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 ainway 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-nitrosothiolmodified proteins is poorly understood in airway epithelial cell cultures in general, and has not been studied in primary, pseudostratified columnar ainway epithelium. In order to begin to understand NOS-dependent Snitrosothiol formafion in the ainways 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 Snitrosylation 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 ainway epithelial cells;and because decreased INOS expression in the CF ainway 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 Snitrosoglutathione treatment;and 3) its S-nitrosylation appears to be important to ainway 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 ainway 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.

Agency
National Institute of Health (NIH)
Type
Research Program Projects (P01)
Project #
5P01HL101871-05
Application #
8646968
Study Section
Heart, Lung, and Blood Program Project Review Committee (HLBP)
Project Start
Project End
Budget Start
Budget End
Support Year
5
Fiscal Year
2014
Total Cost
Indirect Cost
Name
Case Western Reserve University
Department
Type
DUNS #
City
Cleveland
State
OH
Country
United States
Zip Code
Zaman, Khalequz; Sawczak, Victoria; Zaidi, Atiya et al. (2016) Augmentation of CFTR maturation by S-nitrosoglutathione reductase. Am J Physiol Lung Cell Mol Physiol 310:L263-70
Raffay, Thomas M; Dylag, Andrew M; Di Fiore, Juliann M et al. (2016) S-Nitrosoglutathione Attenuates Airway Hyperresponsiveness in Murine Bronchopulmonary Dysplasia. Mol Pharmacol 90:418-26
Marozkina, Nadzeya V; Gaston, Benjamin (2015) Nitrogen chemistry and lung physiology. Annu Rev Physiol 77:431-52
Sawczak, Victoria; Getsy, Paulina; Zaidi, Aliya et al. (2015) Novel Approaches for Potential Therapy of Cystic Fibrosis. Curr Drug Targets 16:923-36
Palmer, Lisa A; Kimberly deRonde; Brown-Steinke, Kathleen et al. (2015) Hypoxia-induced changes in protein s-nitrosylation in female mouse brainstem. Am J Respir Cell Mol Biol 52:37-45
Sun, Yuansheng; Periasamy, Ammasi (2015) Localizing protein-protein interactions in living cells using fluorescence lifetime imaging microscopy. Methods Mol Biol 1251:83-107
Wallrabe, Horst; Sun, Yuansheng; Fang, Xiaolan et al. (2015) Three-color confocal Förster (or fluorescence) resonance energy transfer microscopy: Quantitative analysis of protein interactions in the nucleation of actin filaments in live cells. Cytometry A 87:580-8
Marozkina, Nadzeya V; Wang, Xin-Qun; Stsiapura, Vitali et al. (2015) Phenotype of asthmatics with increased airway S-nitrosoglutathione reductase activity. Eur Respir J 45:87-97
Davisson, Robin L; Bates, James N; Johnson, Alan Kim et al. (2014) Effects of intracerebroventricular injections of 5-HT on systemic vascular resistances of conscious rats. Microvasc Res 95:116-23
Periasamy, Ammasi (2014) Advanced light microscopy. Methods 66:121-3

Showing the most recent 10 out of 35 publications