Exposure of tropoelastin to peroxynitrous acid gives high yields of nitrated tyrosine residues, di-tyrosine cross-links and altered protein structure and function

Abstract

Elastin is an abundant extracellular matrix protein in elastic tissues, including the lungs, skin and arteries, and comprises 30-57% of the aorta by dry mass. The monomeric precursor, tropoelastin (TE), undergoes complex processing during elastogenesis to form mature elastic fibres. Peroxynitrous acid (ONOOH), a potent oxidising and nitrating agent, is formed in vivo from superoxide and nitric oxide radicals. Considerable evidence supports ONOOH formation in the inflamed artery wall, and a role for this species in the development of human atherosclerotic lesions, with ONOOH-damaged extracellular matrix implicated in lesion rupture. We demonstrate that TE is highly sensitive to ONOOH, with this resulting in extensive dimerization, fragmentation and nitration of Tyr residues to give 3-nitrotyrosine (3-nitroTyr). This occurs with equimolar or greater levels of oxidant and increases in a dose-dependent manner. Quantification of Tyr loss and 3-nitroTyr formation indicates extensive Tyr modification with up to two modified Tyr per protein molecule, and up to 8% conversion of initial ONOOH to 3-nitroTyr. These effects were modulated by bicarbonate, an alternative target for ONOOH. Inter- and intra-protein di-tyrosine cross-links have been characterized by mass spectrometry. Examination of human atherosclerotic lesions shows colocalization of 3-nitroTyr with elastin epitopes, consistent with TE or elastin modification in vivo, and also an association of 3-nitroTyr containing proteins and elastin with lipid deposits. These data suggest that exposure of TE to ONOOH gives marked chemical and structural changes to TE and altered matrix assembly, and that such damage accumulates in human arterial tissue during the development of atherosclerosis.

Chlorination and oxidation of human plasma fibronectin by myeloperoxidase-derived oxidants, and its consequences for smooth muscle cell function

“…The extent of such ECM deposition has been reported to be a critical factor in lesion stability and propensity to rupture, and therefore the occurrence of heart attacks and strokes [11] . Previous studies have provided data consistent with damage to, and destabilization of, components of atherosclerotic plaques by oxidants generated by inflammatory cells [17] , [18] , with oxidant-mediated damage detected on ECM perlecan [49] , [50] , laminin [51] , (tropo)elastin [52] and fibronectin [53] in human atherosclerotic lesions, as well as lesion lipoproteins (e.g. [36] , [37] , [39] , [54] ).…”

Chlorination and oxidation of the extracellular matrix protein laminin and basement membrane extracts by hypochlorous acid and myeloperoxidase

“…Recent studies have shown that the ECM is highly susceptible to oxidative damage due to its high abundance, its low rate of turnover (which can results in accumulation of damage during ageing and disease) and the relatively low levels of extracellular antioxidant, repairs and catabolic systems [5] , [6] , [7] . Considerable data has been presented that demonstrates that oxidants generated by activated leukocytes (neutrophils, monocytes, tissue macrophages) can induce structural and functional changes to ECM proteins and proteoglycans (proteins with covalently attached GAGs), with damage evident in multiple tissue samples, including within human atherosclerotic lesions [7] , [8] , [9] , [10] , [11] .…”

Modifying the Tumour Microenvironment: Challenges and Future Perspectives for Anticancer Plasma Treatments

“…HOCl causes FN oxidation, which modulates cell adhesion, proliferation, and mRNA expression [177,178] Elastin Entropic elastic behaviour, its stretch is limited by its association with collagen [179] Fibroblast activation in mouse skin to produce elastin [169] ROS destroy elastin network integrity, influence production of ECM proteins [180]. ONOOH induces tyrosine nitration and crosslinking in topoelastin, alters its structure, function, and changes matrix assembly [181] Adhesion to ECM…”