PP33 - Impact of tyrosine nitration on cellular glutamine synthetase turnover and functionality

Abstract

During oxidative damage to proteins by reactive nitrogen species (RNS), post-translational modifications can lead to dysfunctional proteins and promote their aggregation over time. One of the protein modifications via RNS, such as peroxynitrite (ONOO-), is protein tyrosine nitration. It is based on an initial one-electron oxidation of the aromatic phenol ring of tyrosine followed by the addition of ONOO--derived products, such as nitric dioxide. For glutamine synthetase (GS), an important enzyme for the detoxification of ammonia in brain, it has been shown that ONOO- is able to impair enzyme activity. Thus, we are investigating the link between protein nitration and the loss of enzyme function, to clarify under which conditions the protein activity is declining. Therefore, cellular GS from human astrocytes as well as the human recombinant protein (rGS) were studied. Nitration was induced by the ONOO- donor SIN-1 and quantified immunochemically by an anti-protein-3-Nitrotyrosine (3-NT) antibody. Besides the studies on the formation of nitrated GS and its nitration state after a recovery time, we further followed the impact of SIN-1 on enzyme functionality by a GS activity assay. In order to follow the degradation of GS, we added RAW 264.7 cell lysates (w and w/o LPS) to nitrated GS and analyzed it on protein and activity level. Preliminary results showed an increased formation of ONOO- and an induction of 3-NT after incubation with SIN-1. Further, GS activity was decreased by SIN-1 in both, cellular GS and rGS. After a 3h-recovery period, GS activity increased again in cells. Removing of 3-NT-modified proteins by a putative denitration system might be a possibility to maintain protein function and impede protein aggregation.