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: jsouza@fmed.edu.uy

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?