Intracelulární enzymatické zdroje volných radikálů NADPH Oxidázy Myeloperoxidáza Lukáš Kubala kubalal@ibp.cz Biological Sources of Oxidants/Radicals • Leukocyte NADPH oxidase • Non-Phagocytic NADPH oxidases (Nox family) • Xanthine Oxidase • Cytochrome P450 enzymes • Uncoupled NO synthase • Mitochondria - electron respiratory chain leak • Cyclooxygenase • Lipoxygenase • Heme oxygenase O2 O2 •- H2O2 •OH Diatomic oxygen Superoxide Hydrogen Peroxide Hydroxyl Radical e- e- (O2 2-) e- H2O e- ROS generally Superoxide • Mitochondria - electron respiratory chain leak Complex I, II, III,IV – Function is to reduce O2 to H2O • Complex I (NADH-Ubiquinone reductase complex), • Complex II (succinate dehydrogenase complex). • Ubiquinone, also known as coenzyme Q, accepts electrons from both complexes and is sequentially reduced, one electron at a time, to ubisemiquinone and ubiquinol • Complex III (ubiquinol-cytochrome c reductase) • Complex IV (cytochrome c oxidase) • Mitochondria - electron respiratory chain leak Cytochrome oxidase is estimated to account for 90-95% of the total oxygen uptake in most cells • What happens to other 5-10% • formation of free radicals Which complex is responsible for free radical leak? • This electron is thought to come from the one-electron reduction of ubiquinone. • Instead of accepting another electron and proton to form ubiquinol, ubisemiquinone may leak its unpaired electron to O2, forming O2 •-. Xanthine Oxidase/Dehydrogenase Xanthine Xanthine dehydrogenase (XD) NADH + Uric Acid Xanthine Xanthine oxidase (XO) O2 .- + Uric Acid Hypoxia Cytokines XD XO Flavoprotein enzyme containing iron and molybdenum that promotes the oxidation especially of hypoxanthine and xanthine to uric acid and of many aldehydes to acids Hypoxanthine Xanthine oxidase (XO) O2 .- + Uric Acid Uncoupling of NO synthase Oxidative stress Pathological conditions leading to eNOS uncoupling Struktura a aktivace fagocytární NADPH oxidázy (NOX2) Transport elektronů NADPH oxidázou Homology NADPH oxidáz a jejich struktura Homology NADPH oxidáz a jejich struktura Přehled homologů NADPH oxidáz Prokázaná existence řady orthologních NADPH oxidáz u myší, krys, Drosophil, Caenorhabditis elegans, a Diclostelium. NADPH oxidázy také objeveny u kvasinek a rostlin Aktivace NADPH oxidáz a jejich podjednotky - Obranná funkce (fagocyty, střevní, plicní, ledvinný epitel, keratinocyty) - Signální transdukce (mitogení stimulace, apoptóza, senescence) - Metabolismus látek (biochemické reakce spojené se syntézou thyroidních hormonů a přestavbou kostí) -Regulace krevního tlaku v cévním sytému - Snímání koncentrace kyslíku v kůře ledvin Předpokládané funkce NADPH oxidáz Myeloperoxidase - Heme peroxidase ~ 150 kD - Pair of protomers -  (heavy) subunit and  (light) subunit -  subunit - two hemes and mannose-reach carbohydrate - Single gene located on chromosome 17 - MPO is up to 5% of total neutrophil proteins - High quantities of MPO are released and accumulated at the site of acute inflammation Human Myeloperoxidase MPO is a Highly Cationic Protein Arginine Lysine pI ~ 10 Promyelocyte Neutrophil Myelocyte Metamyelocyte - Blood Monocytes Tissue macrophages - Kupffer cells - Alveolar macrophages - Microglia … Oxidative Burst of Neutrophil Phagosome O2 O2 NADPH + H+ NADP+ NADPH Oxidase Stimulant PKC (PMA) H2O2HOCl MPO MPO MPO-I H2O2 Cl- HOCl Phagocytes Utilize Myeloperoxidase to Form Bleach • Host Defense • Tissue Injury Chronic inflammation MPO is an important factor in the pathophysiology of various disorders connected with chronic inflammation - cardiovascular diseases - renal diseases - asthma - obstructive pulmonary disease - …. Myeloperoxidase & Vascular diseases Baldus, et al. (2003). “Myeloperoxidase serum levels predict risk in patients with acute coronary syndromes” Circulation 108:1440-1445 Brennan, M. L. et al. (2003). "Prognostic value of myeloperoxidase in patients with chest pain." N Engl J Med 349(17): 1595-604. Potential mechanisms of MPO mediated alterations of physiological functions - Posttranslational modifications of proteins - Modulation of intracellular H2O2 pool - Modulation of availability of biologically active lipids - Catabolism of NO Myeloperoxidase-catalyzed Protein Oxidation • Dityrosine Protein Cross-links Tyr• + Tyr• ---> Tyr-Tyr • 3-Chlorotyrosine HOCl + Tyr ---> Cl-Tyr • 3-Nitrotyrosine •NO2 + Tyr• ---> NO2-Tyr Myeloperoxidase-catalyzed Protein Activation/Inactivation • Deactivation of proteins Phagocytic NADPH oxidase Chemotactic factors Alpha1-proteinase inhibitor Proteases (Matrix metalloproteinase 7) • Activation of proteins Proteases (collagenase, gelatinase) MAP kinases Tumor suppressor proteins Modulation of Intracellular H202 Pool H2O2 - Enzymes with redox sensitive catalytic centers (direct control of enzyme activity) - Redox sensitive transcriptional factors (gene expression) MPO HOCl Radical-Mediated NO Consumption by MPO MPO MPO-I H2O2 H2O MPO-II NO NO NO2 NO2 RH RH R R • • - - • • Eiserich et al. (2002) Science MPO is a catalytic sink for NO 0 50 100 150 200 250 H2O2 MPO C 0 50 100 150 200 250 Time (s) MPO H2O2 N3- D Time (s) 0 50 100 150 200 250 A 0 50 100 150 200 250 H2O2 MPOB Time (s) Time (s) Activated Heme Peroxidases Rapidly Consume •NO Shear GCi eNOS •NO eNOS •NO •NO •NO Agonist GCa cGMP Nitric Oxide-Dependent Signaling in the Vasculature MPO Degranulation Stimulus (ie. IL-8) eNOS •NO NO2 - •NO cGMP PMN Degranulation Results in Intimal Myeloperoxidase Localization 100x 50x Endothelial Transcytosis of MPO 2 min 20 min 120 min Apical Intracellular Basolateral Side View Control Heparin LMWH Chondroitin MPOActivity (Units/ugDNA) 0 0,01 0,02 0,03 0,04 0,05 1 MPO hc 0 0,01 0,02 0,03 0,04 0,05 0,06 1 Control Heparinase Heparitinase Chomdroitinase MPOActivity (Units/ugDNA) Binding of MPO is dependent on Heparin GAGs on cell surface Organ Bath for Isometric Tension Measurements 0 20 40 60 0 20 40 60 Time (min) MPO ± MPO Ach PE PE AchH2O2 H2O2-Activated MPO Inhibits Aortic Relaxation in Response to Acetylcholine 0 20 40 60 80 100 0 25 50 75 100 MPO impairs relaxation of rat aortic rings MPO + H2O2 0 20 40 60 80 100 Control MPO + H2O2 Control 0 10-9 10-8 10-7 10-6 Ach (M) PAPANONOate (nM) Relaxation(%) Relaxation(%) MPO attenuates cGMP levels in cocultures of EC-SMC Ionomycin MPO MPO MPO eNOS cGMP 0 1 2 3 4 cGMP(pmol/mgprotein) Ionomycin - + + + + MPO - - + - + H2O2 - - - + + Heparin blocks MPO uptake into vascular tissue and restores vessel relaxation 0 20 40 60 80 1000 0,002 0,004 0,006 0,008 MPOActivity (Units/mgProtein) Control MPO MPO+LMWH Control MPO + LMWH MPO 0 10-9 10-8 10-7 10-6 Ach (M) Relaxation(%) Baldus, Eiserich et al. (2001) JCI MPO increases levels of anti-inflammatory biologically active lipid metabolites (EpOME) MPO decreased levels of pro-inflammatory biologically active lipid metabolites (LTB) MPO controls progress of inflammatory process by modification of bioavailability of lipid metabolites HO O COX 15-HETE 12-HETE 13-HODE TXA2 TXB2 PGF2 PGF-M PGE-M 6-keto-PGF1 PGI CYP2C,2J EETs EpOME DHETs DHOME sEH CYP2C,2J DEETs sEH THF-Diols 12/15-LOX TXA2 Synthase PGI2 Synthase 5-LOX LTA4 Hydrolase 5-HETE 5-oxo-ETE LTB4 LTA4 LTC4, LTD4, LTE4 LTA4 Synthase PGB2 Target metabolites of the Linoleic and Arachidonic Acid Cascade PGH2 PGE2 CYP4A 20-HETE GST Tri HOME Anexin V and PI staining Myeloperoxidase deficiency delay onset of neutrophil granulocyte apotosis