Protein 3-Nitrotyrosine: Formation, Evaluation and Biological Consequences
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Transcript of Protein 3-Nitrotyrosine: Formation, Evaluation and Biological Consequences
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]
OH
NH2
COOH
OH
NO2
NH2
COOH
Nitrating agent
Formation of 3-nitro-tyrosine
(NO2)4C pH8 (NO2)3C- + 2H+
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
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
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
Radical mechanism of nitration
O
..OH
OH
OH
O
.
OH
OH2
OH O..OH
NO2.
CO3-.
O
.
O
. NO2.
OH
NO2
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+
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
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
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
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
Peroxynitrite PharmacologyPeroxynitrite Pharmacology
.NO + O2.- ONOO-
Oxidations andNitrations
NOS inhibitors
NOX and XO inhibitors
NO scavengers SOD orSOD-mimics
Scavengers
Decompositioncatalysts
Repair
Peroxidases Pharmacology
NO2- + H2O2 + MPO / EPO
.NO O2.-
SOD
NO3-
HbO2
Decompositioncatalysts (catalase or
catalase mimics)
Oxidations andNitrations
Peroxidase InhibitorsPeroxidase knockout
Consequences of 3-nitrotyrosine in proteins
Identification of nitrated proteins in plasma
of ARDS patients
-Ceruloplasmin-Transferrin1antichimotrypsine1protease inhibitor-Fibrinogen
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-
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
Purification of nitrated forms of cytochrome c by cation-exchange chromatrography
Biochemistry (2005) 44, 8038
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
Nitrated cytochome c shows an increase in its peroxidase activity
J.B.C. (2000) 275, 21409 Biochemistry (2005) 44, 8038
Nitrated Fibrinogen shows an increase in its pro-thrombotic properties
J.B.C. (2004) 279, 8820
Scanning EM of:
A- Fibrinogen controlB- + MPO/H2O2/NO2
-
C- + SIN-1D- + MPO/H2O2
J.B.C. (2004) 279, 8820
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
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
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?