Protein 3-Nitrotyrosine: Formation, Evaluation and Biological Consequences Dr. José M. Souza Departamento de Bioquímica Centro de Radicales Libres Facultad. - ppt download
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
José M. Souza Departamento de Bioquímica Centro de Radicales Libres Facultad de Medicina, Universidad de la República Av. Gral. Flores 2125, Montevideo, Uruguay
Nitrating agent Formation of 3-nitro-tyrosine (NO 2 ) 4 C pH 8 (NO 2 ) 3 C - + 2H +
NO. All pathways for 3-nitroTyr formation depend on nitric oxide formation.
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.
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.
Radical mechanism of nitration
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 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 H 2 O NO H +
H2O2H2O2H2O2H2O2 H2OH2OH2OH2O MPO Compound I MPO Ground State Cl - HOCl.
H2O2H2O2H2O2H2O2 H2OH2OH2OH2O MPO Compound I MPO Compound II NO 2 -. NO 2 Tyr Tyr. MPO Ground State.
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
277, 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.
NO + O 2.- ONOO - Oxidations and Nitrations NOS inhibitors NOX and XO inhibitors NO scavengers SOD or SOD-mimics Scavengers Decomposition catalysts Repair.
NO O 2.- SOD NO 3 - HbO 2 Decomposition catalysts (catalase or catalase mimics) Oxidations and Nitrations Peroxidase Inhibitors Peroxidase knockout.
Consequences of 3-nitrotyrosine in proteins
Identification of nitrated proteins in plasma of ARDS patients -Ceruloplasmin -Transferrin - 1antichimotrypsine - 1protease inhibitor -Fibrinogen
J.B.C. (2000) 275, Cytochrome c control Cytochrome c mM ONOO - Cytochrome c + 2 mM ONOO -.
(2000) 275, 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.
Purification of nitrated forms of cytochrome c by cation-exchange chromatrography Biochemistry (2005) 44, 8038
Mapping of 3-nitroTyr in cytochrome c
Three-D view of Tyrosines in cytochrome c Biochemistry (2005) 44, 8038
3-nitroTyrosine may induce a gain of function Two examples: Nitration of Cytochrome c Nitration of Fibrinogen
(2000) 275, Biochemistry (2005) 44,
Nitrated Fibrinogen shows an increase in its pro-thrombotic properties J.B.C. (2004) 279, 8820
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.
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.
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 .
Protein 3-Nitrotyrosine: Formation, Evaluation and Biological Consequences Dr. José M. Souza Departamento de Bioquímica Centro de Radicales Libres Facultad. - ppt download
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