PPT presentation from BIOMARKERS IN RESEARCH AND DEVELOPEMENT field. From my course in CLINICAL AND FUNCTIONAL PROTEOMICS. New dimentions in POSTTRANSLATIONAL MODIFICATIONS OF PROTEINS. THE DARK FACE OF THE MOON for Third Millenium Medicine !!!

1. New types of biomarkers in cardiovascular pathology
-NITRATED PROTEINS-
Florin G. Frunza

2. Content
 Impaired NO bioavailability during vascular oxidant formation
 Nitroxidative stress in vivo
 Tyrosine nitration – the radical pathways
 Protein nitration in the cardiovascular system
 Nitrated proteins in different cardiovascular pathologies
 In vivo determination of tyrosine nitrate
 Denitration
 Nitrotyrosine – mediator or biomaker of cardiovascular disease ?

26. Impaired NO bioavailability during vascular oxidant formation
Nitric oxide is a major contributor to the normal
homeostasis of the cardiovascular system .
Physiological levels of •NO are principal determinants of
endothelium-dependent relaxation and regulator of vascular
tone .
NO inhibits platelet aggregation adhesive molecules
expression , and regulates cell proliferation and differentiation at
the vascular wall .
A decrease in the bioavailability of •NO is associated with
a myriad of cardiovascular disease conditions, including
atheromatosis, heart failure, sepsis, CAD,stroke and myocardial
infarction .

27. Impaired NO bioavailability during vascular oxidant formation
The above mentioned pathologies are associated
with an increase in production of reactive oxygen species.
The net concentrations of ·
NO at the tissue level may
predict its protective or toxic effects.
Many lines of evidence suggest that modulation of ·
NO
concentration will determine whether or not the roles played by
RNS/ROS will be protective or detrimental to the cardiovascular
system.

28. Nitroxidative stress in vivo
Regulation of nitric
oxide output from the
endothelium by
superoxide.

29. Tyrosine nitration – the radical pathways

30. Tyrosine nitration – the radical pathways
NH2
O
OH
-O3PO
Phosphotyrosine
NH2
O
OH
-O2SO
Sulfatyrosine
HO
NH2
O
OH
I
Iodotyrosine
OH
H2N
O
OH
HO
NH2
O
HO
Dityrosine
3-Nitrotyrosine
HO
NH2
O
OH
Cl
3-Chlorotyrosine
Post-translational Modifications of Protein Tyrosine ResiduesPost-translational Modifications of Protein Tyrosine Residues

31. Protein nitration in the cardiovascular system
Nitration in the vessel walls
The artery wall is a major site of nitroxidative protein
modification in several pathologies such as hypertension and
atheromatosis.
Nitration reactions are favoured in the artery
wall given that some of the detoxifying reactions that occur
at the lumen are less relevant for example in the extracellular
matrix due to their low concentration.

32. Protein nitration in the cardiovascular system
Fibrinogen
Clinical trials have shown an association between
nitroxidative stress and fibrinogen in patients with CAD
A 30% increase in nitrated fibrinogen was detected in
plasma of patients with documented CAD
Nitrated fibrinogen from patients or in vitro modified
fibrinogen polymerizes faster than normal fibrinogen.

33. Fibrin network as a result of
nitrated fibrinogen activation

34. Protein nitration in the cardiovascular system
Nitration of LDL
Nitrated LDL triggers the release of TNF-α from non
differentiated human monocytes, consequently amplifying the
inflammatory process in vivo
Nitrated LDL from human thoracic aorta atheroma plaques
has been detected.

35. Protein nitration in the cardiovascular system
Nitration of cyclooxygenase and prostacyclin synthase
Low levels of ONOO− and other peroxides in presence
of arachidonic acidcan activate COX but higher pathophysio-
logical levels Inhibit and nitrate COX
Nitration is associated with inhibition of enzyme activity and
and has been detected in human atheroma plaques

36. Protein nitration in the cardiovascular system
Mn-SOD
Mn-SOD is located in the mitochondrial matrix
It was the first nitrated proteins to be identified in chronic
renal allograft rejection disease in humans.
Nitrated Mn-SOD was identified in vascular aging on rats.
Mn-SOD nitration in endothelial cells after addition of
Cyclosporine A due to peroxynitrite formation, has just been
reported.

37. Protein nitration in the cardiovascular system
Nitration of the myocardium
Nitroxidative stress generated by inflammatory changes
that occur after myocardial infarction, infectious process of the
myocardium such as myocarditis, or other experimental setups,
have been linked to the alteration of energetic balance and
contractile dysfunction of the failing heart.

38. Protein nitration in the cardiovascular system
Sarcoplasmic reticulum Ca2+-
ATPase
Alterations in Ca2+ homeostasis have been recognized for
a long time in heart failure.
Studies with transgenic mice confirm that cardiac contrac-
tility is modulated by SERCA levels.
SERCA-2a, found predominantly in slow twitch skeletal
muscle,vessel smooth muscle and cardiac muscle, is sensitive to
inhibition and nitration by ONOO−.
Recent studies have been devoted to unravel the
connection between nitroxidative modification of SERCA-2a and
functionality in the senescent heart.

39. Nitrated proteins in cardiovascular pathology
Hypertension is associated with endothelial dysfunction and
altered •NO bioavailability in part by an increase in O2- production.
Hypertension
The identity of nitrated proteins in hypertension remains to be
deternimed .

40. Nitrated proteins in cardiovascular pathology
Ischaemia and reperfusion
A Tel-Aviv University team have conducted an experiment in
order to establish whether ischaemia or reperfusion is the main trigger in
changes of NOS mRNA expression.
The results show that ischaemic injury causes down-regulation of
endothelial nitric oxide synthase mRNA expression, which is then
associated with reduction of coronary flow during reperfusion,
representing one possible mechanism of ischaemia/reperfusion injury.
They did not find expected elevations of inducible nitric oxide
Synthase mRNA expression during ischaemia or reperfusion and we
suggest that ischaemia/reperfusion injury is not associated with nitric
oxide overproduction.

41. In vivo determination of nitrated free tyrosine and
protein/peptide bound tyrosine
1. Analytical Methods:
HPLC (UV, Electrochemical Detection)Gas
Chromatography/Mass SpectrometryLC/Mass
Spectrometry
Major concern: artificial formation during acid hydrolysis
Remedy: base hydrolysis, inclusion of uniformly labeled
tyrosine
2. Immunological Methods (Antibodies):
Western Blotting
IImmunocytochemistry/Immunohistochemistry
ELISA
Major concern: antibody specificity
Remedy: raise specific monoclonal antibodies to target
proteins

45. Denitration
Loss of antigenic binding without apparent protein degradation
Exhibit different kinetics towards different nitrated protein substrates
Does not function when 3-nitrotyrosine or 3-nitrotyrosine peptides are
used as substrates
The products of the reaction are not known but it does not appear to
be aminotyrosine

46. Nitrotyrosine: mediator or biomaker of cardiovascular disease ?

47. Nitrotyrosine: mediator or biomaker of cardiovascular disease ?
Protein nitration is a usual process in the living organism
and 3-NO2-Tyr accumulates during the aging process reflecting
the basal nitroxidative stress normally produced.
Nevertheless, as the physiological redox balance is
weakened during the disease state, nitroxidative stress
emerges as a mediator of damage, which includes among
others, tyrosine nitration.
The mechanism of tyrosine nitration can help to
rationalize pharmacological strategies to prevent tyrosine
nitration and therefore recover the cardiovascular lesion, which
is the proof-of-principle of the nitrated protein-mediated damage
hypothesis.

48. References
Ischiropoulos H. Biological selectivity and functional aspects of protein tyrosine
nitration. Biochem Biophys Res Commun 2003;305:776–83.
VadsethC, Souza JM, Thomson L, SeagravesA,NagaswamiC, ScheinerT, et al.
Pro-thrombotic state induced by post-translational modification of fibrinogen by
reactive nitrogen species. J Biol Chem 2004;279:
 Olah GA, Malhotra R, Narang SC. (1989) In: Nitration, Methods and Mechanisms.
Organic Nitro-Chemistry Series, VCH Publishers, Inc. Review Biochemistry of
protein tyrosine nitration in cardiovascular pathology
Gonzalo Peluffo, Rafael RadiKoolman , Colored Atlas of biochemistry , 2005
Ischaemia or reperfusion: which isa main trigger for changes in nitric oxide mRNA
synthases expression?
Pevni D;Frolkis I; Shapira I; Schwartz D; Schwartz IF; Chernichovski T; Lev-
Ran O; Sharony R; Uretzky G Department of Cardiothoracic Surgery, Tel Aviv
University, Tel Aviv, Israel.