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 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 protein metallothionein (FRET-MT), and (b) nitrosyl-heme-Fe, via guanosine 3',5'-cyclic monophosphate (cygnet-2). 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. 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-alpha] quinoxalin-1-one 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 emphasizes the specificity of posttranslational modification as a component of NO signaling.
Department of Environmental and Occupational Health, University of Pittsburgh Graduate School of Public Health, Pittsburgh, PA 15260, USA. cls13@pitt.edu
Claudette M
CM
St  Croix
Molly S
MS
Stitt
Karanee
K
Leelavanichkul
Karla J
KJ
Wasserloos
Bruce R
BR
Pitt
Simon C
SC
Watkins
D000818:Animals; D002478:Cells, Cultured; D006152:Cyclic GMP / Q000378:metabolism; D003545:Cysteine / Q000031:analogs & derivatives* / Q000737:chemistry; D004229:Dithiothreitol / Q000737:chemistry; D004730:Endothelium, Vascular / Q000166:cytology; D004789:Enzyme Activation; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D005456:Fluorescent Dyes / Q000494:pharmacology; D005609:Free Radicals; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006162:Guanylate Cyclase; D006418:Heme / Q000737:chemistry; D008856:Microscopy, Fluorescence / Q000379:methods*; D009569:Nitric Oxide / Q000737:chemistry / Q000378:metabolism*; D009603:Nitroso Compounds / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry; D018160:Receptors, Cytoplasmic and Nuclear / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012756:Sheep; D015398:Signal Transduction; D000071756:Soluble Guanylyl Cyclase; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013438:Sulfhydryl Compounds / Q000737:chemistry*
D005456:Fluorescent Dyes; D005609:Free Radicals; D009603:Nitroso Compounds; D010455:Peptides; D011506:Proteins; D018160:Receptors, Cytoplasmic and Nuclear; D011993:Recombinant Fusion Proteins; C429310:S-nitrosocysteine ethyl ester; D013438:Sulfhydryl Compounds; D049452:Green Fluorescent Proteins; D009569:Nitric Oxide; D006418:Heme; D006162:Guanylate Cyclase; D000071756:Soluble Guanylyl Cyclase; D006152:Cyclic GMP; D003545:Cysteine; D004229:Dithiothreitol