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Protein 3-Nitrotyrosine: Formation, Evaluation and Biological Consequences Dr. José M. Souza Departamento de Bioquímica Centro de Radicales Libres Facultad de Medicina, Universidad de la República Av. Gral. Flores 2125, Montevideo, Uruguay E-mail: jsouza@fmed.edu.uy
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Nitrating agent Formation of 3-nitro-tyrosine (NO 2 ) 4 C pH 8 (NO 2 ) 3 C - + 2H +
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Mechanism of 3-nitroTyrosine Formation Two major pathways have been established: PeroxynitritePeroxynitrite Peroxidases or MPO/H 2 O 2 /NO 2 -Peroxidases or MPO/H 2 O 2 /NO 2 - NO. ?NO. ? All pathways for 3-nitroTyr formation depend on nitric oxide formation
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Oxidation state (n)2345. NO 2 -. 2 ONOO - Intermediates/ catalysts Tyr. H + /HNO 2 H 2 O 2,HOCl Myeloperoxidase Hemeproteins Tyr. CO 2 Me n+ ROH,RCO 2 Myeloperoxidase Eosinophil peroxidase In search of the in vivo nitrating agents Tyrosyl Radical: Prostaglandin H Synthase-2, Ribonucleotide Reductase Peroxidases: Catalysts of both nitrite and peroxynitrite-mediated nitration Hypochlorous acid: Likely not involved in peroxidase-mediated nitration Nitrogen Dioxide: Inefficient in the absence of tyrosyl radical ONO(O)CO 2 - : More efficient nitrating agent than peroxynitrite
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Reactive Pathways ONOO - O 2 -. NO + RC-NO 2 NitrativeStress OxFe-S, Carbonyls OxidativeStress RS-NO, RN-NO NitrosativeStress O 2 Me n+ RSH CO 2 2 ON-OCO 2 - H 2 O 2 Me n+ MPOEPO
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Radical mechanism of nitration
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Peroxynitrite free radical-independent nitration mechanism This mechanism may ocurre within protein metal centers ONOO - + Me n X ONOO-Me n X NO 2 -O-Me n X NO 2 + + O=Me n X Tyr NO 2 -Tyr + O=Me n X + H + O=Me n X + 2H + Me n X + H 2 O NO 2 + + H 2 O NO 3 - + 2H +
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MPO-catalyzed Cl - -mediated oxidation Fe III Fe IV+. H2O2H2O2H2O2H2O2 H2OH2OH2OH2O MPO Compound I MPO Ground State Cl - HOCl
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NO 2 Tyr formation by MPO Fe IV Fe III Fe IV+. H2O2H2O2H2O2H2O2 H2OH2OH2OH2O MPO Compound I MPO Compound II NO 2 -. NO 2 Tyr Tyr. MPO Ground State
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Tyrosine Nitration by Nitric Oxide Nitric oxide may react with stable tyrosyl radical residue that are involved in the catalytic mechanism of ribonucleotide reductase or prostaglandin H synthase, or cytochrome c-H 2 O 2
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A Tale of Two Controversies: Defining both the role of peroxidases in nitrotyrosine formation in vivo using eosinophil peroxidase and myeloperoxidase-deficient mice, and the nature of peroxidase-generated reactive nitrogen species Brennan M-L et al (2001) J.B.C. 277, 17415-17427 Peroxidases Knockout Model 3-Nitrotyrosine Formation from Lung Tissue after Aeroallergen Challenge 3-Nitrotyrosine Formation from Zymosan-induced Peritonitis Lavage protein after 20h thioglycollate and 4h zymosan
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Peroxynitrite Pharmacology. NO + O 2.- ONOO - Oxidations and Nitrations NOS inhibitors NOX and XO inhibitors NO scavengers SOD or SOD-mimics Scavengers Decomposition catalysts Repair
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Peroxidases Pharmacology NO 2 - + H 2 O 2 + MPO / EPO. NO O 2.- SOD NO 3 - HbO 2 Decomposition catalysts (catalase or catalase mimics) Oxidations and Nitrations Peroxidase Inhibitors Peroxidase knockout
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Consequences of 3-nitrotyrosine in proteins
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Identification of nitrated proteins in plasma of ARDS patients -Ceruloplasmin -Transferrin - 1antichimotrypsine - 1protease inhibitor -Fibrinogen
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How could we look at protein 3- nitrotyrosine formation? J.B.C. (2000) 275, 21409 Cytochrome c control Cytochrome c + 0.5 mM ONOO - Cytochrome c + 2 mM ONOO -
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3-nitroTyrosine changes the pI of protein J.B.C. (2000) 275, 21409 Native poliacrylamide electrophoresis Cytochrome c 1- Control 2- one bolus ONOO - 3 mM 3- two bolus “ 4- four bolus “ 5- six bolus “ 6- reverse order addition
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Purification of nitrated forms of cytochrome c by cation-exchange chromatrography Biochemistry (2005) 44, 8038
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Mapping of 3-nitroTyr in cytochrome c
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Three-D view of Tyrosines in cytochrome c Biochemistry (2005) 44, 8038
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3-nitroTyrosine may induce a “gain of function” Two examples: Nitration of Cytochrome c Nitration of Fibrinogen
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Nitrated cytochome c shows an increase in its peroxidase activity J.B.C. (2000) 275, 21409 Biochemistry (2005) 44, 8038
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Nitrated Fibrinogen shows an increase in its pro-thrombotic properties J.B.C. (2004) 279, 8820
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Scanning EM of: A- Fibrinogen control B- + MPO/H 2 O 2 /NO 2 - C- + SIN-1 D- + MPO/H 2 O 2 J.B.C. (2004) 279, 8820
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Why is protein tyrosine nitration important in vivo? 4Selective, not all proteins are modified 4Alter function in some but not all proteins 4 Structural alteration, accelerate protein turn-over 4Increase antigenicity and induce immune responses
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O 2 -. NO + SO D MPO/H 2 O 2 /NO 2 - ONOO - / CO 2 Y Y NO 2 Enzymatic Activity Signal Cascades Immunological Responds Repair Activity ? Proteosome Tyrosine Decarboxilase 3-Nitro-hydroxy-fenilacetaldehyde Y NO 2
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Controversial and Challengers “3-nitrotyrosine is produced in vivo; there is an increase in 3-nitrotyrosine concentration in many pathological situations” Some controversies remain: 1- The biological significance of nitration. 2- The mechanisms of 3-nitrotyrosine formation. 3- Is there a repair mechanism for 3-nitrotyrosine? Is it a signal pathway? 4- Where is nitration produced? Which are the preferential targets?
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