Asthma associated disability, morbidity, and mortality continue to increase. Although the causes remain unknown, asthma is now considered as a chronic inflammatory syndrome. Overproduction of nitric oxide (NO) by inducible nitric oxide synthase (iNOS) has been implicated in the pathogenesis of airway inflammation of asthma. The long-term goal of this research is to understand the regulation of iNOS activity and devise novel methods to regulate it. Although much is known about factors affecting the synthesis and catalytic activity of iNOS, little is known about its degradation mechanisms or its subcellular localization. Potentially, acceleration of iNOS degradation is an appealing approach for NO modulation since the process of targeting a cellular protein for degradation is highly selective. The overall objective of this research proposal is to understand the mechanisms of iNOS degradation in epithelial cells. Our preliminary data suggest that human iNOS is degraded primarily by the ubiquitin-proteasome pathway, a pathway responsible for the selective degradation of a number of short-lived regulatory proteins whose activity must be tightly regulated. We hypothesize that: a) iNOS is regulated by specific degradation mechanisms primarily involving the ubiquitin-proteasome pathway and that degradation occurs at the membrane subcellular component; b) iNOS degradation can be accelerated with inhibitors that prevent iNOS dimerization. To test these hypotheses, we propose studies with the following specific aims: 1) Identification of human iNOS degradation pathway by testing the effect of specific inhibitors of various degradation pathways on iNOS half-life. 2) Characterization of possible role of ubiquitination in iNOS degradation by determining whether or not iNOS is ubiquitinated and if ubiquitination is required for targeting iNOS to degradation. 3) Elucidation of the subcellular localization of human iNOS using fluorescence microscopy in the absence or the presence of inhibitors that block iNOS degradation, leading to its accumulation. 4) Analysis of modulation of iNOS degradation by various classes of iNOS inhibitors. Studies will be conducted in epithelial cell lines expressing human iNOS and in airway bronchial epithelial cells obtained by bronchoscopy from normals. The rational for the proposed studies is that once the degradation mechanisms of iNOS are understood, therapeutic strategies can be designed to alter these pathways and accelerate iNOS degradation. The results of these studies will increase our understanding of the cellular process of iNOS regulation and thus lay the groundwork for future studies aiming at controlling NO synthesis in asthma.
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