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Protein 3-Nitrotyrosine: Formation, Evaluation and Biological Consequences

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. Nitrating agent.

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Protein 3-Nitrotyrosine: Formation, Evaluation and Biological Consequences

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  1. 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

  2. Nitrating agent Formation of 3-nitro-tyrosine (NO2)4C pH8 (NO2)3C- + 2H+

  3. Mechanism of 3-nitroTyrosine Formation All pathways for 3-nitroTyr formation depend on nitric oxide formation • Two major pathways have been established: • Peroxynitrite • Peroxidases or MPO/H2O2/NO2- • NO.?

  4. Eosinophil peroxidase In search of the in vivo nitrating agents Oxidation state (n) 2 3 4 5 . - . - NO NO NO ONOO 2 2 . + . Tyr H / HNO Tyr Intermediates/ CO 2 2 n+ H O , HOCl Me catalysts 2 2 Myeloperoxidase ROH,RCO 2 Hemeproteins Myeloperoxidase 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)CO2-: More efficient nitrating agent than peroxynitrite

  5. .NO + ONOO- - O2 CO2 O2 Men+ RSH 2ON-OCO2- OxFe-S, Carbonyls Oxidative Stress RC-NO2 Nitrative Stress RS-NO, RN-NO Nitrosative Stress Reactive Pathways H2O2 Men+ MPO EPO

  6. Radical mechanism of nitration

  7. Peroxynitrite free radical-independent nitration mechanism This mechanism may ocurre within protein metal centers ONOO- + MenX ONOO-MenX NO2-O-MenX NO2++ O=MenX Tyr NO2-Tyr + O=MenX + H+ O=MenX + 2H+ MenX + H2O NO2+ + H2O NO3- + 2H+

  8. MPO-catalyzed Cl--mediated oxidation H2O H2O2 MPO Compound I Fe IV+. Fe III MPO Ground State HOCl Cl -

  9. NO2Tyr formation by MPO H2O H2O2 MPO Compound I Fe IV+. Fe III MPO Ground State NO2- Tyr. Fe IV .NO2 Tyr MPO Compound II

  10. 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-H2O2

  11. Peroxidases Knockout Model 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 3-Nitrotyrosine Formation from Lung Tissue after Aeroallergen Challenge 3-Nitrotyrosine Formation from Zymosan-induced Peritonitis Lavage protein after 20h thioglycollate and 4h zymosan

  12. Peroxynitrite Pharmacology NOX and XO inhibitors Decomposition catalysts NOS inhibitors Repair Oxidations and Nitrations .NO + O2.- ONOO- Scavengers SOD or SOD-mimics NO scavengers

  13. Peroxidases Pharmacology Peroxidase Inhibitors Peroxidase knockout .NO O2.- SOD HbO2 Oxidations and Nitrations NO2- + H2O2 + MPO / EPO Decomposition catalysts (catalase or catalase mimics) NO3-

  14. Consequences of 3-nitrotyrosine in proteins

  15. Identification of nitrated proteins in plasma of ARDS patients • Ceruloplasmin • Transferrin • 1antichimotrypsine • 1protease inhibitor • Fibrinogen

  16. How could we look at protein 3-nitrotyrosine formation? Cytochrome c control Cytochrome c + 0.5 mM ONOO- Cytochrome c + 2 mM ONOO- J.B.C. (2000) 275, 21409

  17. 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 3-nitroTyrosine changes the pI of protein J.B.C. (2000) 275, 21409

  18. Purification of nitrated forms of cytochrome c by cation-exchange chromatrography Biochemistry (2005) 44, 8038

  19. Mapping of 3-nitroTyr in cytochrome c Biochemistry (2005) 44, 8038

  20. Three-D view of Tyrosines in cytochrome c Biochemistry (2005) 44, 8038

  21. 3-nitroTyrosine may induce a “gain of function” Two examples: Nitration of Cytochrome c Nitration of Fibrinogen

  22. Nitrated cytochome c shows an increase in its peroxidase activity J.B.C. (2000) 275, 21409 Biochemistry (2005) 44, 8038

  23. Nitrated Fibrinogen shows an increase in its pro-thrombotic properties J.B.C. (2004) 279, 8820

  24. Scanning EM of: A- Fibrinogen control B- + MPO/H2O2/NO2- C- + SIN-1 D- + MPO/H2O2 J.B.C. (2004) 279, 8820

  25. Why is protein tyrosine nitration important in vivo? • Selective, not all proteins are modified • Alter function in some but not all proteins • Structural alteration, accelerate protein turn-over • Increase antigenicity and induce immune responses

  26. - O2 NO2 NO2 Y Y Y .NO + SOD ONOO-/ CO2 MPO/H2O2/NO2- Tyrosine Decarboxilase Proteosome 3-Nitro-hydroxy-fenilacetaldehyde Repair Activity ? Enzymatic Activity Signal Cascades Immunological Responds

  27. 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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