LPL increased cholesterol xanthomas TG = triglycerides Bauer JE: JAVMA 224:668-675, 2004 Appetite regulation • Arcuate nucleus of the hypothalamus, two parts: – 1. AgRP/NPY—increases appetite and metabolism » Activated by: Ghrelin R (GhR) (Ghrelin; stomach), Agouti-related peptide (AgRP), NPY » Inhibited by: PYY, YY (gut), leptin (adipose), insulin, amylin, pancreatic polypeptide (pancreas) – 2. POMC/CART—decreases appetite and metabolism » Activated by leptin (adipose), insulin (pancreas), cocaineamphetamine pro-opiomelanocortin amphetamine, pro opiomelanocortin POMC • Nucleus tratus solitarius (NTS), medulla • Receives second order neurons from the arcuate nucleus – Also regulated directly by cholecystikinin (liver) and vagus nerve (for sensing stomach stretching) – Sends signals to the rest of the body • The hypothalamus arcuate nucleus and NTS regulate feed centers and behavior in the hypothalamus nuclei: ventromedial, dorsomedial, and lateral 86 Leptin • Leptin will decrease appetite in those with mutant leptin, but not normal individuals • In other words, excess leptin doesn’t decrease appetite in an average person • Leptin protects against weight loss/starvation – There is decreased leptin with decreased fat, therefore increased eating – As above, excessive leptin doesn’t decrease intake A few obesity targets • Inhibiting hunger – – – Cannibas receptor inhibitor (preventing hunger “munchies”) PYY peptide (decreasing appetite) SlimFast (bulky fiber? poorly degraded?) • Preventing food absorption • Delay gastric emptying • Enhance fat burning – – – – • Alizyme, prevents fat absorption in gut Olestra-WOW! Chips, a fat not absorbed Amylin Human growth hormone, amphetamines Degradation of fats – Lipases • Altering fat metabolism • Nutrisystem, Nurrisystem for men (dan marino), Jenny Craig, Atkins Diet, Scarsdale diet, Grapefruit diet, Slimfast, Trimspa, Houdia, LA weight loss, Inches Away, weight watchers, Exercise – Acetyl Co-Enzyme A carboxylase-induces fatty acid synthesis • • TRB3 induces degradation of ACC thus decreasing synthesis PPAR • PPAR alpha – Fatty acid oxidation in liver (heart, muscle, kidney artery) • Tissues with high fatty acid oxidation • PPAR gamma – Lipid storage in liver and adipose tissue – Activated A i db by TZD (Thi (Thiazolidinedione), lidi di ) an anti-diabetic i di b i d drug • Increases skeletal muscle and liver sensitivity to insulin via activation of PPAR gamma • Also reduces inflammation • Weight gain side effect and sodium retention – Widespread and present in many tissues • PPAR beta/delta – Fatty acid oxidation and energy decoupling in adipocytes • Only weakly activated 87 PPAR Cholesterol synthesis • Cholesterol • 27 carbons, synthesized in liver from acetyl CoA • Acyl transferase mediates acetyl CoA formation • Three acetyl CoA’s join to make HMG CoA – HMG-CoA » Can make acetoacetate and ketone bodies » Can make nevalonate to form cholesterol • High cholesterol levels in hepatocyte – HMG CoA reductase is inhibited and ketones form – No LDL receptors are made (to decrease hepatocellular uptake of LDL and thereby reduce cholesterol intake) • Low cholesterol levels in hepatocyte – HMG-CoA reductase makes nevalonate – Increased LDL receptor to increase uptake of cholesterol • Cholesterol forms: – Bile salts – Steroid hormones Robbins and Contran Pathologic basis of disease, 7th edition 88 Drugs that lower cholesterol • Cholestyramia • Bile acid exchange resin. Binds cholesterol. • Lovastatin • Inhibits HMG-CoA reductase. Prevents cholesterol formation • Bezafibrate • Stimulates lipoprotein lipase. Triggers cholesterol degradation. • Probucol • Lowers triglycerides, mechanism uncertain Utilization of energy • Brown fat • Fidgeting – Levine JA: Nonexercise activity thermogenesis (NEAT): environment and biology. Am J Physiol Endocrinol Metab. 2004 May;286(5):E67585: • Nonexercise activity thermogenesis (NEAT) is the energy expended d d ffor everything thi th thatt iis nott sleeping, l i eating, ti or sports-like t lik exercise. It includes the energy expended walking to work, typing, performing yard work, undertaking agricultural tasks, and fidgeting. • The variability in NEAT might be viewed as random, but human and animal data contradict this. It appears that changes in NEAT subtly accompany experimentally induced changes in energy balance and are important in the physiology of weight change. Inadequate modulation of NEAT plus a sedentary lifestyle may thus be important in obesity. It then becomes intriguing to dissect mechanistic studies that delineate how NEAT is regulated into neural, peripheral, and humoral factors. • Exercise Brown fat utilization and thermogenesis • Cold increases – PGC1 (a powerful transcriptional coactivator) for PPAR gamma (peroxisome proliferator activator receptor) – TR (thyroid hormone receptor) – RAR (retinoic acid receptor) – ER (estrogen receptor) • PGC1 PGC1, TR TR, RAR and d ER activate ti t UCP ((uncoupling li proteins) t i ) and d genes of mitochondrial respiratory chain for ATP synthesis (ATPase and cytochrome oxidase C) • The UCP (uncoupling proteins) are in the inner mitochondrial membrane along with F0/F1 ATPase. • The UCP allow H+ passage with loss of gradient resulting in heat with reduced ATP. – PGC1 also activates NRF 1and 2 (nuclear respiratory factors) for mitochondrial biosynthesis and increases conversion of type I to type II muscle fibers • Increased mitochondria for more heat 89 Hyperglycosylation and hyperglycemic vasculopathy • Hyperglycemic vasculopathy in diabetes mellitus – Persistent levels of blood glucose leads to glycosylation l l ti off vessels l – Glucose attaches to amino groups of proteins • Schiff base is formed and is reversible • Amadori complex – A more stable adherence of glucose to the protein • Advanced glycosylation end products (AGEs) – Irreversible glycosylation Mediation of glycosylation damage • Glycosylation of LDL’s, hemoglobin, albumin, extracellular matrix proteins, and basement membrane (thickens) • AGEs bind RAGE receptors on macrophages – Induction of IL-1, TNF, inflammation • Glycosylation of amino groups in DNA nucleotides – Altered transcription and increased strand breaks Intracellular glucose damage • Occurs in tissues that do not require insulin for glucose entry (in diabetics) – Lens, neurons, blood vessels • Converted to sorbitol by aldose reductase and then sorbitol to fructose by sorbitol dehydrogenase • Sorbitol and fructose induce oncotic pressue, influx of water and swelling – Sorbitol also reduces ATPase pumps in schwann cells (of nerve fibers) and retinal capillary endothelial cells – Also, increased protein kinase C and diacylglycerol can occur 90
