Intracelulární enzymatické zdroje volných radikálů NADPH Oxidázy Myeloperoxidaza Lukáš Kubala kubalal@ibp.cz Biological Sources of Oxidants/Radicals (022) o2- _i_^ h2o2 _f_ -oh _!_ 0 '2' Diatomic Superoxide Hydrogen Hydroxyl oxygen Peroxide Radical • Mitochondria - electron respiratory chain leak • Cyclooxygenase ROS • Lipoxygenase generally • Heme oxygenase • Leukocyte NADPH oxidase • Non-Phagocytic NADPH oxidases (Nox family) • Xanthine Oxidase Superoxide • Cytochrome P450 enzymes • Uncoupled NO synthase Mitochondria - electron respiratory chain leak INTERMEMBRANE SPACE mitochondrial membufít MATRIX SPAQ Complex I, II, 111,1V - Function is to reduce 02 to H20 • 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) 738484 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 1-5% ROS Which complex is responsible for free radical leak? • This electron is thought to come from the one-electron reduction of ubiquinone, which generates the reactive intermediate ubisemiquinone formed by Complex III. • Instead of accepting another electron and proton to form ubiquinol, ubisemiquinone may leak its unpaired electron to 02, forming 02»-. e-+ II' H,C O-CHj HO l-UC hydrophobic hydrocarbon tail e" + H* O-CHj HO »■ » H,C ubiquinone ubisemiquinone ((roc radical) O-CHj OH ubiquinol (dihydroubiquinone) Xanthine Oxidase/Dehydrogenase Flavoprotein enzyme containing iron and molybdenum that promotes the oxidation especially of hypoxanthine and xanthine to uric acid and of many aldehydes to acids Xanthine dehydrogenase (XD) Xanthine ^ NADH + Uric Acid Hypoxia --------------^ XD _^ XQ ^ Cytokines ■ Xanthine oxidase (XQ) Q .. +UricAcid Hypoxanthine---------------------------------------► u2 * uricMcia Xanthine ^"thine oxidase (XQ) ^ Q +UricAcid Uncoupling of NO synthase Reductase Domains NADP+ + H+ + O ,L-Arg aa Ä V \ Homodimer ^d Oxygenase Domains NADP+ H + k \NADPH | ^á ô- Oxidative stress .rT ^ P Homodimer x> v^i Oxygenase Domains What leads to eNOS uncoupling Cardiovascular risk factors Hypercholesterolemia Hypertension Cigarette smoking Upregulation of NADPHoxidase(s) C Organic nitrates Elevated angiotensin II v t ™—FMI L-A'9 + 0!^F Upregulation of eNOS Peroxynitrite formation Increased Oo jxidative stress Oxidative damage to eNOS and/or BH4 Struktura a aktivace fagocytární NADPH oxidázy (NOX2) p22phox Microorganisms and inflammatory mediators o2- Extracellular ooooa ooooa Intracellular Cell activation p22phox tooooooooooooooooooooo Lipid metabolism FtdlnsP FtdlnsP- JOOOOOOOOOOOO PI3K FtdlnsP ^ gpG»1 phox POOOOO Flavocytochrorne bc^g Cytosolic-regulatory proteins Protein kinases Phosphorylation Autoinhibitory conformation £Dfy Guanine-nucleotide RhoGDh exchange Membrane binding Transport elektronů NADPH oxidázou vesicular lumen or extracellular space membrane cytoplasm NADPH Charakteristika jednotlivých podjednotek NOX2 (fagocytární NADPH oxidázy) PrerjiťilT« (iftiii plujfiqyls «ijnnilíDj Tiuj?í1 íľíkIiíé ^jiIiíuí 1 (xnnpímaiti Hrtl'*™ V22i*ia p-it*" pň?"*™ p4í|l,*,1I E*c2 CľäU; ľípľl.l STO IM NCF-ť, Sgl 35* 1.Z3 AKjF-Í ]q25 £6 53y AoľÍ 22^111 1*2 ' 1 ľ.uLít ueiidM: PradJíatod Et«ŕ SUS-PAÍffl «,35« Da -«HD* 20,«* D* 22 Iď» 44^ó£l Dl 47 LD* »,735 Da Jfy03*fcD* «i kDa 21,42*0* 22 kD* íil^ĽíKylalym T* PÜKISJÜKlI^JaiKlIl Y» *26 No No HM No *JÉ Y» No Ĺ12 "Im Hl 7.2S Y» No 7JÍ7 LcicUlvin m PM.N Kť.ťlniu ptasiru inanlir^iť C^ÉÚAOl Cyfcisd l.'yViscil MiTlh ■-■_vV.i-.dl SlĽDllIaVSil PlüKiru ]ncf]nlirjaiť M .:i.::í:l MťinTirjfliť Marrtiiani Meinhuiť All LĽItlJül■-■■=■ pilXll.'l (1 and plia^íminť IjO-2J> í'-1ťnru]iift limd-, IjO-2.0 C-tartnJú*l 6j0(2?J0újVQ P]H.I.-,]l]|'.ir,|jť.Dl sfl«. 1.0(460 úM) ' [iŕtrJl no (rp rqi í d; 1.0(460 Al) PX aid S-M.ľ 2_6(13ŮŮdJ) íiUP.liTP-lniidii^; Tyr-Tyr • 3-Chlorotyrosine HOCI + Tyr -> Cl-Tyr • 3-Nitrotyrosine ♦N02 + Tyr» —> N02-Tyr Myeloperoxidase-catalyzed Protein Activation/Inactivation • Deactivation of proteins Phagocytic NADPH oxidase Chemotactic factors Alpha 1-proteinase inhibitor Proteases (Matrix metalloproteinase 7) • Activation of proteins Proteases (collagenase, gelatinase) MAP kinases Tumor suppressor proteins Modulation of Intracellular H202 Pool MPO H202 -----------------------------1 HOCI - Redox sensitive transcriptional factors (gene expression) - Enzymes with redox sensitive catalytic centers (direct control of enzyme activity) Radical-Mediated NO Consumption by MPO H2O2 MPO MPO-I RH ,R* j^ MPO-I I ^\ Y_ s\ RH R# ^f N02- •NO V^ V ' #NO N02- MPO is a catalytic sink for NO Eiserich et al. (2002) Science Activated Heme Peroxidases Rapidly Consume •NO IVPO HA 50 100 150 200 250 Time (s) 50 100 150 200 250 Time (s) IVPO HÖ2 100 150 200 250 Time (s) IVPO 100 150 200 250 Time (s) Nitric Oxide-Dependent Signaling in the Vasculature Shear •NO •NO Degranulation Stimulus (ie. IL-8) PMN Degranulation Results in Intimal Myeloperoxidase Localization Endothelial Transcytosis of MPO 2 min 20 min 120 min Apical Intracellular Basolateral Side View • ' •; ■ Binding of MPO to GAG Mutants CHO = Wild-type pgsA = no G AGs pgsD = no HS, 3X CS pgsF = no S04-ation MPO he Binding of MPO is de Heparin GAGs on c< ■a q MPOhc úbm on p ill surfac JD Organ Bath for Isometric Tension Measurements H202-Activated MPO Inhibits Aortic Relaxation in Response to Acetylcholine MPO impairs rela MPO + H,a vp20- O 40- Controi o 10-9 10-8 10-7 10-6 Ach (M) ion of rat aorti •w *™^--^j "V ^1 -v 20 -0 4°-CÖ CÖ 60- Q) O" Q. O X < MPO controls progress of inflammatory process by modification of bioavailability of lipid metabolites MPO increases levels of T anti-inflammatory biologically active lipid metabolites (EpOME) MPO decreased levels of I pro-inflammatory biologically active lipid metabolites (LTB) Target metabolites of the LinoCeic and Arachidonic Acid Cascade PGB2 PGE-M PGF-M ^ fl \ PGE, PGF, TXB2 \ 6-keto-PGF | TXA2 \ PGI2 | TXA2 < Synthase pQH Synthase> pG| THF-Diols | COX 20-HETE <- CYP4A CYP2C2J, 5-LOX LTC4, LTD4.^ GST . LTE4 ^ LTÄ4 ^ 12/15-LOX ĽTAÄ Hydrolase LTA4 Synthase V LTB, 5-HETE 5-oxo-ETE 15-HETE 12-HETE IJ-tt&DE A DEETs CYP2C2J EETs EpOME'— sEH DHETs (DHÖME i Tri HOME Myeloperoxidase deficiency delay onset of neutrophil granulocyte apotosis 10" 1i CortHDl - MPO-KO .* :■.■' ■ , .,^Éŕ r li3 ,o s: ANX-BTClFL PMAMPO-KO ■H) ■■■■ iiÉi 1 tu /o to í 50 S 40 ŕ so 20 10 ANX-RTCiFLI-M) Anexin V and PI staining ■ Necrotic ■ Apoptotic WT Necrotic Apoptotic KO 0 40 180 40 BO 120 180 40 BO 120 180 control PMA Ionomycin