Increased levels of nitrotyrosine and nitrated proteins have been detected in a variety of pulmonary and cardiovascular diseases, and in neurodegenerative and chronic inflammatory disorders. The overall objective of this R01 application is to obtain new mechanistic insight into how the hydrophobic interior of biological membranes facilitates oxidation and nitration reactions of reactive nitrogen species (RNS), such as peroxynitrite (ONOO or ONOOH) or nitrogen dioxide radical (NO2). This proposal is based on the following recent discoveries: 1) peroxynitrite can cross lipid membranes through anion transport channels or passive diffusion at rates significantly faster than their reaction with any other target molecule in the aqueous phase. 2) The reaction between NO and O2 is significantly faster in the membrane interior. 3) Peroxynitrite and NO2 cause extensive nitration of alpha-tocopherol in membranes under conditions where tyrosine nitration in the aqueous phase was negligible. The investigators hypothesize that nitration of phenols and nitrosation of thiols by RNS in biological systems is increased in a hydrophobic environment. To investigate the nitration and nitrosation reactions in membranes, they will synthesize tyrosylated lipid and tyrosine- or cysteine-containing peptides that are anchored at defined locations in the lipid bilayer. The investigators will use HPLC, stop-flow spectrophotometry, mass spectrometry, and spin trapping to investigate nitration and nitrosation reactions in membranes. Specifically, the PI will: 1) compare the yields of formation of nitro-gama-tocopherol in membranes and nitrotyrosine in the aqueous phase; 2) detect and characterize nitration products of tyrosylated lipid; 3) determine the mechanism of nitration and nitrosation of tyrosine- and cysteine-containing peptides in membranes; and 4) use nitro-gama-tocopherol or nitrated transmembrane peptide as a marker product to detect peroxynitrite formation from nitric oxide synthase enzymes. This comprehensive study of RNS reactions in simple well-defined model membrane system may provide new mechanistic insight for understanding oxidative and nitrosative stress in pulmonary cardiovascular, neurodegenerative, and inflammatory diseases.
Showing the most recent 10 out of 41 publications
