Protein 3-Nitrotyrosine: Formation, Evaluation and Biological Consequences Dr. José M. Souza...
-
Upload
blaise-hall -
Category
Documents
-
view
214 -
download
0
Transcript of Protein 3-Nitrotyrosine: Formation, Evaluation and Biological Consequences Dr. José M. Souza...
![Page 1: Protein 3-Nitrotyrosine: Formation, Evaluation and Biological Consequences Dr. José M. Souza Departamento de Bioquímica Centro de Radicales Libres Facultad.](https://reader035.fdocuments.net/reader035/viewer/2022062717/56649e555503460f94b4c10a/html5/thumbnails/1.jpg)
Protein 3-Nitrotyrosine: Formation, Evaluation and BiologicalConsequences
Dr. José M. SouzaDepartamento de BioquímicaCentro de Radicales LibresFacultad de Medicina, Universidad de la RepúblicaAv. Gral. Flores 2125, Montevideo, UruguayE-mail: [email protected]
![Page 2: Protein 3-Nitrotyrosine: Formation, Evaluation and Biological Consequences Dr. José M. Souza Departamento de Bioquímica Centro de Radicales Libres Facultad.](https://reader035.fdocuments.net/reader035/viewer/2022062717/56649e555503460f94b4c10a/html5/thumbnails/2.jpg)
OH
NH2
COOH
OH
NO2
NH2
COOH
Nitrating agent
Formation of 3-nitro-tyrosine
(NO2)4C pH8 (NO2)3C- + 2H+
![Page 3: Protein 3-Nitrotyrosine: Formation, Evaluation and Biological Consequences Dr. José M. Souza Departamento de Bioquímica Centro de Radicales Libres Facultad.](https://reader035.fdocuments.net/reader035/viewer/2022062717/56649e555503460f94b4c10a/html5/thumbnails/3.jpg)
Mechanism of 3-nitroTyrosine Mechanism of 3-nitroTyrosine FormationFormation
Two major pathways have been established:Two major pathways have been established:
•PeroxynitritePeroxynitrite
•Peroxidases or MPO/HPeroxidases or MPO/H22OO22/NO/NO22--
•NONO..??
All pathways for 3-nitroTyr formation depend on nitric oxide formation
![Page 4: Protein 3-Nitrotyrosine: Formation, Evaluation and Biological Consequences Dr. José M. Souza Departamento de Bioquímica Centro de Radicales Libres Facultad.](https://reader035.fdocuments.net/reader035/viewer/2022062717/56649e555503460f94b4c10a/html5/thumbnails/4.jpg)
Oxidation state (n) 2 3 4 5.NO NO2
- .NO2 ONOO
-
Intermediates/catalysts
Tyr.
H+/HNO2
H2O2, HOClMyeloperoxidaseHemeproteins
Tyr.
CO2
Me n+
ROH,RCO2
Myeloperoxidase Eosinophil peroxidase
In search of the in vivo nitrating agents
Tyrosyl Radical: Prostaglandin H Synthase-2, Ribonucleotide ReductasePeroxidases: Catalysts of both nitrite and peroxynitrite-mediated nitrationHypochlorous acid: Likely not involved in peroxidase-mediated nitrationNitrogen Dioxide: Inefficient in the absence of tyrosyl radicalONO(O)CO2
-: More efficient nitrating agent than peroxynitrite
![Page 5: Protein 3-Nitrotyrosine: Formation, Evaluation and Biological Consequences Dr. José M. Souza Departamento de Bioquímica Centro de Radicales Libres Facultad.](https://reader035.fdocuments.net/reader035/viewer/2022062717/56649e555503460f94b4c10a/html5/thumbnails/5.jpg)
Reactive PathwaysReactive Pathways
ONOOONOO-- O2
-.NO +
RC-NORC-NO22 NitrativeNitrativeStressStress
OxFe-SOxFe-S, , CarbonylsCarbonyls
Oxidative Oxidative StressStress
RS-NO, RN-NORS-NO, RN-NO
Nitrosative Nitrosative StressStress
OO22
MeMen+n+
RSHRSH
COCO22
22ON-ON-OCOOCO22--
HH22OO22
MeMen+n+
MPOMPOEPOEPO
![Page 6: Protein 3-Nitrotyrosine: Formation, Evaluation and Biological Consequences Dr. José M. Souza Departamento de Bioquímica Centro de Radicales Libres Facultad.](https://reader035.fdocuments.net/reader035/viewer/2022062717/56649e555503460f94b4c10a/html5/thumbnails/6.jpg)
Radical mechanism of nitration
O
..OH
OH
OH
O
.
OH
OH2
OH O..OH
NO2.
CO3-.
O
.
O
. NO2.
OH
NO2
![Page 7: Protein 3-Nitrotyrosine: Formation, Evaluation and Biological Consequences Dr. José M. Souza Departamento de Bioquímica Centro de Radicales Libres Facultad.](https://reader035.fdocuments.net/reader035/viewer/2022062717/56649e555503460f94b4c10a/html5/thumbnails/7.jpg)
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+
![Page 8: Protein 3-Nitrotyrosine: Formation, Evaluation and Biological Consequences Dr. José M. Souza Departamento de Bioquímica Centro de Radicales Libres Facultad.](https://reader035.fdocuments.net/reader035/viewer/2022062717/56649e555503460f94b4c10a/html5/thumbnails/8.jpg)
MPO-catalyzed ClMPO-catalyzed Cl---mediated oxidation-mediated oxidation
Fe Fe IIIIII Fe Fe IV+.IV+.
HH22OO22HH22OO
MPO CompoundMPO Compound I IMPO Ground State MPO Ground State
Cl Cl --HOClHOCl
![Page 9: Protein 3-Nitrotyrosine: Formation, Evaluation and Biological Consequences Dr. José M. Souza Departamento de Bioquímica Centro de Radicales Libres Facultad.](https://reader035.fdocuments.net/reader035/viewer/2022062717/56649e555503460f94b4c10a/html5/thumbnails/9.jpg)
NONO22Tyr formation by MPOTyr formation by MPO
Fe Fe IVIV
Fe Fe IIIIII Fe Fe IV+.IV+.
HH22OO22HH22OO
MPO CompoundMPO Compound I I
MPO MPO
Compound IICompound II
NONO22--
..NONO22TyrTyr
TyrTyr..
MPO Ground State MPO Ground State
![Page 10: Protein 3-Nitrotyrosine: Formation, Evaluation and Biological Consequences Dr. José M. Souza Departamento de Bioquímica Centro de Radicales Libres Facultad.](https://reader035.fdocuments.net/reader035/viewer/2022062717/56649e555503460f94b4c10a/html5/thumbnails/10.jpg)
Tyrosine Nitration by Nitric OxideTyrosine Nitration by Nitric Oxide
Nitric oxide may reactwith stable tyrosyl radicalresidue that are involvedin the catalytic mechanismof ribonucleotide reductaseor prostaglandin H synthase,or cytochrome c-H2O2
O
.
OH
NO.NO
Nitrosotyrosine
O
NO.
OH
NO2
Iminoxylradical
-e- H+
-e-
+H2O
![Page 11: Protein 3-Nitrotyrosine: Formation, Evaluation and Biological Consequences Dr. José M. Souza Departamento de Bioquímica Centro de Radicales Libres Facultad.](https://reader035.fdocuments.net/reader035/viewer/2022062717/56649e555503460f94b4c10a/html5/thumbnails/11.jpg)
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 speciesBrennan M-L et al (2001) J.B.C. 277, 17415-17427
Peroxidases Knockout Model
3-Nitrotyrosine Formation from Lung Tissue after Aeroallergen Challenge
NO2Y/Y(mol/mol)
WT + Ova
336798
EPO-KO + Ova
376504
3-Nitrotyrosine Formation from Zymosan-inducedPeritonitisLavage protein after 20h thioglycollate and 4hzymosan
NO2Y/Y(mol/mol)
WT + Tg/Z
1.10.6114.2
MPO-KO + Tg/Z
1.90.97.43.9
![Page 12: Protein 3-Nitrotyrosine: Formation, Evaluation and Biological Consequences Dr. José M. Souza Departamento de Bioquímica Centro de Radicales Libres Facultad.](https://reader035.fdocuments.net/reader035/viewer/2022062717/56649e555503460f94b4c10a/html5/thumbnails/12.jpg)
Peroxynitrite PharmacologyPeroxynitrite Pharmacology
.NO + O2.- ONOO-
Oxidations andNitrations
NOS inhibitors
NOX and XO inhibitors
NO scavengers SOD orSOD-mimics
Scavengers
Decompositioncatalysts
Repair
![Page 13: Protein 3-Nitrotyrosine: Formation, Evaluation and Biological Consequences Dr. José M. Souza Departamento de Bioquímica Centro de Radicales Libres Facultad.](https://reader035.fdocuments.net/reader035/viewer/2022062717/56649e555503460f94b4c10a/html5/thumbnails/13.jpg)
Peroxidases Pharmacology
NO2- + H2O2 + MPO / EPO
.NO O2.-
SOD
NO3-
HbO2
Decompositioncatalysts (catalase or
catalase mimics)
Oxidations andNitrations
Peroxidase InhibitorsPeroxidase knockout
![Page 14: Protein 3-Nitrotyrosine: Formation, Evaluation and Biological Consequences Dr. José M. Souza Departamento de Bioquímica Centro de Radicales Libres Facultad.](https://reader035.fdocuments.net/reader035/viewer/2022062717/56649e555503460f94b4c10a/html5/thumbnails/14.jpg)
Consequences of 3-nitrotyrosine in proteins
![Page 15: Protein 3-Nitrotyrosine: Formation, Evaluation and Biological Consequences Dr. José M. Souza Departamento de Bioquímica Centro de Radicales Libres Facultad.](https://reader035.fdocuments.net/reader035/viewer/2022062717/56649e555503460f94b4c10a/html5/thumbnails/15.jpg)
Identification of nitrated proteins in plasma
of ARDS patients
-Ceruloplasmin-Transferrin1antichimotrypsine1protease inhibitor-Fibrinogen
![Page 16: Protein 3-Nitrotyrosine: Formation, Evaluation and Biological Consequences Dr. José M. Souza Departamento de Bioquímica Centro de Radicales Libres Facultad.](https://reader035.fdocuments.net/reader035/viewer/2022062717/56649e555503460f94b4c10a/html5/thumbnails/16.jpg)
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-
![Page 17: Protein 3-Nitrotyrosine: Formation, Evaluation and Biological Consequences Dr. José M. Souza Departamento de Bioquímica Centro de Radicales Libres Facultad.](https://reader035.fdocuments.net/reader035/viewer/2022062717/56649e555503460f94b4c10a/html5/thumbnails/17.jpg)
3-nitroTyrosine changes the pI of protein
J.B.C. (2000) 275, 21409
Native poliacrylamide electrophoresisCytochrome c
1- Control2- one bolus ONOO- 3 mM3- two bolus “4- four bolus “5- six bolus “6- reverse order addition
![Page 18: Protein 3-Nitrotyrosine: Formation, Evaluation and Biological Consequences Dr. José M. Souza Departamento de Bioquímica Centro de Radicales Libres Facultad.](https://reader035.fdocuments.net/reader035/viewer/2022062717/56649e555503460f94b4c10a/html5/thumbnails/18.jpg)
Purification of nitrated forms of cytochrome c by cation-exchange chromatrography
Biochemistry (2005) 44, 8038
![Page 19: Protein 3-Nitrotyrosine: Formation, Evaluation and Biological Consequences Dr. José M. Souza Departamento de Bioquímica Centro de Radicales Libres Facultad.](https://reader035.fdocuments.net/reader035/viewer/2022062717/56649e555503460f94b4c10a/html5/thumbnails/19.jpg)
Biochemistry (2005) 44, 8038
Mapping of 3-nitroTyr in cytochrome c
![Page 20: Protein 3-Nitrotyrosine: Formation, Evaluation and Biological Consequences Dr. José M. Souza Departamento de Bioquímica Centro de Radicales Libres Facultad.](https://reader035.fdocuments.net/reader035/viewer/2022062717/56649e555503460f94b4c10a/html5/thumbnails/20.jpg)
Three-D view of Tyrosines in cytochrome c
Biochemistry (2005) 44, 8038
![Page 21: Protein 3-Nitrotyrosine: Formation, Evaluation and Biological Consequences Dr. José M. Souza Departamento de Bioquímica Centro de Radicales Libres Facultad.](https://reader035.fdocuments.net/reader035/viewer/2022062717/56649e555503460f94b4c10a/html5/thumbnails/21.jpg)
3-nitroTyrosine may induce a “gain of function”
Two examples: Nitration of Cytochrome c Nitration of Fibrinogen
![Page 22: Protein 3-Nitrotyrosine: Formation, Evaluation and Biological Consequences Dr. José M. Souza Departamento de Bioquímica Centro de Radicales Libres Facultad.](https://reader035.fdocuments.net/reader035/viewer/2022062717/56649e555503460f94b4c10a/html5/thumbnails/22.jpg)
Nitrated cytochome c shows an increase in its peroxidase activity
J.B.C. (2000) 275, 21409 Biochemistry (2005) 44, 8038
![Page 23: Protein 3-Nitrotyrosine: Formation, Evaluation and Biological Consequences Dr. José M. Souza Departamento de Bioquímica Centro de Radicales Libres Facultad.](https://reader035.fdocuments.net/reader035/viewer/2022062717/56649e555503460f94b4c10a/html5/thumbnails/23.jpg)
Nitrated Fibrinogen shows an increase in its pro-thrombotic properties
J.B.C. (2004) 279, 8820
![Page 24: Protein 3-Nitrotyrosine: Formation, Evaluation and Biological Consequences Dr. José M. Souza Departamento de Bioquímica Centro de Radicales Libres Facultad.](https://reader035.fdocuments.net/reader035/viewer/2022062717/56649e555503460f94b4c10a/html5/thumbnails/24.jpg)
Scanning EM of:
A- Fibrinogen controlB- + MPO/H2O2/NO2
-
C- + SIN-1D- + MPO/H2O2
J.B.C. (2004) 279, 8820
![Page 25: Protein 3-Nitrotyrosine: Formation, Evaluation and Biological Consequences Dr. José M. Souza Departamento de Bioquímica Centro de Radicales Libres Facultad.](https://reader035.fdocuments.net/reader035/viewer/2022062717/56649e555503460f94b4c10a/html5/thumbnails/25.jpg)
Why is protein tyrosine nitration important Why is protein tyrosine nitration important in vivoin 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
![Page 26: Protein 3-Nitrotyrosine: Formation, Evaluation and Biological Consequences Dr. José M. Souza Departamento de Bioquímica Centro de Radicales Libres Facultad.](https://reader035.fdocuments.net/reader035/viewer/2022062717/56649e555503460f94b4c10a/html5/thumbnails/26.jpg)
O2
-.NO +
SOSODD
MPO/HMPO/H22OO22/NO/NO22--ONOOONOO--/ CO/ CO22
Y YNO2
Enzymatic Activity Signal Cascades Immunological Responds Enzymatic Activity Signal Cascades Immunological Responds
Repair Activity ?
ProteosomeTyrosine Decarboxilase
3-Nitro-hydroxy-fenilacetaldehyde
YNO
2
![Page 27: Protein 3-Nitrotyrosine: Formation, Evaluation and Biological Consequences Dr. José M. Souza Departamento de Bioquímica Centro de Radicales Libres Facultad.](https://reader035.fdocuments.net/reader035/viewer/2022062717/56649e555503460f94b4c10a/html5/thumbnails/27.jpg)
Controversial and Challengers
“3-nitrotyrosine is produced in vivo; there is an increase in3-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?