Protein 3-Nitrotyrosine: Formation, Evaluation and Biological
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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. Nitrating agent.

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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: [email protected]


Nitrating agent Biological

Formation of 3-nitro-tyrosine

(NO2)4C pH8 (NO2)3C- + 2H+


Mechanism of 3-nitroTyrosine Formation Biological

All pathways for 3-nitroTyr formation depend on nitric oxide formation

  • Two major pathways have been established:

  • Peroxynitrite

  • Peroxidases or MPO/H2O2/NO2-

  • NO.?


Eosinophil peroxidase Biological

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


. BiologicalNO +

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



Peroxynitrite free radical-independent Biological

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 Cl Biological--mediated oxidation

H2O

H2O2

MPO Compound I

Fe IV+.

Fe III

MPO Ground State

HOCl

Cl -


NO Biological2Tyr formation by MPO

H2O

H2O2

MPO Compound I

Fe IV+.

Fe III

MPO Ground State

NO2-

Tyr.

Fe IV

.NO2

Tyr

MPO

Compound II


Tyrosine Nitration by Nitric Oxide Biological

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


Peroxidases Knockout Model Biological

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


Peroxynitrite Pharmacology Biological

NOX and XO inhibitors

Decomposition

catalysts

NOS inhibitors

Repair

Oxidations and

Nitrations

.NO + O2.-

ONOO-

Scavengers

SOD or

SOD-mimics

NO scavengers


Peroxidases Pharmacology Biological

Peroxidase Inhibitors

Peroxidase knockout

.NO O2.-

SOD

HbO2

Oxidations and

Nitrations

NO2- + H2O2 + MPO / EPO

Decomposition

catalysts (catalase or

catalase mimics)

NO3-



Identification of nitrated proteins in plasma Biological

of ARDS patients

  • Ceruloplasmin

  • Transferrin

  • 1antichimotrypsine

  • 1protease inhibitor

  • Fibrinogen


How could we look at protein 3-nitrotyrosine formation? Biological

Cytochrome c control

Cytochrome c + 0.5 mM ONOO-

Cytochrome c + 2 mM ONOO-

J.B.C. (2000) 275, 21409


Native poliacrylamide electrophoresis Biological

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


Purification of nitrated forms of cytochrome c by cation-exchange chromatrography

Biochemistry (2005) 44, 8038


Mapping of 3-nitroTyr in cytochrome c cation-exchange chromatrography

Biochemistry (2005) 44, 8038


Three-D view of Tyrosines in cytochrome c cation-exchange chromatrography

Biochemistry (2005) 44, 8038


3-nitroTyrosine may induce a “gain of function” cation-exchange chromatrography

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



Scanning EM of: properties

A- Fibrinogen control

B- + MPO/H2O2/NO2-

C- + SIN-1

D- + MPO/H2O2

J.B.C. (2004) 279, 8820


Why is protein tyrosine nitration important propertiesin 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


- properties

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


Controversial and Challengers properties

“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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