NITRIC OXIDE-INDUCED MODIFICATION OF PROTEIN THIOLATE CLUSTERS AS DETERMINED BY SPECTRAL FLUORESCENCE RESONANCE ENERGY TRANSFER IN LIVE ENDOTHELIAL CELLS
Low-molecular-weight S-nitrosothiols are found in many tissues and recognized to affect a diverse array of signaling pathways via decomposition to ?NO or exchange of their -NO function with thiol containing proteins (transnitrosation). We used spectral laser scanning confocal imaging to visualize the effects of D- and L- stereoisomers of S-nitrosocysteine ethyl ester (SNCEE) on fluorescence resonance energy transfer (FRET)-based reporters that are targets for the following NO-related modifications: (a) S-nitrosation, via the cysteine-rich, metal binding protein, metallothionein (FRET-MT); and (b) nitrosyl-heme-Fe- guanosine 3?,5?-cyclic monophosphate (cygnet-2) in live cells. Conformational changes consistent with S-nitrosation of FRET-MT were specific to L-SNCEE. In addition, they were reversed by dithiothreitol (DTT) but unaffected by exogenous oxyhemoglobin (HbO?). In contrast, D- and L-SNCEE had comparable effects on cygnet-2, likely via activation of soluble guanylyl cyclase (sGC) by ?NO as they were sensitive to the sGC inhibitor, 1H-[1,2,4]-oxadiazolo[4,3-?] quinoxalin-1 (ODQ) and exogenous oxyhemoglobin. These data demonstrate the utility of spectral laser scanning confocal imaging in revealing subtle aspects of NO signal transduction in live cells. Stereoselective transnitrosation of MT suggests that the structure of L-SNCEE confers access to critical cysteine(s) in the protein. Such stereo-selectivity underscores the specificity of post-translational modification as a component of NO signaling.
Advisor:Partha Roy; Claudette M. St. Croix; Bruce R. Pitt
School:University of Pittsburgh
School Location:USA - Pennsylvania
Source Type:Master's Thesis
Date of Publication:06/20/2005