and specific aims): This project represents a multidisciplinary approach overlapping chemistry, biochemistry, and cell biology, to study the effect of endogenously and exogenously formed nitric oxide (NO) and other reactive nitrogen species (RNS) upon protein modification in the lung. The overall hypothesis is the glutathione S-transferases are preferential targets for tyrosine nitration and chlorination in the inflamed lung, and hence, may serve as appropriate dosimeters for the production of reactive species in vivo. In addition, nitration and/or chlorination of functional tyrosine residues in GSTs may result in enzyme inactivation and weakened cellular defenses.
The specific aims are: 1) to refine existing analytical methods for the simultaneous and site-specific measurement of multiple protein modifications (tyrosine nitration and chlorination) in glutathione S-transferases using electrospray ionization tandem mass spectrometry; 2) to ascertain whether chemical- and neutrophil-mediated nitration and chlorination of conserved active-site tyrosyl residues in purified rat and human glutathione S-transferases inhibits their activity in vitro, and to determine which isozymes are most susceptible to modification and inactivation; 3) to determine whether reactive nitrogen species or stimulated human neutrophils induce nitration and/or chlorination of active-site tyrosines in glutathione S-transferase isozymes in cultured human tracheobronchial epithelial cells (HTBECs), and to determine whether this results in decreased activity of this cellular defense system; and 4) to determine whether nitrated and chlorinated glutathione S-transferases are present in lung tissue from cigarette smokers and patients with inflammatory lung diseases, and to correlate the extent to which these modifications impair the function of this critical cellular defense system.
