Effect of Second-Hand Tobacco Smoke on the Nitration of Brain Proteins: A Systems Biology and Bioinformatics Approach

Abstract

Second-hand smoke (SHS) exposure leads to the death of approximately 48,000 nonsmokers per year in the United States alone. SHS exposure has been associated with cardiovascular, respiratory, and neurodegenerative diseases. While cardiac function abnormalities and lung cancer due to SHS have been well characterized, brain injury due to SHS has not undergone a full systematic evaluation. Oxidative stress and nitration have been associated with smoking and SHS exposure. Animal studies suggest that exposure to tobacco smoke increases oxidative stress. Oxidative stress is characterized by an increase in reactive oxygen and nitrogen species (ROS/RNS). Among the oxidative mechanisms affecting protein functionality is the posttranslational modification (PTM)-mediated tyrosine nitration. Protein tyrosine nitration, a covalent posttranslational modification, is commonly used as a marker of cellular oxidative stress associated with the pathogenesis of several neurodegenerative diseases. In our previous published work, the utility of a targeted proteomic approach has been evaluated to identify two brain abundant proteins in an in vivo SHS rat model namely the GAPDH and UCH-L1. In this current study, mass spectrometric-based proteomic and complementary biochemical methods were used to characterize the SHS-induced brain nitroproteome followed by bioinformatics/systems biology approach analysis to characterize protein interaction map. Sprague Dawley rats were exposed to SHS for 5 weeks and then cortical tissues were collected. Nitroprotein enrichment was performed via 3-Nitro tyrosine (3-NT) immunoprecipitation of brain lysates proteins. Protein nitration was validated via Western blotting to confirm the presence of nitroproteins complemented by gel-free neuroproteomic analysis by data-dependent LC-MS/MS. We identified 29 differentially expressed proteins in the 3-NT-enriched samples; seven of these proteins were unique to SHS exposure. Network analysis revealed an association of the proteins to different cellular processes including oxidative stress, ROS generation, and cell death-related pathway. This confirms the association of oxidative stress mechanisms with SHS which may contribute to neuronal injury, an area that has not been well studied in the area smoking.