Protein 3-Nitrotyrosine: Formation, Evaluation and Biological
This presentation is the property of its rightful owner.
Sponsored Links
1 / 27

Protein 3-Nitrotyrosine: Formation, Evaluation and Biological Consequences PowerPoint PPT Presentation


  • 56 Views
  • Uploaded on
  • Presentation posted in: General

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.

Download Presentation

Protein 3-Nitrotyrosine: Formation, Evaluation and Biological Consequences

An Image/Link below is provided (as is) to download presentation

Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.


- - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - -

Presentation Transcript


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: [email protected]


Protein 3 nitrotyrosine formation evaluation and biological consequences

Nitrating agent

Formation of 3-nitro-tyrosine

(NO2)4C pH8 (NO2)3C- + 2H+


Protein 3 nitrotyrosine formation evaluation and biological consequences

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.?


Protein 3 nitrotyrosine formation evaluation and biological consequences

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


Protein 3 nitrotyrosine formation evaluation and biological consequences

.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


Protein 3 nitrotyrosine formation evaluation and biological consequences

Radical mechanism of nitration


Protein 3 nitrotyrosine formation evaluation and biological consequences

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+


Protein 3 nitrotyrosine formation evaluation and biological consequences

MPO-catalyzed Cl--mediated oxidation

H2O

H2O2

MPO Compound I

Fe IV+.

Fe III

MPO Ground State

HOCl

Cl -


Protein 3 nitrotyrosine formation evaluation and biological consequences

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


Protein 3 nitrotyrosine formation evaluation and biological consequences

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


Protein 3 nitrotyrosine formation evaluation and biological consequences

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


Protein 3 nitrotyrosine formation evaluation and biological consequences

Peroxynitrite Pharmacology

NOX and XO inhibitors

Decomposition

catalysts

NOS inhibitors

Repair

Oxidations and

Nitrations

.NO + O2.-

ONOO-

Scavengers

SOD or

SOD-mimics

NO scavengers


Protein 3 nitrotyrosine formation evaluation and biological consequences

Peroxidases Pharmacology

Peroxidase Inhibitors

Peroxidase knockout

.NO O2.-

SOD

HbO2

Oxidations and

Nitrations

NO2- + H2O2 + MPO / EPO

Decomposition

catalysts (catalase or

catalase mimics)

NO3-


Protein 3 nitrotyrosine formation evaluation and biological consequences

Consequences of 3-nitrotyrosine in proteins


Protein 3 nitrotyrosine formation evaluation and biological consequences

Identification of nitrated proteins in plasma

of ARDS patients

  • Ceruloplasmin

  • Transferrin

  • 1antichimotrypsine

  • 1protease inhibitor

  • Fibrinogen


Protein 3 nitrotyrosine formation evaluation and biological consequences

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


Protein 3 nitrotyrosine formation evaluation and biological consequences

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


Protein 3 nitrotyrosine formation evaluation and biological consequences

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

Biochemistry (2005) 44, 8038


Protein 3 nitrotyrosine formation evaluation and biological consequences

Mapping of 3-nitroTyr in cytochrome c

Biochemistry (2005) 44, 8038


Protein 3 nitrotyrosine formation evaluation and biological consequences

Three-D view of Tyrosines in cytochrome c

Biochemistry (2005) 44, 8038


Protein 3 nitrotyrosine formation evaluation and biological consequences

3-nitroTyrosine may induce a “gain of function”

Two examples: Nitration of Cytochrome c

Nitration of Fibrinogen


Protein 3 nitrotyrosine formation evaluation and biological consequences

Nitrated cytochome c shows an increase in its peroxidase activity

J.B.C. (2000) 275, 21409 Biochemistry (2005) 44, 8038


Protein 3 nitrotyrosine formation evaluation and biological consequences

Nitrated Fibrinogen shows an increase in its pro-thrombotic properties

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


Protein 3 nitrotyrosine formation evaluation and biological consequences

Scanning EM of:

A- Fibrinogen control

B- + MPO/H2O2/NO2-

C- + SIN-1

D- + MPO/H2O2

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


Protein 3 nitrotyrosine formation evaluation and biological consequences

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


Protein 3 nitrotyrosine formation evaluation and biological consequences

-

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


Protein 3 nitrotyrosine formation evaluation and biological consequences

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?


  • Login