Mechanisms and Biological Consequences of Damage to Extracellular Matrix Proteins by Peroxynitrite

Abstract

Peroxynitrite (ONOO–) is a potent oxidizing and nitrating agent, formed under inflammatory conditions by the diffusion-controlled reaction of superoxide radicals (O2•–) with nitric oxide (•NO). This species reacts rapidly, via both non-radical and radical reactions, with proteins resulting in protein damage. As there are limited antioxidant and repair enzymes extracellularly, damage to extracellular matrix (ECM) proteins is likely to persist and accumulate over time. However, there is a lack of detailed mechanistic information on how ONOO– oxidation and nitration affects ECM proteins. This Thesis investigates the significance of these reactions and how this modulates the structure and function of three different isolated ECM proteins, laminin-111, plasma fibronectin, recombinant tropoelastin and a complex mixture of basement membrane proteins. Exposure of ECM proteins to ONOO– at physiological relevant concentrations (10 μM) lead to protein aggregation and fragmentation. Significant amounts of nitration products (3-nitroTyr, 6-nitroTrp) in the millimolar-range and depletion of corresponding parent Tyr and Trp were detected on all analysed ECM proteins with this effect being modulated by CO2. Furthermore biological function and activity was compromised post ONOO– exposure. Overall, the studies presented in this Thesis provide new information about the reactivity of ONOO– with ECM proteins, and how this affects the structure and the function of these proteins.