Leelavanichkul, Karanee
(2005)
NITRIC OXIDE-INDUCED MODIFICATION OF PROTEIN THIOLATE CLUSTERS AS DETERMINED BY SPECTRAL FLUORESCENCE RESONANCE ENERGY TRANSFER IN LIVE ENDOTHELIAL CELLS.
Master's Thesis, University of Pittsburgh.
(Unpublished)
Abstract
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.
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| Item Type: |
University of Pittsburgh ETD
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| Status: |
Unpublished |
| Creators/Authors: |
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| ETD Committee: |
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| Date: |
20 June 2005 |
| Date Type: |
Completion |
| Defense Date: |
24 February 2005 |
| Approval Date: |
20 June 2005 |
| Submission Date: |
14 March 2005 |
| Access Restriction: |
No restriction; Release the ETD for access worldwide immediately. |
| Institution: |
University of Pittsburgh |
| Schools and Programs: |
Swanson School of Engineering > Bioengineering |
| Degree: |
MSBeng - Master of Science in Bioengineering |
| Thesis Type: |
Master's Thesis |
| Refereed: |
Yes |
| Uncontrolled Keywords: |
5Œ-cyclic monophosphate; Free radicals; Green fluorescent protein; Guanosine 3Œ; Live cell imaging; Metallothionein; Nitrosothiol; S-nitrosation |
| Other ID: |
http://etd.library.pitt.edu/ETD/available/etd-03142005-104552/, etd-03142005-104552 |
| Date Deposited: |
10 Nov 2011 19:32 |
| Last Modified: |
15 Nov 2016 13:37 |
| URI: |
http://d-scholarship.pitt.edu/id/eprint/6494 |
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