Abstract 11370: Quantification of S-Nitrosation Indicates Thiol Reactivity Can Gauge the Magnitude of Oxidative Stimuli
Oxidative species modify critical cysteine thiols, known as redox-switches, which sense and respond to the cell's fluctuating environment. Depending on the magnitude, these fluctuations can regulate metabolism, activate protective mechanisms or be cytotoxic. Redox-signaling is thought to derive from an integration of oxidant species, its solution chemistry and cellular localization. However, less is known about the nature of the cysteine residues being targeted or how the oxidative signals are interpreted. Here we tested the hypothesis that protein thiols have variable reactivity which can detect the magnitude of oxidative stimuli.
Methods: S-nitrosation (SNO) was measured in human pulmonary arterial endothelia cells in vitro and in vivo using a novel cysTMT6 quantitative biotin switch assay and mass spectrometry. Slope (m) of individual thiol reactivitives were determined by quantitatively assessing the response of these sites to various NO-donor concentrations (2, 10, 20 µM).
Results: The in vitro approach led to the identification of 220 SNO-modified cysteine residues on 179 different proteins. Modified sites displayed a continuum of thiol reactivity (m=0.5 most to 3.5 least). Treatment of living cells with 50 and 200 µM SNO-Cys revealed, that in vivo thiol reactivity correlated with the magnitude of oxidative stimuli (50 µM, m=0.78 to 1.18; 200 µM, m=0.78 to 2.33). SNO-sites obtained from a previous study using 1 mM SNO-Cys were found to modify further insensitive sites (m=0.61 to 3.5) (see figure). An additional pool of cysteines was found to be available but unmodified suggesting capacity for greater oxidative input.
Conclusions: The variable thiol reactivity of cellular cysteine sensors can gauge the magnitude of oxidative stimuli constituting a new mechanism in redox-signaling. Based on this, we propose the progressive and specific accumulation of modified redox-switches contributes to the cell's detection and response to oxidative signals.
- © 2011 by American Heart Association, Inc.