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New types of biomarkers in the cardiovascular sistem corectat

Florin George Frunza
Florin George Frunza
Florin George FrunzaLecturer in Functional and Clinical Proteomics at University of Medicine and Pharmacy Iasi

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 !!!

New types of biomarkers in the cardiovascular sistem corectat

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New types of biomarkers in cardiovascular pathology
-NITRATED PROTEINS-
Florin G. Frunza
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 ?
New types of biomarkers in the cardiovascular sistem corectat
New types of biomarkers in the cardiovascular sistem corectat
New types of biomarkers in the cardiovascular sistem corectat
New types of biomarkers in the cardiovascular sistem corectat

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New types of biomarkers in the cardiovascular sistem corectat

  • 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.