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 (HbO₂). 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-α] 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.
| Identifer | oai:union.ndltd.org:PITT/oai:PITTETD:etd-03142005-104552 |
| Date | 20 June 2005 |
| Creators | Leelavanichkul, Karanee |
| Contributors | Partha Roy, Claudette M. St. Croix, Bruce R. Pitt |
| Publisher | University of Pittsburgh |
| Source Sets | University of Pittsburgh |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.library.pitt.edu/ETD/available/etd-03142005-104552/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to University of Pittsburgh or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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