Overproduction of nitric oxide (NO) by inducible nitric oxide synthase (iNOS) has been implicated in the pathogenesis of many pathological disorders, including airway inflammation associated with asthma. The long-term goal of this research is to understand the regulation of iNOS activity and to devise novel methods to regulate it. Selective modulation of iNOS requires better understanding of its cellular biology. Although much is known about factors affecting the synthesis and catalytic activity of iNOS, little is known about its posttranslational regulation. The overall objective of this research proposal is to understand the posttranslational mechanisms of iNOS turnover in epithelial cells. We have recently shown that iNOS is primarily degraded through the 26S proteasome. In addition, we have shown that iNOS ubiquitination is required for its degradation. However, the molecular mechanisms by which this process is regulated remain to be studied. Our preliminary data suggest that iNOS is subject to phosphorylation and that this process precedes its ubiquitination. They further suggest that iNOS monomers (inactive form) may be preferentially targeted for degradation compared to dimers (active form). We hypothesize that A): phosphorylation on specific iNOS residue(s) regulates iNOS ubiquitination and hence its degradation. Furthermore, specific lysine residue(s), which are involved in iNOS ubiquitination lie within the oxygenase domain (dimer interface), thus allowing for faster turnover of iNOS monomers, with exposed lysine residues. B) Cells maintain a tight control over the regulation of NO synthesis by maintaining a robust rate of iNOS turnover, thus allowing rapid modulation of iNOS degradation by phosphorylation and by other cellular factors that relate to cell type, availability of substrate and/or cofactors. To test these hypotheses we propose studies with the following Specific Aims: 1) Elucidation of the role of phosphorylation in iNOS turnover. 2) Characterization of specific ubiquitin ligase activity responsible for iNOS ubiquitination. 3) Determination of specific sites of iNOS ubiquitination. 4) Characterization of the rate of iNOS turnover by examining factors that modulate it. Studies will be conducted in epithelial cells expressing iNOS and in primary bronchial epithelial cells cultured at the air/liquid interphase. The rational for the proposed studies is that once these mechanisms are understood, therapeutic strategies can be designed to modulate iNOS turnover.
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