Nitric oxide synthase (NOS) activation can result in formation of relevent nitrogen oxide diseases, other than nitric oxide radicals. One of these is nitrite. Additionally, covalent cysteine modifications form Snitrosothiol bonds. When this cysteine modification signals a change in protein function, it is often termed Snitrosylation. These reactions are increasingly recognized to represent metabolically regulated cell signaling processes. Disorders of airway epithelial S-nitrosylation signaling have been observed in a range of diseases, including asthma and cystic fibrosis (CF). However, the formation and location of S-nitrosothiol modified proteins is poorly understood in airway epithelial cell cultures in general, and has not been studied in primary, pseudostratified columnar airway epithelium. In order to begin to understand NOS-dependent S-nitrosothiol formation in the airways as it relates to disease, we will test the three hypotheses that 1) specific proteins in normal human airway epithelial cells are S-nitrosylated by NOS (Aim 1);2) protein S-nitrosylation occurs in specific subcellular locations in human airway epithelial cells (Aim 2);and 3) S-nitrosylation signaling is disordered in the human CF airway epithelium (Aim 3). We chose to study S-nitrosylation by the inducible and endothelial NOS (iNOS and eNOS) isoforms because each is expressed and active in normal human airway epithelial cells;and because decreased INOS expression in the CF airway epithelium may have important disease implications. Hsp70/Hsp90 organizing protein was chosen to study the paradigm of S-nitrosothiol signaling downstream from iNOS and eNOS activity because 1) it is S-nitrosylated at baseline;2) its S-nitrosylation increases with S-nitrosoglutathione treatment;and 3) its S-nitrosylation appears to be important to airway epithelial cell biology in general, and to the pathobiology and treatment of CF in particular. We have chosen to perform our studies primarily in human airway pseudostratified columnar epithelial cultures because these most closely to recapitulate the human airway in vivo. We will do additional in vivo studies in a mouse model. This project will make extensive use of interactions with investigators on the other projects in this program, and each aim will make use of one or more cores in the program. At the conclusion of this project, we anticipate that we will have 1) a functional model of mechanisms by which NOS activation leads to Snitrosylation of specific proteins in specific cellular locations in human pseudostratified columnar epithelium;and 2) the relevance of disorders of S-nitrosothiol formation to the development of new therapies for CF.
The mechanisms by which NOS signals S-nitrosylation in the human airway epithelium are poorly understood. Our preliminary data suggest that disorders of S-nitrosylation signaling are relevant to the development of new corrector therapies for CF patients. Additionally, this mechanism?and the targets of S-nitrosylation we will study?may be relevant to the underlying pathophysiology not only of CF, but of other pulmonary diseases.
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